A simple manual rosetting method for absolute CD4+ lymphocyte counting in resource-limited countries

A CD4 monoclonal antibody which reacts with CD4+ lymphocytes but does not react significantly with monocytes was generated and used to develop a simple method for CD4 count. In a comparison with standard flow cytometry, high correlation was obtained. The developed method can be an alternative to flow cytometry for resource-limited countries.     

A subset of functional effector-memory CD8+ T lymphocytes in human immunodeficiency virus-infected patients

CD8(+) T cells provide protective immune responses via both cytolytic and non-cytolytic mechanisms in subjects infected with human immunodeficiency virus (HIV). In the present study, we investigated the CD28 expression of CD8(+) T cells present in the peripheral blood lymphocyte subset isolated from chronically HIV-infected subjects. Using flow cytometric analysis, a continuous spectrum of CD28 intensity ranging from negative to high, which could be separated into CD28-negative, intermediate (int) and high, was seen for CD8(+) T cells. Our study focused mostly on the CD28(int) CD8(+) T cells. The CD28(int) CD8(+) T cells are CD57(-) CD27(+) CD45RO(+) CD45RA(-) CCR7(low) CD62L(int). The proliferative capacity of CD28(int) CD8(+) T cells was intermediate between those of CD28(-) CD8(+) T cells and CD28(high) CD8(+) T cells. The CD28(int) CD8(+) T cells are specific for HIV, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) antigens as measured by human leucocyte antigen pentamer binding and produce both intracellular interferon-gamma and tumour necrosis factor-alpha in response to their cognate viral peptides. The CD28(int) CD8(+) T cells have HIV-specific, CMV-specific and EBV-specific cytotoxic activity in response to their cognate viral peptides. These findings indicate that a subset of functional effector-memory CD8(+) T cells specific for HIV, CMV and EBV antigens may contribute to an efficient immune response in HIV-infected subjects.     

Novel low-cost assay for the monitoring of CD4 counts in HIV-infected individuals

Several CD4 lymphocyte enumeration methods have been developed to be used as alternatives to conventional flow cytometry. However, no consensus has been reached on which technique should be widely adopted. We introduce here a novel nonflow cytometric method, called CD4 Select, for enumeration of CD4 lymphocytes. Instead of using a flow cytometer, the method requires only an automatic hematoanalyzer (complete blood count [CBC] machine). The correlations of the percentage of CD4 lymphocytes and absolute CD4 counts with the CD4 Select method and standard flow cytometry were 0.932 and 0.922, respectively. The CD4 Select method yielded percentage of CD4 lymphocytes greater than flow cytometry by a mean of 0.06%, and the limits of agreement at 95% confidence interval (CI) were 9.46% to -9.56% using Bland-Altman analysis. The CD4 select method is cheap, valid, and simple enough to be conducted locally at small facilities. The method yields not only absolute CD4 lymphocytes, but also percentage of CD4 lymphocytes. The use of a hematoanalyzer also gives this method relatively high sample throughput and makes it less labor-intensive than other nonflow cytometric methods.     

Factors associated with CD4 lymphocyte counts in HIV-negative Senegalese individuals

CD4+ lymphocytes are a primary target of the human immunodeficiency virus (HIV), and CD4 counts are one of the factors used to measure disease progression in HIV-positive individuals. CD4 counts vary in uninfected individuals and across populations due to a variety of demographic, environmental, immunological and genetic factors that probably persist throughout the course of HIV infection. This study sought to determine reference levels and identify factors that influence lymphocyte counts in 681 HIV-uninfected adults in Senegal, where residents are exposed to a variety of infectious diseases and other conditions that may affect CD4 counts. Lymphocyte counts were assessed in commercial sex workers, symptomatic men and women presenting to the University of Dakar infectious disease clinic for out-patient care and women seeking family planning services. CD4 and CD3 lymphocyte counts differed between the four study groups (P < 0.01). Men had the lowest mean CD4 count (711.6 cells/microl), while commercial sex workers had the highest levels (966.0 cells/microl). After adjustment for age and other behavioural and clinical factors, the difference in CD4 counts between the three groups of women did not remain. However, both gender and smoking were associated independently with CD4 counts, as men maintained lower mean CD4 counts (beta = -156.4 cells/microl, P < 0.01) and smokers had higher mean CD4 counts (beta = 124.0 cells/microl, P < 0.01) than non-smokers in multivariable analyses. This study is the first to explore factors that may influence CD4 levels in Senegal and to estimate baseline CD4 levels among HIV-negatives, information that may guide clinicians in interpreting CD4 counts.     

Comparison of absolute CD4+ lymphocyte counts determined by enzyme immunoassay (TRAx CD4 test kit) and flow cytometry.

Currently, CD4+ lymphocyte counts are one of the most widely used surrogate markers for monitoring disease progression in and initiating therapy for human immunodeficiency virus-infected individuals. However, the process of obtaining lymphocyte subset counts can be complex and expensive, often rendering the test inaccessible to many patients. In contrast to standard laser-based flow cytometry, the TRAx CD4 Test Kit utilizes an enzyme-linked immunoassay format to provide CD4+ lymphocyte counts by a simple and more cost-effective means. In order to evaluate the utility of the TRAx CD4+ assay in comparison with flow cytometry, heparinized blood samples were drawn from 188 infected and uninfected adult patients and 24 infected pediatric patients and evaluated by both assays. The correlation coefficient for all adult individuals tested was 0.94, and the mean absolute counts (in cells per milliliter, +/- standard deviation) were 510 +/- 358 for TRAx and 480 +/- 361 for flow cytometry. The correlation for the pediatric group was 0.93, with mean absolute counts of 956 +/- 767 for TRAx and 1,521 +/-a 1,438 for flow cytometry. Overall the TRAx CD4 Test Kit performed well in comparison to flow cytometry, and its lower cost and ease of use make it an encouraging alternative for the routine determination of CD4+ lymphocyte counts.     

A hereditary immunodeficiency characterized by CD8+ T lymphocyte deficiency and impaired lymphocyte activation.

An unusual form of severe combined immunodeficiency in children from two different families was associated with absence of CD8+ T lymphocytes and normal numbers of CD4+ T lymphocytes that did not respond to stimulation by non-specific mitogens, specific antibodies against T cell receptor or specific antigens. The defect in the CD4+ cells was bypassed by activating agents which are independent of the T cell receptor. The combination of an activation defect and selective depletion of CD8+ T lymphocytes suggests that the defective pathway is important in the differentiation of immature thymocytes as well as the proliferation of mature lymphocytes.     

Simultaneous determination of absolute total lymphocyte and CD4+ lymphocyte levels in peripheral blood by flow cytometry

A distinguishing feature of the Spectrum III flow cytometer is its capacity to analyze a constant volume of cell suspension. The authors have capitalized on this feature to directly and simultaneously measure the absolute numbers of all lymphocytes and CD4+ lymphocytes per microliter of peripheral blood. The study group consisted of 42 hospital patients, 12 former blood donors seropositive for human immunodeficiency virus (HIV), and 12 HIV-seronegative donors. Regression analysis revealed a highly significant correlation (r = 0.97) between Spectrum lymphocyte counts and lymphocyte counts determined by a Coulter Counter S-Plus IV. Similarly, Spectrum CD4 cell counts were significantly correlated (r = 0.98) with CD4 cell counts calculated from the Coulter lymphocyte counts and % CD4+ lymphocytes determined by flow cytometry. These findings indicate that the absolute numbers of lymphocytes and subsets of lymphocytes in peripheral blood can be rapidly and simultaneously measured by flow cytometry. Such an assay should prove useful in studies of HIV infection, where total lymphocyte and CD4 cell levels are important parameters for clinical staging and assessing responses to treatment.     

Function and phenotype of immature CD4+ lymphocytes in healthy infants and early lymphocyte activation in uninfected infants of human immunodeficiency virus-infected mothers.

The function and phenotypes of CD4+ lymphocytes in infants are different than in adults and are modulated by maturational changes and exposure to environmental antigens. Infants of non-human immunodeficiency virus (HIV)-infected mothers and uninfected infants of HIV-infected mothers, 0 to 6 months of age, were examined for CD4+ lymphocyte function by in vitro interleukin-2 (IL-2) production and for CD4+ phenotypes by three-color flow cytometry. A minority of these uninfected infants (28%) had functional responses similar to those of healthy adult women (IL-2 production in response to anti-CD3, alloantigen, and mitogen), while the remainder were capable of responding to alloantigen and mitogen but not to anti-CD3. We did demonstrate reduced phytohemagglutinin-stimulated IL-2 production in uninfected infants born to HIV-seropositive mothers compared to that in infants from seronegative mothers. The proportions of CD3+ CD4+, CD4+ HLA-DR- CD38+, and CD4+ CD45RA+ RO- (naive) lymphocytes were much higher in infants than in adults, and the proportions of CD4+ CD45RA- RO+ (memory) and CD4+ CD25+ (IL-2 receptor-bearing) lymphocytes were lower in infants than in adults. The proportions of activated (CD4+ HLA-DR+ CD38+) and memory (CD4+ CD45RA- RO+) lymphocytes were increased in uninfected infants of HIV-infected mothers compared to infants of uninfected mothers. Therefore, T-helper-cell function is immature in many infants, but the CD4+ lymphocytes of some HIV-exposed, uninfected infants have been stimulated by antigen at an early age.     

Correlates of CD4+ and CD8+ lymphocyte counts in high-risk immunodeficiency virus (HIV)-seronegative women enrolled in the women's interagency HIV study (WIHS)

Studies of human immunodeficiency virus (HIV) infection often compare values from HIV-uninfected controls, including CD4 and CD8 lymphocyte counts. Nonetheless, little is known regarding factors associated with CD4 and CD8 cell numbers in HIV-uninfected individuals. To ascertain potential factors associated with differences in CD4 and CD8 cells among HIV negative women, we studied these cells in a group of 953 women, enrolled as HIV-negative comparators in the Women's Interagency HIV Study. Using standard techniques, we measured CD4 and CD8 cells obtained during study-related visits every six months through visit 20 (maximum of 9.5 years). Results were correlated with demographic and behavioral factors, and data were analyzed using a multiple linear regression approach with generalized estimating equations. At baseline, the median age was 32.4 years, body mass index (BMI) was 26.4 kg/m(2), CD4 cell count was 1010 (range 214-2705)/microL, and CD8 cell count was 542 (range 72-2448)/microL. African-Americans comprised 54%, 24% were Hispanic, and 19% were Caucasian. In multivariate analysis, increasing age (p = 0.0006), increasing BMI (p = 0.001), and current smoking status (p = 0.03) were independent predictors of higher CD4 counts. Multivariate analyses of CD8 cells revealed that lower age (p = 0.001), higher BMI (p = 0.03), Hispanic race/ethnicity (p = 0.01); current smoking (p = 0.006), injection drug use (p = 0.02), and Hepatitis C infection (p = 0.01) were independent predictors of higher CD8 cell counts. Multiple demographic and behavioral factors may influence CD4 and CD8 counts in HIV negative women. These factors must be considered in future analyses comparing lymphocyte subsets in HIV positive and negative women.     

Alterations in T-cell receptor Vbeta repertoire of CD4 and CD8 T lymphocytes in human immunodeficiency virus-infected children

Perturbations in the T-cell receptor (TCR) Vbeta repertoire were assessed in the CD4 and CD8 T lymphocytes of human immunodeficiency virus (HIV)-infected children who were receiving therapy during the chronic phase of infection by flow cytometry (FC) and PCR analysis. By FC, representation of 21 TCR Vbeta subfamilies was assessed for an increased or decreased percentage in CD4 and CD8 T cells, and by PCR, 22 TCR Vbeta subfamilies of CD4 and CD8 T cells were analyzed by CDR3 spectratyping for perturbations and reduction in the number of peaks, loss of Gaussian distribution, or clonal dominance. The majority of the TCR Vbeta subfamilies were examined by both methods and assessed for deviation from the norm by comparison with cord blood samples. The CD8-T-lymphocyte population exhibited more perturbations than the CD4 subset, and clonal dominance was present exclusively in CD8 T cells. Of the 55 total CD8-TCR Vbeta families classified with clonal dominance by CDR3 spectratyping, only 18 of these exhibited increased expression by FC. Patients with high numbers of CD8-TCR Vbeta families with decreased percentages had reduced percentages of total CD4 T cells. Increases in the number of CD4-TCR Vbeta families with increased percentages showed a positive correlation with skewing. Overall, changes from normal were often discordant between the two methods. This study suggests that the assessment of HIV-induced alterations in TCR Vbeta families at cellular and molecular levels yields different information and that our understanding of the immune response to HIV is still evolving.     

A biometrical view on normal values of CD4 and CD8 lymphocyte counts in peripheral blood

Statistical methods have been used to determine an optimal approach to the definition of the reference range for CD4 ("helper") and CD8 ("suppressor/cytotoxic") T cell numbers and the CD4/CD8 ratio in the peripheral blood. A graphical presentation of the absolute values for CD4 and CD8 for 85 healthy blood donors showed that a reference ellipse defined by fitting a gaussian distribution to logarithmically transformed data for absolute counts of CD4 and CD8 cells was superior to the fitting of an ellipse to untransformed data. Further analysis for another 147 subjects showed that the 95% tolerance prediction for the CD4/CD8 ratio in health could be stated with 95% confidence as 0.6 to 5.0. This approach allows clear definition of reference ranges for T cell tests in health and would also be applicable to results for patients with a disease such as HIV 1 infection in which a reference range for "well" patients exists and a change in the T cell ratio is of prognostic significance.     

Elevated soluble CD8 levels in sera of human immunodeficiency virus-infected populations

Soluble CD8 levels in sera were quantitated in asymptomatic intravenous drug abusers, homosexuals, and patients with lymphadenopathy or acquired immunodeficiency syndrome. Soluble CD8 levels were elevated in human immunodeficiency virus-seronegative intravenous drug abusers and homosexuals, probably reflecting infections like cytomegalovirus. The sera of human immunodeficiency virus-seropositive groups of patients with human immunodeficiency virus infection also had elevated levels of soluble CD8, reflecting infections like cytomegalovirus and human immunodeficiency virus infection.     

Estimation of CD4+ and CD8+ T-lymphocytes in human immunodeficiency virus infection and acquired immunodeficiency syndrome patients in Manipur

Purpose: To estimate and stratify CD4⁺and CD8⁺T-lymphocyte levels in human immunodeficiency virus (HIV) infected (asymptomatic) and acquired immunodeficiency syndrome (AIDS) patients (symptomatic) and correlate the clinical features of the patients with CD4+ and CD8+ lymphocyte level. Methods: : Between April 2002 and September 2003, a total of 415 HIV seropositive adult patients (297 males and 118 females) attending Regional Institute of Medical Sciences (RIMS) hospitals were tested for CD4+ and CD8+ T-lymphocytes by fluorescent activated cell sorter (FACS) counter (Becton Dickinson). Symptomatic patients were diagnosed as per NACO clinical case definition. Results: Ranges of 0-50, 51-100, 101-200, 201-300, 301-400, 401-500 and above 500 CD4+ T-lymphocyte per microlitre were seen in 68, 52, 101, 73, 47, 31 and 43 patients respectively whereas CD8+ T-lymphocyte ranges of 0-300, 301-600, 601-900, 901-1500, 1501-2000, 2001-3500 per microlitre were seen in 29, 84, 92, 145, 40 and 25 patients respectively. One hundred and fifty patients were asymptomatic and 265 were symptomatic. CD4/CD8 ratio in asymptomatics and symptomatics were 0.13-1.69 and 0.01-0.93 respectively. Tuberculosis and candidiasis occurred in CD4+ T-lymphocyte categories between 0-400 cells per μL in symptomatics. However, cryptosporidiosis, toxoplasmosis, herpes zoster, cryptococcal meningitis, Pneumocystis carinii pneumonia, penicilliosis and cytomegalovirus retinitis were seen in patients having CD4+ T-lymphocyte less than 200 per μL. Conclusions: CD4+ T-lymphocyte was decreased in both asymptomatic and symptomatic HIV patients, The decrease was greater in symptomatics while CD8+ T-lymphocyte was increased in both except advanced stage symptomatics. CD4: CD8 ratio was reversed in both groups. Opportunistic infections correlated with different CD4+ T-lymphocyte categories.     

Association of immune complexes and plasma viral load with CD4+ cell depletion, CD8+ DR+ and CD16+ cell counts in HIV+ hemophilia patients. Implications for the immunopathogenesis of HIV-induced CD4+ lymphocyte depletion

OBJECTIVE: There is evidence that HIV induces CD4+ depletion in part by the formation of immune complexes (IC) that attach to CD4+ blood lymphocytes. In the present study we examined the relationship of IC-coated CD4+ blood cells with retroviral replication in HAART-treated patients. PATIENTS And METHODS: 52 hemophilia patients were studied from 1997 to 1999. Lymphocyte subsets, IgM, IgG and gp120 on CD4+ blood cells, in vitro responses of lymphocytes to mitogens, plasma neopterin and plasma viral load were measured. RESULTS: Patients with detectable viral replication and without ICs on CD4+ blood lymphocytes had a lower viral load (4100 versus 21000 HIV-1 mRNA copies/ml; P = 0.079) and higher CD4+ cell counts (310/microl versus 161/microl; P = 0.035) than patients with ICs on circulating CD4+ lymphocytes. Among patients with < 80 HIV-1 mRNA copies/ml, IC- individuals had slightly higher CD4+ lymphocyte counts than IC+ patients (384/microl versus 316/microl; n.s.). Further evidence for the clinical relevance of the ICs was obtained when 18 patients who had an undetectable viral load at previous investigations were analyzed. Among patients with a stable undetectable viral load, CD4+ counts increased in 6 of 8 IC- but in none of 2 IC+ individuals. In patients whose viral load increased during the observation period, 5 of 6 IC- but none of 2 IC+ individuals showed higher CD4+ cell counts. Impaired virus killing is suggested by lower CD16+ (35/microl versus 107/microl; P = 0.016), higher CD3+ DR+ (178/microl versus 66/microl; P = 0.006), and higher CD8+ DR+ (142/microl versus 34/microl; P = 0.017) cell counts in IC(-) patients compared to IC- patients without detectable viral load. Strong retroviral replication induced strong T cell dysfunctions. Fewer CD3+ 25+ blood lymphocytes (19/microl versus 47/microl; P = 0.006) and a lower in vitro response of T lymphocytes to the mitogens Con A (RR: 0.3 versus 1.2; P=0.023) and CD3 mab (RR: 0.5 versus 2.4; P = 0.012) was observed in IC+ patients with detectable versus undetectable viral load. CONCLUSION: Our data suggest that ICs on circulating CD4+ blood lymphocytes are primarily associated with CD4+ lymphocyte depletion whereas the plasma viral load is primarily associated with decreased T lymphocyte activation, lower CD16+ counts, and higher CD8+ DR+ lymphocytes which might be the effector cells for virus elimination.     

Improving CD4+ lymphocyte counts in HIV-infected hemophilia patients. A favorable prognostic indicator?

CD4+ lymphocyte counts of 91 HIV+ hemophilia patients were monitored for a mean of 4 years (range: 15-69 months). CD4+ lymphocytes decreased in 55 but increased in 36 patients over time. The CD4+ cell increases were persistent in 5 patients, whereas they fluctuated in 31. Of the 36 patients with increasing CD4+ counts 3 developed AIDS and 1 LAS. The other 32 patients were clinically asymptomatic (CDC II), but had immunological abnormalities, such as increased serum neopterin (N = 18) and impaired in vitro T cell responses to pooled allogenic stimulator cells (N = 15) or mitogens (N = 18). In contrast, of the 55 patients whose CD4+ cells decreased, 24 developed AIDS and 5 ARC (P less than 0.0005). Only 2 of these 55 patients had normal mitogen stimulation in vitro and normal serum neopterin levels.     

Screening for hepatitis C virus in human immunodeficiency virus-infected individuals

Immunosuppression from human immunodeficiency virus (HIV) may impair antibody formation, and false-negative hepatitis C virus antibody (anti-HCV) tests have been reported in individuals coinfected with HIV and HCV. It is unknown if the frequency of false-negative tests is sufficiently high to change screening recommendations in this setting. Thus, the prevalence of false-negative results for anti-HCV by third-generation tests was determined with samples from HIV-infected individuals. Sera from 559 HIV-infected and 944 HIV-negative prospectively followed injection drug users were tested for anti-HCV by a third-generation enzyme immunoassay and for HCV RNA by using a branched DNA assay and the HCV COBAS AMPLICOR system. Of 559 HIV-infected participants, 547 (97.8%) were anti-HCV positive. One of the remaining 12 anti-HCV-negative participants was HCV RNA positive, and she later developed detectable anti-HCV. Of the 944 HIV-negative participants, 825 (87.4%) were anti-HCV positive. One of the remaining 119 anti-HCV-negative participants was HCV RNA positive, and she also developed detectable anti-HCV at a later visit. These data indicate that HIV infection does not alter the approach to hepatitis C virus screening, which should be performed with third-generation assays for anti-HCV unless acute infection is suspected.     

Elevated levels of CD4 antigen in sera of human immunodeficiency virus-infected populations.

CD4 antigen levels in sera from asymptomatic intravenous drug users and homosexuals and patients with lymphadenopathy, acquired immunodeficiency syndrome-related complex, or acquired immunodeficiency syndrome were quantitated. Like soluble CD8, CD4 antigen levels were elevated in human immunodeficiency virus-seronegative asymptomatic intravenous drug users and homosexuals, probably reflecting infections such as cytomegalovirus, Epstein-Barr virus, and hepatitis B virus infections. The sera from human immunodeficiency virus-seropositive groups of patients with human immunodeficiency virus infection also had elevated levels of CD4 antigen, presumably reflecting infections like cytomegalovirus and human immunodeficiency virus infections.     

Influence of CD4+ lymphocyte counts on GB virus C/hepatitis G virus carriership in HIV-positive individuals

The duration of the GB virus C or hepatitis G virus (GBV-C/HGV) carriership varies according to the patient group studied. The immune competence of the host may be important. GBV-C/ HGV was studied in human immunodeficiency virus (HIV)-infected persons and an attempt was made to correlate the presence of viral RNA or E2 antibodies with CD4+ lymphocyte counts. Of 138 HIV-positive subjects, 30 were GBV-C/HGV RNA-positive and 20 others were E2 antibody-positive, whereas in healthy GBV-C/HGV-infected persons, the proportion of E2 antibody carriers was much higher. On the other hand, a relationship was not found between CD4+ lymphocyte counts and the presence of GBV-C/HGV RNA in the HIV-infected persons. This result does not necessarily imply that the CD4+ lymphocyte count does not affect viral clearance, but the results could be due to the trans-sectional nature of this study. A longitudinal assessment should clarify this point.     

CD4+ and CD8+ T cell responses in Helicobacter pylori-infected individuals

In order to characterize T cell responses in human Helicobacter pylori infection, we have examined proliferative responses and cytokine production by CD4+ and CD8+ T cells isolated from duodenal ulcer patients and asymptomatic H. pylori carriers, after activation with some H. pylori antigens that may be important in disease development. For control purposes, T cells from uninfected volunteers were also examined. The different H. pylori antigens induced only modest proliferative responses in circulating CD4+ and CD8+ T cells from both H. pylori-infected and uninfected individuals. However, circulating T cells from H. pylori-infected subjects produced larger amounts of interferon-gamma (IFN-gamma) in response to the Helicobacter antigens than did T cells from uninfected volunteers. Furthermore, CD8+ T cells produced larger amounts of IFN-gamma than did CD4+ T cells, on a per cell basis. Most IFN-gamma-producing cells from both infected and uninfected volunteers appeared to be naive T cells expressing CD45RA. Increased production of IL-4 and IL-5 was, on the other hand, only seen in a few instances after stimulation of isolated CD4+ and CD8+ T cells. Stimulation of freshly isolated gastric T cells with the different H. pylori antigens did not result in increased proliferation or cytokine production. In conclusion, our results show that several different purified H. pylori antigens induce production of IFN-gamma, preferentially by CD8+ cells. Therefore, they suggest that IFN-gamma-secreting CD8+ cells contribute significantly to the cytokine response induced by H. pylori infection.