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