Table of contents :

• hypoplasias (leukopenia (< 4 ^. 10³ / mL) : granulocytopenia (neutropenia + eosinopenia + basophilopenia) + lymphopenia)
• hyponeocytosis : leukopenia with immature forms of leukocytes present in the blood
• hypo-orthocytosis : leukopenia in which the proportion of the various forms of leukocytes is normal
• lymphocytes (lymphopenia / lymphocytopenia (< 1.5 ^. 10³ / mL in adults or < 3 ^. 10³ / µL in children < 2 yr))

Aetiology :
• sarcoidosis
• systemic lupus erythematosus (SLE)
• autoimmune lymphopenia
• immunodeficiencies
• Salmonella typhi
• influenzavirus
• B lymphopenia
• T lymphopenia : apoptosis of T lymphocytes in vitro, linked to transient T cell loss in vivo, is a consequence of certain viral infections, including lymphocytic choriomeningitis^ref and vaccinia^ref in mice and EBV^ref in humans. It has also been reported in association with HIV-1^{ref1, ref2} in humans, although whether or not direct HIV infection is required for this effect, and its specificity for CD4⁺ T cells, are subjects of debate^ref
• CD4⁺ lymphopenia

Aetiology :
• neoplasia
• HIV-1
• type II BLS / class II MHC deficiency
• idiopathic CD4⁺ lymphocytopenia (ICL) / severe unexplained HIV-seronegative immune suppression (SUHIS) : CD4⁺ count <300 cells/ml or a CD4⁺ count that is <20% of the total T cell count on 2 occasions, with no evidence of retroviral infections (HIV-1 or -2, HTLV-1 or -2) on testing, and absence of any defined immunodeficiency or therapy that depresses the levels of CD4⁺ T cells (unaccompanied by increased CD8⁺ T lymphocytes and hypergammaglobulinemia) either alone (ICL according to CDC, 1992^ref) or accompanied by clinical signs and symptoms of cellular immune deficiency (SUHIS according to WHO^ref). Cases have been identified since 1983^ref. It is likely that others could have been identified much earlier; however, the determination of CD4⁺ T-cell counts has been routine only since the early to mid-1980s and the beginning of the HIV epidemic. ICL differs from HIV infection by stable levels of CD4⁺ T cell counts in contrast to the progressive loss of this subpopulation observed in the course of HIV disease^{ref1, ref2, ref3}. Anyway recently a subset of ICL patients mimicking HIV disease has been reported: severe, prolonged decrement in peripheral CD4⁺ T cells with progressive decline in such counts documented in some; CD4/CD8 T cell ratios <= 1; and a history of opportunistic infections^{ref1, ref2}. Moreover, a great heterogeneity in the symptomatology and the T cell phenotypic abnormalities have been reported among patients with ICL.

Epidemiology : 0.5–2% of adults^{ref1, ref2, ref3}. 30% of the patients are women, as compared with 11% among those with HIV or AIDS in the USA
Aetiology :
• although Laurence et al.^ref and Gupta et al.^ref suggested in 1992 that ICL could be due to a new retrovirus distinct from HIV-1 and HIV-2 (a human intracisternal A-type retrovirus, HIAP-II, was detected in a subset of patients with ICL : most of them were also ANA positive^ref), epidemiologic studies showed no evidence for a transmissible agent : all close contacts and sexual partners who were studied were clinically well, including 31 sexual partners^{ref1, ref2, ref3} in whom serologic, immunologic, and virologic studies for HIV were negative. Indeed, ICL can be diagnosed with the onset of opportunistic infections or among autoimmune disorders, whereas it remains asymptomatic in other patients^{ref1, ref2, ref3, ref4}. In addition to the CD4⁺ lymphocytopenia, several patients also display a CD8⁺ lymphocytopenia while low B or NK cell counts have also been reported in others^{ref1, ref2, ref3}. The heterogeneity of ICL does not favor the hypothesis of a unique cause^ref (Moore, J. P. and Ho, D. D. 1992. HIV-negative AIDS. Lancet 340:475). CD4 cell counts of < 500 cells/ml were, however, associated with subsequent HIV seroconversion^ref. A subset of patients has anyway antibodies against retroviral proteins and nuclear antigens^ref . Low CD4⁺ counts are rare among anti-HIV-1-negative volunteer blood donors and are generally associated with transient illnesses. If any unknown virus progresses similarly to HIV-1, CD4⁺ count donor screening would be a poor surrogate for its detection^ref. There is also no epidemiologic evidence to suggest that a transmissible microbe is involved. The cases of idiopathic CD4⁺ T-lymphocytopenia were widely dispersed, with no clustering.
• some authors have suggested ICL to be classified among common variable immunodeficiency^ref. Similarly, a case of ICL associated with recurrent opportunistic infections could be attributed to a primary immunodeficiency disorder^ref
• ICL was found in 16% of anti-SSA seropositive primary Sjogren's syndrome patients^ref
• 2 familial cases have been reported^{ref1, ref2}
• a recent study of "normal" persons reported a substantial proportion of subjects with CD4⁺ T lymphocytopenia, suggesting that this condition may occur as a result of the inherent variability in the measurement of T lymphocyte subsets^ref
• it is hypothesised that CD4⁺ T lymphocytopenia represents the tail end of natural statistical variation in CD4+ cell counts^ref
Pathogenesis : increased spontaneous and activation-induced apoptosis was associated with enhanced expression of Fas and FasL in unstimulated cell populations, and partially inhibited by soluble anti-Fas mAb^ref. Antihistone autoantibodies, presumably induced by fragmented chromatin, were detected in some of these sera, with reactivity predominantly to type H2B, a pattern identical to that found in HIV disease^{ref1, ref2} and SIV infection in macaques^ref, both of which have been associated with CD4⁺ T cell apoptosis: in vitro in the case of HIV^{ref1, ref2}, and in vivo in macaques^ref. Alternatively, other studies reported proliferative T cell defects to mitogens or antigens in patients with ICL and opportunistic infections^{ref1, ref2, ref3, ref4}. Major reduction of the proliferative response to CD3-TCR stimulation that affected only the depleted T-cell subpopulation^ref. Abnormality of the PTK p56^Lck in CD8⁺ T cells might play a role : a full-length p56^Lck was expressed in T lymphocytes which rather displayed a 50% decrease in autophosphorylation and in in vitro kinase activity. This observation contrasted with the conserved protein tyrosine phosphorylation process induced by CD3 triggering in the patient's CD4⁺ and CD8⁺ T cells^ref.  In HIV patients a CD4⁺ T cell proliferation deficiency is associated with an impaired CD3-induced tyrosine phosphorylation process, and altered levels of p56^Lck and p59^{Fynref1, ref2, ref3, ref4}.
Symptoms & signs : the CD4 deficit in these patients is not associated with other cellular and/or humoral immunological anomalies and the clinical manifestations, essentially of scant importance, have not shown signs of progression towards severe immunodeficiency syndromes. Anyway severe ICL predisposes to the same opportunistic infections as AIDS :
• dementia and encephalopathy^ref
• viral infections :
• 3 cases of PML have been reported in ICL^{ref1, ref2, ref3}
• EBV and CMV coinfection of the central nervous system^ref
• empyema thoracis and cytomegaloviral retinitis^ref
• cutaneous infections by papillomavirus, relapsing generalized herpes zoster^ref and Candida albicans^ref
• juvenile laryngeal papillomatosis (JLP)^ref
• bacterial infections :
• chronic mucocutaneous candidiasis (CMC), recurrent abscesses, and relapsing aphthous and ulcerous lesions. In addition to ICL the patient frequently showed a panlymphocytopenia. An increased percentage of gd⁺ T lymphocytes and IgD⁺ IgM⁺ B lymphocytes, and a decreased percentage of CD21⁺ B lymphocytes, were observed. In vitro assays showed normal T-cell responses to candidin and T-cell mitogens, but impaired B-cell responses to PWM. B-cell maturation after stimulation with Staphylococcus aureus Cowan I (SAC) and IL-2 was nearly normal^ref.
• chronic severe mycobacterial disease^ref and disseminated infection with Mycobacterium avium^ref
• fungal infections :
• pleural effusion due to Histoplasma capsulatum^{ref1, ref2}
• cryptococcal infections :
• pulmonary cryptococcosis and lung cancer^ref
• cryptococcal meningitis^{ref1, ref2, ref3}
• recurrent oral candidiasis who subsequently developed cryptococcal meningitis^ref
• active intestinal tuberculosis with esophageal candidiasis^ref
• episodic erythematous candidiasis, persistent angular cheilitis, lingua exfoliativa areata, and teleangiectasia of facial skin and buccal mucosa^ref
• Pneumocystis carinii and Hemophillus influenzae pneumonia^ref
• protozoal infections :
• toxoplasmic myositis^ref
• cancers : 6 cases with NHL, including 1 with primary leptomeningeal lymphoma^ref
• epilepsy^ref
• intracranial haemorrhage^ref
Association with selective IgA deficiency has been reported^ref
Therapy : weekly subcutaneous polyethylene glycol (PEG)-IL-2 injections 50 000 U/m² for 5.5 years^{ref1, ref2} and antimicrobials^ref
Prognosis : ICL patients have a longer survival time than AIDS cases without HAART
• CD8⁺ lymphopenia

Aetiology :
• type I BLS / class I MHC deficiency
Analytic variability : the temperature of the specimen in transit, the type of anticoagulant used, and the delay in analysis are of some importance [9]; however, the major source of analytic variability is the actual phenotype measurement, which is typically done using murine monoclonal antibodies and flow cytometric analysis. Absolute subset values are a product of three components: the total leukocyte count, the lymphocyte differential, and the percentage of CD3+ T lymphocytes that express membrane CD4 or CD8. Published guidelines have minimized technical difficulties, including problems of coexpression of CD4 and CD8, contamination of preparations by CD4+ but CD3- non-T cells, and genetic polymorphisms among lymphocyte antigenic determinants [9]. No mandatory standards exist, however, for immunophenotyping by flow cytometric analysis, and limited information is available on the degree of interlaboratory variability. Indeed, both analytic and biologic variability exist in all three test components. One quality assessment found that no technical change related to instrument, monoclonal antibody, or fluorochrome label would significantly improve interlaboratory agreement on CD4 measurements [10]. Yet, it is heartening that a recent multicenter proficiency test of 13 laboratories found that the analytic variability for the percentage and absolute number of CD4+ T cells using specimens from normal controls was 4.1% and 8.4%, respectively, compared with values of 6% and 29.4%, respectively, measured 4 years previously [9].
Biological variability may be even greater in magnitude than analytic variability. First, circannual (seasonal) rhythms may occur, with a 13% change from week to week in total lymphocyte counts [11] and with substantial alteration in absolute CD4 and CD8 counts from month to month [12]. No such variability was seen in the CD4:CD8 ratio or in the total number of CD3+ T lymphocytes [12], however, and persons maintained a fixed, discrete range of CD4 values when followed for periods of 2 [13] or 5 [14] years, even among those with initial values less than 300/mm3. Other quantifiable factors that can influence these cell populations include age, sex, ethnic origin, circadian rhythm, physical and psychological stresses, drugs (such as zidovudine, cephalosporins, cancer chemotherapeutic agents, nicotine, adrenal and gonadal steroids), antilymphocyte autoantibodies, and splenectomy [7, 9, 15, 16] (Table 1). Sex need not be taken into account when assessing CD8 counts. In one study, however, the mean CD4 percentages were greater for women than for men by 3.5% (P = 0.0001), and the CD4:CD8 ratio was 0.17 units greater for women than for men [17]. Age also does not affect CD8 values, but an increase of 1.1% per decade occurs in the percentage of CD4+ cells, and a 0.09-unit-per-decade increase in CD4:CD8 ratio in persons older than 20 years [17]. Without appropriate correction, as many as 10% of elderly patients (>70 years) would be classified as having values higher than published "normal ranges" [17].
Effect of Psychological and Physical Stressors and Splenectomy on T-Cell Subpopulations*

Standard values for T-cell subsets have been generated using a Monte Carlo procedure for nongaussian distribution and the best-fit distribution of each parameter [17]. However, the literature offers contradictory assessments, usually based on small samples. For example, although most studies have described age-associated increases in the percentage of CD4+ T cells with no effect on CD8 values, a few noted decreases in the percentage of CD8+ cells [18-20]. Some studies have reported that the absolute number of CD4+ T cells remains stable with advancing age [20], whereas others have shown a decline as lymphocytes constitute a lower percentage of peripheral leukocytes [19]. In one large survey of adolescents between ages 11 and 16 years, absolute counts did not differ from adult values [14]. In addition, the inclusion of heterosexual women did not statistically affect the overall mean values for the various subsets [14]. An earlier report supported these basic tenets, except for the fact that the "adult pattern" of CD4 counts and CD4:CD8 ratios was not seen in adolescents between ages 12 and 16 years [21]. It should also be recognized that some blacks and Asians may lack or be heterozygous for one CD4 epitope, defined by the OKT4A monoclonal antibody, but not by the Leu-3a reagent [4, 18]. Otherwise, race does not appear to significantly influence CD4 or CD8 determinations, at least in the absence of physiologic lymphopenia [18, 21].

Proper accounting of these variables is important in evaluating an individual patient, although these factors are unlikely to lead to a diagnosis of ICL/SUHIS or to be associated with a progressive decrease in any cell population. Biologic variability due to diurnal rhythms may be of greater significance, however. From a nadir at approximately 12:30 hours, CD4 and CD8 counts increase to a peak at about 20:30, whereas CD3 counts reach a zenith somewhat later, at 4:30 [22, 23]. The increment in absolute CD4 counts can be as high as a cumulative 60% [22]. Many laboratories recommend that serial T-cell subset analyses be done on blood samples taken at a standard time of the day [22], but this admonition is rarely addressed in clinical practice or in small studies. Although these changes persist in HIV-positive persons, their magnitude is greatly attenuated [22]. The physiology of this response is unclear, given that neither the circadian organization of steroid secretion from the adrenal cortex nor testis appears to correlate with the 12-hour harmonic of T-lymphocyte circulation [23]. Circadian fluctuations in growth hormone may play a role. Changes in T-cell subsets might also be expected in association with the menstrual cycle, but such alterations have not yet been well documented. Changes linked to pregnancy are noted below.
 

Effects of Pharmacologic, Psychological, and Physical Stressors
 

Changes in T-cell subsets may result from the exogenous administration of adrenal or gonadal steroids. Acute glucocorticoid treatment of three volunteers led to suppression of both CD4 and CD8 counts, from a baseline of 920 ± 33 and 510 ± 31, respectively, to 270 ± 4 and 240 ± 0.14 (mean ± SE), respectively, with a return to normal values within 48 hours [24]. These changes were probably secondary to redistribution of leukocytes among the periphery, bone marrow, lymph node, and spleen, with a decreased efflux from lymphoid organs [25]. Chronic changes secondary to long-term steroid use are less dramatic [26] and must be distinguished from the disease process that prompted use of the drug.

Transient changes may also be seen after severe physical or psychological stress. In one study of 15 healthy persons [27], cognitive stressors caused elevations in heart rate and blood pressure, without affecting serum cortisol or catecholamine levels or absolute T-lymphocyte subsets (Table 1). Physical stress (ergometry), leading to elevation in heart rate, blood pressure, and serum adrenaline and noradrenaline, has been associated with a concomitant increase in the absolute number of CD8+ T cells relative to CD4+ T cells, resulting in a decrease in the CD4:CD8 ratio (Table 1). Splenectomy has also been linked to a stable increase in the percentage, but not the absolute count, of CD4+ cells; an increase in absolute CD8+ cells; and a decrease in the CD4:CD8 ratio Table 1 [28].

Other medical conditions accompanied by severe stress may have immediate effects on peripheral T-cell subsets without long-lasting sequelae. The most dramatic changes have been reported after acute myocardial infarction. In one study [29], although absolute CD4 and CD8 counts did not differ statistically between infarct and control groups, a decrease in CD4:CD8 ratios was noted among infarct patients, with these low values typically persisting for 3 or more days after the event. The CD4:CD8 ratios among the 11 infarct patients (0.83 ± 0.43) differed from both control (2.12 ± 1.13, P = 0.001) and acute sepsis cases (1.76 ± 1.05, P = 0.004); however, no statistical difference was seen in ratios between the control and acute sepsis groups [29].
 

Ranges for T-Lymphocyte Subsets in "Normal Controls"
 

Table 2 shows studies of unselected, asymptomatic adults that provided means, standard deviations, and ranges or 95% CIs for absolute CD4 and CD8 counts. All but three included screening for HIV-1 infection by enzyme-linked immunosorbent assay (ELISA). The largest studies [14, 30, 31] specified that attention was paid to the time of day at which blood was drawn, with duplicate determinations and quality control measures in place; all participants were HIV seronegative. The smaller studies usually did not provide specific information about analytic or biologic variables for controls; these data are included for comparison with the more established normal ranges and as specific controls for the studies listed in Tables 3 and 4. (When HIV serologies were not done in these latter instances, a notation has been included in the appropriate table.) Companion data are given for HIV-seronegative pregnant women as well as for HIV-seronegative controls from the two major risk groups for HIV infection: homosexual men and intravenous drug abusers.
 
 
 

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   Table 2. T-Lymphocyte Subsets: Asymptomatic, Predominantly HIV-Seronegative Controls
 
 
 
 
 

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   Table 3. T-Lymphocyte Subsets: Infectious Diseases in Previously Healthy Persons*
 
 
 
 
 

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   Table 4. Miscellaneous Conditions Associated with Changes in T-Lymphocyte Subsets

The means for CD4+ and CD8+ T-cell counts presented in each of these reports are remarkably similar, despite differences in sex and the broad age groups included as adults. In one study [14], a small number of these ostensibly healthy persons had stable but very low CD4 counts during a 5-year period, technically fulfilling the criteria for idiopathic CD4+ T lymphocytopenia. Clearly, other phenomena must be considered when evaluating CD4+ T-cell counts below the lowest limits of normal. The homeostatic control of peripheral T lymphocytes is susceptible to the various internal and environmental influences described above and conditioned by circulating hormones, cytokines, and other lymphocyte products [46]. In addition, the total number of peripheral T cells appears to be independent of cellular input. In experimental systems, in the absence of either the CD4+ or CD8+ T-cell population, cell loss is routinely compensated for by the remaining subset [46]. This phenomenon is also seen in patients with HIV, given that throughout much of the clinical course the total T-cell levels (CD3+) remain constant in the face of declining CD4 cells, secondary to CD8+ T lymphocytosis [47]. Regulation of these T-cell populations is rapid and flexible, adapting to environmental changes through selection and amplification of appropriate T-lymphocyte clonal specificities. In adults, T cells are replaced primarily by cell proliferation at the periphery. Although more than 30% of such cells are renewed every 3 days, total numbers are relatively fixed, with lymphocytes at varying stages of differentiation having different probabilities of survival, modulated by the environment [46]. Evaluation of T-cell population kinetics, with documentation of stability in absolute values and attention to the subset ratio, is thus important in defining "normal" fluctuations in T-cell counts. Unlike HIV, which involves a progressive decline in CD4 counts that is typically accompanied by a depressed CD4:CD8 ratio, combined changes of "physiologic" CD4+ T lymphopenia (low CD4 percentage and absolute counts <400/mm3), and an inverted ratio occurred in only 0.6% of 500 persons enrolled in one large study [14], and even these markedly depressed values were stable during a 5-year period. This observation has been supported by other studies: only 1 of 275 healthy blood donors had a CD4 count of less than 300/mm3 (CD4:CD8 ratio not reported) [48], and none of 2284 HIV-seronegative homosexual men had CD4 counts of less than 300/mm3 with multiple determinations during a 10-year period [49]. It has been suggested that such persons with an absolute CD4 count physiologically "set" significantly below the means outlined in Table 2, if infected with HIV, might be expected to progress to a clinical definition of the acquired immunodeficiency syndrome (AIDS) at a more rapid rate than a patient whose baseline CD4 counts were significantly greater than the mean [13]. Some anecdotal data support this contention [50], but no evidence exists that these persons are otherwise compromised immunologically. Unless long-term stability of CD4 counts less than the 95% CI and CD4:CD8 ratios of greater than 1.0 are documented, such persons with low CD4 cell counts warrant further evaluation. Indeed, before the identification of HIV-1 and HIV-2 as the primary etiologic agents of AIDS, the possibility of segregating healthy homosexual men from those at potential risk for disease, using a combination of depressed absolute CD4 counts and CD4:CD8 ratio, was suggested [34]. One blood center even conducted a T-lymphocyte subset analysis as an interim screening procedure for a putative "AIDS agent" [51]. Nearly 2% of 8715 consecutive volunteer blood donors between 17 and 77 years old had CD4:CD8 ratios  0.85, and blood from these persons was not used for clinical purposes. Most had concomitant low CD4 values, and follow-up showed that some belonged to AIDS risk populations, despite denials at the time of donation [51]. Other studies examining persons at high risk for HIV who were repeatedly seronegative for HIV by ELISA and immunoblotting but who had HIV-1 proviral DNA detectable by polymerase chain reaction [35, 52], support the use of CD4 counts in conjunction with the CD4:CD8 ratio. Similarly, among male homosexual couples discordant for HIV-1 antibodies, those without evidence for HIV infection by polymerase chain reaction amplification of proviral DNA had stable CD4+ T-cell counts and CD4:CD8 ratios greater than 1, regardless of the absolute number of cells in these subsets [53]. This finding further supports our recommendation that all persons with a CD4 count of less than 400/mm3 and a CD4:CD8 ratio less than 1.0 should be investigated for HIV, as well as for other causes of immune deficiency, and that the definition of ICL/SUHIS be restricted to patients with less than 300 to 400 CD4+ cells/mm3, an inverted ratio, and evidence of a progressive decline in CD4+ cells. A similar approach, together with the aggressive tracing of donors and recipients, has been recommended within the transfusion community to investigate cases of idiopathic CD4+ T lymphocytopenia [54] and is further discussed here.
T-Lymphocyte subsets in infectious disease : as reported in Table 3, common pathogenic and opportunistic bacterial, viral, parasitic, and fungal diseases can cause transient alterations in T-lymphocyte subsets. These changes may be superimposed on various functional immune defects associated with such infections [68] as well as on ill-defined syndromes of putative infectious etiology sometimes linked to decrements in CD4 counts, such as the chronic fatigue syndrome [69]. Even immunizations may affect absolute numbers of T-cell subsets, transiently but significantly depressing CD4:CD8 ratios to less than 1.0 in 25% of cases in one study [70]. 2 risk factors for HIV, intravenous drug abuse (see Table 2) and clotting disorders (see Table 4), are not necessarily associated with significant alterations in absolute peripheral CD4+ or CD8+ T-cell counts, despite the fact that chronic antigenic exposure might have been expected to render these patients particularly susceptible to lymphocyte subset alterations. Infections linked to at least transient depression in CD4 counts (including tuberculosis, hepatitis B, and Epstein-Barr virus-associated mononucleosis) are usually associated with a CD4:CD8 ratio greater than 1.0, even if it is statistically lower than control ratios. This finding also appears to be typical of other opportunistic infections seen in patients with HIV, including toxoplasmosis and P. carinii pneumonia (see Table 3). For oral candidiasis [64, 65] and cryptococcosis [66, 67], the numbers of HIV-seronegative patients studied are still too small to permit definitive conclusions. The major exception to the generalization that infectious disorders do not cause a low CD4 count in conjunction with a CD4:CD8 ratio of less than 1.0 is acute cytomegalovirus infection, in which depression of CD4 counts is typically accompanied by a marked increase in CD8 values (see Table 3). Both usually return to baseline after resolution of cytomegalovirus disease, with no statistical difference in absolute CD4+ or CD8+ T-cell counts in cytomegalovirus-seropositive compared with seronegative persons (see Table 2). Human T-cell lymphotropic virus type II (HTLV-II) occurs in a substantial portion of intravenous drug abusers and some homosexual men at risk for HIV and is capable of altering CD4 counts for prolonged periods. In HTLV-II-positive persons whose T cells do not exhibit spontaneous proliferation in vitro, T-cell subsets do not differ from those of normal controls [61]. Among most HTLV-II- positive persons whose cells do exhibit such spontaneous growth, however, a significant increase in both CD4+ and CD8+ T-cell subsets, without alteration in CD4:CD8 ratio, has been reported (see Table 3).  Lymphopenia, with artificial lowering of absolute counts, affects T-cell phenotype in the presence of certain infectious diseases or congenital disorders. Reference ranges for analysis of disease-related variations by T-cell subset percentages may be more appropriate in this setting [17, 71], but these results do not alter our approach to assessing T-cell changes. Transient alterations in CD4 values in infectious diseases also occur in HIV-seropositive persons. These changes may have clinical relevance, although their pathophysiologic nature is incompletely understood. For example, primary cytomegalovirus infection in HIV-positive persons may initiate a more rapid and substantial decline in CD4+ T-cell counts than in HIV-positive controls not exposed to cytomegalovirus [72]. The risk for advanced HIV disease in cytomegalovirus-seropositive persons was 2.5 times that of a similar cytomegalovirus-negative cohort [72].
Congenital conditions that may be recognized late in life : common variable immunodeficiency may lead to altered CD4 counts recognized in later life. It is an important exclusion criterion for ICL/SUHIS [4, 8]. Although progressive decreases in CD4+ T cells were not documented during a 2-year follow-up of HIV-seronegative and culture-negative patients with common variable immunodeficiency [73], including those with low baseline CD4 values [39], initial absolute counts may be substantially less than the usual mean. Although CD4: CD8 ratios of less than 1.0 are unusual in common variable immunodeficiency [73], they have been reported [39, 74], unfortunately, in the absence of documentation of HIV serostatus. In these cases, CD4+ T-cell subset analysis may be illuminating because decrements in CD4 count appear to be secondary to a dramatic deficit in those cells that induce CD8+ suppressor cells, the CD4+ CD45RA+ population (126 ± 91 compared with 384 ± 142 in controls; P < 0.001), whereas the CD4+ CD29+ "memory" subset, which induces helper cells, remains unaffected [39].  All of these analyses beg the issue of qualitative defects in CD4+ T-cell function occurring in the absence of quantitative changes. Such alterations are seen in the early stages of HIV infection [75] and may underlie increased susceptibility to opportunistic infections occurring in the absence of changes in absolute CD4 count. They are beyond the scope of this review.
Importance of biologic variability in assessing CD4+ T lymphocytopenia and severe unexplained HIV-negative immunosuppression : because myriad factors can affect T-cell subsets, changes in CD4+ T-cell counts should be investigated over time, together with the CD4:CD8 ratio, particularly in the context of intercurrent disease. This is especially important in evaluating patients with ICL/SUHIS and should aid in refining its definition. For example, case 5 from our original five reports of idiopathic CD4+ T lymphocytopenia [3] was a sexually active homosexual man, negative for HIV by serologic testing and DNA amplification by polymerase chain reaction, who had pulmonary tuberculosis, a persistent but stable CD4 count of less than 300/mm3, and a CD4: CD8 ratio of less than 1.0. Six months after successful therapy for his tuberculosis, his CD4 counts increased to more than 600/mm3, making the diagnosis of ICL/SUHIS untenable. Indeed, in all of the few patients with ICL/SUHIS investigated by reverse transcriptase measurements in viral cultures [4, 6], no evidence for retroviral activity has been found. These patients have not shown progressive declines in their CD4 counts, however, and thus have been accurately described as not having an "HIV-like" immunologic picture. It has been estimated recently that more than 300 000 persons in the USA alone would meet the current definition of idiopathic CD4+ T lymphocytopenia, with the possible involvement of a novel agent(s) lost within this mixture of uncertain lower truncation point for CD4 distribution and statistical variations [76]. Thus, the failure to detect a lymphocytopathic or retrovirus or other micro-organisms should not discourage a thorough analysis of that subset of ICL/SUHIS patients with T-cell changes more characteristic of HIV. They should undergo extensive evaluation for HIV or other recognized or novel infectious causes of immune deficiency [3, 77]. Such patients represent a very small fraction of the total cases reported to the Centers for Disease Control and Prevention and the World Health Organization [3-8]. I believe that it is these persons, however, for whom pneumocystis prophylaxis should be considered after CD4 counts decrease to less 200/mm3, regardless of whether a retrovirus or other infectious agent has been identified. In the absence of clear epidemiologic support for a transmissible agent, however, any recommendation concerning ICL/SUHIS must be presented with great reserve. For example, a "novel retrovirus" was reported to have been isolated from two HIV-seronegative patients with common variable immunodeficiency [78] who, unlike the typical cases summarized here, had very low CD4 counts as well as depressed CD4:CD8 ratios. Follow-up showed that these patients were actively infected with HIV-1, even if incompetent to mount a serologic response to it [73]. Finally, apart from T-cell subset analyses, AIDS in the "pre-AIDS era" had been described [79] before recognition of ICL/SUHIS, but in only 1 of these 19 patients with opportunistic infections were CD4 counts measured. Indeed, my colleagues and I had previously documented P. carinii pneumonia in patients with normal CD4 counts and CD4:CD8 ratios [63], and similar reports of AIDS-linked illnesses in patients with normal CD4 counts and T-cell subset ratios have been published [80]. It is only through careful follow-up of patients screened in the manner suggested here, using historical knowledge of the effects of various infectious diseases and conditions on immunophenotyping, that complex issues such as physiologic CD4+ lymphopenia, ICL/SUHIS, requirements for institution of prophylactic antibiotics, and potential new infectious agents associated with alterations in T-cell subsets can be assessed.

• granulocytes (granulocytopenia)
• agranulocytosis / agranulocytic or neutropenic angina / malignant or pernicious leukopenia / Schultz's angina or syndrome / idiopathic or malignant neutropenia : any condition involving greatly decreased numbers of granulocytes

Aetiology :
• sensitization to drugs
• type II hypersensitivity to sulfonamide
• chemicals
• vesnarinone is an important new drug that significantly decreases mortality rates in severe congestive heart failure; however, its use is associated with a relatively high incidence (approximately 1%) of agranulocytosis^ref
• radiation affecting the bone marrow and depressing granulopoiesis
Symptoms & signs : severe neutropenia results in lesions of the throat, other mucous membranes, gastrointestinal tract, and skin
• neutropenia : absolute neutrophil count (ANC) <  2s below the age-related mean
• peripheral neutropenia : decrease in the number of neutrophils in the circulating blood.
Grading :
• mild neutropenia : ANC = 1000-1500/mm³
• moderate neutropenia : 500-1000/mm³
• severe neutropenia : < 500/mm³
Epidemiology : 1-2 cases per million population; incidence is the same in males and females.
Onset :
• congenital neutropenia / chronic hypoplastic neutropenia : former names for infantile genetic agranulocytosis. The 2 mains forms of hereditary neutropenia are cyclic neutropenia, also known as cyclic hematopoiesis, and severe congenital neutropenia (SCN), sometimes referred to as Kostmann syndrome. Other syndromes can feature neutropenia as a component :

syndrome	inheritance	gene	clinical features	animal model	animal model phenotype
cyclic neutropenia	autosomal dominant	ELA2	alternate 21 day cycling of neutrophils and monocytes	mouse  knock-out	no neutropenia resistance to smoking-induced COPD vulnerability to infection
severe congenital neutropenia (SCN)	autosomal dominant	ELA2 (35–84%)	static neutropenia MDS and AML	mouse knock-in (V72M)	no obvious phenotype
	autosomal dominant	Gfi1  (rare)	static neutropenia circulating myeloid progenitors lymphopenia	mouse  knock-out	resembles human Gfi1 deficiency
	sex-linked	wASP  (rare)	neutropenic variant of  Wiskott-Aldrich syndrome	mouse  knock-out	lymphopenia thrombocytopenia colitis
	autosomal dominant	G-CSFR  (rare)	G-CSF refractory neutropenia no documented MDS or AML	mouse  knock-out	moderate neutropenia decreased progenitors in bone marrow increased apoptosis in circulating neutrophils
Kostmann  syndrome	autosomal recessive	unknown	static neutropenia without  MDS or AML
Hermansky Pudlak  syndrome,  type 2	autosomal recessive	AP3B1	SCN platelet dense body defects oculocutaneous albinism	gray collie  syndrome  of dogs	14-day cycles of pancytopenia coat and eye color changes
				mouse pearl mutation mouse knockout	no documented neutropenia platelet dense body defects coat and eye color changes
Chediak-Higashi syndrome	autosomal recessive	LYST	neutropenia oculocutaneous albinism giant lysosomes lymphohistiocytic infiltration impaired platelet function	blue-smoke Persian cat	neutropenia coat and eye color changes giant lysosomes lymphohistiocytic infiltration impaired platelet function
				mouse beige mutation cattle Aleutian mink	no neutropenia coat and eye color changes giant lysosomes lymphohistiocytic infiltration impaired platelet function
Barth syndrome	sex-linked	TAZ	neutropenia, often cyclic dilated cardiomyopathy methylglutaconic-aciduria
Cohen syndrome	autosomal recessive	COH1	mental retardation neutropenia dysmorphism

Aetiology :
• cyclic or periodic neutropenia / cyclic hematopoiesis / gray collie syndrome : a chronic type of neutropenia that abates and recurs, accompanied by malaise, fever, stomatitis, and various types of infections. A rare autosomal recessive disorder of gray collie dogs in which there is a bone marrow stem cell defect with periodic fluctuations in numbers of circulating neutrophils, platelets, and reticulocytes; during the episodes animals have lethargy, fever, arthralgias, and bacterial infections that can be fatal
• cyclic neutropenia : in cyclic neutropenia, peripheral blood neutrophil counts oscillate with approximately 21 day frequency, with a nadir approaching zero and a peak near normal^ref. Monocytes cycle but do so in a phase opposite to that of neutrophils. Similar periodicity may occur in acquired diseases, including chronic myelogenous leukemia (CML), large granular lymphocytosis (LGL), and hypereosinophilic syndrome. Life-threatening infections can accompany the 3- to 4-day neutropenic nadir of the cycle, with frequent aphthous stomatitis, periodontitis, typhlitis, and occasional sepsis. There may be particular vulnerability to infection with anaerobic bacteria, suggesting that the deficiency in neutrophils is not merely one of low numbers. Most cases respond to G-CSF treatment, administered at about 2–3 µg/kg at 1- to 2-day intervals^ref. G-CSF does not abrogate cycling, but instead reduces infectious complications by shortening the cycle period and increasing the amplitude of the waves. Genetic transmission is autosomal dominant. As with other dominant disorders, sporadic cases commonly arise from new mutations. Genetic linkage analysis and positional cloning demonstrated that heterozygous, germline mutations of the ELA2 gene, encoding neutrophil elastase, explain many cases of cyclic neutropenia^ref. There are many different alleles, but the most common are intronic substitutions that destroy a splice donor site in intron 4. This forces the utilization of an upstream, cryptic splice donor site resulting in an internal deletion of 10 amino acid residues from the protein (V161-F170).
• Kostmann's neutropenia / infantile genetic agranulocytosis / severe familial congenital cyclic neutropenia

Epidemiology : most of the initial patients reported by Kostmann were seen in Överkalix parish in northern Sweden, a geographic region with excessive inbreeding.
Aetiology : autosomal recessive mutations leading to increased SHP-1 and SHP-2 in neutrophils
Symptoms & signs : temperature instability in newborn period, fever, irritability, localized site(s) of infection, oral ulcers, gingivitis, pharyngitis, sinusitis, otitis media, lymphadenopathy and/or lymphadenitis, bronchitis and/or pneumonia, cellulitis, boils, cutaneous abscess, omphalitis, perianal abscess, lung abscess, liver abscess, peritonitis, enteritis with chronic diarrhea and vomiting, bacteremia and/or septicemia, urinary tract infection, fractures. Bone demineralization resulting in bone pain and unusual fractures occur in approximately 50% of patients, either as a part of the pathophysiology of the disease or potentially from either endogenous or exogenous G-CSFs by increased bone resorption. Acute myeloid leukemia may develop in approximately 5-7% of patients, which suggests that Kostmann disease is a preleukemic syndrome. Because of the prolonged survival rate of patients with G-CSF therapy, the frequency of leukemias may increase. Although G-CSF receptor mutation does not appear responsible for the initial neutropenia in Kostmann disease, leukemic transformation is associated with this spontaneous mutation.
Laboratory examinations : ANC < 500/mm³, monocytosis and eosinophilia => normal TLC, mild anemia may be present from chronic inflammation, hypergammaglobulinemia, normal complementemia, no ANCA. Neutrrophils are CD16 / FcgRII^low and CD64 / FcgRI^high normal ROS generation and intracellular killing of bacteria; decreased intracellular calcium mobilization in response to fMLP or IL-8. Bone marrow aspiration or biopsy reveals an arrest of neutrophil precursor maturation at the promyelocyte or myelocyte level. Cytogenetic analysis typically reveals a normal bone marrow karyotype.
Differential diagnosis : GSD-1, SCID, Chediak-Higashi syndrome, myelokathexis
Therapy :
• prophylactic antibiotics may be considered but are not usually required
• steroids and testosterone is not effective.
• drain abscess as needed.
• splenectomy is not effective
• G-CSF
• hematopoietic stem cell transplantation (HSCT) only in patients unresponsive to therapy with G-CSF or in those with leukemic transformation.
• severe congenital neutropenia (SCN) / Kostmann congenital agranulocytosis^ref when he reported static neutropenia accompanied by a promyelocytic maturation arrest in the bone marrow in a consanguineous family in Sweden, with apparent autosomal recessive inheritance. However, SCN is genetically heterogeneous, and most cases seem to arise sporadically, consistent with its transmission as an often lethal, autosomal dominant disorder. A rare case of sex-linked recessive inheritance has been described and constitutes an allelic variant of the Wiskott-Aldrich syndrome^ref. SCN usually responds to G-CSF but requires higher doses than those used to treat cyclic neutropenia^ref. Addition of corticosteroids may benefit cases otherwise not responding to G-CSF^ref. In the past, most patients died from infections (and many still do). With improved survival because of G-CSF, myelodysplastic syndrome (MDS) and acute myeloid leukemia have emerged as significant complications, occurring in about 10% of all SCN patients^ref. In general, patients with cyclic neutropenia do not develop leukemia, although there are a few exceptions^ref. Mutations of G-CSFR, the gene encoding the G-CSF receptor, were initially reported as causative of some SCN cases^{ref1, ref2}. Later, it was appreciated that G-CSFR mutations represent acquired, nonheritable, somatic events in the bone marrow, accumulating as SCN progresses to MDS and AML, although the mutations do not invariantly occur in AML and may also appear in the absence of neoplasia. Leukemia in SCN may also show acquired monosomy 7, trisomy 21, and ras mutations. Subsequently, there have been reports of 2 sporadic SCN patients, resistant to G-CSF therapy, with constitutional heterozygous G-CSFR mutations that do appear to represent new pathogenetic germline mutations. A candidate gene study determined that constitutional heterozygous ELA2 mutations are present in DNA extracted from the peripheral blood of 35%–84% of SCN cases^ref. Most cases are sporadic, but ELA2 mutations segregate with multigenerational transmission of the disease in several pedigrees where there are multiple affected family members, thus indicating that the mutations, at least in familial cases, occur constitutionally in the germline. The possibility of somatic ELA2 mutations, similar to the case for G-CSFR, remains unexplored. Mutations causing SCN are generally distinct from those responsible for cyclic neutropenia. The genotypes and phenotypes can overlap^{ref1, ref2}, however, and the mutation P110L appears roughly equally among both cyclic neutropenia and SCN patient populations. Chain terminating nonsense and frameshift mutations near the carboxyl terminus are the most common SCN mutations. SCN patients whose disease is the result of ELA2 mutations represent a subset with worse disease: lower neutrophil counts, requirement for higher doses of G-CSF to achieve a response, and higher rate of neoplastic progression^ref. Some ELA2 mutations are particularly severe. G185R occurs in 4 SCN patients known in the French neutropenia registry^ref and among our samples, each of whom has failed G-CSF treatment and has developed MDS or AML. Homozygous mutation of ELA2 is unknown, and this gene would be an unlikely candidate for the recessive syndrome first reported by Kostmann, where the responsible gene remains unidentified. The possibility of somatic ELA2 mutations in MDS or AML arising in the absence of hereditary neutropenia has not been addressed.
• Gänsslen familial neutropenia
• Hitzig chronic benign familial neutropenia : a rare familial type of peripheral neutropenia, probably transmitted as an autosomal dominant trait, related to but less severe than agranulocytosis. It is usually seen in children and is characterized by recurrent infections with eventual spontaneous remission, but in a few cases it has persisted into adulthood
• reticular dysgenesia
• Shwachman-Diamond syndrome (SDS)
• Whim syndrome : myelokathexis (the occurrence of neutropenia and retention of bone marrow neutrophils has been called myelokathexis (kathexis = retention)), combination of chronic papillomavirus and bacterial sinopulmonary infections, low immunoglobulin levels.
Genetic data supporting the role of ELA2 mutation in the pathogenesis of cyclic neutropenia and SCN have been confirmed independently in several laboratories^ref. Clinical Laboratory Improvement Amendment (CLIA)-certified tests to detect ELA2 mutation are commercially available. Nevertheless, the finding that such a pedestrian enzyme as neutrophil elastase causes hereditary neutropenia was greeted with skepticism. At least two further observations unambiguously establish causality. First, the probability of identifying by chance a new mutation—an extremely rare occurrence—when screening a single gene, from among > 20,000 human genes, in a sporadic case whose unaffected parents lack the illness, is infinitesimal. Yet, for sporadic cases, new mutation of ELA2 occurs invariantly in cyclic neutropenia and commonly in SCN. Second, germline mosaic individuals^ref who have fathered children with SCN demonstrate ELA2 mutations in myeloid progenitors, but not neutrophils, indicating that the mutation alone is sufficient to prevent the maturation of stem cells into neutrophils (or to direct them to an alternate cell fate, such as monocytes). A debate focusing on the origins of MDS and AML in SCN has drawn two sides. One argues that malignant evolution is a consequence of bone marrow failure per se, noting that clinically and genetically distinct cytopenic disorders, such as Shwachman-Diamond syndrome, also undergo such transformation. The other centers on the possible contributions of G-CSF treatment. In fact, neither argument may be correct. Epidemiological data do not reveal an association between G-CSF dose or duration of treatment and neoplasia^ref. Recent observations indicate that even though SCN patients without ELA2 mutations generally have clinically indistinguishable disease, MDS or AML arises almost exclusively in the subset of SCN patients whose illness is caused by ELA2 mutations^ref. Neutrophil elastase could thus be the first protease known to act as an oncoprotein. In fact, it may have a role in other malignancies. Genetic deficiency of a₁-antitrypsin, the major inhibitor of neutrophil elastase, is associated with an increased risk of (in addition to pulmonary emphysema) lymphoma and carcinoma of the lung, liver, gall bladder, and bladder^ref. Common sequence variants of the ELA2 promoter that cause its overexpression are found at higher frequency in lung cancer patients. The beige mouse, a genetic model of the human neutropenic Chediak-Higashi syndrome resulting from LYST mutation and leading to secondary deficiency of neutrophil elastase, is resistant to UV- and benzopyrene-induced skin cancer. ELA2 is expressed only in promyelocytes and promonocytes, but the neutrophil elastase protein persists through the cell divisions of terminal differentiation to neutrophils and monocytes, respectively^ref. The mature protein is 218 amino acids in length (following removal of pre-pro amino terminal sequences and a carboxyl tail). Neutrophil elastase predominately resides in granules but is also present in the plasma membrane and released into serum. As a chymotryptic protease, it is capable of digesting many substrates, including matrix components such as elastin, clotting factors, immunoglobulins, and complement. Intriguingly, with respect to disease pathogenesis, neutrophil elastase also cleaves G-CSF, the G-CSF receptor, the c-KIT receptor, and Notch family receptors^ref. Nevertheless, in crude recombinant expression assays, the mutations have varying effects on catalytic activity, with some markedly reducing activity and others appearing to be largely inconsequential^ref. Given this background, determining how the mutations cause disease has proven to be elusive. The proposal that these ELA2 mutations cause accelerated apoptosis has been called into question^ref. Cyclic neutropenia in dogs, also known as the gray collie syndrome, differs from the human form of the illness because it features autosomal recessive inheritance, oculocutaneous albinism, cycling of all blood lineages, and a periodicity closer to 2, instead of 3, weeks. A candidate gene approach found that canine cyclic neutropenia is the equivalent of the rare human Hermansky Pudlak syndrome type 2 (HPS2), with both diseases resulting from homozygous inactivating mutations of AP3B1, encoding the beta subunit of the adaptor protein 3 (AP3) trafficking complex^ref. There are just 4 patients from 3 families known to have HPS2; all are neutropenic, and, in the only patient in whom cycling was investigated, the neutropenia was severe and static^ref. As recently reviewed^ref, there are 4 heterotetrameric adapter protein complexes, and all are involved in the intracellular transport of luminal "cargo" proteins within membrane-bound organelles. AP3 specifically shuttles cargo proteins from the trans-Golgi network to lysosomes, which, in neutrophils, generally correspond to granules. The mu or beta subunits recognize tyrosine or dileucine based peptide motifs, respectively, within cargo proteins. In the absence of AP3, cargo proteins are routed to a default destination in the plasma membrane. Mutations yielding absence of beta subunits lead to disassembly and decay of the entire complex. Several lines of evidence suggest that neutrophil elastase is a cargo protein for AP3^ref. First, its localization in granules is compatible with the distribution of other known AP3 cargo proteins, and mutations in either cause similar diseases. Furthermore, neutrophil elastase is deficient in canine cyclic neutropenia, even though the canine ELA2 gene is intact and appropriately expressed. Finally, neutrophil elastase (processed free of its carboxyl tail) and the AP3 mu subunit interact in a yeast two-hybrid assay, and a tyrosine residue in neutrophil elastase (NE) is required for their association. The most common category of SCN mutations—those that delete the carboxyl terminus—also remove the tyrosine residue required for association in vitro between neutrophil elastase and the mu subunit of AP3 and redirect neutrophil elastase to the plasma membrane in transfected cells. Nevertheless, these observations raise a potential biological problem. AP3 coats the cytoplasmic (outer) surface of membrane-bound organelles, and cargo proteins, within the interior of such vesicles, must extrude through the membrane in order to interact with AP3. Thus, if neutrophil elastase is a genuine AP3 cargo protein, then it must have at least one transmembrane segment. Neutrophil elastase, a textbook serine protease, has been extremely well studied. Although routinely appearing on membranes, it had generally been regarded as a soluble protein. Somewhat surprisingly, several, though not all, computer algorithms designed to predict transmembrane domains detect two such segments in neutrophil elastase^ref. Interestingly, when the location of mutations is superimposed on the predicted transmembrane domains, a striking pattern emerges : mutations capable of causing cyclic neutropenia approximately overlap with predicted transmembrane segments. Experimentally, expression of neutrophil elastase representing cyclic neutropenia mutations in proposed transmembrane segments appears to cause enhanced granular accumulation of the protein, whereas wild-type protein also shows some distribution in the plasma membrane. Fit of ELA2 mutations into 1 of 3 proposed functional categories.

The linear sequence of the protein is marked with respect to the processed pre-pro amino and carboxyl termini, predicted transmembrane domains (TM-1 and TM-2), cryptic transmembrane (TM-cryptic) domain predicted as a result of some mutations, and proposed recognition site of the AP3 mu subunit. Squares represent mutations exclusively causing severe congenital neutropenia (SCN). Circles indicate mutations found in cyclic neutropenia patients (but that may also appear in SCN patients). Horizontal lines depict deletions. Lines connected by right angles reveal disulfide bonds that generally bracket predicted transmembrane domains, with mutated cysteine residues at their corners. Missense mutations generally aligning with transmembrane domains are colored black. Mutations destroying disulfide bonds are shaded light gray. Chain terminating nonsense and frameshift mutations that delete the AP3 mu recognition signal are shaded dark gray. Each mutation^ref is shown once. Mutations unaccounted for by this classification scheme are listed in the text, but not charted. Gfi1 encodes a zinc finger transcriptional repressor oncoprotein identified in a retroviral screen for IL-2 growth-independence of lymphomas. It regulates a subset of genes governing myeloid differentiation, including ELA2^ref. Gene targeting unexpectedly revealed neutropenia in Gfi1-deficient mice. Consequently, a screen of 105 neutropenic individuals (49 with SCN and 56 with nonimmune chronic idiopathic neutropenia of adults (NI-CINA), incorporating milder neutropenia diagnosed as an adult) lacking ELA2 mutations led to the identification of two different, heterozygous, autosomal dominant Gfi1 zinc finger missense mutations in a family of 3 SCN patients and in an NI-CINA patient^ref. One mutation, N382S in the fifth zinc finger, disrupts DNA binding and another, K403R, perturbs a lysine residue that may serve as a site for posttranslational modification with the SUMO polypeptide. SUMO is involved in DNA replication and repair, nuclear-cytoplasmic transport, and subnuclear localization. The clinical features of human Gfi1 mutation resemble the mouse knock-out and, in addition to neutropenia, consist of circulating primitive myeloid cells and B cell and CD4 T cell lymphopenia. A group has proposed a somewhat speculative model for how mutations in neutrophil elastase, adaptor protein 3, and Gfi1 cause cyclic and congenital neutropenia and HPS2. Neutrophil elastase can exist in both soluble and transmembrane conformations. (In the soluble isoform, the transmembrane segments are folded into disulfide-bonded loops.) Processing of the carboxyl tail exposes a tyrosine-based signal permitting its recognition and transport to granules as an AP3 cargo protein. There are 4 categories of mutations^ref. Proposed model of normal and pathological processing and transport of neutrophil elastase :

The product of the ELA2 gene, neutrophil elastase (NE), is shown in the membrane of the trans Golgi network (TGN). If the C-terminus is cleaved, then NE normally interacts with AP3 (via the mu subunit of AP3 recognizing a tyrosine residue, depicted by a black dot, in the cytoplasmic tail of NE), which transports it to granules, where NE re-equilibrates into a soluble form. If the C-terminus remains intact, then interaction with AP3 is blocked and NE is routed to a default destination in the plasma and other membranes. In SCN, deletions of the AP3 recognition signal or missense mutations that favor a transmembrane configuration of NE increase trafficking through the membrane pathway. Mutations of AP3 itself, as in canine cyclic neutropenia and HPS2, act similarly. In cyclic neutropenia, mutations disrupting the transmembrane segments favor a shift in equilibrium to soluble forms accumulating in granules. Mutations of Gfi1 lead to overexpression of ELA2, overwhelming normal AP3-mediated trafficking and diverting excess NE to membranes. The most commonly occurring ELA2 mutations in SCN prematurely terminate neutrophil elastase and delete the AP3 recognition signal, thereby sending neutrophil elastase to the plasma membrane, the default destination for cargo proteins in the absence of AP3. In HPS2, the absence of AP3 similarly redirects neutrophil elastase to the plasma membrane. Mutations of Gfi1 lead to overexpression of neutrophil elastase, which overwhelms normal AP3-based granular transport pathways and leads to excessive accumulation in the plasma membrane. Upregulating ELA2 promoter variants may act similarly to cause SCN^ref. Mutations that can produce cyclic neutropenia tend to disrupt transmembrane segments, thus favoring a shift toward a soluble form of neutrophil elastase, predominately localizing intraluminally within granules. It is possible that mistrafficking of neutrophil elastase causes neutropenia in other syndromes in which neutropenia is a component feature. For example, in the beige mouse model of the human neutropenic disorder Chediak-Higashi syndrome, posttranslational processing and trafficking of neutrophil elastase is disturbed^ref. The autosomal recessive Cohen syndrome of mental retardation, dysmorphic features, and neutropenia results from mutation of COH1, encoding a protein with homology to the yeast protein VPS13, involved in vesicle sorting and intracellular protein transport^ref. Finally, as outlandish as it may be to propose that neutrophil elastase leads a secret double life as a transmembrane protein, it may have even more tricks up its sleeve. Neutrophil elastase appears to cleave the PML/RAR fusion gene, the product of the t(15;17) translocation in FAB M3 AML, and ELA2-deficient mice expressing a PML/RAR transgene are resistant to the leukemia that otherwise would develop^ref. Even more bizarre, a recent report suggests that neutrophil elastase and chromatin are expulsed together from neutrophils to form net-like, extracellular traps for bacteria^ref. Neutrophil elastase is turning up in some surprising places.
• primary splenic neutropenia / hypersplenic neutropenia : a syndrome characterized by splenomegaly, hypercellular bone marrow, profound leukopenia and neutropenia, and susceptibility to infection, occasionally with anemia and thrombocytopenia
• acquired neutropenia : is a relatively rare disorder
• immune neutropenia is caused by antibodies directed against neutrophil-specific antigens^ref. Antineutrophil antibodies are directed against a defined group of neutrophil-specific glycoproteins^{ref1, ref2, ref3, ref4}

antigens	previous nomenclature	glycoprotein	allele frequency (%)
HNA-1a	NA1	FcgIIIb (CD16)	58
HNA-1b	NA2	FcgIIIb (CD16)	58
HNA-1c	SH, NA3	FcgIIIb (CD16)	8-38
HNA-2a	NB1	CD177(gp50-64)	94
HNA-3a	5b	Gp70-95	97
HNA-4a	MART	CD11a	99
HNA-5a	OND	CD11b	96

Immune neutropenia can be alloimmune or autoimmune.
Laboratory examinations : detection of antineutrophil antibodies.
• granulocyte immunofluorescence test (GIFT) : detection of neutrophil-bound antibody by binding of glutaraldehyde-

fixed patient neutrophils to fluorescently labeled anti-human IgG
• granulocyte indirect immunofluorescence test (GIIFT) : detection of serum antibody by incubation with normal neutrophils, or specifically phenotyped neutrophils, with subsequent staining with fluorescently labeled anti-human IgG
• granulocyte agglutination test (GAT) : incubation of granulocytes with patient sera and microscopic evaluation of agglutination
• enzyme linked immunoassays (ELISA) : detection of antibody in serum by binding to glutaraldehyde-fixed normal neutrophils on microtiter plates with detection by conjugated anti-human IgG
• monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) : simultaneous incubation of defined neutrophils with patient serum and monoclonal antibodies directed against neutrophil-specific antigens. Antigens are then immobilized on column coated with anti-mouse ab, and assayed for presence of human antibody
Tests for anti-neutrophil antibodies parallel tests that have been done to detect antibody against red cells. The most widely performed assays are the granulocyte agglutination test (GAT) and the granulocyte immunofluorescence test (GIFT). The GAT was one of the first tests to be developed and depends on the detection of the functional effect of antibody binding resulting in visible agglutination of neutrophils. Results with this assay are reported to have widely varied rates of sensitivity, depending on the procedures used. The GIFT detects neutrophil-bound antibody by binding of glutaraldehyde-fixed neutrophils to fluorescently labeled anti-human IgG. Glutaraldehyde fixation prevents spontaneous fluorescence of neutrophils, which can confound interpretation of the test. The assay can be performed directly on patient neutrophils, although this may be difficult if the patient is profoundly neutropenic. Alternatively, detection of circulating antibodies in serum can be detected by incubating glutaraldehyde-fixed heterologous neutrophils with patient serum and then assaying with fluorescently labeled antihuman IgG. This can be further adapted by using typed neutrophils homozygous for known neutrophil antigens, allowing identification of the target antigen of the patient’s antibody. Fluorescence can be detected by inspection under a microscope or by flow cytometry. More refined tests have been developed, including enzyme-linked immunoassays (ELISA), in which glutaraldehyde-fixed normal neutrophils are fixed onto microtiter plates, incubated in serum, and antibody binding detected with conjugated anti-human IgG. Perhaps the most specific test employs monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA). In this assay, neutrophils with defined antigen specificity are incubated with patient serum and monoclonal antibodies directed against an antigen on the neutrophil surface. Antigens are then isolated on an affinity column coated with anti-mouse IgG and assayed for the presence of human antibody^ref. This allows for the direct and simultaneous determination of antibody specificity directed at a variety of antigens. Tests of neutrophil antibodies are less widely performed and are more difficult to interpret than comparable tests on erythrocytes. Direct assay for the presence of antibody bound to neutrophils, either on patient neutrophils or on heterologous neutrophils incubated with patient serum or plasma, is fraught with difficulty^ref. Because neutrophils have abundant Fc receptors, false positive results may complicate interpretation of any of these studies, especially in the presence of high levels of circulating antibodies (as in myeloma or HIV infection) or in the setting of immune complex disease. Furthermore, neutrophils are fragile, tend to aggregate spontaneously in vitro, and often lyse upon manipulation, complicating interpretation of direct assays further. The degree to which these difficulties are estimated to complicate the interpretation of the assays varies among investigators. However, it may explain the presence of detectable antibodies in certain nonneutropenic study populations as well as the lack of correlation between the level of detected antibody and the degree of neutropenia reported by most investigators. The most common antigens defined to cause immune neutropenia were defined in 1998 by the Granulocyte Antigen Working Party of the International Society of Blood Transfusions^ref. They include the glycoprotein modifications of FcRIIIb, termed human neutrophil antigen 1 (HNA-1) with 3 defined alleles (a, b, and c), as well as determinants on gp50-64, 70-95, and CD11a and b, for which no allelic diversity has been defined. In addition, investigators have sought potential nuclear antigens that can be expressed on the cell surface that may play a role in the secondary autoimmune neutropenia seen in patients with systemic lupus erythematosus (SLE). One such antigen that may play an important role in SLE-associated neutropenia is SSB/La, as discussed below with secondary autoimmune neutropenia.
• alloimmune or isoimmune neonatal neutropenia (AINN) : neutropenia in the newborn due to in utero incompatibility between its paternal neutrophil antigens and those of the mother's blood; the mother's blood produces IgG antineutrophil antibodies that cross the placenta and sensitize fetal neutrophils. Affected infants may have fever, pneumonia, septicemia, and other infections that can be fatal. The condition eventually resolves itself as the infant's immunoglobulin replaces that from the mother. Newborns develop transient neutropenia that recovers spontaneously after an average of 11 weeks. In general, infectious complications are minor, and most series report no septic deaths^ref. When necessary, patients respond well to G-CSF^ref. The majority of patients with AINN develop neutropenia in response to antibodies directed against antigens of HNA-1. The HNA antigens are located FcgIIIb (CD16). Pan-FcgRIIIb antibodies can arise in individuals who lack the receptor altogether as the result of a gene deletion; this is a rare cause of AINN^ref. Although FcgRIII deficiency was first discovered in the evaluation of patients who had newborns with AINN, the incidence of the development of antineutrophil antibodies was actually quite low. For example, in a study of 21 patients with FcgRIIIb deficiency, among 3 patients with 10 at-risk pregnancies, there were no newborns with neutropenia^ref. Interestingly, the incidence of AINN appears to be lower than the incidence of detectable granulocyte-specific antibodies in the population. In one survey of over 1000 postpartum women, 1.1% demonstrated granulocyte-specific antibodies, but no newborns had neutropenia^ref. Whether the detected antibodies were false positives inherent in the test or whether relative clinical silence reflects the biology of antineutrophil antibodies is unknown
• autoimmune neutropenia (AIN)
• primary AIN is a rare disorder. It occurs predominantly in early childhood: one study of 143 patients with AIN demonstrated that of 101 patients with primary AIN, 76 patients were under age 3^ref. The average age of onset is 6–12 months, and patients develop a moderate to severe chronic neutropenia. Infections are usually mild to moderate, and serious infections are unusual. Spontaneous remission occurs in 95% of childhood AIN patients over the course of 2 years^ref, with one group suggesting that the level of detected antibody is predictive of both infectious complications and time to remission^ref. Treatment with prophylactic antibiotics ameliorates infectious complications. Although patients are almost uniformly responsive to G-CSF, chronic administration is usually unnecessary and should be reserved for recurrent or severe infections^ref. Tests for antineutrophil antibodies in AIN are nearly always detected by GIFT, but in about 3% of cases may be positive only by GAT. Antibodies are uniformly IgG, and are directed primarily against HNA1 and 2, with rarer cases associated with antibodies to CD11b (HNA-5a) or pan-FcgIIIb (CD16)^{ref1, ref2}. This is in contrast to secondary AIN, where pan-FcgRIIIb antibodies are frequently detected. Primary AIN is rare in adults, where autoimmune neutropenia is more often secondary to underlying rheumatologic syndromes. In adults, infectious complications are also frequently absent or mild, although the disease is usually chronic and spontaneous recovery is unusual^ref. Again, since symptoms may be minimal, treatment should be based on the patient’s clinical course rather than on the absolute level of the neutrophil count
• secondary AIN : secondary AIN in adults is usually associated with systemic autoimmune disease, predominantly rheumatoid arthritis (RA) and SLE^ref. Neutropenia in RA is usually attributable to :
• Felty’s syndrome (FS) typically occurs in patients with longstanding RA associated with end-organ manifestations of RA, including pulmonary fibrosis, vasculitis, rheumatoid nodules, and splenomegaly. Patients may also have Sjögren’s syndrome. Patients may have considerable morbidity from bacterial infection and may in rare cases succumb to overwhelming sepsis^ref. Laboratory evaluation of patients with FS demonstrates high levels of rheumatoid factor, circulating immune complexes, and hypergammaglobulinemia. In addition, many patients may be antinuclear antibody (ANA)⁺. 90% of patients with FS are HLA-DR4⁺
• T-LGL leukemia : interestingly, patients with LGL leukemia share this incidence of HLA-DR4. This and other pathophysiologic features of the disease have prompted some investigators to suggest that FS and LGL leukemia represent a spectrum of the same disease process^ref.
• SLE-associated neutropenia : neutropenia occurs in approximately half of patients with SLE. It is rarely severe and serves more as a marker of disease activity than as a clinically important complication. Neutropenia has little impact on the course of the disease and does not appear to predispose to an increase in infectious complications. The incidence of infectious complications is more reflective of immunosuppressive therapy than the height of the neutrophil count^ref. Neutropenia in SLE has been attributed to neutrophil-specific antibodies, to increased apoptosis of neutrophils, and to decreased marrow neutrophil production. All of these effects appear to be antibody-mediated. Increased neutrophil-associated IgG has been detected in half of patients diagnosed with SLE, but not all patients are neutropenic^ref. This further supports the observation that interpretation of increased neutrophil-associated IgG is especially difficult in the presence of immune complex disease. Both immune complexes and neutrophil antigen-specific antibodies have been implicated in the pathogenesis of SLE-associated neutropenia, but the correlation between laboratory testing and clinical neutropenia is poor. Some investigators have hypothesized that antinuclear antibodies may crossreact with neutrophil surface antigens, either because of crossreactive epitopes or because the nuclear antigens themselves are expressed on the cell surface. Neutropenia in SLE has been hypothesized to be pathogenetically related to the presence of anti-SSA (Ro) and anti-SSB (La) antibodies. Anti-Ro antibodies have been shown to bind a crossreacting antigen on the neutrophil cell surface and to fix complement. In another study^ref, immunoscreening of a leukocyte expression library identified La as an antigen bound by antineutrophil-positive sera from patients with SLE; this too was demonstrated to increase neutrophil apoptosis, as well as decreasing phagocytosis and increasing IL-8 production^ref. Finally, some antibodies have been demonstrated to be reactive against early myeloid progenitors, leading to decreased neutrophil production^ref
• secondary AIN in childhood is rare and may be associated with autoimmune lymphoproliferative syndrome (ALPS). This disorder is caused by heterozygous mutations in the fas gene, leading to abnormalities of lymphoid apoptosis. ALPS is associated with autoimmune cytopenias in association with adenopathy and splenomegaly. Patients have increased numbers of circulating double negative (CD4^–, CD8^–) T cells. ALPS is associated with a markedly increased incidence of non-Hodgkin’s lymphoma^ref
• drug-induced neutropenia is an idiosyncratic reaction that results in profound neutropenia or agranulocytosis^ref. Unlike the chronic immune neutropenias, which have a surprisingly low rate of morbidity and mortality, drug-induced neutropenia is associated with a high rate of infectious complications and has a mortality rate of approximately 10%^{ref1, ref2}. The most common drugs associated with agranulocytosis are antithyroid medications and sulfonamides. The most common mechanisms are immunological (formation of antibodies destructive to neutrophils or of immune complexes that bind to neutrophils), followed by inhibition of granulopoiesis and direct damage to bone marrow or precursor cells of the granulocytic series.
• antithyroid medications :
• carbimizole
• methimazole
• thiouracil
• antibiotics :
• cephalosporins
• penicillins
• sulfonamides
• chloramphenicol
• anticonvulsants :
• carbamazepine
• valproic acid
• antiplatelet agents
• antivirals :
• zidovudine
• acyclovir (at high doses)
• antibacterials :
• isoniazid
• rifampicin
• NSAIDs
• sulphasalazine
• antipsychotics
• clozapine^ref : the usual recommendation is to discontinue treatment with the drug when the peripheral neutrophil count drops < 1,500/ml. The therapeutic procedures described (symptomatic treatment of neutropenia by co-administration of lithium or G-CSF, management of the adjunctive medication) seem to be efficient strategies that allow continuation of clozapine treatment despite the occurrence of neutropenia^ref.
• anticancer drugs
• SSRI : fluoxetine^ref
• immunomodulators :
• azathioprine
• lenalidomide
The pathogenesis of drug-induced neutropenia is poorly understood. Investigation is limited because cases are rare, sporadic, and transient. In some cases, anti-neutrophil antibodies are detected and have been characterized as both autoantibodies and drug-dependent antibodies detectable only in the presence of the offending drug^ref. Some of these antibodies have been demonstrated to bind complement. In the setting of Graves’ disease, antineutrophil antibodies have been associated with an antigen that is crossreactive with thyroid-stimulating hormones (TSH)^ref as well as with antigens related to antineutrophil cytoplasm antibodies (ANCA)^ref. Clozapine-induced agranulocytosis has a unique etiology that appears to be genetically determined. Clozapine is associated with a high rate of agranulocytosis, which can be seen in 1% of patients receiving the drug. There is no evidence for an immune etiology. It is thought to be caused by accumulation of nitrenium ion, a metabolite of clozapine, which in turn causes depletion of ATP and reduced glutathione, rendering the neutrophils highly susceptible to oxidant-induced apoptosis^ref. The reaction is linked to the MHC locus and has been most closely associated with polymorphism of the TNF genes, which are in linkage disequilibrium with HLA alleles^{ref1, ref2}
• nonimmune chronic idiopathic neutropenia (NI-CINA) : a subset of patients with chronic neutropenia has no evidence of immune-mediated disease. NI-CINA is an acquired syndrome associated with chronic neutropenia, normal marrow cytogenetics, and no evidence for underlying autoimmune disease, nutritional deficiency, or myelodysplasia^ref. The marrow findings are variable, ranging from a hypoplastic to a hyperplastic myeloid series. The clinical course is usually quite benign, and many of these patients are diagnosed by examination of routine laboratory tests in the absence of any history of infection or other symptoms^ref. The pathogenesis of NI-CINA is poorly understood. The pathophysiology of the disease has been hypothesized to reflect decreased neutrophil production, excessive neutrophil margination, and increased peripheral neutrophil destruction. There has been a suggestion that patients with this syndrome have an undiagnosed underlying inflammatory illness, with increased production of inflammatory cytokines. A recent study by Papadaki et al concentrated on those patients with myeloid hypoplasia on marrow examination. Investigation of marrow progenitors and colony forming unit–granulocyte macrophage (CFU-GM) production documented a selective decrease in CD34⁺/CD33^– myeloid progenitors, with evidence for increased fas-mediated apoptosis as the cause for the reduction in CFU-GM. In addition, they demonstrated that stromal cell layers produced increased amounts of TNF^{ref1, ref2}. Finally, the same group has previously demonstrated that increased risk of developing NI-CINA may be related to HLA phenotype^ref. It should be noted that the TNF locus lies within the HLA cluster, and the apparent HLA predilection for the development of clonazapine-induced agranulocytosis actually was more tightly linked to TNF microsatellite polymorphisms^ref. Hence, it is tempting to speculate that perhaps a predisposition to NI-CINA is also linked to polymorphisms of the same locus. Finally, 2 patients with NI-CINA have been found to have heterozygous mutations in Gfi-1^ref.
• anticancer drugs

Prophylaxis :
• oral antibiotic therapy :
• levofloxacin 500 mg 1 cps/day 4-5 days before expected onset of severe neutropenia and until hematological recovery (ANC > 1,000); no prophylaxis in patients with quinolone allergy; it is an effective and well-tolerated way of preventing febrile episodes and other relevant infection-related outcomes in patients with cancer and profound and protracted neutropenia. The long-term effect of this intervention on microbial resistance in the community is not known^ref
• cotrimoxazole 960 mg 2 times a day for 3 consecutive days a week until end of therapy
• oral antifungal therapy : itraconazole (oral solution or caps) 200 mg 2 times a day (alternatively fluconazole 200 mg/day)
• hygiene and prophylaxis of oral cavity :
• nystatin (oral suspension) 2 tablespoons at meals (wash and swallow)
• clorhexidine (wash without swallowing)
• G-CSF (only for non-myeloid malignancies)
• for weekly chemotherapy : 150 mg/m²/day for 4 days in the interval
• for monthly chemotherapy : 150 mg/m²/day since day + 5 of end of therapy until nadir of ANC (generally 4-7 days)
• copper deficiency (associated with macrocytic anemia, normal platelet count, and elevated serum ferritin and EPO levels)^ref
• folic acid deficiency
• vitamin B₁₂ deficiency
• hairy cell leukemia
• ethanol
• aplastic anemia
• MDS
Therapy : G-CSF. Nearly all studies demonstrate that in primary and secondary immune neutropenia and in NI-CINA, response to G-CSF is rapid and occurs in nearly all patients^{ref1, ref2}. Treatment is frequently unnecessary, however, and is usually reserved for recurrent or serious infections. In drug-induced neutropenia, most studies have shown that G-CSF shortens the time to neutrophil recovery, although several authors have commented that evidence-based data are lacking to justify its use^{ref1, ref2}. One study suggested that drug-induced neutropenia induced by antithyroid medication does not improve time-to-neutrophil recovery, but the dose used was low and the study size was small^ref. Given the rarity and heterogeneity of drug-induced neutropenia, it seems unlikely that an evidence-based algorithm for G-CSF use will ever be validated. However, because drug-induced neutropenia is an acute, life-threatening complication of therapy, with a mortality of 10%, the demonstrated safety and apparent effectiveness of G-CSF in hastening neutrophil recovery justifies its use in this setting.
• pseudo-neutropenia
• myeloperoxidase deficiency that occurs when the automated instrument employs a peroxidase reaction for the identification of neutrophils, eosinophils, and monocytes
• neutrophil or platelet clumping
• platelet satellitism.
Symptoms & signs : when blood neutrophils are < 500 / mL, likelihood of infections from Staphylococcus epidermidis > Escherichia coli > Pseudomonas spp. > Candida spp. > Aspergillus spp. increases.
Therapy :
• G-CSF
• hematopoietic stem cell transplantation (HSCT)
• fluoroquinolones are highly effective in preventing Gram-negative infections in neutropenic cancer patients, but offer inadequate coverage for Gram-positive infections. Considering the lack of cut-clear benefit on some parameters of morbidity and mortality, routine use of Gram-positive prophylaxis is not advisable. This strategy, however, should be particularly valuable in subgroups of patients at high risk of streptococcal infection (eg, those with severe and prolonged neutropenia or mucositis, and those receiving cytarabine). Problems of tolerability and the potential for the emergence of resistant microorganisms should be considered when prescribing prophylaxis with enhanced Gram-positive activity to neutropenic patients.
Prognosis : mortality rate is 70% within the first year of life if untreated
• eosinopenia : abnormal deficiency of eosinophils in the blood

Aetiology :
• acute bacterial infection (Simon's septic factor : decrease of eosinophils and increase of neutrophils in the blood in pyogenic infections)
• GR agonists
• hairy cell leukemia
• basophilopenia / basophilic leukopenia : abnormal reduction in the number of basophils in the blood

Aetiology :
• hypothyroidism
• stress
• a few other conditions
• monocytopenia : abnormal reduction in the number of monocytes in the blood

Aetiology :
• hairy cell leukemia
• hyperplasias (leukocytosis (> 10 ^. 10³ / mL))
• hyperleukocytosis :
• absolute leukocytosis : increase in the total number of leukocytes in the blood
• relative leukocytosis : increase in the proportion of just one type of leukocyte in the blood, without increase of the total number of leukocytes.
• physiologic leukocytosis : that caused by nonpathologic factors such as strenuous exercise
• pathologic leukocytosis : that occurring as the result of some morbid condition, such as infections, inflammation, fever, trauma or hemorrhage.
• toxic leukocytosis : leukocytosis occurring in septicemia and other toxic conditions.
• agonal or terminal leukocytosis : leukocytosis occurring just before death
• granulocytes (granulocytosis)
• neutrophilia / pure leukocytosis / neutrophilic leukocytosis
• pseudoneutrophilia
• masked neutrophilia
• leukemoid reactions : metamyelocytes, myelocytes and rarely promyelocytes
• eosinophilia / eosinophilic leukocytosis : the formation and accumulation of an abnormally large number of eosinophils in the blood (> 450 / mL; [eosinophil]_blood inversely relates with [cortisol]_blood)

Aetiology :
• drugs :
• IL-2
• GM-CSF
• NSAIDs
• heparin
• type I hypersensitivities :
• allergic rhinitis (rare)
• allergic bronchial asthma (rare)
• atopic dermatitis (rare)
• allergic bronchopulmonary aspergillosis (ABPA)
• drug allergies :
• pulmonary infiltration with eosinophilia (PIE) syndrome / eosinophilic pneumonia
• drug rash with eosinophilia and systemic symptoms (DRESS)
• type III hypersensitivity :
• serum sickness
• autoimmune disease :
• pemphigus
• Churg-Strauss syndrome (CSS) / allergic granulomatous angioitis
• eosinophilic fasciitis
• panarteritis nodosa (PAN)
• inflammatory bowel disease
• connective tissue disorders
• rheumatoid arthritis (RA)
• sarcoidosis
• hypoadrenalism
• infestations (expecially in patients with HIV-1 infection) :
• protozoa :
• Isospora spp. (rarely)
• Sarcocystis spp.(rarely)
• Toxoplasma gondii (low and discontinous)
• Aspergillus spp.
• Coccidioides immitis
• helminths (related to level of tissue spreading ; normal eosinophil concentration if the helminth persists in cysts or lumens)
• myiasis
• scabies
• pulmonary infiltration with eosinophilia (PIE) syndrome / eosinophilic pneumonia
• Langerhans cell histiocytosis (LCH) / eosinophilic granuloma of bone
• neoplasms :
• Hodgkin's lymphoma (15%)
• non-Hodgkin lymphomas (NHLs) (5%)
• mycosis fungoides
• chronic myeloid leukemia (CML)
• g-chain disease / Franklin disease
• lung cancer
• gastric cancer
• pancreas cancer
• ovarian cancer
• cervical cancer
• cholesterol embolization (e.g. renal atheroembolism)
• radiation exposure
• Job syndrome
• chronic granulomatous disease (CGD)
• eosinophilia-myalgia syndrome (EMS)
• toxic oil syndrome (TOS)
• hypereosinophilia : extreme eosinophilia
• filarial hypereosinophilia / tropical pulmonary eosinophilia
• enteropathy-associated T-cell-lymphoma (EATCL)
• chronic eosinophilic leukemia (CEL) (and the hypereosinophilic syndrome (HES))
Symptoms & signs :
• lymph node or spleen enlargement
• pruritic skin ulcers or erythroderma
• eosinophilic pneumonia
• interstitial nephritis
• endocarditis
• restrictive cardiomyopathy
• retinitis
• peripheral polyneuropathy
• gastrointestinal disorders
Laboratory examinations : a minimum standard for all patients should include a complete history and physical examination, a complete blood count and differential, routine chemistries, serum IgE and vitamin B₁₂ levels, HIV serology, electrocardiogram, echocardiogram, pulmonary function tests, chest and abdominal CT, and bone marrow aspirate and biopsy. Additional organ-specific evaluations should be guided by the clinical manifestations. Testing for occult parasitic infection with stool examination for ova and parasites and/or specific serologic tests is warranted in the setting of an appropriate exposure history
Therapy : GR agonists
• basophilia / basophilism / basophilic leukocytosis : an abnormal increase of basophils in the blood (> 150 / mL)

Aetiology :
• hypothyroidism
• ulcerative colitis
• certain types of anemia
• chronic myeloproliferative disorders
• myelodysplastic sydromes
• lymphomas
• mastocytosis : mast cell proliferative disorders are characterized by the abnormal growth and accumulation of mast cells (MCs) in one or more organs. Clinical symptoms occur from the overwhelming and sometimes devastating infiltration of tissues by neoplastic cells and/or the release of chemical mediators from MCs. Mast cells are metachromatic cells that, when mature, reside in vascularized tissues in most organs. In contrast to other hematopoietic cells, MCs are extremely long-lived cells with an estimated life span of months to years. MCs are derived from pluripotent and MC-committed hematopoietic progenitors that are detectable in the bone marrow as well as in the peripheral blood^ref. Circulating MC progenitors have the capability to transmigrate from the blood into tissues where differentiation and maturation occurs^ref. Cytokines and other factors are involved in the regulation of growth and differentiation of MCs. The most important cytokine is stem cell factor (SCF), which induces development of MCs from uncommitted progenitors^ref. The effects of SCF on MC-progenitors and mature MCs are mediated through CD117 / c-KIT, a tyrosine kinase receptor encoded by the c-kit proto-oncogene^ref. SCF and KIT are considered essential for the development and differentiation of MCs. Supporting this concept, defects in either the c-kit or scf gene in mice are associated with MC-deficiency. By contrast, "gain-of-function mutations" in c-kit^ref are associated with enhanced survival (and under certain circumstances with autonomous growth) of MCs and their progenitors^ref. Such "transforming" mutations, particularly D816V, are often detected in patients with SM^{ref1, ref2, ref3}. The common pathogenetic hallmark of mastocytosis, shared by all disease variants, is the focal accumulation (clustering) of MCs in various tissues and organs^ref. Depending on the disease variant, MCs and their progenitors may also show enhanced survival and an increased proliferative capacity. In contrast to CM, monoclonality of SM is well established. Notably, the c-kit point mutation D816V is detectable in the majority (> 80%) of all adult patients with SM^{ref1, ref2, ref3}. The D816V mutation of c-kit is considered to represent an important "hit" contributing to disease development and possibly to the abnormal clustering of neoplastic MCs. However, c-kit D816V alone is unable to act as a fully transforming oncogene^ref. Rather, in ASM and MCL, in which the c-kit mutation D816V is also detected and MCs, in addition to clustering, show increased proliferative capacity^ref, additional gene defects are likely to be responsible for uncontrolled MC growth. In this regard it is noteworthy that apart from c-kit D816V, other c-kit mutations (rarely occurring) have been reported in SM as well as chromosomal defects and gene polymorphisms^{ref1, ref2, ref3, ref4}. However, the factors determining the course of SM (indolent versus aggressive/leukemic) remain at present unknown. Similarly, little is known about factors contributing to abnormal clustering of MCs in mastocytosis. One hypothesis is that aberrant expression of cell-cell adhesion molecules on MCs, such as CD2, contributes to abnormal clustering. Whether the c-kit mutation D816V or other defects are responsible for abnormal expression of adhesion molecules on MCs in patients with SM remains unknown.

Recognized gene defects, gene polymorphisms, and karyotype abnormalities in patients with (systemic) mastocytosis :

finding		reported in patients with:	frequency in patients with SM
gene defects	c-kit D816V	all variants of SM (rarely in CM)	> 80% (the presence of D816V KIT mutation in virtually all adults (93%) with indolent and aggressive forms of SM, except well-differentiated SM (29%), while other KIT mutations were rarely (<3%) detected. In around one third of patients with mutated MC, the KIT mutation was also detected in CD34+ haematopoietic cells and eosinophils, and, to a lower extent, in monocytic, neutrophil-lineage BM precursor cells and lymphocytes. Most poor-prognosis SM cases (81%) carried the KIT mutation in two or more BM myeloid cell populations while this was detected in a smaller proportion (27%) of indolent cases. These results would support the notion that KIT mutation is a hallmark of adult SM where it targets a pluripotent haematopoietic stem cell and may contribute to explain previously observed discrepancies in the literatureref)
	c-kit D816Y	CM, SM, SM-AHNMD	< 5%
	c-kit D816F	CM, SM	< 5%
	c-kit D816H	SM-AHNMD	< 5%
	c-kit D820G	ASM	< 5%
	c-kit V560G	SM	< 5%
	c-kit F522C	SM	< 5%
	c-kit E839K	CM	< 5%
	c-kit V530I	SM-AML	< 5%
	c-kit K509I	SM (familial type)	< 5%
	FIP1L1/PDGFRA	SM-HES, SM with eosinophilia	< 5%
gene polymorphisms	IL-4Ra Q576R	CM, indolent SM (ISM)	unknown
karyotype abnormalities	del 20(q12)	SM, SM-AHNMD	< 5%
	+9	SM, SM-AHNMD	< 5%
	t(8;21)	SM-AML M2	< 5%

Diagnostic criteria and WHO classification : mastocytosis can be separated into CM and SM. Localized mast cell tumors (mastocytomas and mast cell sarcoma) are very rare. In contrast to CM, SM is a persistent disease with established monoclonality, marked by c-kit D816V, in most cases. Remarkably, in several patients with SM, the c-kit mutation D816V is found not only in MCs, but also in non-MC-lineage hematopoietic cells.12 Based on such data, SM is now considered a myeloproliferative disorder. This concept is consistent with the observation that MCs derive from myelopoietic progenitors and with the relatively high incidence of AHNMDs, including secondary acute myeloid leukemia, that occur in these patients. The World Health Organization (WHO) consensus classification of mastocytosis is based on specific criteria that help in the delineation between SM and CM, between SM and myelomastocytic disorders, and between SM and a reactive increase in MCs^ref (Valent P, Horny H-P, Li CY, et al. Mastocytosis (mast cell disease). In: Jaffe ES, Harris NL, Stein H, Vardiman JW (eds). World Health Organization (WHO) Classification of Tumours. Pathology & Genetics. Tumours of Haematopoietic and Lymphoid Tissues. Vol 1. 2001:291–302). Respective criteria have been termed SM-criteria and are divided into major SM-criteria and minor SM-criteria^ref. If at least 1 major and 1 minor criterion or at least 3 minor criteria are fulfilled, the diagnosis of SM can be established^ref.
• major : multifocal dense infiltrates (>15 MCs in aggregate) of MCs within a bone marrow biopsy section^{ref1, ref2}. The most suitable marker for MC detection in such biopsies is tryptase. Compact MC infiltrates can also be detected in extra-medullary extra-cutaneous organs in SM^ref. However, the primary organ to be examined in suspected SM is the bone marrow
• minor^{ref1, ref2, ref3} :
• MCs in bone marrow or other extracutaneous organ(s) show an abnormal morphology (> 25%) (spindle shaped, atypical MCs)
• c-kit mutation at codon 816 (Activating mutations at codon 816; in most cases, c-kit D816V) in extracutaneous organ(s)
• MCs in bone marrow express CD2 and/or CD25
• serum total tryptase > 20 ng/mL (does not count in patients who have AHNMD-type disease)
Differential diagnosis : the application of such criteria is considered crucial in the diagnostic work-up, since MCs may also increase and even form focal infiltrates in reactive MC hyperplasia or in myelomastocytic disorders. For example, advanced myeloid neoplasms may exhibit an increase in diffusely spread MCs in the bone marrow without histological, cytological or biochemical evidence of SM. If MCs in these patients > 10% of all nucleated cells in the bone marrow smear or the differential blood count, it is appropriate to establish the diagnosis of myelomastocytic leukemia^{ref1, ref2}
Subsets of mast cells defined by morphological criteria :
• metachromatic blast : blast-like, few metachromatic granules
• promastocyte = atypical MC type II : mostly immature mast cells with bi- or multi-lobed nuclei, often hypogranulated
• atypical mast cell type I : mast cells in which 2 of the 3 following morphological aspects are found:
• cytoplasmic extensions (spindle shape)
• hypogranulated cytoplasm
• oval decentralized nucleus
• mature mast cell = typical tissue mast cell : round cell with round central nucleus and well-granulated cytoplasm
Categories of mastocytosis : based on consensus criteria, a number of different subvariants of CM and of SM have been recognized by the WHO^ref :
• cutaneous mastocytosis (CM)is considered a benign disease that is confined to the skin and often regresses spontaneously: the delineation of subcategories of CM is based on inspection and biopsy of the dermis. Based on these aspects, 3 major variants have been defined:
• maculopapular CM (MPCM) / urticaria pigmentosa (UP)
• diffuse CM (DCM)
• solitary mastocytoma of skin
Most patients are children. The serum total tryptase level in CM is usually < 20 ng/mL^ref. In many children, skin lesions regress during puberty. Progression of pediatric CM to SM is unusual. In adults, however, the disease persists and often progresses to SM^ref. In some patients with CM, skin lesions are extensive and accompanied by symptoms which require management. This includes the use of antihistamines, application of skin care, and sometimes the use of psoralen-ultraviolet A (PUVA). In severe cases, topical glucocorticoids may be required.
• systemic mastocytosis (SM) appears as a clonal persistent disease of myelomastocytic progenitors. The somatic c-kit mutation D816V is detectable within hematopoietic cells in the majority of SM patients. The clinical course in SM is variable :
• indolent systemic mastocytosis (ISM) is the most frequently diagnosed variant of SM. ISM is associated with SM criteria^ref, presence of urticaria pigmentosa (UP)-like skin lesions, and an indolent clinical course without significant organomegaly (which can anyway occur without impairment of organ function) or organopathy. The prognosis appears to be good. Symptoms like flushing and abdominal cramping are often reported, and less frequently also episodes of hypotension. The bone marrow is almost invariably affected. "B-findings" (for "borderline benign") include:
• infiltration grade (MCs) in bone marrow > 30% in histology and serum total tryptase level > 200 ng/mL
• hypercellular marrow with loss of fat cells, discrete signs of dysmyelopoiesis without substantial cytopenias or WHO criteria for an MDS or MPD
• organomegaly: palpable hepatomegaly, splenomegaly, or lymphadenopathy (on CT or US: > 2 cm) without impaired organ function
• isolated bone marrow mastocytosis (BMM) : in typical ISM, the grade of infiltration is rather low. Typically, MCs in bone marrow smears are spindle-shaped.

Therapy : patients with ISM are treated with "mediator-targeting" drugs including antihistamines as well as epinephrine for severe episodes of hypotension, but usually not with cytoreductive agents. Skin lesions in ISM may require symptomatic therapy. In most cases, transient responses are seen after psoralen-ultraviolet A (PUVA). Isolated bone marrow mastocytosis is a rare subentity of ISM characterized by the absence of skin lesions and lack of multiorgan involvement
• smouldering systemic mastocytosis (SSM) is another subentity of ISM^ref. In contrast to typical ISM, B-Findings (>= 2) are noted in SSM. These B-Findings reflect a high burden of MCs and extension of the clonal disease to several myeloid lineages^{ref1, ref2}. Clinically, the smouldering state has an uncertain prognosis and a variable clinical course. In some cases, the clinical course is long-lasting and silent. In other patients, an AHNMD or ASM develops after some time^ref.

Therapy : watch and wait in most cases. However, in select cases (rapidly progressive B-findings) IFN-a_2b ± glucocorticoids or cladribine (2-CdA) can be considered.
• systemic mastocytosis with an associated clonal hematologic non-mast cell lineage disease (SM-AHNMD) : in these patients, criteria to diagnose an associated clonal hematological non-MC-lineage disease (AHNMD) as well as SM-criteria must be met. Patients with SM-AHNMD are categorized according to the AHNMD and the type of SM^ref. In most cases, a myeloid neoplasm develops^{ref1, ref2}. In contrast, the occurrence of a lymphoid neoplasm is rare
• SM-AML
• SM-MDS
• SM-MPD
• SM-CMML
• SM-NHL
• SM-HES
Therapy : treat AHNMD as if no SM is present and also treat SM as if no AHNMD is found^{ref1, ref2, ref3}. If splenomegaly and hypersplenism prohibit therapy, consider splenectomy
• aggressive systemic mastocytosis (ASM) : a rare aggressive variant of SM characterized by organopathy caused by pathologic infiltration of various organs by neoplastic MCs (has to be confirmed by biopsy in most cases) and the resulting impairment of organ function^{ref1, ref2}. In contrast to MCL, the bone marrow smear shows < 20% MCs. In contrast to ISM and smouldering SM, "C-finding(s)" (for "consider cytoreduction") indicative of devastating organopathy due to MC infiltration is/are detectable^{ref1, ref2}.
• cytopenia(s): ANC < 1000/µL or Hb < 10 g/dL or Plt < 100,000/µL
• hepatomegaly with ascites and impaired liver function
• palpable splenomegaly with hypersplenism
• malabsorption with hypoalbuminemia and weight loss
• skeletal lesions: large-sized osteolyses or/and severe osteoporosis causing pathologic fractures
• life-threatening organopathy in other organ systems that is definitively caused by an infiltration of the tissue by neoplastic MCs
MC infiltration with associated organomegaly should not automatically be assumed to be the same as organopathy^ref. The most commonly affected organs are the liver, bone marrow, and the skeletal system. UP-like skin lesions are usually absent.
• lymphadenopathic SM with eosinophilia (SME) : due to excessive autocrine secretion of SCF; in a few cases, the FIP1L1/PDGFRA-fusion gene may be detected
Therapy : patients with ASM are candidates for treatment with cytoreductive drugs^ref.
• patients with a relatively slow progression are usually treated with glucocorticoids (prednisone) and IFN-a_2b. We recommend initiating prednisone (50–75 mg p.o. daily) a few days before IFN-a is administered (3 million I.U. s.c. 3 times a week)^ref. During the first days of treatment, patients should be carefully monitored. After a few weeks, the IFN dosage can usually be escalated to 3–5 million units per day, and prednisone tapered to a low maintainance dose (12.5 mg/day or less) or even discontinued. In patients with severe osteoporosis, IFN-a can be administered without glucocorticoids. If splenomegaly and hypersplenism prohibit therapy, consider splenectomy. In the absence of c-kit D816V, Imatinib may be considered.
• ASM patients with rapid disease progression, signs of MCL, or failure to respond to IFN-a, are candidates for cladribine (2-CdA) or other chemotherapy^{ref1, ref2, ref3}. Polychemotherapy (± IFN-a_2b); consider bone marrow transplantation in select cases. If splenomegaly and hypersplenism prohibit therapy, consider splenectomy. For select cases, cladribine (2CdA) or other cytoreductive drugs can be considered. Consider hydroxyurea as palliative drug. The use of targeted drugs has also been considered. Imatinib (STI571) has been described to be effective in patients with SM associated with eosinophilia and the FIP1L1/PDGFRA fusion gene^ref. Patients with wild-type c-kit or c-kit mutations other than D816V, may also respond to Imatinib^ref. However, most patients exhibit the D816V c-kit mutation which appears to confer relative resistance against Imatinib^{ref1, ref2, ref3, ref4}. In addition, most patients lack the FIP1L1/PDGFRA fusion gene even if eosinophilia is present. Therefore, it can be expected that imatinib will not show significant beneficial effects in the majority of patients with ASM or MCL, at least when used as single agent. However, a number of drugs are currently being examined for their potential to overcome D816V-based resistance of Kit against STI571.
• mast cell leukemia (MCL) is a rare subentity of SM characterized by circulating MCs and an aggressive clinical course^ref. Patients typically suffer from rapidly progressive organopathy involving the liver, bone marrow, and other organs. The bone marrow typically shows diffuse and dense infiltration and > 20% MCs on bone marrow smears^ref. MCs are often immature with a blast-like morphology, and/or have polylobed nuclei (promastocytes)
• typical MCL : MCs account for > 10% of blood leukocytes
• aleukemic variant of MCL (rare) : MCs account for < 10%^ref
The CD117 / c-kit / SCFR mutation D816V (D814Y in mice) may be detected
Symptoms & signs : mastocytosis syndrome : an episodic syndrome occurring in certain patients with systemic mastocytosis, usually those with skin lesions, bone lesions, and hepatosplenomegaly, presumably associated with histamine release from degranulation of mast cells, and characterized mainly by intense pruritus, flushing, headache, tachycardia, hypotension, and syncope.
Therapy : polychemotherapy (± cladribine (2-CdA); ± IFN-a_2b). Consider bone marrow transplantation. If splenomegaly and hypersplenism prohibit therapy, consider splenectomy. Consider hydroxyurea as palliative drug.
Prognosis : poor. Most patients survive < 1 year and respond poorly to cytoreductive drugs or chemotherapy. A curative therapy is currently not available.
• mast cell sarcoma (MCS) is an extremely rare form of mastocytosis. To date, we are aware of only 3 well-documented cases^ref. The disease is defined by a local destructive sarcoma-like growth of a tumor consisting of highly atypical MCs. At initial diagnosis, no systemic involvement is found. However, the terminal phase may be indistinguishable from ASM or MCL. The prognosis in patients with MCS is grave.
• extracutaneous mastocytoma
Symptoms & signs : urticaria pigmentosa (in 90%, Darier's sign : urtication and itching occurring on rubbing the lesions of urticaria pigmentosa), face flushing, cardiocirculatory shock, abdominal cramping, persistent macular, erythematous teleangiectasia (1%), cognitive impairment
Laboratory examinations : [histamine]_{urine, 24h}, [histamine, a-tryptase]_serum.
Therapy : H₁ antagonists, H₂ antagonists, anticancer drugs.
• monocytosis / histiocytosis / mononuclear leukocytosis / mononucleosis (> 10³ / mL) : a condition marked by the abnormal appearance of histiocytes (macrophages) in the blood. Disorders arising from cells of histiocytic and dendritic derivation are the epitome of confusing medical terminology, incomplete disease definitions and partial biologic and immunologic understanding. They are among the most rare of disorders of the hematopoietic system and thus, among the most difficult to study. They comprise a spectrum from benign proliferations of unknown etiology to frankly malignant tumors. The scarcity of markers specific to histiocytes, the lack of consistent means for detection of monoclonality, and the clinocopathologic overlap with reactive and infectious proliferations further complicate understanding their pathogenesis. Formulating a classification that incorporates the heterogeneity of histiocytic disorders has been challenging. The classification adopted by the Histiocyte Society separates dendritic cell- and macrophage-related entities of varied biological behavior from those that are malignant^{ref1, ref2}. A recent proposal expounded by the International Lymphoma Study Group classifies tumors of histiocyte and accessory dendritic cells into four groups based on their immunophenotype^ref:
• histiocytic sarcoma
• Langerhans cell tumor/sarcoma (LCH)
• follicular dendritic cell tumor/sarcoma
• interdigitating dendritic cell tumor/sarcoma
This classification only incorporates histiocytic disorders currently recognized as neoplastic. Thus, nonmalignant proliferations of histiocytic derivation, where the histiocyte may be central to the pathogenesis or a mere bystander, are still poorly understood.
Aetiology :
• infective causes
• Mycobacterium leprae
• Mycobacterium tuberculosis
• Brucella melitensis
• Leishmania spp.
• Treponema pallidum
• Toxoplasma gondii
• recovery from neutropenia
• autoimmune diseases
• inflammatory bowel disease
• rheumatoid arthritis
• chemical causes
• malignant histiocytosis : a rare type of histiocytosis found accompanying malignancies such as acute myeloid leukemia; it may be a malignant transformation of stem cells of the monocyte series. It usually affects children or young adults and has a poor prognosis.
• hereditary histiocytosis from lipid storage diseases (LSD)
• Gaucher disease
• sea-blue histiocyte disease or syndrome / sea-blue histiocytosis

Aetiology : unknown, but sometimes it is inherited as an autosomal recessive condition
• primary : D409 apoE mutation
• Niemann-Pick disease type B (NPB)
Symptoms & signs : splenomegaly; may range from a benign course with mild purpura secondary to thrombocytopenia, to progressive cirrhosis, hepatic failure, and death
Laboratory examinations : abnormal histiocytes that stain blue with Wright and Giemsa stains (sea-blue histiocytes)
• unknown causes
• type I histiocytosis / Langerhans cell histiocytosis (LCH) or granulomatosis / histiocytosis X (HX) / eosinophilic granuloma or granulomatosis of bone (EGB)  (CD1a⁺)

Epidemiology : children are more often affected than adults
Aeiology : no adenoviral, parvoviral HHVs, HIVs or HTLVs genome detected by PCR^ref
Pathogenesis : Langerhans cells (LC) were identified over 140 years ago at the dermal/epidermal border of the skin and subsequently as the most efficient antigen-presenting cell of the dendritic cell family. Nezelof et al proved in 1973 that the LC were the principal cells of LCH lesions, although lymphocytes, eosinophils and macrophages are present also. For decades most investigators have assumed the LC was the "pathologic" cell, but there is increasing evidence that the other cells may be playing extremely important roles in controlling the behavior of LC. Geissmann et al found that the LC in bone, lymph node, and some skin lesions were immature dendritic cells lacking expression of CD83, CD86, or DC-LAMP and had intracellular MHC class II antigen^ref. When cells were isolated from the lesions and incubated with CD40L they gained the maturation markers listed above and were better able to function in an allogeneic lymphocyte proliferation assay. Many macrophages in bone or lymph node lesions expressed IL-10, but this was not found in skin lesions. The authors hypothesized that IL-10 as well as TGF-ß could be key factors inhibiting the maturation of LC. IL-10 is noteworthy since it also downregulates B7 and MHC class II antigen expression, thus inhibiting effective antigen presentation. This is consistent with the known defect of this function of LC from LCH patients. The authors concluded that LC in LCH are not intrinsically malfunctioning but are heavily influenced by the microenvironment. Egeler and colleagues had earlier identified the types of cells producing many of the cytokines in the microenvironment^ref. They found LC, macrophages, and T cells produced GM-CSF and IFN-g; IL-1a came exclusively from LC, T cells produced IL-2, IL-4, IL-5 and TNF-a; IL-10 came from LC and macrophages, and eosinophils produced IL-5, IFN-g, GM-CSF, IL-10, and IL-7. In another study this group has reported extensive expression of CD40 on LCH and CD40L on T cells. Since Geissman et al did not stain their specimens for CD40L we do not know if there could have been a deficiency in those lesions. One would expect not and thus hypothesize that pharmacologic doses of exogenous CD40L caused the LC in Geissmann’s in vitro studies to overcome the effects of other cytokines that inhibit LC maturation. The close proximity of T cells and LC suggested that the "cytokine storm" in the lesions resulted from autocrine and paracrine amplification of signals between cell types. Ultimately this would be a favorable environment for proliferation of LC (and other cell types). Not surprisingly many of the cytokines could be responsible for the features of LCH: bone osteolysis, lung necrosis, liver and lung fibrosis. Others have found similar cytokine profiles (without IL-10 expression) in adult pulmonary LCH along with increased expression of the co-stimulatory molecules CD80/86 (B7-1/B7-2)^ref. Additional evidence of the "immature" state of the LC and a possible mechanism for their accumulation in lesions comes from analysis of chemokines on the cell surface. CD1a⁺ cells express the chemokine of iDCs, CCR6, and its ligand CCL20/MIP-3^ref. Among the other cytokines expressed by LC are CCL5/RANTES and CXCL11/I-TAC, which may help recruit eosinophils and CD4⁺ T cells. Thus, the abnormal CD1a⁺ cells would remain at the site of origin and recruit T cells that are also retained by the expression of CXCR3 and CCR6. Conversely, others have found that CCR7 was also expressed on the LC, suggesting they could migrate to skin and bone or to lymph nodes^ref.
• LCH = malignancy? : a central controversy about LCH is whether this is a malignant or inflammatory disorder. Clonal expansion of LC, but not lesional T cells, was defined by the human androgen receptor (HUMARA) DNA assay as well as TcR analysis^ref. These findings have led many aficionados of LCH to strongly state that it is a malignant proliferation. Clinical data that are considered supportive of this concept include the finding of familial cases and the higher than expected incidence of malignancies in LCH patients. Until recently there had been scant data on chromosome analysis. Now there are 2 papers in the literature showing molecular cytogenetic abnormalities in LCH by comparative genomic hybridization (CGH) and loss of heterozygosity (LOH) analysis^{ref1, ref2}. The first study evaluated 7 bone lesions and found losses of DNA sequences on chromosomes 1p, 5, 6, 9, 16, and 22q^ref. There was gain of DNA copy number on 2q, 4q, and 12. The highest frequencies of LOH (3/7 cases) were found on chromosome 1p and chromosome 7 (4/7 cases). Allele loss occurred on chromosomes 9 (2 cases) and 22q (1 case). The authors noted that 1p35-p36.3 contains several putative tumor suppressor genes and 22q12 contains the NF2 gene. They hypothesized that gains of DNA at sites similar to those found in malignancies (4q and 12) could relate to presence of a proliferation sequence. The above CGH data seem supportive of the malignancy theory given that cells from bone lesions are clonal by the HUMARA assay. However, analysis of adult pulmonary LCH by HUMARA has determined that it is not a clonal proliferation. Dacic et al reported that by CGH/LOH analysis of pulmonary LCH there was allelic loss at 1 or more tumor suppressor gene loci in 80% of the nodules dissected from 14 patients using essentially the same polymorphic microsatellite markers as noted in the first CGH study^ref. Given similar CGH/LOH results from clonal versus nonclonal LCH, the controversy on the malignant nature of LCH seems far from settled.
• LCH = inflammation? : for those who like the "inflammatory" disease label, the collection of morphologically normal LC along with other players in common inflammatory lesions (macrophages, lymphocytes, eosinophils) augur for a nonmalignant biology. Until recently there had been few data linking a specific viral infection to LCH. Glotzbecker et al found immunohistochemical evidence for HHV-6 in 71% of LCH biopsies^ref. However, there was no serologic evidence to show that a recent infection had occurred. Given the latent nature of herpes viruses and their propensity for reactivation, the link of HHV-6 and LCH is controversial. Another argument against a transformed LC phenotype is the fact that no one has been able to propagate these cells in vitro for more than a few days or successfully maintain LCH cells in immune-deficient mice. Furthermore, the generation of "clonal proliferations" does not always signal a malignancy. Clonal proliferation of T cells in the peripheral blood of normal individuals or those with cutaneous lymphomas have been termed "clonality of uncertain significance." The familial LCH cases are extraordinarily rare, and HLA data show that siblings of LCH patients who don’t have the disease share same HLA type^ref. A study of Scandinavian LCH patients revealed immunogenetic heterogeneity (as did the US study) with the HLA-DRB1*03 phenotype present in 38% of single-system LCH and only 3% of multisystem LCH^ref. It is well known that individuals with immune deficiencies have high rates of malignancies so it is difficult to use this data to argue that LCH is an a priori (pre)malignant condition. Strong TGFß receptor expression and heterogeneous staining for MDM2, p53, and p21 in LCH lesions are thought to be evidence of a normal cellular response, not of mutational damage^ref. Given the tumor suppressor actions of TGFß, p53, and p16 it seems unlikely that all of these would be functional in a truly malignant cell. High expression of Bcl-2 in LCH lesions has been reported by more than one group and is thought to enhance cell survival. A Danish group has evaluated the expression of p53, Fas and Fas-ligand in LCH and found strong expression of p53 and some cells co-expressing Fas/Fas-ligand creating an "autocrine apoptotic shortcut." This mechanism could possibly explain the spontaneous regression of some LCH lesions.
Symptoms & signs : lesions may be unifocal or multifocal and may involve the bone marrow, endocrine system, or lungs (the last being more common in adults than in children).
• unifocal LCH : occurring as a single osteolytic lesion, usually in a long or flat bone; it may be asymptomatic or may produce bone pain, tenderness, and swelling and, sometimes, pathologic fracture
• floating tooth sign : on radiographic examination of the mandible, erosion of the bony alveoli around the teeth so that they seem to be floating in space; it occurs in some forms of histiocytosis X.
• type II or multifocal LCH : erosive accumulations of proliferating Langerhans' cells, commonly within the medullary cavities of bones, but also affecting the skin, gingiva, lungs, and stomach. It most commonly affects children age 2-6 years and is accompanied by seborrheic eruptions, fever, frequent occurrences of otitis media, mastoiditis, and URT infection, often with sclerosing cholangitis, lymphadenitis and splenomegaly. When the triad of calvarial bone defects, exophthalmos, and diabetes insipidus is present (sometimes with accumulation of cholesterol), it is referred to as Hand-Schüller-Christian disease / chronic idiopathic xanthomatosis (Hand A. Jr. Polyuria and tuberculosis. Arch.Pediatri. 10:673-675 (1893); Schuller A. Uber eigenartige Schadel-defekte im Jugendalter. Fortschr.Rontgenstr. 23:12-18 (1915); Christian HA. Defects in membranous bone, exophthalmos and diabetes insipidus. Contribut.Med. Biol.Res., Hoeber Inc. 199 p.320)
• acute disseminated LCH / Letterer-Siwe (L-S) disease (Letterer E. Aleukemische Retikulose (Ein beitraf zu den proliferativen Erkrankugen des Retikulo-endothelial Apparates). Zeitschr. Pathol. 30:377-394 (1924). Siwe SA. Die Reticulo-Endotheliosis. Ein neues  Krankheitsbild unter den Heptosplenomegalien. Eur. J.Ped. 55:212-230 (1933))

Aetiology : autosomal recessive inheritance
Epidemiology : children < 2 years
Symptoms & signs : fever, pulmonary involvement, thrombocytopenia, cutaneous lesions resembling seborrheic dermatitis, hemorrhagic tendency, hepatosplenomegaly with lymph node enlargement, and progressive anemia
Prognosis : if untreated it is rapidly fatal
• reticuloendotheliosis / hemohistioblastic syndrome : hyperplasia of reticuloendothelial tissue.

Laboratory examinations : Custer cells (cells with long delicate protoplasmic processes replacing the lymphoid tissue of lymph nodes)
• disseminated lipogranulomatosis
Laboratory examinations :
• in bone and chronic lymph node lesions, LCH cells have immature phenotype (CD1a⁺14⁺40⁺68⁺83^-86^-LAMP^-langerin⁺ IL-10⁺class II MHC intracellular) and function (poorly stimulate allogeneic T-cell proliferation in vitro, but treatment with CD40L induces the expression of membrane class II and CD86 and strongly increases LCH cell allostimulatory activity to a level similar to that of mature DCs)
• in patients with self-healing and/or isolated cutaneous disease, LCH cells have more mature phenotype (CD14^-86⁺ IL-10^-)^ref
• in florid (exudative and granulomatous) nonfibrotic lung LCH lesions, LC, particularly those in contact with lymphocytes, are S100⁺CD1a⁺4⁺11a⁺11c⁺16⁺25⁺54⁺, MHC Class II⁺. Lymphocytes around LC are CD3⁺25⁺45RO⁺HLA-DR⁺ with a "memory" phenotype^ref
• eosinophilia
• Touton giant cell : a large vacuolated cell with numerous nuclei surrounding a peripheral rim of foamy cytoplasm


• jaw lesions can be divided into 2 types:
• type I (alveolar type) : the bone was destroyed along the alveola. This type is seen more frequently in the disseminated cases than in the EGB, and the majority of the lesions in this type manifested as ulcerative lesions clinically
• type II (jaw body type) : the mandibular body was destroyed in two ways which manifested as bony radiolucence with or without partitions. Most of the lesions had local swelling or massive manifestations clinically, and almost all lesions occurred in mandible, particularly in the cases of EGB.
Prognosis :
• congenital self-healing Langerhans cell histiocytosis or reticulohistiocytosis (CSHH / CSHRH / CSHLCH) / Hashimoto-Pritzker disease (CD1a/c⁺, CD1b^-, CD4^+/-, HLA-DR/DQ⁺, S-100⁺) : complete involution of the eruptions, leaving atrophic lesions in the sites corresponding to the initial findings^ref; consisting of precursor Langerhans cells^ref; may contain a large number of mast cells, leading to confusion with mastocytosis expecially when accompanied by urticaria^ref
• Lavin-Osband criteria : age of onset of the disease, number of involved organs, function of organs, characters of the traditional classification
Therapy : the focus on clinical features and response to therapy has sharpened because of international cooperative studies of childhood LCH. Hopefully an adult trial will commence soon. The clinical classification of pediatric LCH patients has been divided between :
• "low risk" sites: skin, bone, lymph nodes, and pituitary
• "high risk" sites: lung, liver, bone marrow, and spleen.
Since adult pulmonary disease is closely linked to smoking and has different biologic characteristics it should not be automatically put in the latter group. Analysis of data from the second international study for pediatric LCH (LCH-II) has shown some "Special Site" lesions of the skull (mastoid, orbit, temporal bones) that are associated with much higher frequency of central diabetes insipidus and parenchymal brain lesions. Should all LCH patients receive therapy? The simple answer is no when including single skull lesions in the frontal, parietal, or occipital areas and other skeletal lesions. However, it would be a mistake to say LCH is a slowly progressing disease in which a "wait and see" approach should be taken. This is especially true of pulmonary, jaw, and skin disease of adults
• smoking cessation may be effective in some patients with lung disease, but they need to be monitored carefully since the insidious progression of cystic changes and fibrosis can rob the patient of vital lung function.
• feroic surgery for jaw disease results in disfigurement and loss of teeth, whereas a 6-month course of vinblastine and prednisone can cure the disease and allow reformation of the bone with no loss of dentition.
• painful and disturbing perineal ulcers of LCH in women are best treated with chemotherapy or thalidomide, not radiation.
LCH-II results helped settle some questions, but left several others for the current (LCH-III) and future studies to answer^ref. LCH-II compared 24 weeks of treatment with vinblastine/prednisone to etoposide/prednisone for treatment of multisystem LCH. There was no difference in survival, number of reactivations, or toxicity between the 2 regimens. Thus, etoposide is no longer considered a reasonable therapeutic agent for LCH because of the known complication of causing secondary acute promyelocytic leukemia (APL)^ref. It was concerning that 50% of patients in either treatment arm had at least one recurrence. Patients with bone, skin, and/or lymph node involvement were all eventually cured with 1-2 salvage regimens. Earlier trials (DAL-HX 83/90) that extended therapy to 1 year and were more intense (additional maintenance therapy with mercaptopurine plus oral methotrexate in addition to vinblastine/prednisone pulses) had only a 32% recurrence rate^ref. The current LCH-III study is randomizing low-risk patients to 6 or 12 months of therapy with vinblastine/prednisone alone and treating all high-risk patients for 1 year. The randomization for high-risk patients is between vinblastine/prednisone (Arm A) vs vinblastine/prednisone/IV methotrexate (Arm B) in the first 6 weeks followed by maintenance with vinblastine/prednisone pulses every 3 weeks plus daily oral mercaptopurine for 1 year with oral methotrexate weekly for patients in Arm B. 2 other important results of the LCH-II trial were that age was not a prognostic factor, but response at week 6 was. High-risk patients who had not improved by the sixth week had only a 17% chance of survival in contrast to the 88% survival of those who had a good initial response. Further analysis of the patients with only bone LCH who had 1 year of therapy (DAL-HX 83/90 Trial) revealed that 18% had recurrences, with essentially all occurring in the first 2 years off therapy^ref. There was no difference in the frequency of recurrence whether the patients had unifocal or multifocal bone lesions. All patients with multifocal bone disease had the multi-agent chemotherapy mentioned above with the modest recurrence rate that is strikingly better than the 50–80% recurrence rate for patients treated with only single agent chemotherapy, radiotherapy or only observed. Another group of patients for whom multi-agent chemotherapy treatment is definitely indicated are those with lesions in the orbit, mastoid, or temporal bones. 40% of these individuals will develop central diabetes insipidus if they receive single agent therapy or surgery only as opposed to 20% who are treated with multi-agent chemotherapy. Patients with these bone lesions also may have parenchymal brain disease, which could be masses or infiltrative, that causes a neurodegenerative process. IFN-a₂^ref
Prognosis : late effects of the disease and treatment occur in 50% of LCH patients, making long-term follow-up a necessity^ref. These include diabetes insipidus (24%), orthopedic abnormalities (20%), hearing loss (13%) and neurologic problems (11%). The latter is characterized by cerebellar symptoms that may not be manifest until 10 or more years after diagnosis. Over 70% of multisystem patients had one or more of the late effects compared to 24% of single system patients. Another study of 59 patients with diabetes insipidus and serial magnetic resonance imaging (MRI) studies suggested that those with a thickened pituitary stalk were at higher risk of developing anterior pituitary deficiencies that ultimately occurred in 58% of all those with diabetes insipidus^ref. Adults with LCH continue to have difficulty in finding physicians familiar with the disease or comfortable with choosing treatments. A survey of adult LCH patients treated at 18 different centers throughout the world revealed a median latency time to diagnosis of 4 months, but was 11 months for those initially presenting with diabetes insipidus^ref. Unfortunately there are patients with pulmonary LCH who may not be diagnosed for years and then die of lung or secondary cardiac failure. Seventy-seven percent of pulmonary LCH patients were smokers compared to 44% of those with nonpulmonary LCH. Although only 16% of patients had isolated lung disease, it was present in 62% of those with multisystem disease. The optimal therapy for pulmonary LCH is not known^ref. Hence, in the adult LCH trial that should open soon, we will be assessing the natural history of patients who quit smoking and then offer a short course of steroids for progressive disease. An organized trial of chemotherapy for these patients must await a better understanding of the time course of the disease. Patients with lung plus LCH in other sites who receive chemotherapy may show us if there is some benefit for treatment of nodular or progressive cystic lung changes. Results of salvage therapy studies for LCH have been frustratingly slow coming to the literature because of insufficient accrual. Cladribine (2-CdA) has been used effectively for patients with bone disease^ref, but is probably not adequate for patients with "risk organ" involvement. The addition of cytosine arabinoside with 2-CdA is being considered for the next salvage protocol. Stem cell transplants have been successful in a number of patients, but the true rate of efficacy remains unknown since the total number of attempted stem cell transplants is not available^ref. Obviously, it is preferable to use this modality after only one or two salvage attempts since the patients are better able to withstand the rigors of the procedure. Antibodies to the CD1a epitope have been developed and may be available for diagnostic and therapeutic use in the future. Recently we found that the pathologic, but not normal LC express CD52 so a trial of alemtuzumab in patients refractory to standard therapy is needed^ref.
• type II (non-Langerhans cell) histiocytosis
• Erdheim-Chester disease (ECD) is a rare histiocytosis first described in 1930, histologically characterized by xanthogranulomatous infiltrates of foamy, lipid-laden histiocytes in all involved sites

Symptoms & signs : it may affect the bones and multiple extraosseous organs, most commonly the eyes, lungs, pleura, pericardium, pituitary gland, and kidneys, and, rarely, myocardium, liver, retroperitoneum, periadventitial tissue of the aorta (cystic medial necrosis / Edrheim's disease) and major arteries, brain (intracranial mass with potential to mimic glial neoplasms^ref) and retroorbital tissue. In the majority of patients, orbital infiltration is bilateral. The clinical orbital manifestation of ECD occurs in 2 different forms: one presenting as a mild impairment of visual function, while the second, clinical form is characterized by a progressive loss of visual acuity despite therapeutic efforts such as immunosuppressive systemic therapy, radiation, and surgery.
Laboratory examinations : radiologic evidence of cortical expansion of long bones
• disseminated xanthoma / xanthoma disseminatum / xanthoma multiplex : a rare form of non-Langerhans histiocyte proliferation, described by Montgomery in 1938

Symptoms & signs : normolipoproteinemic skin and mucous membrane xanthomatosis manifested by the development of reddish yellow to brown papules and nodules that may coalesce to form furrowed plaques and have a predilection for flexural creases, mucous membranes of the mouth and respiratory tract, cornea, sclera, and CNS, including the pituitary gland, the involvement of which often produces central diabetes insipidus
Therapy : surgical excision or laser therapy can improve physical and functional aspects but the evolution is characterized by very frequent relapses. Treatment with antimitotic drugs seems to be ineffective in many cases, but vinblastine may work^ref
Prognosis is related to the mucous membrane manifestations (50% of cases) and involvement of the upper respiratory tract
• sinus histiocytosis : a disorder of the lymph nodes in which the distended sinuses are completely, or nearly completely, filled by histiocytes, as a result of active multiplication of the littoral cells
• sinus histiocytosis with massive lymphadenopathy (SHML) / Rosai-Dorfman-Destombes disease (RDD) is a rare disorder characterized by a nonmalignant proliferation of distinctive histiocytic/phagocytic cells within lymph node sinuses and lymphatics in extranodal sites. Much of what is known of SHML today was pioneered by 2 pathologists, Juan Rosai and Ronald Dorfman. They recognized SHML as a distinct clinicopathologic entity and established a registry to collect and catalogue cases of SHML worldwide such that this unusual disorder may be better studied^ref. This scholarly contribution is well recognized today by the popularity of the eponym Rosai-Dorfman disease (RDD), particularly to refer to the extranodal form of this disorder. Since its initial description in 1969, SHML remains a disorder defined primarily by its histopathologic features. Due to its rarity, neither community-based surveys nor systematic treatment studies have been undertaken. Although a few hundred publications related to SHML can be found in the literature, many are single case reports or descriptions of small series of patients, limiting comprehensive appraisal of the subject.

Epidemiology : SHML occurs worldwide and is primarily a disease of childhood and early adulthood. No specific gender, ethnic or socioeconomic predilection is encountered, and the 423 cases in the SHML registry reflect the fact that people of Caucasian and African descent are equally affected while the disease is less common in those of Asian descent^ref. Occasional familial cases have been reported.
Pathogenesis : since its original description in 1969, SHML has remained a disorder of unknown etiology. Infectious agents have been suspected, but no clinical response to antibacterial or anti-tubercular drugs has been documented. Although serologic evidence of EBV was found in 3 of 6 cases, no evidence of latent or lytic EBV infection was identified within histiocytes of SHML^ref. Thus, it is unlikely that EBV plays a causative role in SHML. In situ DNA hybridization has demonstrated HHV-6-specific DNA within histiocytes of SHML in 7 of 9 cases^ref. In addition, IHC for the ORF-1 protein of HHV-6 demonstrates viral protein in the SHML histiocytes in both cases investigated^ref. In the latter study, the expression of HHV-6–specific p101K antigen was also found in follicular dendritic cells (FDC) in SHML, the relevance of which is unclear. The unfavorable clinical outcome in patients with associated immune dysfunction has led to the consideration that SHML results from an exuberant response of the hematopoietic system to an as yet undetermined immunologic trigger. Recently, cases of SHML associated with autoimmune lymphoproliferative syndrome (ALPS) have been described^ref. ALPS occurs primarily in early childhood and is an inherited disorder of lymphocyte programmed cell death orchestrated by mutations in death receptor genes that specifically abrogate apoptosis in lymphocyte subsets. First described by Canale and Smith in 1967 with the constellation of findings that include lymphadenopathy, splenomegaly and autoimmune cytopenias, ALPS shares similarities with mouse models of defective apoptosis (MRL/lpr phenotype) and shows an increase in mature TcRa/b⁺, CD4^-, CD8^- (double-negative, DN) T cells. The classical form of ALPS harbors specific mutations of Fas and FasL, although additional ALPS-like syndromes have since been identified and involve mutations of genes downstream of the Fas receptor^{ref1, ref2, ref3}. Recent data suggest that mutations of the Fas gene are found in a small subset of patients with SHML unassociated with ALPS. Thus, SHML may represent an acquired disorder of deregulation of apoptotic signaling pathways. Additionally, familial hemophagocytic lymphohistiocytosis (FHL), a rare and often acutely fatal autosomal recessive disorder, results from gene defects in perforin, a central mediator in lymphocyte cytotoxicity^ref. FHL is characterized by deregulated proliferation and accumulation of CTLs and histiocytes accompanied by overproduction of inflammatory cytokines leading to fever, pancytopenia, coagulopathy and neurologic deficits. Similar to the Fas/FasL pathway, the perforin pathway is another mediator of cytotoxic activation and a potential target for study in unraveling the pathogenesis of SHML. SHML is characterized by a rare, acquired, nonmalignant proliferation of distinctive histiocytes that presents with lympadenopathy or extranodal disease primarily in children and young adults. The causative agent is unknown. However, its diverse clinical manifestations and frequent association with subtle or severe immunologic abnormalities suggest an immune-mediated etiology. Recent studies of other immune-mediated histiocytic and CTL proliferations have raised interesting parallels worthy of further investigation in SHML. Defective Fas/FasL signaling leading to defective apoptosis, as demonstrated in patients with ALPS, may be an important mechanism whereby the uncontrolled histiocytic proliferation of SHML is triggered. However, a single gene defect is unlikely to explain the various clinical manifestations of SHML, which are most likely modulated by other immunologic factors or infectious agents. Additional studies are needed to characterize the interplay between death receptors and cytotoxic mediators and to further elucidate the loss of immune hemostasis that may underlie idiopathic histiocytic proliferations such as SHML.
Symptoms & signs : frequency (RDD patients from the SHML registry^ref with involvement at multiple extranodal sites are included under each anatomic site. The rarity of SHML precludes accurate statistics on the incidence of SHML or the frequency of specific clinical findings in the general community)
• lymph nodes (87%) : massive painless adenopathy: cervical most frequent (axillary, para-aortic, inguinal and mediastinal lymph nodes are also commonly affected), may be bilateral, occasionally accompanied by pain or tenderness  and contain large numbers of histiocytes
• extranodal disease (43%), in some without associated lymphadenopathy which may or may not develop later in the disease course^ref : sometimes with fever, anemia, neutrophilia, elevated ESR, and hypergammaglobulinemia.
• skin and soft tissue (16%) : broad range of non-specific presentations: typically painless, maculopapular rash, yellow xanthomatous solitary or multiple, usually raised reddish or bluish lesions, scaly
• subcutaneous nodules or soft tissue masses
• nasal cavity/paranasal sinuses (16%) : mucosal thickening, nasal polyps (may be recurrent) or masses causing mild or complete nasal obstruction, epistaxis, displacement of septum, infiltrative mass extending into maxillary, ethmoid, or sphenoid sinuses
• eye/orbit/ocular adnexa (11%) : eyelid or orbital mass with proptosis; may require enucleation
• bone (11%) : bone pain with osteolytic lesions in any bone of the axial or appendicular skeleton; on radiography, may be purely lucent or mixed with sclerosis or exhibit irregular borders or a broad sclerotic rim
• salivary gland (7%) : bilateral parotid enlargement or submandibular mass; may be associated with dry mouth
• central nervous system (7%) : intracranial epidural or dural, single or multiple masses, spinal cord masses, associated lytic bone lesions in skull or spine, cranial or spinal nerve palsies, weakness, syncope, headache
• oral cavity (4%) : infiltrative mass of soft or hard palate, diffuse mucosal thickening, papillomatosis, gingival swelling
• kidney/genitourinary tract (3%) : unilateral or bilateral renal masses accompanied by obstruction: hydronephrosis, hydroureter, ureteral obstruction, glomerulonephritis, chronic pyelonephritis
• testicular, scrotal or epididymal masses with intermittent penile or pubic edema
• respiratory tract/larynx/lungs (3%) : laryngeal obstruction, thickening and fibrosis associated with hoarseness
• liver (1%) : enlargement with congestion, milliary infiltrates, masses
• tonsil (1%) : enlargement, may be accompanied by tonsillitis
• breast (< 1%) : predominantly subcutaneous involvement, rarely involves breast parenchyma, associated axillary adenopathy
• gastrointestinal tract (< 1%) : hyperplastic mucosal folds, single or multiple exophytic masses
• heart (< 1%) : subendocardial or valvular infiltrates causing arrythmias
Simultaneous involvement of multiple extranodal sites is not unusual. Involvement of the kidney, lower respiratory tract and liver are associated with a worse clinical outcome as is the number of extranodal sites involved. Hepatosplenomegaly is uncommon.
SHML exhibits a broad range of clinical presentations and thus, elicits a wide differential diagnosis. Although not comprehensive, the salient clinical features of SHML affecting various anatomic sites are provided to illustrate its heterogeneous presentations. SHML in lymph nodes may clinically mimic metastatic carcinoma or melanoma, Hodgkin and non-Hodgkin lymphoma, infectious processes, granulomatous lymphadenitis, reactive lymphadenopathy or other histiocytoses including Langerhans cell histiocytosis. Histiocytic disorders, particularly Langerhans cell histiocytosis must be distinguished from RDD affecting skin, soft tissue and bone. In the nasopharynx, nasal polyps, nasopharyngeal carcinoma, lymphoma and rhinoscleroma enter the spectrum of diagnostic considerations. In the lung, the differential diagnosis includes granulomatous inflammation such as sarcoid, infectious processes, Erdheim-Chester disease, foreign-body giant cell reaction and aspiration pneumonia. In addition to these entities, RDD affecting the heart may resemble giant cell myocarditis, granulomatous myocarditis or a foreign-body giant cell response. Tonsilar involvement may simulate EBV-associated lymphoproliferative disorders and infectious mononucleosis. Since its recognition 35 years ago, the constellation of clinical findings has formed an ever-expanding almanac as more and more cases of SHML are diagnosed.
Laboratory examinations :
• clinical laboratory findings include hematologic abnormalities such as normocytic or microcytic anemia. Immunologic abnormalities are found in a significant number of patients and often lead to a clinically unfavorable disease course. Up to 90% of patients demonstrate an elevated erythrocyte sedimentation rate. The most frequent immune dysfunction is autoimmune hemolytic anemia, which leads to severe hemolysis or fatality in a subset of patients^{ref1, ref2}. Polyarthralgia, rheumatoid arthritis, glomerulonephritis, atopic asthma and diabetes mellitus are also known to complicate SHML. Polyclonal hypergammaglobulinemia is found in 90% of patients. Rarely, patients exhibit rheumatoid factor, antinuclear antibodies, and a reversal of CD4/CD8 ratio among peripheral lymphocytes. A small subset of patients also has concurrent neoplasia including NHL, other histiocytic proliferations, multiple myeloma,melanoma and carcinoma. Consistent serologic findings are lacking, although antibodies to pathogenic organisms including EBV and HHV-6 have been reported^{ref1, ref2, ref3}
• surgically excised lymph nodes are yellow-white with frequent capsular and pericapsular fibrosis. Generally, normal lymph node architecture is preserved and effacement is only seen in patients with longstanding lymphadenopathy. Typically, the lymph node sinuses are expanded by a proliferation of distinctive histiocytes exhibiting enlarged round or oval vesicular nuclei with well-defined, delicate nuclear membranes and a single prominent nucleolus. Multilobulated nuclei, nuclei with multiple nucleoli and nuclear atypia are rare. Mitoses are infrequent, although increased mitotic activity can be apparent occasionally. The histiocytes possess abundant pale eosinophilic cytoplasm; however, on occasion numerous histiocytes with foamy cytoplasm may predominate the cellular milieu. The hallmark of the SHML histiocyte is the presence within the cytoplasm of variable numbers of intact lymphocytes, a phenomenon referred to as lymphophagocytosis or emperipolesis, defined as lymphocytic penetration of and movement within another cell, in this case, the histiocyte^ref. These lymphocytes are often housed within vacuoles and escape degradation during their transit through the histiocyte. Plasma cells, neutrophils and red blood cells may also occupy this unique intracytoplasmic niche. Associated with the histiocytic proliferation are numerous, morphologically typical plasma cells often aggregated around post-capillary venules. Eosinophils are not a salient feature of SHML as compared with LCH, classical Hodgkin or T-cell lymphoma. Collections of neutrophils, eosinophilic microabscesses and reactive germinal centers are occasionally seen but are not prominent findings in SHML. Rosai-Dorfman disease involving extranodal sites shows similar morphologic features to its nodal counterpart although more fibrosis and fewer histiocytes with emperipolesis are encountered.
• the histologic differential diagnosis includes LCH, histiocytic sarcoma, storage disorders such as Gaucher’s disease, classical Hodgkin lymphoma, metastatic melanoma and carcinoma, and infections caused by Histoplasma and mycobacterial organisms involving the lymph node. In the nasal cavity, rhinoscleroma may morphologically resemble RDD but is distinguished by the identification of Klebsiella rhinoscleroma. Perhaps the most frequently faced histologic challenge is that of distinguishing SHML from reactive sinus histiocytic proliferations which occur as nonspecific responses to a variety of instigating agents. Emperipolesis is rare or absent in reactive histiocytic proliferations. Although erythrophagocytosis is seen in reactive and neoplastic histiocytic proliferations, including LCH, emperipolesis in a setting outside of SHML is extremely rare. The appearance of lymph node sinuses expanded by a histiocytic proliferation is a feature common to LCH and SHML. However, unlike SHML, Langerhans cells have smaller, frequently folded or grooved nuclei and are associated with eosinophilic microabscesses.
• immunohistologic studies : the most useful immunohistologic marker for SHML histiocytes is the expression of the S100 protein^{ref1, ref2}. In addition, SHML histiocytes express pan-macrophage antigens (CD68, HAM 56, CD14, CD64, CD15), antigens associated with phagocytosis (CD64 or Fc receptor for IgG), lysosomal activity (lysozyme, a₁-antitrypsin) and immune activation (transferring receptor, IL-2R). We have recently found that SHML histiocytes express CD163, a hemoglobin scavenger receptor and acute phase-regulated transmembrane protein found on tissue macrophages and monocytes^ref. These studies support the hypothesis that the effector cell in SHML is a functionally activated macrophage and is distinct from Langerhans cells, FDCs and interdigitating dendritic cells. While Langerhans cells stain for the S100 protein and CD1a, only rare CD1a⁺ cells are found in SHML. In addition, SHML histiocytes do not express CD21, CD23, or CD35 (markers of dendritic differentiation). The overlap in morphologic and immunophenotypic features among reactive and neoplastic histiocytic proliferations, especially in unusual nodal presentations or extranodal sites, can make the diagnosis of SHML particularly problematic.
Therapy : the disease is usually chronic with spontaneous remission and is refractory to treatment. Partial removal of tumor masses is a workable way to improve visual acuity and correct corneal exposure if optic neuropathy occurs. Treatment is not necessary in the majority of patients as the disease is self-limiting in most cases. Only in a minority of patients where massive nodal or extranodal enlargement infringes on organ function or threatens life is treatment warranted to halt the natural progression of SHML. As randomized clinical studies of treatment for SHML have not been undertaken, precise and objective measures of therapeutic response are unavailable. In a recent review of treatment strategies for SHML^ref, 50% of patients (40/80) did not require any treatment. Patients given antibiotics or anti-tuberculosis drugs showed no response. Steroid therapy resulted in reduction of lymphadenopathy and associated fevers. Of 9 patients treated with radiation therapy for lymphadenopathy 3 achieved complete remission, 3 had persistent SHML and 3 died of disease. Of 12 patients treated with currently used chemotherapy regimens involving a combination of Vinca alkaloids, anthracyclines and alkylating agents, 10 showed no response while 2 others achieved complete and durable remission with methotrexate and 6-mercaptopurine. Surgery and radiation therapy employed in 8 patients led to complete remission in 1 case and partial responses in 6 cases. High-dose interferon- was given to 1 patient with long-term remission. In a recent report, 2 children with intracranial RDD were treated with the purine analog cladribine (2-CdA) found to be of clinical utility in the treatment of refractory LCH; although short-term symptomatic relief was obtained there was no objective response^ref. The ideal treatment for SHML is as yet undefined. For SHML that does not involve or compress vital organs the patient may be followed with observation. For patients with high fever without other symptomatology steroid therapy may be instituted, although a concurrent infection should be carefully investigated before the administration of steroid therapy. Surgical debulking, radiation therapy or a combination of both may be used in cases where vital organ function is compromised. The lack of response to high-dose chemotherapy for severe SHML suggests that chemotherapy should not be a mainstay of therapy in the management of this disease. More data are needed on newer agents such as IFN-a to permit evaluation of their efficacy in the treatment of SHML.
Prognosis : the clinical course of SHML is characterized by spontaneous resolution in most cases, especially in those patients presenting with localized lymph node involvement. Some patients have episodes of exacerbation alternating with periods of remission that continue for many years, where the timing and duration of each phase is entirely unpredictable. Persistence of lymphadenopathy or progression to widespread dissemination may occur, particularly in SHML involving the kidney, lower respiratory tract or liver with associated immunologic dysfunction. Of 423 cases in the SHML registry 17 patients died of the disease or with persistent disease^ref.
• reticulohistiocytoma : a granulomatous proliferation of lipid-laden histiocytes and multinucleated giant cells with pale eosinophilic cytoplasm having a ground-glass appearance. It occurs in 2 clinically different but histologically indistinguishable forms
• reticulohistiocytic granuloma / reticulohistiocytoma sensu strictu : a solitary reticulohistiocytoma, seen mainly in men, which is not associated with systemic involvement, as in multicentric reticulohistiocytosis
• multicentric reticulohistiocytosis / lipid or lipoid dermatoarthritis : a rare systemic disease seen mainly in women, characterized by the formation of multiple reticulohistiocytomas, polyarthritis of the hands and large joints with non pruriginous and hyperchromic papulonodular rash in the skin, bone, and mucous and synovial membranes and sometimes soft cystic ganglialike swellings over the extensor and flexor surfaces of the wrists; there may be evidence of internal malignancy. It may become quiescent, leaving only crippling arthropathy and disfigured skin, or it may progress to multiple organ involvement and eventual death

Therapy : first GR agonists and secondary chloroquine^ref
Pathogenesis : severe loss of thymic cortical and medullary structures rather than stress type changes in which only the cortical structure changes. Severe dysplastic changes equivalent to those seen in congenital immune deficiency are only seen in those patients with Type I histiocytic disease, while severe nonspecific involution occurrs in patients with Type II histiocytic disease. Dysmorphic change (dysplasia + smaller lobules + interstitial histiocytic infiltration) is apparent in both, but it had a much higher incidence in Type II^ref
Web resources : Histiocytosis Association of America
• lymphocytosis
• polyclonal lymphocytosis
• polyclonal B-cell lymphocytosis (PPBL) : 84 cases have been published since 1983 to 2000. This rare hematological disorder of unknown etiology is characterized by morphologically atypical lymphocytes, polyclonal IgM production in association with smoking, female gender, and HLA-DR7 phenotype. In contrast to normal B-cells, PPBL cells showed no response to IL-4 with regard to CD23 and HLA-DR expression. F2mu antibodies failed to co-stimulate IL-4-mediated CD23 expression. Crosslinking membrane IgM receptors by F2mu resulted in elevated HLA-DR expression but did not induce in vitro proliferation of PPBL cells. This indicates a different activation and differentiation status than normal B-cells^ref

Differential diagnosis : B-cell chronic lymphocytic leukaemia (CLL)
• atypical lymphocytosis
• acute infectious lymphocytosis : an acute, benign infectious disease of children characterized by an excess of normal small lymphocytes in the blood without lymphadenopathy or splenomegaly, and with varying degrees of clinical expression and constitutional response.
• Adenovirus
• HHV-2 / HSV-2
• absolute large granular lymphocytosis (ALGL)
• plasmacytosis
• plasma cell leukemia
• reactive plasmacytosis :
• viral infections :
• infectious mononucleosis
• acute parvovirus B₁₉ infection^ref
• dengue virus^ref
• rubella infection^ref
• staphylococcal sepsis^ref
• inflammation^ref
• medication
• serum sickness syndrome^ref
• acalculous cholecystitis^ref
• reactive plasmacytosis of the thyroid gland in a patient with disseminated tuberculosis^ref
• primary Sjogren's syndrome(skin^ref, lung^ref and bone marrow^{ref1, ref2})
• drug hypersensitivity
• mucous membrane plasmacytosis is a rare, idiopathic condition consisting of a dense plasma-cell infiltrate of the mucous membranes. The phenomenon of plasma-cell infiltrate was first described by Zoon^ref in 1952 when he described balanitis plasma cellularis. Since then, plasma-cell infiltrates have been found on the vulva, buccal mucosa, palate, nasal aperture, gingiva, lips, tongue, epiglottis, larynx, and other orificial surfaces. These conditions have been described with a variety of different terms. During the late 1960s and early 1970s, cases of plasma-cell infiltrates of the lips, gums, and tongue were described primarily in the dental literature under the names atypical gingivostomatitis^{ref1, ref2}, idiopathic gingivostomatitis^ref, and allergic gingivostomatitis^ref. The lesions were thought to be a result of a reaction to chewing gum, dentifrices, and other foreign substances^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8}, although extensive allergy testing has been inconclusive^{ref1, ref2}. Sherman (Schuermann H. Plasmacytosis circumorificialis. Dtsch Zahnarztl 1960; 15: 601-610) and Luders (Luders G. Plasmocytosis mucosae: Ein oft verkanntes neues Krankheitsbild. Munch ed Wochenschr 1972; 114: 8-12) simplified the nomenclature in 1960 and 1973, respectively, by grouping the infiltrates by anatomy under the titles plasmacytosis circumorificialis and plasmacytosis mucosae. However, additional terms have been used in the literature to describe plasma-cell infiltrates of the aerodigestive tract, such as plasma cell gingivitis^{ref1, ref2, ref3, ref4}, plasmacytosis of the gingival^ref, and plasma-cell cheilitis^{ref1, ref2}. In 1986, White et al.^ref grouped all plasma-cell infiltrates of the aerodigestive tract under the name plasma-cell orificial mucositis because of the fact that all the cases reported had clinical and histologic findings that were indistinguishable from one another, eliminating the need for separate names for each anatomic area. Since that time, plasma-cell orificial mucositis, or variants of the name such as mucous membrane plasmacytosis and plasma-cell mucositis, have been used to describe benign plasmacytic lesions of the aerodigestive tract in the majority of cases reported^{ref1, ref2, ref3, ref4, ref5, ref6}. Clinically, the lesions of mucous membrane plasmacytosis may present with a variety of symptoms and morphologies depending on the area of the aerodigestive tract in which it occurs. When appearing on the lips and gums, patients complain of sore, swollen lips and gums. Examination reveals a well-circumscribed, soft, slightly elevated, edematous mass in which the surface is red and glistening but not ulcerated. When occurring in other parts of the aerodigestive tract, such as the larynx, pharynx, palate, buccal mucosa, trachea, and distal airways^ref, patients complain of oral pain, dysphonia, dysphasia and difficulty breathing. On examination the lesions typically appear cobblestone- or wart-like^ref. Several cases have been reported in which patients demonstrate simultaneous plasma-cell infiltrate lesions of the gingiva and the pharynx or larynx^{ref1, ref2, ref3, ref4}. Histologically, the majority of lesions are described as having an acanthotic epidermis with narrow and elongated rete ridges, spongiosis and a dense subepithelial cellular infiltrate composed largely of mature plasma cells, but also including a few polymorphonuclear leukocytes and lymphocytes. Occasional Russell bodies (accumulation of immunoglobulin within cytoplasm of plasma cells) were occasionally noted^ref. The plasma cells do not show anaplasia or prominent nucleoli. In addition, there are reports of plasma-cell orificial mucositis that show epidermal atrophy (Luders G. Plasmocytosis mucosae: Ein oft verkanntes neues Krankheitsbild. Munch ed Wochenschr 1972; 114: 8-12) and eroded epidermis^{ref1, ref2, ref3}. Immunoperoxidase staining show mixed population of k and l light chains and various heavy chains, demonstrating the polytypic and benign nature of the condition.

Differential diagnosis : several other disorders may present with lesions that appear similar clinically or histologically including fungal infection, carcinoma, syphilis, lichen planus, cicatricial pemphigoid, allergic or contact mucositis, sarcoidosis, cheilitis granulomatosa, plasma-cell granuloma, plasmoacanthoma, rhinoscleroma, Rosai-Dorfman disease, Melkerson-Rosenthal syndrome, and extramedullary plasmacytoma. Several of these diagnoses can be ruled out by histology or further testing for an infectious process
• fungal infection can mimic mucous membrane plasmacytosis but is usually distinguished by absence of fungal hyphae on microscopic examination, negative KOH preparation, no growth on culture, or a lack of response to treatment with nystatin. Histologic examination of the tissue can exclude carcinoma from the differential diagnosis.
• mucous membrane lesions are present in one-third of patients with secondary syphilis, and histology can show superficial and deep perivascular infiltrate of plasma cells, lymphocytes and macrophages distributed in a bandlike pattern in the dermis, accompanied by psoriasiform epidermal hyperplasia and hyperkeratosis (Odom, RB, James, WD, Berger, TG. Andrew's Diseases of the Skin Clinical Dermatology, 9th Edition. Philadelphia: W.B. Saunders Co., 2000). However, negative serologic tests for syphilis and absence of spirochetes on a silver stain of tissue sections can rule out secondary syphilis as the cause for the mucous membrane lesions.
• when lichen planus occurs on mucous membranes, these often painful lesions are usually white plaques, typically with a lacelike pattern. Other presentations of mucous membrane lichen planus include atrophy, erosions or ulcers, and bullae. Microscopically, lichen planus is characterized by a superficial, bandlike, lymphocytic infiltrate and occasionally hyperkeratosis, but no significant accumulation of plasma cells.
• cicatricial pemphigoid (benign mucosal pemphigoid) can clinically resemble mucous membrane plasmacytosis. The oral mucosa of patients with cicatricial pemphigoid is almost always involved, including the nasal mucosa, nasopharynx, larynx, oropharynx, hypopharynx, and esophagus. Oral lesions are characterized by evanescent vesicles that quickly rupture and leave erosions and ulcers, as opposed to the raised and cobblestone-appearing lesions of mucous membrane plasmacytosis. Cicatricial pemphigoid typically affects middle-aged to elderly women whereas mucous membrane plasmacytosis tends to affect elderly men. Histologically, there is subepithelial disruption or ulceration but no psoriasiform changes or plasmacytosis as expected with mucous membrane plasmacytosis^ref (Odom, RB, James, WD, Berger, TG. Andrew's Diseases of the Skin Clinical Dermatology, 9th Edition. Philadelphia: W.B. Saunders Co., 2000)
• allergic or irritant contact dermatitis can initially be ruled out by history and by distribution of the lesions. Dentures or foreign objects can be removed to rule out contact dermatitis and patch testing can be done to detect any other contact or allergic factors. Histologically, lesions of allergic contact dermatitis reveal spongiosis with infiltrate of monocytes and histiocytes in the dermis. Primary irritant dermatitis presents with superficial vesicles containing polymorphonuclear leukocytes.
• chronic, granulomatous disease sarcoidosis can affect mucous membranes, including the palate, tongue, buccal mucosa or posterior pharynx. In the oral cavity, the lesions are papules that may coalesce to form a flat plaque (Odom, RB, James, WD, Berger, TG. Andrew's Diseases of the Skin Clinical Dermatology, 9th Edition. Philadelphia: W.B. Saunders Co., 2000). Microscopic examination reveals large, epithelioid histiocytes with a few giant cells and lymphocytes, and occasional asteroid bodies with no evidence of a plasma-cell infiltrate.
• mucous membrane plasmacytosis can clinically resemble cheilitis granulomatosa; however, histopathology shows evidence of a granulomatous infiltrate rather than a plasma-cell infiltrate.
• plasma-cell granulomas tend to locate in the oral cavity, primarily on the periodontal tissue. These lesions are often single; whereas, the lesions of mucous membrane plasmacytosis tend to be multiple. On histologic evaluation, plasma cells are prominent but are intermixed with abundant other cellular elements and usually surrounded by connective-tissue septae, distinguishing it from mucous membrane plasmacytosis^ref
• plasmoacanthoma is described as a discrete, verrucous tumor with hyperkeratosis, pronounced acanthosis of the epidermis and plasmacytic infiltrate in the dermis on histologic examination. Plasmoacanthoma is considered a separate disease entity from plasma-cell orificial mucositis; however, in 1995 van de Kerhof and van Baar^ref described the simultaneous occurrence of these conditions in a patient and suggested that both conditions are part of one single disease entity.
• rhinoscleroma is a chronic, inflammatory, granulomatous disease of the upper aerodigestive tract, associated with Klebsiella rhinoscleromatis infection, which can have a clinical presentation similar to that of mucous membrane plasmacytosis. Rhinoscleroma affects both sexes equally, most commonly during the third and fourth decades in persons from Russia, Central Europe, Egypt, Pakistan, Africa, and South and Central America. Histology reveals a granulomatous tumor consisting primarily of plasma cells, an occasional Russell body, a few spindle cells and hypertrophic collagenous tissue. Warthin-Starry silver stain can allow for visualization of the bacilli of rhinoscleroma within the foamy macrophages (Mikulicz's cells). Culture and serologic tests can also be performed to rule out rhinoscleroma (Odom, RB, James, WD, Berger, TG. Andrew's Diseases of the Skin Clinical Dermatology, 9th Edition. Philadelphia: W.B. Saunders Co., 2000)
• Rosai-Dorfman disease (sinus histiocytosis with massive lymphadenopathy) can involve the head and neck^ref. Most patients who present with skin lesions are 40 years old or older at presentation. The lesions can be isolated or disseminated yellow-brown papules or nodules or macular erythema. The histopathologic features of Rosai-Dorfman disease are a superficial and deep, perivascular infiltrate of lymphocytes and plasma cells, in addition to a diffuse dermal infiltrate of large, foamy histiocytes. Additionally, fibrosis and lymphophagocytosis are characteristically present, features not common in mucous membrane plasmacytosis. The elements of epithelial psoriasiform changes and dyskeratosis typical of mucous membrane plasmacytosis are not seen in Rosai-Dorfman disease (Odom, RB, James, WD, Berger, TG. Andrew's Diseases of the Skin Clinical Dermatology, 9th Edition. Philadelphia: W.B. Saunders Co., 2000)
• Melkerson-Rosenthal syndrome consists of a triad of recurring facial paralysis or paresis; soft, nonpitting edema of the lips; and scrotal tongue. Usually starting in adolescence, the first symptoms can be the swelling of the skin and mucous membranes of the face and mouth. A tuberculoid type of granuloma with lymphedema and a banal perivascular infiltrate are seen on microscopic examination (Odom, RB, James, WD, Berger, TG. Andrew's Diseases of the Skin Clinical Dermatology, 9th Edition. Philadelphia: W.B. Saunders Co., 2000)
• on the basis of histology, extramedullary or primary cutaneous plasmacytoma needs to be considered. This tumor is found in the upper respiratory tract in approximately 80% of cases, especially in the nasal cavity and sinuses, nasopharynx, and larynx and can be sessile, polypoid or pedunculated^ref. Histologically, plasmacytomas are composed of a diffuse infiltrate of plasma cells in the dermis and subcutaneous tissue. There is minimal to prominent nuclear atypical of the plasma cells and on immunohistochemistry they are monoclonal, distinguishing plasmacytoma from plasmacytosis. Gene-rearrangement studies can be done if immunohistochemistry is inconclusive.
Therapy : although several treatment modalities have been tried, including corticosteroids (topical, intralesional, and systemic), antibiotics, destruction of the tissue (liquid nitrogen, CO₂ laser and electrocoagulation), excision of the tissue, and radiation therapy, no treatment clearly stands out as consistently effective
• corticosteroids have been used with the most frequency but with inconsistent results. A review of the literature by White et al.^ref in 1986 revealed that topical and intralesionally administered steroids were of no benefit and other studies have reported some symptomatic improvement but no true regression of the disease^{ref1, ref2, ref3, ref4, ref5}. Additionally, the patient reported in this case report has not responded to topical or systemic corticosteroids. A few cases in which corticosteroids induced marked regression with residual scarring^{ref1, ref2}, as well as total eradication of the disease have been reported^{ref1, ref2, ref3}
• antibiotics have also been used to treat mucous membrane plasmacytosis. Treatment with oral nystatin was found to be ineffective in the patient described in this case report as well in another case report^ref. Ferrerio et al.^ref report on 2 patients treated with antibiotics (type unspecified) without effect. In one case, 2% fusidic acid was found to be an effective treatment^ref and ineffective in another^ref. Tamaki et al.^ref described two patients with plasma-cell cheilitis who responded to oral griseofulvin with complete remission of lesions.
• Fogarty^ref reports on a case of gingival plasmacytosis that had progressed to involve the larynx and was treated with systemic chemotherapy (cyclophosphamide, vincristine, and prednisolone) with temporary improvement, but symptoms progressed after therapy was discontinued. Eventually, this patient was treated with low-dose radiation therapy, which subsequently caused symptomatic improvement, which was maintained at 12month followup.
• several authors report on resorting to debulking procedures, either surgical excision, electrocoagulation, carbon dioxide laser, or cryotherapy, to reduce the mass effect of the mucous membrane plasmacytosis involving the pharynx and larynx^{ref1, ref2, ref3, ref4, ref5}. These procedures resulted in improvement of symptoms; however, the procedures are a temporary measure as the lesions and symptoms recurred. In fact, a few patients have required a tracheostomy, owing to the progression of the mucous membrane plasmacytosis lesions causing subglottic strictures^{ref1, ref2}
Prognosis : long-term prognosis for patients with mucous membrane plasmacytosis appears good. Ferreira^ref reports that the majority of the cases he followed (seven of nine) had no progression nor regression of their disease, and the long-term survival appeared good with the longest survivor living 16 years with the disease. However, a few studies have demonstrated progression of the disease from the oral mucosa to the larynx^{ref1, ref2, ref3, ref4, ref5, ref6}, occasionally causing sufficient airway obstruction to require a tracheostomy^{ref1, ref2}. No studies to date report a progression of benign mucous membrane plasmacytosis to a malignancy of any type.
Mucous membrane plasmacytosis is a diagnosis of exclusion, distinguished primarily on the histologic finding of a marked submucosal plasma-cell infiltrate in addition to psoriasiform epithelial changes, after conditions such as infection and plasmacytoma have been eliminated. The etiology of this condition is unclear but is believed to be a nonspecific inflammatory response, in the form of a plasma-cell infiltrate, to an unknown exogenous agent. Attempts to induce plasma-cell infiltrations on mucosal and nonmucosal surfaces by allergic and irritant stimuli were not successful^ref. Roman^ref hypothesized that plasma-cell gingivitis may be associated with low levels of serum IgA and secretory IgA, which allows localized, repetitive, subclinical infections that could lead to the plasma-cell infiltrate. Aiba^ref points out that a plasma-cell infiltrate is a rare histologic feature in ordinary inflammatory dermatoses but is often found around such epidermal neoplasms as actinic keratosis, Bowen disease, squamous cell carcinoma, and syringocystadenoma papilliferum. The authors hypothesize that, although the inciting factor of the plasma-cell infiltrate is unknown, it is plausible that similar mechanisms are involved in both the mucosal and skin plasma-cell infiltrate conditions. The best treatment for mucous membrane plasmacytosis is also unclear, with inconsistent data from trials with corticosteroids, antibiotics, radiation, ablative therapy, and surgical excision. Mucous membrane plasmacytosis should be included in the differential diagnosis of swelling or lesions of the aerodigestive tract; typical histology can easily confirm the diagnosis.
• Castleman's disease (CD) / angiofollicular lymphoid hyperplasia / giant lymph node hyperplasia : a polyclonal, nonneoplastic condition resembling lymphoma but without recognizable malignant cells; there are isolated masses of lymphoid tissue and lymph node hyperplasia. Understanding CD and the literature about CD requires not only recognizing these very different types of CD but also recognizing that, because CD in many circumstances is a diagnosis made by exclusion, there can easily be overusage of this diagnosis by pathologists, and, at the same time the diagnosis can also be easily overlooked in certain circumstances. CD is a set of complex disorders that range from localized benign tumor masses to extremely aggressive disseminated proliferations. Although the major subtypes of CD do have some features in common, suggesting a possible relationship to one another, their differences are much greater and must be recognized both for clinical purposes and to best study their underlying biologic features.

Histology :
• hyaline vascular or hyalinovascular type (HV-CD) (more common) is a disorder of stromal cells, which presents as a solitary mass that is usually curable surgically. It can be complicated by the development of soft tissue sarcomas. It presents numerous small hyalinized and hypervascular GCs with hypervascular interfollicular stroma and sinus effacement. It  was described in 1956 by Benjamin Castleman and colleagues as a localized mediastinal mass in otherwise usually asymptomatic patients^ref
• plasma cell type (PC-CD) (less common) is a plasma cell disorder with elevated levels of IL-6. It presents fewer but larger (hyperplastic) GCs with plasma cell aggregates in lymph node paracortex and partially spared sinuses. It is associated with more generalized lymphadenopathy and immunological abnormalities. This disease occurs more frequently in older individuals and is more common in men. It  was described that was also localized but had very different clinical and histopathologic features^ref
Localizations :
• localised Castleman's disease (LCD) : single lymph node in mediastinum, pulmonary hilum, or extrathoracic localization
• HV-LCD

Epidemiology : occurs in children and adults (median age in the 30s)
Aetiology : viral infection including HHV-4 / EBV or HHV-8 / KSHV and IL-6 overexpression are not considered to play a role in at least most cases of HV-CD^{ref1, ref2}.
Pathogenesis : the vascular proliferation may, in some cases, be due to abnormal, usually intrafollicular, VEGF expression^ref. A clonal cytogenetic abnormality has been reported in at least 2 cases of HV-CD including one where the abnormality that involved the HMGIC gene was documented in the CD21⁺ FDC^ref. HMGIC (high mobility group protein I-C) abnormalities are reported in a variety of benign mesenchymal neoplasms. Although this suggests that the FDC in HV-CD are neoplastic, it could be a secondary event unrelated to the basic pathogenesis of the CD. FDC dysplasia, however, is a relatively frequent finding in HV-CD^{ref1, ref2}. In addition, like many FDC sarcomas and unlike normal germinal centers, there is often moderate or strong expression of EGFR in the FDC of CD, a feature that seemed to correlate with the degree of FDC dysplasia present^ref. Consistent with the importance of FDC and other stromal elements in HV-CD is the development of FDC sarcomas and vascular neoplasms in some patients with HV-CD.
Symptoms & signs : large solitary masses of the mediastinum, or markedly enlarged single peripheral or abdominal lymph nodes but in > 90% of cases without constitutional symptoms^{ref1, ref2, ref3}. Although most cases are believed to arise in lymph nodes, HV-CD can also form soft tissue masses^ref.
Laboratory examinations : HV-CD is a lymphoid proliferation that demonstrates characteristic abnormalities both in the lymphoid follicles and in the interfollicular regions. The follicles have widened mantle zones composed of concentric rings of small lymphocytes that surround one or more abnormal small regressively transformed germinal centers. The regressively transformed germinal centers have lymphoid depletion, vascular proliferation often with penetrating small hyalinized vessels and prominent follicular dendritic cells (FDC) that sometimes appear dysplastic or show an expanded/disrupted pattern^{ref1, ref2}. The mantle zone cells have been reported to have an abnormal immunophenotype^ref. The variably expanded interfollicular region is composed of small lymphocytes, mostly of T cell type, a variably hyalinized vascular proliferation including many high endothelial venules, and frequently foci of plasmacytoid dendritic cells (also known as plasmacytoid monocytes). The lymphoid cells in the vast majority of cases are polyclonal. Plasmacytoid dendritic cells, identified morphologically and based on their CD68⁺, CD45RA⁺, CD123⁺ phenotype, produce large amounts of type 1 interferon (IFN-a/b) after stimulation and may play a role in the response to viral infections or prolong survival of antigen-activated T cells^ref. There is a variably prominent interfollicular stromal component including fibroblastic reticulum cells, vessels, myoid cells and a variety of dendritic cells. Some cases have a predominance of the interfollicular component, and the stromal components can even form nodular masses. Other inflammatory cells, such as plasma cells, eosinophils or histiocytes, may be present but are not numerous. Significant fibrosis and calcification can also be seen. The explanation for these varied abnormalities remains uncertain.
Therapy : generally surgical with only rare recurrences^{ref1, ref2, ref3}. Radiation therapy has also been utilized^ref.
• PC-LCD

Epidemiology : occurs in children and adults (median age in the 20s)
Pathogenesis : in complete contrast to HV-CD and normal lymph nodes, there is abnormal IL-6 expression by cells in the germinal centers and elsewhere in the lymph nodes that may be very important in the pathogenesis of this disorder^{ref1, ref2}. IL-6 levels are also increased in the serum. IL-6 leads to the differentiation of B-cells into plasma cells, may play a role in vascular proliferation and also induces acute phase reactant proteins. It thus can account for many of the pathologic findings, systemic symptoms and laboratory abnormalities in PC-CD. Abnormal expression of VEGF by interfollicular plasma cells may also be of importance^ref. VEGF expression in fibroblast-like cells in germinal centers of CD of uncertain type has also been reported^ref.
Symptoms & signs : a mass lesion that, in contrast to HV-CD, is typically an aggregate of lymph nodes, most frequently in the mediastinum or abdomen^{ref1, ref2, ref3}. Unlike those with HV-CD, these patients often present with constitutional symptoms. Other disorders have occasionally been associated with PC-CD, such as growth retardation^ref
Laboratory examinations : 50–90% demonstrate hematologic disorders, including anemia and a leukocytosis, with many having hypergammaglobulinemia and hypoalbuminemia. In contrast to HV-CD, the follicles are mostly hyperplastic rather than regressively transformed, FDC patterns are usually unremarkable, and there are sheets of usually polyclonal but sometimes monoclonal plasma cells. The distinction of the latter cases from a small B cell lymphoma with plasmacytic differentiation is problematic. In addition to the sometime similar follicular abnormalities, the mantle zones in PC-CD have been reported by one group of investigators to have the same phenotypic abnormality as in HV-CD^ref and others have reported similar upregulation of the EGFR in the follicles (including at least some cases that are probably not localized)^ref
Prognosis : rare cases of lymphoma or plasmacytoma have been described following localized CD^ref.
Therapy : as with HV-CD, most cases are cured by surgical excision including resolution of the systemic signs and symptoms^{ref1, ref2}. Some patients have also received steroids and/or radiation therapy
Epidemiology : more common than MCD
Symptoms & signs : asymptomatic or aspecific signs of inflammation
Therapy : surgical ablation
• multicentric Castleman's disease (MCD) / multicentric angiofollicular hyperplasia : isolated masses of lymphoid tissue and lymph node hyperplasia, usually in the abdominal or mediastinal area (mainly involves lymph nodes and spleen). They may develop autoimmune phenomena, cytopenias, skin rashes, and intercurrent infections. Patients with MCD frequently develop malignancies, most commonly KS and non-Hodgkin's lymphoma. Notably, MCD is characterized by vascular proliferation in the germinal centers which is reminiscent of KS. However, this virus is present in MCD in patients with AIDS whether accompanied by KS or not. It was first described in 1980's

Epidemiology : more rare than LCD; M-CD is a systemic disorder that is reported to occur in children^ref but which most typically occurs in older, immunocompromised individuals (median age 50s)^{ref1, ref2, ref3}. A specific plasmablastic type (PB-M-CD) has been recognized more recently^{ref1, ref2}. These latter cases are undoubtedly present and not distinguished in many series of M-CD, thus complicating interpretation of this literature
Aetiology : M-CD occurs in patients with HIV infection, in older individuals with Kaposi’s sarcoma (KS) and in others as well. It has been suggested that, independent of HIV infection, M-CD "probably arises in a setting of immunoregulatory deficit" perhaps related to immune senescence^ref. MC-CD is associated with HHV-8 / KSHV infection in the HIV⁺ patients and in about 40% of HIV-negative cases. M-CD is not generally considered to be associated with HHV-4 / EBV; however, EBV⁺ cases have been reported^ref.
Pathogenesis : as in PC-LCD, IL-6 is of pathogenetic importance and VEGF may be as well^{ref1, ref2}.
Symptoms & signs : > 1 site of disease including nodal involvement plus frequent hepatosplenomegaly together with the types of constitutional symptoms and laboratory abnormalities as seen in PC-LCD^{ref1, ref2, ref3, ref4}.
Laboratory examinations : perhaps with the exception of PB-M-CD, M-CD is a diagnosis made by exclusion as the pathologic findings are non-specific. Most cases closely resemble PC-CD except for the presence of intact and often dilated sinuses in lymph nodes. There may be significant fibrosis. Spleens demonstrate numerous plasma cells in the white pulp surrounded by a zone of fibrosis^ref. Most but not all cases are polyclonal.
Therapy : aggressive systemic therapy; as noted recently "...no standard of care for the treatment of MCD has been established...."^ref Numerous modalities including steroids (prednisone), radiation therapy and chemotherapeutic agents^{ref1, ref2} as well as more targeted therapies have been utilized. Use of a humanized anti-IL-6R antibody has led to symptomatic relief and partial or complete regression of the adenopathy^ref. The status of this investigational agent, atlizumab, was recently reviewed^ref. Anti-IL-6 antibodies and rituximab have also been variably successful, although the latter led to exacerbation of KS^ref. Both decreases in viral load and clinical responses to antiviral agents such as ganciclovir have been reported in HHV-8/HIV–associated disease, although antiviral therapy, like many other therapies, is not always successful^ref.
• plasmablastic MCD (PB-MCD)^ref

Aetiology : HHV-8 / KSHV infection, both in people with (100% of cases) and without HIV (50% of cases) infection (Chadburn, A,, 109; Soulier, J., L, 1275-1280; A. Gessain, A. Sudaka, J. Brière, N. Fouchard, M.-A. Nicola, B. Rio, M. Arborio, X. Troussard, J. Ausouin, J. Diebold, and G. de Thé, letter to the editor, Blood 87:414-416, 1995). In HIV⁺ patients, MCD has been found to be frequently associated with KS and is usually observed in men infected with HIV by sexual contact^ref. KSHV sequences have also been detected in PBMCs of patients with MCD
Pathogenesis : it is not clear whether the KSHV⁺ cases differ clinically from KSHV^- ones in individuals without AIDS. It has been shown that the germinal centers of hyperplastic lymph nodes of patients with PB-MCD produce large quantities of IL-6. This may explain the large proportion of plasma cells in these lymph nodes, since IL-6 induces B-cell differentiation. Notably, KSHV encodes a viral IL-6 homologue, which is also expressed in MCD in scattered cells surrounding the lymphoid follicles^{ref1, ref2}. KSHV has been found in mantle zone large immunoblastic B cells by using an ORF-73 mAb^ref. The infected cells are naïve CD20⁺ plasmablastic/immunoblastic B-cells (their rearranged Ig genes lack somatic mutation) that usually express IgMl but are not monoclonal^{ref1, ref2}. They express high levels of cytoplasmic Ig and surface CD27, therefore resembling mature B cells. CD20^- infected cells are also described and, in some studies of HHV-8⁺ CD, many infected cells are small. Some cases are associated with small aggregates of polyclonal or, less often, monoclonal plasmablasts that have been termed "microlymphomas." A significant number of cases have simultaneous or subsequent overt plasmablastic lymphomas and some have an associated PEL. Many of these patients also have or develop KS.Whether, as in the classic description of PB-M-CD, HHV-8 positivity in M-CD is synonymous with the plasmablastic variant remains to be determined. These cells are monotypic for l light chain and can sometimes proliferate to give rise to monoclonal expansions and frank lymphomas, which are called plasmablastic lymphomas and are distinct from PEL^ref. KSHV⁺ cells in MCD are invariably negative for HHV-4 / EBV. PB-MCD is distinguished by the presence of plasmablasts that express the HHV-8 latent nuclear antigen and are present mostly in follicular mantles^{ref1, ref2}. A subset of the HHV-8 infected cells in M-CD express other HHV-8 encoded proteins including v-IL-6 and lytic antigens^ref. HHV-8 infected cells in KS or primary effusion lymphomas (PEL) have more restricted protein expression^ref. The v-IL-6 not only contributes to the plasmacytosis but can induce VEGF and enhance angiogenesis^ref. Most of the infected cells also express the human IL-6R, suggesting the importance of this pathway^ref. HIV⁺ patients with HHV-8⁺ M-CD also have high plasma IL-6(and IL-10) levels^ref. Other HHV-8 encoded proteins may play an important role in the pathogenesis of these lesions (a subject beyond the scope of this discussion)^ref. HHV-8⁺ CD has also been reported to show EGFR overexpression in the FDC^ref.
Differential diagnosis : it is important to distinguish PB-M-CD from other HHV-8–associated disorders includingAIDS-related body cavity-based B-cell primary effusion lymphomas (PEL) that is also often found in HIV⁺ individuals and germinotropic lymphoproliferative disorder (GLD), both of which are most typically EBV^+ref. The latter is a monotypic but polyclonal/oligoclonal plasmablastic proliferation that occurs largely in germinal centers. It presents with localized adenopathy in immunocompetent patients who respond well to therapy.
Prognosis : both KSHV-associated PEL and MCD usually pursue an aggressive clinical course^{ref1, ref2}. PB-M-CD have behaved in a very aggressive fashion; in one study most patients died within 1 year, suggesting that it is more aggressive than HHV-8– M-CD^ref. This conclusion would be consistent with the reportedly better prognosis of "idiopathic plasmacytic lymphadenopathy with polyclonal hypergammaglobulinemia," an entity described in Japan that has many features of M-CD but is HHV-8^–ref.
Prognosis : although often considered to be an aggressive disorder, the reported course is variable, with some cases that may wax and wane over long periods of time. Pediatric cases are described as having a better prognosis^ref.  The development of lymphoma is an important complication of M-CD.
Differential diagnosis : there are several disorders that have unique clinical features but in which lymph node biopsies may resemble or possibly truly represent CD. CD is considered a minor criteria for the diagnosis of the POEMS (polyneuropathy, organomegaly, endocrinopathy, M protein and skin changes) syndrome, with the reported estimate of 11–30% of cases having CD considered a conservative estimate^ref. As in PB-M-CD, the POEMS syndrome is a -restricted "plasmaproliferative" disorder^ref and most POEMS-associated CD is HHV-8^+ref. These cases are included in some CD series but probably should be separated as this is a well-defined, distinct clinicopathologic entity. "Cutaneous plasmacytosis," a chronic benign disorder usually but not exclusively reported in Japanese patients who present with multiple cutaneous plaques with numerous polyclonal plasma cells, is another entity that can be associated with lymphadenopathy and other signs and symptoms seen in PC or M-CD^ref. Hodgkin lymphoma is sometimes described with adjacent CD-like changes or in association with CD or CD-like changes, perhaps reemphasizing the nonspecific nature of many of the pathologic findings of CD and the need to exercise caution before making the diagnosis^ref.
• during hypersensitivities
• during autoimmunity
• hairy cell leukemia
• monoclonal lymphocytosis
• lymphomas
• monoclonal lymphocytosis of undetermined significance (MLUS)
• benign monoclonal B-cell lymphocytosis (MBL) (BMBL) / B-cell monoclonal lymphocytosis of undetermined significance (B-MLUS)  : very low levels of circulating monoclonal B-cell subpopulations can now be detected using flow cytometry, in apparently healthy individuals with a prolonged asymptomatic or benign clinical course, and essentially normal humoral and cellular immunity and normal karyotype. The majority of individuals with MBL will have cells that are indistinguishable from B-cell chronic lymphocytic leukaemia (CLL). However, this blood cell clonal expansion of CD5⁺ or CD5^- B-lymphocytes is age-dependent and immunophenotypic heterogeneity is common. Longitudinal studies are required to determine whether MBL is a precursor state to CLL or other B-lymphoproliferative disease in a situation analogous to a monoclonal gammopathy of undetermined significance and myeloma. Future studies of MBL should be directed towards determining its relationship to clinical disease, particularly in individuals from families with a genetic predisposition to developing CLL^{ref1, ref2}

Several investigators have demonstrated that patients fitting within these criteria had disease that was stable for long periods of time, and have used a variety of different names to denote this situation. It has also been noted that patients with CD5^- B-cell disorders may have stable disease. These studies have been critical to the understanding that there is a spectrum of disease in CLL and other B-cell lymphoproliferative disorders, both in terms of the tumour burden at presentation and the probability that the disease will progress to a stage that requires treatment. The application of basic diagnostic flow cytometry to the general population has led to the detection of a MBL in otherwise healthy individuals. Monoclonal B cells in healthy individuals with normal peripheral blood counts were first noted in studies of unaffected siblings families with a genetic predisposition to CLL^{ref1, ref2} (Marti, G.E. (1993) Mechanisms of a B cell neoplasia. In: Workshop at the Basel Institute for Immunology, April 4–6, 1993 organized by Fritz Melchers and Michael Potter (ed. by M.F. Melchers), pp. 129–136, Editiones Roche, Basel, Switzerland). In 1995, a US Public Health Service (USPHS) Workshop reviewed the laboratory, clinical, and population data and recognized that both CD5⁺ and CD5^- MBL could be identified in healthy individuals and outpatients with no clinically apparent evidence of haematological disease (Cartwright, R.A. (1997) Summary of epidemiology and immunobiology; Marti, G.E., Muller, J., Stetler-Stevenson, M. & Caporaso, N. (1997) B-cell monoclonal lymphocytosis in three individuals living near a hazardous waste site, Vogt, R.F., Meredith, M.N.K., Powell, J., Ethridge, S.F., Whitfield, W., Henderson, L.O. & Hannon, W.H. (1997) Results in eleven individuals with B-CLL-like phenotypes detected in environmental health studies, and Sarasua, S.M., Vogt, R.F., Middleton, D.C., Slade, B.A, McGeehin, M.A. & Lybarger, J.A. (1997) ‘CLL-like’ B-cell phenotypes detected in superfund studies: epidemiologic methods and findings. In: Proceedings of a USPHS Workshop on Laboratory Approaches to Determining the Role of Environmental Exposures as Risk Factors for B-cell Chronic Lymphocytic Leukemia and Other B-cell Lymphoproliferative Disorders (ed. by G.E. Marti, R.F. Vogt & V.E. Zenger), pp. 7-18, 19-35, 37–50, 215–222. USPHS, Atlanta, GA). In a large cross-sectional population study of 1491 individuals, US investigators at the Agency for Toxic Substances and Disease Registry, Centres for Disease Control and the Food and Drug Administration found evidence of a MBL phenotype in 11 individuals (0.8%). Research from the Haematological Malignancy Diagnostic Service Laboratory using 3-colour flow cytometry demonstrated a higher prevalence of approximately 1.7% in an outpatient clinic population (Jack, A., Richards, S., Evans, P. & Wilks, C. (1997) Population screening for B-cell monoclonal lymphocytosis using PCR and flow cytometry to determine immunophenotypes. In: Proceedings of a USPHS Workshop on Laboratory Approaches to Determining the Role of Environmental Exposures as Risk Factors for B-cell Chronic Lymphocytic Leukemia and Other B-cell Lymphoproliferative Disorders (ed. by G.E. Marti, R.F. Vogt & V.E. Zenger), pp. 93–96. USPHS, Atlanta, GA.). Following recommendations from the USPHS Workshop, Slade (1999) selected patients with an absolute B-cell lymphocytosis or relative increase in the proportion of CD5⁺ B cells for monoclonality assessment, and demonstrated a prevalence of 0.6% (13/1985). More recently, Rachel et al (Rachel, J.M., Zucker, M.L., Plapp, F.V., Fox, C.M., Marti, G.E., Abbasi, F. & Menitove, J.E. (2002) B cell monoclonal lymphocytosis in blood donors (abstract). Blood, 100, 590a.) examined MBL using a similar screening strategy in a study of mid-Western US blood donor population. Among donors aged 39–80 years, they found a prevalence of MBL of 0.14% (7/5138). These studies demonstrated that an MBL may be detected in the absence of an absolute lymphocytosis and in individuals with a normal proportion of CD5⁺ B cells. However, the reported prevalence varied dependent on the technique used, and the development of CLL-specific assays suitable for minimal residual disease detection that are more sensitive than polymerase chain reaction for immunoglobulin heavy chain gene rearrangement (IgH-PCR)^ref potentially allows the detection of monoclonal B cells in individuals with an apparently normal k:l ratio. Using this assay, Rawstron et al^ref detected CLL phenotype cells in 2.7% of adults aged over 18 years (0.3% in individuals younger than 40 and 5.2% in individuals older than 60). Incidental to CLL-specific assay, these studies identified CD5) non-CLL phenotype MBL in nine of 910 (1.0%) individuals over 40^ref). Using a similar approach in a population-based study of 500 normal Italian subjects over the age of 65 years with a normal blood cell count, Ghia et al^ref found 29 individuals with a clonal expansion of lymphocytes with either a CLL-phenotype (n = 22/442, prevalence 5.5%) or CD5) non-CLL-phenotype (n = 7/500, 1.4%) showing good consensus between the two independent studies. All cases, regardless of the phenotype, were CD10 negative and BCL1 or BCL2 rearrangements were not found. In both studies, a monoclonal IgH rearrangement was confirmed by IgH-PCR in the majority of cases. The absolute monoclonal B-cell count ranged from 0.002–1.5 · 10⁹/l. There was a male predominance, and an increasing prevalence with age similar to that observed in clinical disease. It has also been demonstrated that the CLL-phenotype cells in otherwise haematologically normal outpatients have similar karyotypic abnormalities to clinical disease with respect to 13q14 deletions (O’Connor, S.J.M., Rawstron, A.C., Plummer, M., Hillmen, P., Owen, R.G. & Jack, A.S. (2003) Demonstration of the genotypic relationship between CLUS and clinical B-CLL. Leukemia and Lymphoma, 44(Suppl. 2): S13). In summary, studies assessing light chain clonality only will identify MBL in approximately 0.5–1% of the adult population. Studies combining a disease-specific phenotype with clonality assessment detect MBL in approximately 2–3% of the population. The prevalence increases with age, rising to over 5% for adults aged 60 years or older.
Diagnostic criteria : the diagnosis of MBL is based on the identification of a clonal lymphocyte population by immunophenotypic characterization. Different laboratories have used diverse approaches toidentify minimal B-cell monoclonal lymphocytosis, making comparisons across geographic, ethnic, and in different risk groups difficult. In order to standardize and facilitate future studies, we propose the following set of guidelines for the diagnostic characterization of a blood B-cell monoclonal lymphocytosis.
• detection of a monoclonal B-cell population in the peripheral blood with
• overall k:l ratio >3:1 or <0.3:1, or
• > 25% of B cells lacking or expressing low level surface immunoglobulin or
• a disease-specific immunophenotype.
• repeat assessment should demonstrate that the monoclonal B-cell population is stable over a 3-month period.
• exclusion criteria
• lymphadenopathy and organomegaly, or
• associated autoimmune/infectious disease, or
• B-lymphocyte count >5 · 10⁹/l, or
• any other feature diagnostic of a B-lymphoproliferative disorder. However, a paraprotein may be present or associated with MBL and should be evaluated independently.
• subclassification:
• CD5⁺23⁺: this is the major subcategory and corresponds to a CLL immunophenotype (Cheson et al, 1996).
• CD5⁺23^-: correlate moderate level of CD20 and CD79b expression with atypical CLL.
• CD5^-: corresponds to non-CLL lymphoproliferative disease (LPD).
Table I. Reported studies of subclinical chronic lymphocytic leukaemia (CLL) states and previous terminology to describe monoclonal B-cell lymphocytosis (MBL)*.

Notes:

• 1 > 1 set of k/l light chain reagents may be used, the detection of any B-cell monoclonal population by light chain restriction is sufficient. Confirmation with IgH-PCR may be helpful but is not essential.
• 2 The monoclonal B-cell population may represent a minority of total B cells when identified by a disease-specific immunophenotype. These may be demonstrable even if the overall k:l ratio is normal, although clonality must be demonstrated within the cellular population identified by the disease-specific phenotype.
• 3 MBL lacking surface immunoglobulin is associated with CD5⁺CD23⁺ MBL.
• 4 A minimum of 3 colours (CD19 or CD20, anti-k and anti-l) should be used to confirm clonality, although four or more colours are preferable.
• 5 The fluorescence intensity of surface immunoglobulin, CD20 and CD79b expression if moderately increased should be noted.
• 6 The number of cells analysed should allow the formation of a cluster containing at least 50 events.
• 7 Repeat flow cytometric analysis is not necessary for research applications if monoclonality is confirmed by other approaches, e.g. fluorescence in situ hybridization or PCR, but may be useful for monitoring.
• 8 A disease-specific phenotype exists for hairy cell leukaemia (CD5^-CD103⁺CD11c⁺CD25^±) but it is probable that a full diagnosis of hairy cell leukaemia will be made in the presence of any level of circulating disease.
• 9 Other subclassifications may be included if sufficiently specific tests with evidence of a clinical association can be confirmed.
Natural history of the disorder : as MBL has a greater prevalence than CLL, even if it were to be a precursor of CLL, a significant proportion of cases must either regress or remain static. This is in keeping with the finding that a large proportion of patients with early stage CLL show stable disease and, occasionally, decreasing CLL cell counts. Faguet et al (Faguet, G.B., Agee, J.F. & Marti, G.E. (1992) Clone emergence and evolution in chronic lymphocytic leukemia: characterization of clinical, laboratory, and immunophenotypic profiles of 25 patients. Leukemia & Lymphoma, 6, 345–356) reported a series of 39 patients with a slight increase in their absolute lymphocyte count; 24 had an abnormal B-cell clone and 8 of the 24 patients progressed to clinical B-cell CLL over 5 years. In the limited NCI follow-up of familial MBL, progression to CLL has not yet been observed. Rawstron et al (2003) have begun to assess outcome in CD5⁺23⁺ MBL in a clinical population of patients who do not meet the diagnostic criteria for CLL. These are patients with an incidental finding of MBL meeting the criteria outlined above. Initial data (5-year median follow-up) suggests that approximately 60% have stable or regressive lymphocytosis; 35% show increasing lymphocyte cell counts, and 5% have required treatment. Of note, 6 of 47 patients showed bi-phasic progression kinetics, with initially stable disease followed by a more pronounced increase in lymphocyte count at a median 3.5 years (range 0.5–7.0) from presentation, suggestive of a secondary event. These data reinforce the concept that CLL presents with a very broad spectrum of tumour burden, and that risk of progression is independent of the absolute level at presentation. There is currently no evidence that early diagnosis of CLL is of any benefit to the patient. However, identifying CLL at its earliest stages is likely to be of significant value in identifying and defining the biological mechanisms responsible for disease aetiology and progression.
Populations to consider : one group^{ref1, ref2} established a significant relationship between multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). Over a 30-year period, there is a 25-fold relative risk of MGUS progressing to MM and a 40-fold relative risk of developing macroglobulinaemia in subjects with IgM MGUS. At present, the notion that MBL is a precursor of CLL and other non-Hodgkin lymphomas (NHLs) is presumptive and a major goal of future studies should be to define this relationship. However, given the high population prevalence, screening of the general population would be prohibitively expensive and clinically uninformative and is not recommended outside of research applications. It is likely that, as with MGUS, MBL will predominantly be identified as an incidental finding in individuals being found medically for other reasons. Epidemiological studies have shown that the risk of CLL in relatives of patients with CLL is increased and it is likely that a subset of the disease is caused by a genetic susceptibility^{ref1, ref2, ref3, ref4, ref5, ref6, ref7} (Caporaso, N.E., Whitehouse, J., Bertin, P., Amos, C., Papadopoulos, N., Muller, J., Whang-Peng, J., Tucker, M.A., Fleisher, T.A. & Marti, G.E. (1991) A 20 year clinical and laboratory study of familial B-chronic lymphocytic leukemia in a single kindred. Leukemia & Lymphoma, 3, 331–342; Caporaso, N., Fontaine, L., Whitehouse, J. & Marti, G.E. (1997) Familial B-CLL: review of literature and the NCI familial B-CLL registry. In: Proceedings of a USPHS Workshop on Laboratory Approaches to Determining the Role of Environmental Exposures as Risk Factors for B-cell Chronic Lymphocytic Leukemia and Other B-cell Lymphoproliferative Disorders (ed. by G.E. Marti, R.F. Vogt & V.E. Zenger), pp. 173–180. USPHS, Atlanta, GA.; Ishibe, N., Sgambati, M.T., Fontaine, L., Goldin, L.R., Jain, N., Weissman, N., Marti, G.E. & Caporaso, N.E. (2001) Clinical characteristics of familial B-CLL in the National Cancer Institute Familial Registry. Leukemia & Lymphoma, 42, 99–108). As CD5⁺23⁺ MBL may be a precursor of CLL, it has been postulated that MBL might be overrepresented in apparently healthy relatives of familial CLL cases. Such a proposition has recently been examined by two research groups. Rawstron et al^ref found that 14% of relatives of familial cases had MBL compared with 3% of outpatient controls. Marti et al^ref also reported overrepresentation of MBL in relatives of familial cases (18% compared with 0.7% in controls). These studies provide strong support for the notion that MBL with a CLL-like phenotype may be a surrogate marker for carrier status. Therefore, there is a powerful rationale for research into the risk of progression for individuals with MBL in CLL families. However, it is still far from clear whether the detection of MBL predicts a higher probability of eventually developing CLL within CLL family members. Therefore, as with the general population, screening apparently healthy members from CLL families is currently not clinically informative. It is recommended that relatives who are potential donors for allogeneic transplant be screened for MBL. This is primarily because the transplanted MBL is likely to impact on residual disease monitoring in the donor, although the potential for transplanted MBL to expand and or transform in an immunocompromised host is also of concern. Genetic counselling for a familial disorder could be considered for high-risk individuals in certain settings^ref.
Recommendations for follow-up : there are 2 key problems that arise in the area of asymptomatic or subclinical disorders: the first is what toinform the patient or individual, and the second is how closely the patient should be investigated and monitored. The risk of progression to clinical disease is not known, and even if an association with clinical disease is convincingly demonstrated, it will be sometime before any risk of progression can be calculated. In the absence of precise clinical information about outcome, the adverse consequences of inducing anxiety by informing an individual that they might have a preclinical blood cancer or precursor condition; the lack of any effective early intervention; and the potential for adverse individual health insurance concerns, it may be unethical to inform patients of their disorder when it would result in little or no clinical benefit whilst incurring the risk of significant adverse effects. It is therefore acceptable in a research setting, where extremely low levels of monoclonal B cells may be identified, to not provide the results of investigations to patients. If MBL is diagnosed after specific investigation, it is important to stress that the risk of progression is not known but is likely to be very low and it may be helpful to offer periodic monitoring in these cases. How closely patients are investigated and monitored is a significant problem. Although population screening is not clinically indicated, a substantial and increasing number of individuals are demonstrated to have MBL as an incidental finding during routine medical assessment. As this report is aimed at providing diagnostic criteria, it is not possible to provide evidence-based guidelines for monitoring patients with MBL. However, some suggestions can be provided based on initial studies. The close relationship between CD5⁺23⁺ MBL and CLL suggests that the levels in the peripheral blood are likely to be indicative of bone marrow involvement. Therefore bone marrow investigation is unlikely to be of any value for these patients. B-lymphoproliferative disorders with a non-CLL phenotype may show much greater tissue or bone marrow
involvement than the peripheral levels suggest, and therefore marrow investigation may be of value in patients with CD5⁺23^- or CD5^- MBL. The presence of peripheral lymphadenopathy or organomegaly will have been excluded as part of the initial physical examination. However, in selected cases, a baseline chest X-ray, abdominal ultrasound and or a computed tomography scan may be indicated. And a rapid rise in the absolute lymphocyte count might warrant a diagnostic/prognostic bone marrow aspirate and biopsy.
For MBL patients identified during investigation of a mild lymphocytosis, it may be helpful to confirm whether the monoclonal B-cell counts are regressive, stable or progressive over a 3–6-month period. Long-term prospective studies will be required to determine whether periodic monitoring (i.e. as in MGUS, approximately yearly assessment of the absolute lymphocyte count and/or flow cytometry) will be of value in identifying the small proportion of MBL patients who will show progression to clinical disease. Monoclonal B cells that are indistinguishable from early CLL/NHL may be detected in up to 4% of the general population. We propose the term MBL to describe this phenotype. MBL is detectable at a higher frequency in apparently unaffected members of CLL kindreds and is more
prevalent with advancing age. Appropriate studies are needed to establish the natural history of MBL in both the general population and high-risk settings. The relationship of MBL to other non-CLL LPD, MGUS and reactive lymph node hyperplasia^ref remains to be defined. Continued investigation of the human B-cell repertoire in a defined MBL population may reveal further abnormalities relevant to the aetiology and pathogenesis of CLL.
Laboratory examinations :
• L chain restriction^ref
• absolute lymphocyte count (ALC) in PB < 5 · 10⁹/l
• immunophenotype : patients with a prominent lymphadenopathy and/or splenomegaly had CD22⁺ leukemic cells while in patients with a progressive lymphocytosis the B-cell clone expressed Leu-8. Thus, CD22 might be related to the homing capacity of B lymphocytes for lymphnodes and spleen, while Leu-8 might define a circulating B-cell subset. In B-MLUS about 50% of the monoclonal B cells co-expressed Leu8 which is consistent with a more differentiated phenotype compared to B-CLL with progressive lymphocytosis. The CD22 expression was mostly low in B-MLUS although a few patients showed high values. The expression of receptors for growth factors (CD23, CD25, CD71) was higher in B-CLL compared to B-MLUS patients (p < 0.001), which is consistent with a difference in lymphocyte activation stage and/or response to growth factors^ref
• monoclonal T-cell dyscrasia of undetermined significance (MTUS)

Laboratory examinations : immunophenotype : CD3⁺, CD4⁺, CD7^-, CD26^-ref
• chronic granular T cell lymphocytosis of undetermined significance

Laboratory examinations : immunophenotype : CD3⁺, CD4^-, CD8⁺, Leu7^+ref
Therapy : GR agonists
• lymphohistiocytosis : lymphocytosis + histiocytosis.
• hemophagocytic or hemophagocytosis syndrome (HPS / HS) / erythrophagocytic or hemophagocytic lymphohistiocytosis (HLH) / hemophagocytic lymphohistiocytic syndrome (HLS) / macrophage activation syndrome (MAS)

Epidemiology : usually seen in children, but occurs in all age groups. There are no data about the incidence of acquired HLH in children or in adults. From numerous case reports, however, it appears that acquired HLH may be more common than previously believed.
Aetiology :
• genetic, hereditary, primary or familial HLH (a few cases) autosomal recessive inheritance. Both genetic subgroups are associated with impaired NK cell function
• familial HLH (FHLH) (Farquhar disease, first described by Farquhar and Claireaux in 1952^ref) / familial erythrophagocytic lymphohistiocytosis / familial erythrophagocytic, hemophagocytic or histiocytic medullary reticulosis / familial reticuloendotheliosis with eosinophilia / Omenn's syndrome : a fatal hereditary disorder transmitted as an autosomal recessive trait, characterized by anemia, granulocytopenia, thrombocytopenia, intense phagocytosis of red blood cells, diffuse proliferation of histiocytes of various organs, and enlargement of the liver, spleen, and lymph nodes. FHLH is estimated to occur with a frequency of 1 in 50,000 births. The onset of the disease is during the first year of life in 70% of the children. A symptom-free interval after birth is typical.
• known gene defects (perforin, munc 13-4, syntaxin 11)
• unknown gene defects
• immune deficiency syndromes with distinctive clinical features in which the development of HLH is sporadic, though frequent. HLH is often the presenting symptom but may also occur later during the course of disease
• Chédiak-Higashi syndrome (CHS) show albinism and frequent pyogenic infections. Their white blood cells exhibit decreased chemotaxis and characteristic giant inclusion bodies (lysosomes)
• Griscelli syndrome (GS) also have hypopigmentation and various degrees of neutrophil dysfunction but lack the giant granules
• Griscelli syndrome type 2 (GS2) / partial albinism with immunodeficiency : mutation in RAB27A
• X-linked lymphoproliferative syndrome (XLP) is mainly characterized by a predisposition for Epstein-Barr virus (EBV)-associated HLH. XLP patients may develop lymphomas and dysgammaglobulinemia.
• acquired, reactive, or secondary HLH :
• malignancies
• lymphoma-associated hemophagocytic syndrome (LAHS) is a well-known entity in adults but is rare in children. Cases formerly diagnosed as histiocytic medullary reticulosis or malignant histiocytosis included patients with LAHS, but also IAHS. Interestingly, in a recent review of patients with LAHS from Japan, the EBV genome was detected only rarely in patients with B-cell lymphoma but was demonstrated in > 80% of patients with T/NK cell lymphoma^ref. EBV-infected T/NK cells appear to play a major role in the development of LAHS as well as EBV-associated HLH without lymphoma^ref, and in both LAHS and EBV-HLH the infected T/NK cells show a monoclonal or oligoclonal proliferation. Hemophagocytosis and symptoms of HLH have also been described in association with inborn errors of metabolism, including lysinuric protein intolerance and multiple sulfatase deficiency. In such cases, it is not clear what role metabolic products may play as triggers to the immune response.
• T-cell lymphomas (e.g. hepatosplenic gd- T-cell lymphoma with isochromosome 7q and trisomy 8^ref)
• autoimmune disease
• systemic juvenile rheumatoid arthritis (expecially after administration of etanercept)
• adult onset Still's disease (AOSD)
• acute systemic lupus erythematosus (SLE) HPS^ref
• exogenous agents (infectious organisms, toxins)
• infection-associated hemophagocytic syndrome (IAHS) : whereas in the first report by Risdall et al the majority of cases had an acquired iatrogenic immune deficiency, most patients in subsequent reports had no known genetic or acquired immune defect. A review of the published cases in children diagnosed with IAHS before 1996 reported that more than half of them were from the Far East. The identification of an infectious organism does not help to differentiate FHLH from acquired HLH since the former is also usually triggered by infectious agents. This cannot be emphasized enough, since appropriate therapy should not be withheld when an infectious agent has been found
• Plasmodium vivax^ref
• Mycoplasma pneumoniae^ref
• adrenal gland Mycobacterium tuberculosis^ref
• virus-associated hemophagocytic syndrome (VAHS) : the patients in the original report by Risdall and colleagues were mostly adults with a viral infection following organ transplantation^ref
• smoldering adult T-cell leukemia/lymphoma from HTLV-1^ref
• acute fulminant HHV-4 / EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) (74%^ref) : although the majority of EBV-HLH cases develop in apparently immunocompetent children and adolescents, it also occurs in association with infectious mononucleosis, chronic active EBV infection, familial HLH, X-linked lymphoproliferative disease, lymphoproliferative disease like peripheral T-cell lymphoma and NK cell leukemia. The majority of successfully treated EBV-HLH patients have a good outcome and remain disease-free^ref.
• fulminant ulcerative colitis and HHV-5 / CMV infection^ref
In a series, most patients received only supportive care and the mortality was 50%^ref
• endogenous products (tissue damage, metabolic products)
• rheumatic diseases
• macrophage activation syndrome (MAS)
Pathogenesis : low or absent CTL and NK cell activity (defective function of perforin) and hyperactivation of T lymphocytes (lymphocytosis) and macrophages (histiocytosis), with consequent excessive hemophagocytosis in bone marrow, the lymphoreticular system and/or the CNS and proinflammatory cytokine storm
Symptoms & signs : non-remitting high fever, lymphadenitis, hepatosplenomegaly, neurological manifestations in many cases
The cardinal symptoms of HLH are prolonged high fever, hepatosplenomegaly and cytopenias. Lymphadenopathy, icterus, or neurological symptoms such as cranial nerve palsies or seizures may also be present. Characteristic laboratory findings include high triglycerides, ferritin, transaminases and bilirubin and decreased fibrinogen. Hemophagocytosis, although it has given the disease its name, is found at presentation in only the minority of cases, but usually develops as the disease progresses. A hallmark of HLH is impaired or absent function of natural killer (NK) cells and cytotoxic T-cells (CTL)^{ref1, ref2, ref3}. While many of these cardinal symptoms are found in immune-competent patients in response to an infectious organism, they are more pronounced in patients with HLH. In particular, the progression of organomegaly, blood count changes and biochemical parameters should alert the physician that this could be an unusual response to an infectious agent. Without treatment, the uncontrolled inflammatory response leads to sustained neutropenia and death from bacterial or fungal infections as well as from cerebral dysfunction.
Genetic defects in FHLH elucidate the pathophysiology of the spectrum of HLH : the cytotoxic activity of NK cells and CTLs is mediated by the release of cytolytic granules (containing large amounts of perforin, granzymes and other serine like proteases) via the immunological synapse to the target cell. Several independent genetic loci related to this activity have been implicated in the pathophysiology of genetic HLH. In 1999 mutations in the perforin gene at locus 10q24 were described in 8 patients with FHLH^ref. The overall frequency of perforin mutations in FHLH is between 15% and 50% and depends on the geographical and ethnic origin of the patients^ref. UNC13D, at locus 17q25, was the second gene associated with FHLH. The encoded protein (Munc13-4) is important for cytolytic granule exocytosis^ref. UNC13D mutations do not affect docking of secretory vesicles (also known as lytic granules or secretory lysosomes) at the plasma membrane, but they impair priming of these vesicles and the subsequent release of cytolytic enzymes. The frequency of UNC13D mutations in FHLH was estimated to be around 30%^ref. Our group recently identified a third FHLH associated gene on chromosome 6q24 with mutations in STX11^ref. The encoded protein, t-SNARE syntaxin 11, also plays a role in intracellular trafficking, but its precise role is not known. The protein is strongly associated with intracellular membrane fractions and is detectable only in monocytes and not in lymphocytes. So far, mutations in the coding exon of syntaxin 11 seem to be restricted to patients from Turkey. Linkage analysis in 4 HLH families of Pakistani origin revealed a fourth putative disease gene for familial HLH on chromosome 9q21.3-22 but the gene has not yet been identified^ref. Genetic defects in hemophagocytic lymphohistiocytosis :

disease	chromosome location	associated gene	gene function
FHLH-1	9q21.3-22	Not known	not known
FHLH-2	10q21-22	PRF1	induction of apoptosis
FHLH-3	17q25	UNC13D	vesicle priming
FHLH-4	6q24	STX11	vesicle transport; t-SNARE
GS-2	15q21	RAB27A	vesicle transport; small GTPase
CHS-1	1q42.1-q42.2	LYST	vesicle transport; not further defined
XLP	Xq25	SH2D1A	signal transduction and activation of lymphocytes

In patients with GS type 2 (GS-2), immune dysfunction results from mutations in RAB27A on chromosome 15q21^ref. Activated T cells of Rab27-deficient patients are unable to dock their secretory granules at the plasma membrane, thereby impairing secretion via the immunological synapse^ref. A direct interaction between Munc13-4 and Rab27 has recently been demonstrated, and the complex seems to be an essential regulator of secretory granule fusion with the plasma membrane^ref. CHS is caused by mutations in the LYST gene, located on chromosome 1q42.1-q42.2, which encodes a 3801 amino acid protein required, by an unknown mechanism, for the final steps of granule secretion^{ref1, ref2}. XLP results from mutations in the SH2D1A gene^ref. SH2D1A(SAP) seems to be required for the signaling lymphocytic activating molecule (SLAM), which itself is important for signal transduction in immune cells and for activation of granule-mediated cytotoxicity of NK cells and CTLs^ref. Mutation screening in male patients with HLH may be useful as some of them harbor mutations in SH2D1A^ref.
Defective cytotoxic function is crucial in the pathophysiology of HLH : the clinical picture of HLH is due to an increased inflammatory response caused by hypersecretion of pro-inflammatory cytokines such as interferon  (IFN), TNF-a, IL-6, IL-10 and M-CSF. These mediators are secreted by activated T-lymphocytes and histiocytes that infiltrate all tissues, and lead to tissue necrosis and organ failure. The pivotal role of IFN together with CD8⁺ T cells for the development of HLH was demonstrated in a perforin knock-out mouse model^ref. Inflammatory cytokines are responsible for the characteristic disease markers such as cytopenias, coagulopathy and high triglycerides. In spite of the excessive expansion and activation of cytotoxic cells, patients with HLH have severe impairment of the cytotoxic function of NK cells and CTLs. Impaired NK cell activity is seen in FHLH, GS, CHS and XLP as well as in acquired HLH. In the latter, impaired NK cell activity may be due in part to reduced numbers of NK cells and is usually transient. Defective cytolytic activity thus seems to be the common denominator that predisposes to HLH. NK cells and cytotoxic T lymphocytes kill their targets through cytolytic granules containing perforin and granzyme. Upon contact between the effector killer cell and the target cell, an immunological synapse is formed and cytolytic granules have to traffic to the contact site, dock and fuse with the plasma membrane and release their contents^ref. All known defects in HLH seem to be involved in this process: LYST mutations impair granule secretion, RAB27 deficiency leads to impaired docking at the membrane, mutations in UNC13D do not impair docking but cause defective granule priming at the immunological synapse. Finally, lack of PRF1 leads to loss of cytolytic activity. In XLP, granule-mediated cytotoxicity is defective through impaired lymphocyte activation. Syntaxin 11 mutations might theoretically impair cytolytic activity involving the interaction of dendritic and killer cells^ref.

Molecular mechanisms based on the identification of genetic defects associated with the clinical picture of familial hemophagocytic lymphohistiocytosis (FHLH), Griscelli syndrome (GS-2) and Chédiak-Higashi syndrome (CHS). Perforin is secreted via cytotoxic granules and leads to disruption of the target cell. Cytotoxic granule processing occurs by means of a complex that contains at least a Rab27/Munc13-4 complex and several other unknown proteins. The exact functions of LYST and syntaxin 11 are not known. In case of syntaxin 11, monocytes or macrophages/dendritic cells may interact with cytotoxic cells by an unknown mechanism.
The mechanisms leading to cytolytic defects in immune competent patients with acquired HLH are less clear. Viruses may interfere with CTL function by specific proteins^ref, and high levels of cytokines may have the same effect^ref. The prevalence of EBV-associated HLH in Asia suggests that a specific genetic susceptibility may result in an abnormal immune response to the virus. HLH cases associated with genetic defects in the granule exocytosis pathway demonstrate a critical role of the granule-dependent cytotoxic activity in T lymphocyte homeostasis^ref. Activated NK cells and CTLs can kill infected cells and antigen-presenting cells, thus reducing the antigen load; however, more complex mechanisms seem to be involved in the downregulation of the immune response. Studies in mice indicate that NK cells and perforin-based systems play an important role in this process^ref.
Macrophage activation syndrome is closely related to HLH : the macrophage activation syndrome (MAS) occurs in children and adults with autoimmune diseases. It is most commonly seen in association with systemic onset juvenile arthritis (sJRA) or adult-onset Still’s disease, but also occurs rarely with systemic lupus erythematosus or other entities^{ref1, ref2, ref3}. The clinical picture has all of the characteristic features of HLH including clinical symptoms, laboratory findings (especially very high ferritin levels), and hemophagocytosis. Pronounced coagulopathy and severe cardiac impairment are common and potentially life-threatening^ref. Patients with MAS exhibit the defective NK cell function common to other patients with HLH. They may also have decreased expression of perforin or SAP, mimicking the defects associated with FHLH and XLP, respectively^{ref1, ref2,ref3}. Furthermore, low NK cell function and perforin expression was found to distinguish patients with sJRA from those with other clinical forms of JRA^ref. The overall incidence of MAS is not known, but about 100 cases have been published to date. It generally develops in the earlier and active phases of the underlying disease. Viruses have been identified as triggering factors, but other inciting factors that have been implicated include nonsteroidal anti-inflammatory drugs, methotrexate, and gold-salt injections. Mortality of patients with MAS is between 10% and 20%. It has been suggested by some rheumatologists that MAS be classified as a form of secondary HLH^{ref1, ref2}.
Diagnostic Problems in HLH : many patients with HLH present with mild signs of an upper respiratory or gastrointestinal infection and a high fever. The fever often subsides spontaneously and recurs after some days to weeks. Transient improvements in cytopenias, especially thrombocytopenia, with unspecific measures such as antibiotics and transfusions are seen frequently. Organomegaly, anemia or other changes commonly persist. In babies, the initial suspicion often focuses on a metabolic disorder due to organomegaly, disturbed liver function or excessively high triglycerides. Severe, fulminant liver failure with coagulopathy or neurological symptoms may dominate the presentation and thereby delay the diagnosis of HLH. Several patients, including 2 with a novel perforin mutation, have been described with isolated CNS involvement mimicking chronic encephalitits and preceding systemic HLH by several months^{ref1, ref2, ref3}. The characteristic laboratory findings of high triglycerides and ferritin and a low fibrinogen may be absent; therefore, repeat studies are indicated when HLH is suspected (Janka G, Henter JI, Imashuku S. Clinical aspects and therapy of hemophagocytic lymphohistiocytosis. In: Histiocytic Disorders of Children and Adults: Cambridge University Press; 2005:353–37). A high lactate dehydrogenase may suggest a hemolytic anemia, but most patients with HLH characteristically have only moderately elevated reticulocytes in the presence of severe anemia, compatible with ineffective erythropoiesis. Highly activated immature-looking lymphocytes in lymph node biopsies may suggest a malignant process, and periportal infiltrates in a liver biopsy are often interpreted as viral hepatitis.
Laboratory examinations : when a patient presents with prolonged fever, hepatosplenomegaly and cytopenias, the diagnosis of HLH should be considered. Minimal diagnostic requirements are a complete blood count, liver enzymes, bilirubin, triglycerides, ferritin and a coagulation profile including fibrinogen. All patients should have a bone marrow aspirate; however, this test is insensitive. In the majority of cases, hemophagocytosis is not observed in the initial bone marrow aspirate and only increased monocytes and monohistiocytic cells may be present. A myelodysplastic syndrome is sometimes suspected due to marked dysplastic changes in the red cell precursors. Lumbar puncture is also indicated and more than half of the patients will have an elevated cell count or protein or both, even in the absence of clinical symptoms. 2 highly diagnostic disease parameters are an increased plasma concentration of the  chain of the soluble IL-2R (sCD25) and impaired NK cell activity^ref. The latter is usually not available in time but should be measured in all patients. It is persistently decreased to absent in nearly all genetic cases. In acquired cases it usually reverts to normal, but some patients show prolonged impairment. In these patients genetic testing is recommended.
The patients should be screened for an underlying disease like GS, XLP, CHS, autoimmune diseases or malignancy by detailed clinical history, physical examination and other appropriate studies. The search for a triggering infectious agent like EBV, cytomegalovirus (CMV), herpes simplex virus (HSV), adenovirus, varicella zoster virus (VZV) and leishmania is important since most of these organisms are treatable. However, it should be emphasized that, with the possible exception of leishmaniasis, anti-infectious therapy alone is not sufficient to control HLH. Viral PCRs are more helpful than serology. Positive results are useful to follow during antiviral treatment. Expression of perforin by flow cytometry can identify patients with perforin defects and is also desirable in patients with MAS or other acquired forms of HLH in which data on perforin expression are still scant. In proven familial cases or in presumed familial cases with persistent or relapsing disease material for genetic analysis should be obtained.
Diagnostic criteria are based primarily on the findings in familial cases but also apply to the diagnosis of infection-associated acquired HLH. For patients with systemic onset juvenile arthritis who develop MAS some criteria may not be relevant. For example pre-existing inflammation is associated with a higher white blood count and platelets as well as fibrinogen. Accordingly, for MAS, other diagnostic criteria have recently been advocated that take the specific characteristics of these patients into account^ref.^ref
• familial disease/known genetic defect
• clinical and laboratory criteria (5/8 criteria)
• fever
• splenomegaly
• cytopenia >= 2 cell lines
• hemoglobin < 90 g/L (below 4 weeks < 120 g/L)
• neutrophils < 1 x 10⁹/L
• hypertriglyceridemia and/or hypofibrinogenemia
• fasting triglycerides >= 3 mmol/L
• fibrinogen < 1.5 g/L
• ferritin  500 µg/L
• sCD25 >= 2400 U/mL (Janka G, Henter JI, Imashuku S. Clinical aspects and therapy of hemophagocytic lymphohistiocytosis. In: Histiocytic Disorders of Children and Adults: Cambridge University Press; 2005:353–37)
• decreased or absent NK-cell activity
• hemophagocytosis in bone marrow, CSF or lymph nodes
Supportive evidence are cerebral symptoms with moderate pleocytosis and/or elevated protein, elevated transaminases and bilirubin, LDH > 1000 U/L
Therapy : the immediate aim in the treatment of any patient with HLH is to suppress the severe hyperinflammation that is responsible for the life-threatening symptoms. Another aim is to kill pathogen-infected antigen-presenting cells and thus remove the stimulus for the ongoing but ineffective activation of T cells. It has to be specifically emphasized that pathogen-directed therapy is usually not sufficient to control hyperinflammation with the possible exception of leishmaniasis, in which most patients promptly respond to liposomal amphotericin B. In genetic HLH the ultimate aim must be stem cell transplantation to exchange the congenitally defective immune system with normal functioning immune effector cells. In our experience treatment should be guided mainly by the severity of clinical symptoms, but age of the patient, presence of familial disease, underlying secondary disease, and the identification of an infectious organism also should be taken into account. Hyperinflammation can be treated with corticosteroids, which are cytotoxic for lymphocytes and inhibit expression of cytokines and differentiation of dendritic cells. Since dexamethasone crosses the blood brain barrier better than prednisolone it is the preferred corticosteroid. Cyclosporin A (CSA) prevents T-lymphocyte activation and immunoglobulin infusions probably act by providing cytokine-and pathogen-specific antibodies. The cytostatic drug etoposide has high activity in monocytic and histiocytic diseases and it inhibits EBNA synthesis in EBV-infected cells. In patients with less severe symptoms, corticosteroids and immunoglobulin infusions are sometimes sufficient to control hyperinflammation and to reverse the clinical and laboratory symptoms. However, if symptoms progress, the treating physician should not hesitate to use therapy with dexamethasone, etoposide and CSA. The risk of etoposide, even if given for 8 weeks, is by far exceeded by the risk of losing a patient through inadequate treatment. Etoposide may be life-saving, especially in patients with HLH due to EBV infection. Mortality was 14 times higher for patients with EBV-associated HLH who did not receive etoposide within the first 4 weeks^ref. Since initially mild cases may progress rapidly it is our opinion that the indication for the use of etoposide should be determined liberally. Several problems may arise during the initial treatment of patients with HLH. Although symptoms and laboratory abnormalities usually improve within 2–3 weeks of treatment, persistent cytopenias could indicate either non-response to therapy or myelosuppression by etoposide. A repeat bone marrow examination may be helpful. However, since uncontrolled hypercytokinemia also leads to bone marrow aplasia, at least 3–4 doses of etoposide should be given before a decision is made to interrupt the drug, while continuing with CSA and dexamethasone. The close monitoring of other parameters associated with disease activity is important in this situation. If there is no response to treatment after 4 weeks it is unlikely that the patient will have a benefit from continuing the same combination. There is no established salvage regimen; in single patients there has been anecdotal evidence of transient responses to daclizumab or alemtuzumab. Antithymocyte globulin, which may be effective in initial treatment, rarely produces a response in patients with no response to an etoposide-containing combination. If a donor is available and the clinical condition is not prohibitive, stem cell transplant could be tried, although there is little experience proving its benefit in this situation. Reactivation during treatment is a frequent problem, either systemically or in the CNS. Immunosuppressive treatment should be reinforced in this situation. Intrathecal therapy with methotrexate +/– corticosteroids has been beneficial for some patients with recurrent CNS involvement and is recommended in view of the deleterious late effects of uncontrolled CNS disease. Study HLH 94, which enrolled children up to the age of 15 years, used a combination of dexamethasone, CSA and etoposide for the initial treatment of HLH. Familial cases or patients with recurrences and therefore presumed genetic disease, subsequently received a bone marrow transplant from related or unrelated donors. Probability of survival at 3 years in 113 children was 55% for all cases and 51% for proven familial cases^ref. Based on these data, patients with known familial disease, suspected genetic disease due to age below 1 year, or those with a severe clinical picture such as marked cytopenias, coagulation problems and CNS symptoms should receive treatment according to the present HLH protocol (available at www.histio.org/society/protocols). In familial cases or presumed familial cases (no complete resolution of symptoms, reactivations during or after cessation of therapy), active therapy should be continued until stem cell transplantation can be performed. A similar approach should be used for patients with the accelerated phase of CHS, GS or XLP, based on documented responses to HLH therapy. If a treatable infectious agent is identified, appropriate antimicrobial or antiviral therapy should be given. The management of patients with MAS is usually based on the administration of high doses of corticosteroids. CSA has also proven effective in treating severe or corticosteroid resistant cases. Whereas immunoglobulin infusions failed in children with MAS, responses were observed in adult patients^ref. Limited experience exists with etoposide in MAS; however, similarities in pathophysiology between MAS and other forms of HLH^ref suggest that it might be beneficial in cases refractory to CSA and corticosteroids.
Laboratory examinations : haemopoietic cells that are actively ingested by monocytes and macrophages in lymph nodes, bone marrow (hypocellular with an increase in histiocytes), liver and spleen, liver dysfunction, pancytopenia, hyperferritinemia with glycosylated ferritin < 20%, hypertriglyceridemia, hypofibrinogenemia => coagulopathy, and progressive pancytopenia (including anemia), pulmonary infiltrates, renal failure and an inappropriate antidiuretic state
Therapy : in most cases there is no response to treatment and the evolution is fatal
• intensive supportive therapy
• treatment of coagulopathy disorders
• specific antiviral therapy
• pulse therapy with high-dose GR agonists^ref and immunosuppressive treatment
• effective control of disease in some patients with infection-associated HPS can be achieved with 20 to 25 mg/kg chloroquine per day orally for 7 days^ref
Therapy : lymphocytapheresis

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