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The CCAS EXPERT SUMMIT convened an array of international experts in Barbados on August 27–31, 2017 under the theme “From Care to Cure–Shifting the HIV Paradigm.” The Caribbean Cytometry & Analytical Society (CCAS) partnered with the Joint United Nations Programme on HIV/AIDS (UNAIDS) to deliver a program that reviewed the advances in antiretroviral therapy and the public health benefits accruing from treatment as prevention. Particular emphasis was placed on reexamining stigma and discrimination through a critical appraisal of whether public health messaging and advocacy had kept pace with the advances in medicine. Persistent fear of HIV driving discriminatory behavior was widely reported in different regions and sectors, including the healthcare profession itself; continued fear of the disease was starkly misaligned with the successes of new medical treatments and progress toward the UNAIDS 90-90-90 targets. The summit therefore adopted the mantra “Test-Treat-Defeat” to help engage with the public in a spirit of optimism aimed at creating a more conducive environment for persons to be tested and treated and, thereby, help reduce HIV disease and stigma at the individual and community levels.

It has been suggested that the range of AIDS-defining conditions witnessed in patients with HIV infection has changed since the early years of the HIV epidemic. In this paper we consider the range of AIDS-defining conditions in a cohort of 111 HIV-positive men with haemophilia registered at the Royal Free Hospital Haemophilia Centre. In particular we assess whether the incidence of Pneumocystis carinii pneumonia (PCP) has changed over time. The men were all infected between 1979 and 1985 after treatment with infected blood products and have now been followed prospectively for up to 13 years from HIV seroconversion. By the end of 1992, 44/111 patients had developed AIDS. Of the 44 men, 18 (41%) presented with PCP as their first AIDS-defining condition (ADC), mainly before the initiation of primary prophylaxis in 1989. The remaining 26 patients presented with a range of conditions as their first ADC, but there were no more than four cases in any one disease category. It is estimated that patients suffer from 0.7 further ADCs per year after being diagnosed with AIDS. After taking account of the increased levels of immuno-suppression in the cohort with time, it appears that the incidence of PCP, both as the first ADC or as any ADC, has declined since the introduction of primary prophylaxis for the disease in 1989. However, non-compliance with prophylaxis for PCP appears to have played a major role in the continuing occurrence of PCP since 1988. Improvements in compliance with therapy should result in a further reduction in the incidence of PCP both as a first ADC and as any ADC.

Background: CD38 expression on CD81 T lymphocytes in HIV-infected patients is monitored by flow cytometry (FCM). There is however no consensus re CD38 protocols, analyses or result reporting within/ between laboratories. Internal quality control measures (QC) were established for a standardized CD38 protocol and a system proposed for reporting CD38 fluctuation in longitudinal HIV1 patient monitoring. Methods: A single-platform (SP) CD38/CD8 protocol was ‘‘piggy-backed’’ onto the standardized ‘‘panleucogating’’ CD45/CD41 protocol. A weekly QC was established to monitor instrument stability (FlowSET TM ) and absolute cell count accuracy and reproducibility (stabilized blood product, Immuno-Trol TM ). The Mean Fluorescence Intensity (MFI) of CD38 expression on CD8 1 -lymphocytes was monitored on both stabilized blood and HIV-control samples. Linearized MFI values were determined from biological controls, i.e. healthy donor monocytes and granulocytes, and tested as a method of reporting CD38 expression on selected HIV1 patients on ART. Results: The CD45/CD4/CD8/CD3 method for lymphocyte enumeration compared well with the CD38 protocol (CD45/CD4/CD8/CD38) with excellent similarity (6100%) and precision for absolute CD4 and CD8 counts (CVs < 5%). Fluorosphere MFI- (FlowSet TM , FlowCount TM ) and color compensation values were exceptionally stable over time. CD38 MFI values established on monocytes as biological control was 4.0 and

Summary One hundred and eleven men with haemophilia at the Royal Free Hospital Haemophilia Centre, London, became infected with HIV between October 1979 and July 1985. This paper describes the incidence of HIV-related disease in this cohort in the absence of pre-AIDS treatment and assesses the effect of antiretroviral and prophylactic therapies on this. In particular the relationship between the CD4 count and the development of HIV-related disease is investigated. Before the introduction of pre-AIDS treatment in November 1988, 60 patients (54%) had developed some type of HIV-related pre-AIDS condition or AIDS, a cumulative incidence of 77% (95% Confidence Interval 63% to 91%) by nine years after seroconversion. The probability of developing such conditions was associated with falling CD4 lymphocyte counts and an estimated 51% of patients (95% Confidence Interval 38% to 63%) would be expected to develop some manifestation of HIV-related disease before their CD4 count has fallen to 0.2 x 109/1. Consequently, only conditions which develop later in HIV infection (AIDS, oral candida, herpes zoster) are likely to be influenced by treatment. Since November 1988, there has been a reduction in these conditions, although the reduction in the rate of new AIDS cases was small (P= 0.16). The risk of developing oral candida has been reduced at low CD4 counts from 12 cases in 76 years of experience below 0.2 x 109/1, prior to November 1988, to to cases in 81 years after November 1988, (P= 0.005). Similarly, since that date no herpes zoster infections were observed in our cohort.

Objectives: Subcutaneous administration of interleukin-2 (IL-2) has been shown to increase CD4 counts in HIV-infected patients. It remains unclear whether this effect is associated with a clinical benefit. Patients and methods: We conducted a long-term follow-up in the cohort of the UK-Vanguard study in which three groups of 12 antiretroviral-naive subjects with CD4 cell counts >350 cells/mm 3 received no treatment or IL-2 at either 4.5 or 7.5 MIU twice daily in 5 day cycles, respectively. Results: Mean follow-up was 376 weeks. IL-2 therapy was associated with a higher area under the curve of CD4 cell count change from baseline at week 48 but not thereafter. HIV-RNA levels were unaffected. Highly active antiretroviral therapy (HAART) was initiated after a mean of 172, 175 and 152 weeks in the control group, low-dose and high-dose IL-2 treatment group, respectively, a statistically non-significant difference. There was a tendency to start HAART soon after discontinuation of IL-2 therapy which may have been triggered by the steep decay of CD4 counts. There were two serious adverse events in the control group, seven in the low-dose IL-2 group and eight in the high-dose IL-2 group. No pattern of disease was detected, making an association with IL-2 therapy unlikely. Conclusions: We could detect neither a benefit of IL-2 therapy after week 48 nor delayed initiation of HAART. This is currently the longest follow-up data comparing IL-2 therapy with no therapy in antiretroviral-naive HIV-infected patients and does not show a persistent benefit of the intervention.

Bone marrow graft rejection following HLA-matched bone marrow transplantation (BMT) for leukaemia has been a rare problem. However, with the introduction of T-lymphocyte depleted BMT, graft rejection is recognized as a new complication. At the Royal Free Hospital (RFH) in London T-depletion is achieved using two monoclonal antibodies with complement mediated lysis. The methodology was extended to other centres and in total 56 patients have received T-depleted, HLA matched BMT. Twelve of 56 patients have had graft rejection. At the RFH three of 41 (7%) patients have had rejection whereas at collaborating centres nine of 15 (60%) patients have had rejection. We have investigated these rejections in order to identify factor(s) responsible. Rejection was not restricted by patient or donor characteristics, nor disease status. Patient management, chemotherapy conditioning, efficiency of T-depletion, graft versus host disease (GvHD), and infection post BMT, were not consistently implicated. The major difference between the RFH and all other centres was in the radiotherapy (RT) conditioning: The RFH prescribed a single fraction of 7.5 Gy total body irradiation (TBI) whilst collaborating centres gave 10 or 12 Gy fractionated TBI. We conclude that the different incidence of rejection (7% v. 60%) relates primarily to the RT conditioning although the mechanisms(s) of rejection remain unknown. We conclude that where T-depleted BMT is used, compensation by more intensive RT conditioning is required in order to avert graft rejection.

In order to expand the treatment of human immunodeficiency virus-1 (HIV) infected patients in Africa, millions will require cost-effective CD4 counts. Supporting laboratories therefore, need to move away from crisis management and haphazard practices to organized pathology services. The authors reviewed the performance of the simplified single platform (SP) PanLeucogated (PLG) CD4 methodology, introduced into 52 laboratories across the South African National Health Laboratory Service (SA-NHLS), with a proactive approach to training, internal quality control (IQC), and external quality assessment (EQA).Two-color flow cytometry for SP PLG (CD4/CD45) was combined with the sample-by-sample bead-count-rate (BCR) IQC for bead pipetting. PLG + BCR was validated versus conventional predicate SP and dual-platform (DP) 4-color flow cytometric methods used in the first world-on 1181 samples from 250 HIV+ patients followed longitudinally on anti-retroviral therapy (ART). EQA (accuracy) was performed through the United Kingdom National External Quality Assessment Scheme (UK-NEQAS). Further EQA was performed across the 52 SA-NHLS SP-PLG laboratories participating on the CD4 African Regional External Quality Assessment Scheme (AFREQAS), to assess both accuracy and/precision between NHLS PLG laboratories.There was virtually no bias noted between SP PLG and SP predicate methods. On DP, bias and variability increased but the errors introduced were minor without affecting CD4-related clinical decisions. The simpler 2-color PLG was less expensive with additional advantages: CD4+ T-cells were discriminated from monocytes without a need for CD3-staining, and the training was faster and easier for the trainees and trainers alike. The accuracy of SP-PLG was satisfactory: all PLG results submitted to the UK NEQAS were within +/-1 Trimmed Standard Deviation (SD) of the UK NEQAS CD4 Pool Trimmed Mean. Further, on the CD4 AFREQAS, the SA-NHLS laboratories using SP-PLG + BCR showed better precision (mean %CV = 7.2%) than the CD4 methods employed in other laboratories in Africa (mean %CV = 10.7%) or on other continents (mean %CV = 12.9%). PLG + BCR accommodated high workloads, exceeding 3,000 tests/laboratory/month, with capacity for further growth around 10% per month across the SA-NHLS.The superior performance of PLG + BCR over other methods has been demonstrated. In resource-conscious countries, where large-scale ART is being introduced, flow-cytometry using PLG + BCR can make a significant impact-due to simpler operation, easier training, stricter quality assurance, and better cost-efficiency. These cost-effective flow methods can legitimately replace the more cumbersome predicate technology of the first world for ART monitoring whilst accommodating an ever-expanding national ART program and consequent extremely high workloads reached country-wide.

Background The affordable, reliable, and simplified flow cytometric method on single platform with 2-color (CD45+, CD4+) Panleucogating (PLG-CD4) is used to monitor HIV+ patients on antiretroviral therapy (ART) in South Africa (SA). Viral load (VL) assays are also used to monitor response to ART but are labor-intensive with costs that, in the long run, may remain unsustainable. Cheaper and quicker alternatives to VL such as CD38 tests on CD8+ T cells may therefore play a role in securing the continuation of ART programs in high-volume resource-restricted settings. Methods The single-tube PLG assay (CD45/CD4) was modified to use the full capacity of flow cytometers for 4-color immunofluorescence by including two more parameters: the CD38-activation marker on CD8 T-cells (CD8/CD38). This was introduced at baseline prior to the start of ART and then the changes of CD38+ mean fluorescent intensity (MFI) on CD8+ T-cells were then regularly assessed during follow-up—in comparison with the VL. Results A total of 103 patients received ART. By the end of their 4-, 8-, 12-, and 24-week observation periods, 29 (32.2%), 58 (64.4%), 74 (82.2%), and 83 (92.2%) had undetectable VL. This was also reflected by the gradual decrease of CD38 MFI expression on CD8+ T cells, irrespective of whether patients were “high,” “moderate,” or “low” CD38 responders at baseline. As expected, the CD4+ T cell counts substantially increased during the first 4 weeks from baseline 186 ± 8.3 (SEM) to 267 ± 12.3 cells/μl blood. But the recovery was slower over the rest of the 1-year follow-up to reach 334 ± 18.2 cells/μl at week 48. As these CD4 increments were meager, the longitudinal follow-up of the continuously decreasing CD38 MFI values has become a particularly useful laboratory parameter to ascertain that the patients had indeed been responding well to ART. This monitoring protocol, in uneventful cases, may assist in reducing the frequency of VL testing. Conversely, a rise of CD38 MFI, if significantly higher than that seen at the previous visit even without fully reverting back to high baseline CD38-MFI values, provides an immediate indication for VL testing to judge whether or not the rebound of immune-activation is due to HIV VL-related irregularities. Therefore the CD8/CD38 test can provide the early, albeit not fully HIV-specific, warning signs about nonadherence to ART and/or developing drug resistance. Conclusion This single-tube 4-color PLG-CD4 + CD8/38 activation assay (CD4/CD45/CD8/CD38), can replace the conventional 4-color CD4 protocols by substituting the redundant CD3 reagent with the informative CD38 antibody. This modification carries virtually no extra costs, while adding extra value to CD4 monitoring and enabling real-time, practical management of patients on ART. As a result, the numbers of VL tests required in patients on ART can be reduced—to save costs across our national treatment program which is already equipped with the necessary flow-cytometric screening capacity. © 2008 Clinical Cytometry Society

Background The global struggle with human immunodeficiency virus (HIV) and the battle to develop affordable CD4 T-cell counting technology are both unfulfilled goals in 2008. The need for such instrumentation is more critical now as implementation of antiretroviral therapy (ART) is in progress in many resource limited regions. Major scaling-up efforts in rural situations are difficult to implement without laboratory infrastructure. CD4 T-cell counting is especially critical when trying to reach individuals with HIV to have them enrolled in ART as soon as they qualify for treatment based on CD4 count. Method This review covers both the chronological evolution and the scientific milestones of technological development of affordable immunophenotyping. It is more focused on flow cytometry but does consider the potential contribution by digital image cytometry. Results Thus far flow cytometry offered only modest progress toward affordable immunophenotyping. A list with desirable features is offered for side by side comparison. Digital image cytometry has yet to show its enormous affordable market potential. Conclusions It is possible to develop truly affordable, portable flow cytometry but it is not here yet. There are some hopeful signs as there are innovative and practical technical components appearing at regular intervals. However, so far the technical breakthroughs have been fragmented efforts without any attempts to consider intercorporate collaboration to optimize critical mass and synergy. The smaller players in the industry have made some progress toward meeting the monumental needs in Africa and Asia. Digital image cytometry may well be the ultimate winner in the affordable technology race. © 2008 Clinical Cytometry Society

TB remains uncontrolled. In resource-rich countries, only approximately 60% of diagnoses are confirmed by culture. The number is lower in resource-poor environments. Huge scope therefore exists for alternative diagnostic strategies. Counting antigen-specific lymphocytes by virtue of cytokine production following 8-16 h stimulation with tuberculosis antigens is currently the strategy of choice. Several methods exist, including ELISA, ELISpots, and flow cytometry. Although it is clear that blood samples stimulated by ESAT-6 and CFP-10 antigens discriminate between TB infection and BCG vaccination, it is flow-cytometry that seems to be able to distinguish active TB disease from mere TB exposure. Of the various flow-protocols including four-color tests (CD45-CD3-CD4-IFNgamma), three-color tests (CD3-CD4-IFNgamma) and two-color tests (CD4-IFNgamma), even the simplest is performing well, provided that the results are expressed as percentage of IFN-gamma+ cells per CD4+ lymphocytes (%IFNgamma/CD4+). Studies using broncho-alveloar lavage (BAL) and Induced-Sputum (ISp) show that TB-specific CD4+IFN-gamma+ T cells accumulate in the lung in pulmonary and extra-pulmonary TB at frequencies >5-20-fold more frequent than in blood. This pulmonary homing is absent following BCG immunization. The use of PPD to stimulate CD4+IFN-gamma+ cells in the lung in active TB leads to >3-12-fold greater responses than seen with CFP-10 or ESAT-6, and any interference from BCG vaccination is absent. This method is unaffected by HIV coinfection, which has always been the problem for other immune-based diagnostics. Further, lung-based samples provide material for rapid tests of both the IFN-gamma assay and bacteriology, and importantly, these tests are amenable for future simplification with automated fluorescence-image cytometers.Another development of the multiparameter analytical power of flow-cytometry is to use markers for "lung-seeking" populations of CD4+ T cells in blood, obviating lung sampling. In active TB, but not in BCG vaccinees, TB-specific memory CD4+ T cells can be found in blood that are dominantly CD27-negative and probably lung seeking and can be diagnostically useful.

Background: Immunological ex vivo assays to diagnose tuberculosis (TB) have great potential but have largely been blood-based and poorly evaluated in active TB. Lung sampling enables combined microbiological and immunological testing and uses higher frequency antigen-specific responses than in blood. Methods: A prospective evaluation was undertaken of a flow cytometric assay measuring the percentage of interferon-γ synthetic CD4+ lymphocytes following stimulation with purified protein derivative of Mycobacterium tuberculosis (PPD) in bronchoalveolar lavage fluid from 250 sputum smear-negative individuals with possible TB. A positive assay was defined as >1.5%. Results: Of those who underwent lavage and were diagnosed with active TB, 95% (106/111) had a positive immunoassay (95% CI 89% to 98%). In 139 individuals deemed not to have active TB, 105 (76%) were immunoassay negative (95% CI 68% to 82%). Of the remaining 24% (34 cases) with a positive immunoassay, a substantial proportion had evidence of untreated TB; in two of these active TB was subsequently diagnosed. Assay performance was unaffected by HIV status, disease site or BCG vaccination. In culture-positive pulmonary cases, response to PPD was more sensitive than nucleic acid amplification testing (94% vs 73%). The use of early secretory antigen target-6 (ESAT-6) responses in 71 subjects was no better than PPD, and 19% of those with culture-confirmed TB and a positive PPD immunoassay had no detectable response to ESAT-6. Conclusions: These findings suggest that lung-orientated immunological investigation is a potentially powerful tool in diagnosing individuals with sputum smear-negative active TB, regardless of HIV serostatus.

The composition of the mononuclear cell infiltrate in the liver was studied in patients with autoimmune and hepatitis B virus (HBV)-induced liver disease. The ratio of inducer to cytotoxic/ suppressor cells was greater in patients with lupoid chronic active liver disease, primary biliary cirrhosis, and HBeAb positive HBV-induced chronic active liver disease than in patients with HBeAg positive HBV-induced chronic hepatitis. In patients with chronic HBV-induced (HBeAb positive) liver disease, this ratio was greater in the periportal/portal area than in the lobule. These data are consistent with a relative deficiency of the cytotoxic/suppressor population of T cells in autoimmune liver diseases and possibly in HBeAb positive HBV-induced chronic active liver disease. In the latter patients, different ratios in the periportal and centrilobular zones suggest different mechanisms for periportal and lobular hepatitis.

Alterations in bronchoalveolar lavage (BAL) CD4/CD8 T cell subset ratios have been demonstrated in a variety of different respiratory disorders and the measurement of these changes may be diagnostically helpful. Flow cytometry (FCM) is a precise technology that offers many advantages over conventional cytospin techniques to determine T cell subset ratios in tissue fluids such as BAL. However, the optimum gating strategies for evaluating these parameters by FCM have not been evaluated. Here, the CD4/CD8 ratios in 33 BAL samples were compared using three different methods by FCM with two different flow cytometers. Bland Altman analysis demonstrated clinically insignificant differences between two simplified staining and gating strategies and a more complex ‘gold standard’ method. These findings confirm the precision of FCM for BAL T cell subset ratio analysis and suggest that the optimal gating strategy may be a simple panel using only CD45, CD4 and CD8.

One mechanism of HIV pathogenesis has been proposed to be the activation of T lymphocytes, resulting in proliferation and decreased survival of these activated cells. Others have suggested that co-infections may exacerbate this process. These hypotheses were tested by examining the relationship between HIV blood viral load with the proportion of activated and proliferating CD8+ lymphocytes in lung and blood. In the lung these responses were compared in patients with or without respiratory pathogens. Thirty-five HIV-positive patients and five controls underwent bronchoalveolar lavage (BAL). BAL and blood samples were fixed and permeabilized and the proportions of memory CD8+ lymphocytes that expressed the activation marker CD38 and the cell cycling marker Ki67 were measured by flow cytometry. CD38bright CD8+ lymphocytes from both sites increased with increasing blood viral load. In BAL there was no significant difference between the CD38 activation status in those with respiratory pathogens compared with those without. More CD38bright CD8+ lymphocytes expressed Ki67 when compared with the CD38dim lymphocytes. These findings provide evidence that HIV is the primary factor in stimulating CD8+ cell activation.

Inexpensive antiretroviral therapy (ART) might soon be available to treat human immunodeficiency virus (HIV) infections in resource-restricted areas of the globe. The number of CD4+ T-cells in the blood is the single most important laboratory parameter to select patients for therapy at the right time and to monitor the effect of ART. The question is asked whether flow cytometry is adaptable to change from an expensive and complicated scientific analytical instrumentation to become a practical diagnostic tool that can be widely operated. Recent studies indicate that a branch of clinical/practical flow cytometry is gaining a new identity by a confident simplification and improvement of clinical protocols. These new observations can be marshaled into three new areas of development. First, the changes in the choices of reagents and staining protocols have been initiated by the preferred use of primary immunological gating during the flow-cytometric analysis, exploiting the exquisite discriminating power of antibodies. The recent National Institutes of Health (NIH) and Centers for Disease Control and Prevention (CDC) guidelines introduced CD45-assisted protocols that are also at the heart of the PanLeucogating protocol that revitalized the testing of absolute CD4 counts on double-platform. With CD45 staining the leucocyte populations can be reliably studied in aging samples. It is now also documented that minimal technology, that is primary gating with CD4 (or CD8) antibodies, provides excellent CD4 (or CD8) T-cell counts. Consequently the optimal affordable protocol uses CD45 and CD4 antibodies in combination. The second area of development is the absolute counting facility on flow cytometers. Most frequently, microbeads are added to blood to define the sample volume in instruments that are not equipped with a volumetric microsyringe. The beads are, however, expensive, and the utilization of the stable flow rate of the excellent instruments might soon provide an answer to replace dear microbeads. With stable flow, the time span of analysis tells the sample volume and the absolute counts can be calculated. Finally, stabilized blood cell standards and blood stabilizing fluid, referred to as Transfix, have recently been introduced for quality assurance. These products are important to prove the precision and accuracy of the new affordable protocols and also to check, and guide if necessary, the performance of the laboratories even in remote areas. These recent developments provide evidence about the renaissance of affordable flow cytometry, a precise, costeffective and quantitative technology that is capable of providing CD4+ T-lymphocyte counts in as high volume as >400 patient assessment per day at the fraction of the cost of the current Western prices. These recent achievements need to be taken into consideration when alternative, nonflow CD4-counting methods are assessed.

BACKGROUND Measuring CD4 T-cell counts at low cost is relevant in dealing with the human immunodeficiency virus (HIV) epidemic throughout the developing world. The recently introduced novel concepts in gating strategies and sample stabilization facilitate affordable immunophenotyping by flow cytometry. However, the impact of these developments is still limited by the high cost of currently available flow cytometers. Methods: Diode lasers emitting 10–15 mW at 635 nm are one-tenth the size and cost and require one thousandth the power of an equivalent 488-nm argon ion laser. We used the available 635-nm diode-based flow cytometers, including PA-II, Luminex 100, SuperMot, and FACSCalibur, to investigate whether these instruments can generate reliable CD4 counts when used with allophycocyanin (APC) and cyanin-5 (Cy5)-labeled CD4 antibodies. Results: We document the feasibility of obtaining leucocyte differential counts using orthogonal side scatter (SSC) without the need for forward scatter (FSC). Accurate CD4% values among lymphocytes and leucocytes can be obtained by primary CD4 gating using a single CD4 monoclonal antibody conjugated to APC or Cy5. Double immunofluorescence (IF) staining with CD4-APC (FL1) and CD45-APC-Cy7 (FL2) introduces pan-leucogating for a convenient assessment of absolute CD4 counts on double platforms. We demonstrate that small flow cytometers with laser diodes are capable of delivering absolute CD4 T-cell counts with a precision similar to the performance of the current state-of-the-art single-platform instruments (e.g., the CytoronAbsolute; R2 = 0.961). In this respect, they appear to be superior to the nonflow CD4 counting techniques. Conclusions: Accurate CD4 counts can be generated at minimal cost on red diode laser-operated flow cytometers, retaining the potential for high throughput capacity without compromising precision. With further improvements in volumetric technology and clinical software, these cytometers may develop into a new generation of inexpensive battery-operated laboratory hardware that combines cellular phenotyping with bead-based multiplexing immunoassays for (HIV) serology. Cytometry (Clin. Cytometry) 50:78–85, 2002. © 2002 Wiley-Liss, Inc.

The story of T-lymphocyte subset immunophenotyping technology is reviewed on the occasion of the 20th anniversary of CD4 T-cell enumeration. Over time, immunophenotyping has evolved into precise, reliable, but complicated and expensive technology requiring fresh blood samples. The gating technologies that were universally adapted for clinical flow cytometry for the past decade relied on rapidly deteriorating morphological scatter characteristics of leukocytes. This special issue dedicated to CD4 T-cell enumeration features most of the available new options that will have a significant impact on how this technology will be implemented within the first decade of the 21st century. In a series of original publications, including the new NIH guideline for T-cell subset enumeration, contemporary gating protocols that use immunologically logical parameters are presented as part of the more reliable and affordable immunophenotyping alternative. Some of the improvements addressed here include the costs of the assays and the capacity to monitor interlaboratory and intralaboratory performances. It is clear that an effective attack on the human immunodeficiency virus (HIV) epidemic has to embrace resource-poor regions. Reducing the cost of the assay while improving reliability and durability is a move in the right direction. Cytometry (Clin. Cytometry) 50:39–45, 2002. © 2002 Wiley-Liss, Inc.

Most cortical thymocytes die within the thymus. It is proposed here that lethally high intracellular levels of deoxynucleosides are the cause of death. Developing thymocytes appropriately equipped for self-recognition are protected because contact with stromal cells prevents the lethal accumulation of deoxynucleosides.

We tested the feasibility and precision of affordable CD4+ T cell counting in resource-poor settings using a recently standardised fixative, TransFix in whole blood (WB) by flow cytometry (FCM). The precision of the assays was established under optimal conditions for single-platform FCM such as the volumetric CytoronAbsolute and the bead-based FACSCan. Fresh WB samples from HIV-seropositive and seronegative patients were tested in Tanzania and South Africa, fixed and sent to the UK for reanalysis 7 days later. Correlation, bias and limits of agreements were analysed by linear regression and the Bland-Altman test. Absolute CD4+ T cell counts remained stable for at least 10 days when TransFix was added to WB in 1:10 dilution at 20-25 degrees C, and for 7 days when added in 1:10 or 1:5 dilution to samples stored to mimic 'tropical' conditions at 37 degrees C. Higher temperatures such as 42 degrees C were tolerated for only short periods since the recovery had decreased to 63% by day 3. The reproducibility of lymphocyte subset analysis remained unchanged by TransFix with coefficient of variations

By protecting and stimulating HIV-specific CD4 cell responses, treatment of primary HIV infection (PHI) with potent quadruple HAART could lead to prolonged suppression of HIV replication after cessation of antiretroviral therapy. The QUEST trial investigates this hypothesis and aims to determine whether addition of a therapeutic vaccine to HAART increases the likelihood of prolonged viral suppression compared to HAART alone.148 patients with PHI were recruited. Participants were treated with open-label HAART for at least 76 weeks. Participants with sustained viremia50 copies/mL were randomized to one of three 5-month, double-blinded study treatment groups: HAART alone, HAART + ALVAC-HIV (vCP1452), or HAART + ALVAC-HIV (vCP1452) + Remune. After a further month of HAART alone, all treatment was stopped where plasma HIV-1 RNA remained at50 copies/mL. Intensive virologic and immunologic monitoring during a 24-week observation period followed treatment interruption. Patients who met treatment reintroduction criteria were offered HAART rescue.

Objective: To describe the short-term changes in CD4 lymphocyte counts after the interruption of antiretroviral HIV therapy in order to increase the understanding of CD4 lymphocyte dynamics, and so that appropriate monitoring strategies can be designed.Methods: We studied 35 HIV-infected patients with late-stage disease who had therapy interruptions leading to high viral load levels, median greater than 750 000 RNA log(10) copies/ml, and in whom two CD4 cell counts (median 28 days apart) were available before beginning a salvage regimen.Results: Overall, there was a substantial decline in CD4 cell counts from a median of 125 to 83 cells/mm(3) in the average 28 day period, with median proportionate and absolute losses of 26% and 24 cells/mm(3) per month, respectively (P < 0.008). This tended to be greater in individuals studied sooner after interrupting therapy (P = 0.03) and in those with CD4 cell counts above the pre-therapy baseline (P = 0.06). There was a strong negative correlation between the proportionate increase in viral load and the absolute change in CD4 cell count (-0.66, P = 0.0002).Conclusion: Patients with relatively advanced HIV infection interrupting antiretroviral therapy after failing a protease inhibitor-containing regimen require frequent monitoring because CD4 cell counts appear to fall quite rapidly, at least in the first few weeks after interruption (C) 2000 Lippincott Williams & Wilkins.

HIV-1 RNA levels are routinely monitored as part of patient management. However, little is known about the course of HIV-1 RNA levels over the entire period of infection. The aim of this study was to investigate the course of HIV-1 RNA levels in a cohort of men with hemophilia who were observed for up to 17 years after HIV-1 seroconversion, and to assess the risk of HIV disease progression at any HIV-1 RNA level. Viral loads were measured on annual stored serum samples in 107 men with hemophilia A using the Roche Amplicor Monitor assay with non-B primers. On average, HIV-1 RNA levels increased significantly by 0.11 log10 per year over the course of HIV infection. This rate of increase was significantly faster in those who developed AIDS or died over the subsequent 12 to 17 year period, and in those who were older at HIV- 1 seroconversion. The risk of developing AIDS and death remained low when the HIV-1 RNA level was below 4 log10 copies/ml, but increased rapidly thereafter, supporting current guidelines for the initiation of antiretroviral therapy after the viral load has exceeded this level.

In humans, blood is commonly monitored to provide surrogates of disease progression and assess immune status. However, the varied, rapid and atypical alterations in lymphocyte subsets which may occur in blood in response to pathogens, are not predictive of changes in the bulk of the immune system. A hallmark of human and simian immunodeficiency virus (SIV) infections is the profound loss of blood CD4+ lymphocytes, a feature widely accepted as being a consequence of direct or indirect viral killing of CD4+ cells throughout the body. However, in recording declining CD4 counts and CD4/8 ratios in the blood, little attention has been paid to migratory behaviour or the composition and tissue distribution of various lymphocyte subsets. This article compares the lymphocyte subsets in blood and various tissues in normal and virus-infected individuals prior to and following drug treatment and indicates an absence of selective CD4+ cell decreases or increases, highlighting the importance of lymphocyte trafficking and compartmentalization in regulating blood T cell levels and suggesting a reevaluation of the currently favoured paradigm.

As part of the effort to promote elimination of global health care disparities, this special supplement compiled 19 articles about practical diagnostic cytometry to recognize the recent achievements of laboratory scientists working in the shadow of the HIV/AIDS epidemics in resource limited settings. First, the clinical significance, diagnostic utility (as governed by international guidelines), and the historical perspectives of CD4+ T cell enumeration are reviewed. Then successful large-scale implementations of cost-effective CD4 counting are described for parts of Africa, USA, and the Caribbean. These activities are linked with both the training of personnel in fledgling laboratories as well as with external quality assessment implementations. Some of the more recent solutions related to pediatric CD4 testing using CD4% values are covered. Nevertheless, the need for further simplification and parsimony is still immense, and the potential solutions are catalogued in the articles written by experts operating in truly challenging rural environments. Cytometry is considered to be an expandable flexible technology for other assays beyond CD4 assessment, particularly within organized laboratory services in the Third World. These include haematological measurements, CD38/CD8 lymphocyte activation for viral load-related assessments, diagnosis of active tuberculosis and malaria, and bead-based serological assays for a variety of infectious diseases. The development and support of these emerging technologies by affluent countries is not entirely altruistic but is likely to be beneficial for both the Third and the First Worlds. © 2008 Clinical Cytometry Society

We have investigated the regulation of adult and cord blood CD45RA+ T cell proliferation and apoptosis to identify factors that may control the naive T cell pool. Cord CD45RA+ T cells were highly susceptible to spontaneous apoptosis as compared with CD45RA+ T cells from adults. Apoptosis was prevented by the addition of IL-2, IL-4, IL-7, and IL-15 which signal via the γ-chain of the IL-2 receptor. IL-7 prevented the decrease in Bcl-2 and Bcl-xL and induced cell cycling in up to 20% of cord T cells after 8 days, resulting in a threefold increase in cord T cell numbers. However, the expanded cells retained a CD45RA+CD45RO− phenotype. Similar results were obtained with adult CD45RA+ T cells. IL-7-expanded CD45RA+RO− T cells expressed CD45RO after stimulation through the TCR. Investigations into the regulation of replicative senescence showed that after 12 days in culture with IL-7, cord blood CD45RA+ T cell proliferation resulted in telomere shortening. Nevertheless, IL-7-expanded cord blood T cells still maintained longer telomeres than unstimulated adult T cells. IL-7 but not IL-2 could directly induce high telomerase activity which probably retarded the rate of telomere shortening in cord blood T cells. These results suggest that proliferation induced by IL-7 may be important for extrathymic expansion of neonatal CD45RA+ T cells and may also contribute to the maintenance of the adult CD45RA+ T cell pool.

Quantitation of immunofluorescence intensity serves to estimate the number of defined molecules expressed on or in cells. Clinical applications of this diagnostic tool are increasing, e.g., aberrant expression of various antigens (Ag) by leukemic blasts or lymphoma cells, intensity of CD38 expression by CD8+ T-lymphocytes to monitor activation status, and intensity of CD62P to detect platelet activation. In this report we discuss the quality-control measures required for quantitation of fluorescence intensity, and we review seven concepts that have been developed to quantify fluorescence intensity during the past 15 years. Initial work addressed the conversion of logarithmic channel numbers into units of relative fluorescence. The design and use of calibration beads labeled with predefined amounts of dye allowed instrument-independent expression of fluorescence intensity in units of molecules of equivalent soluble fluorochrome (MESF). This method was refined by the combined use of such standards with monoclonal antibodies (mAb) conjugated 1:1 with phycoerythrin (PE), allowing translation of fluorescence intensity into numbers of antibodies bound per cell. Alternatively, the use of 1:1 PE-conjugated mAb under the assumption that CD4+ lymphocytes reproducibly bind 50,000 CD4 mAb molecules was proposed to convert units of relative fluorescence intensity into units of antibodies bound per cell. The use of antibody-binding capacity as a surrogate marker for quantification of Ag expression was addressed more directly by the development of antibody-binding standards. The quantitative indirect immunofluorescence assay is based on beads labeled with various amounts of CD5 mAb that calibrate the binding of the secondary antibody in units of antibody-binding capacity. Alternatively, goat anti-mouse-labeled calibration beads have been developed. Published results obtained with the latter calibrators showed an unexpected inaccuracy. The different ways in which calibrators and cells under study bind mAb (i.e., Fab mediated versus Fc mediated) may have contributed to this variation. Recently, the use of stabilized cell populations expressing Ag in a specified range of concentrations has been proposed as an Ag-specific calibration system of mAb binding. We identify several issues on the level of instrumentation, reagents, and cells under study that should be solved to allow standardization of quantitative assessments of immunofluorescence intensity.

Objective: To assess the prognostic value of HIV RNA levels measured shortly after HIV seroconversion and whether markers of immune response (CD4+ and CD8+ T-cell counts, IgA and IgG) measured at the same time, continue to provide prognostic information once the HIV RNA level is known. Design and methods: HIV RNA levels were measured approximately 2.5 years after seroconversion in 97 haemophilic men followed for up to 17 years. Levels of CD4 + and CD8+ T cells, IgA and IgG were measured within 1 year of the HIV RNA level. The relationships between these markers and progression to AIDS and death were studied using Kaplan-Meier plots and proportional hazards regression models. Results: High HIV RNA levels were associated with faster progression to AIDS and shorter survival in univariate Cox regression models. High IgA and IgG levels were also associated with faster disease progression. In multivariate models, high HIV RNA levels remained independently associated with faster disease progression [relative hazard (RH), 1.86; P = 0.01 for AIDS; RH, 1.66; P = 0.05 for death]. However, high IgA and IgG levels provided strong independent prognostic information for AIDS and death (IgA: RH, 1.38; P = 0.006 for AIDS; RH, 1.33; P = 0.07 for death; IgG: RH, 1.10 ; P = 0.02 for AIDS ; RH, 1.12; P = 0.01 for death). Conclusions: Our results confirm the importance of the HIV RNA level in assessing the long-term prognosis in individuals infected with HIV. However, our results suggest that immune activation markers, rather than merely reflecting high HIV RNA levels are important in assessing prognosis in their own right. These findings may improve our understanding of HIV pathogenesis and may aid clinical management of patients.

Umbilical cord blood (CB) transplantation is thought to be associated with a reduced risk of severe graft-versus-host-disease (GVHD) compared with bone marrow transplantation (BMT). The cytokine cascade is known to be important in the pathogenesis of GVHD; however, previous studies investigating the cytokine secretion pattern of CB cells have been contradictory because of variations in experimental techniques. In this study, the cytokine profile of cord and adult blood lymphocytes and lymphocyte subsets has been assessed at the single-cell level by flow cytometry, using CD4/CD8 and CD45RA/CD45RO markers. Cord and adult blood mononuclear cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of monensin. After 4 to 24 hours of incubation, interleukin-2 (IL-2), IL-4, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) production was measured by three-color flow cytometry. The results show that cord blood lymphocytes (CBL) produce less IL-2, IL-4, IFN-γ, and TNF-α than adult peripheral blood lymphocytes (ABL). Further subset analysis showed that in CBL the majority of cytokine producing cells were CD4+CD45RA+, whereas in ABL the cytokine-producing cells were both CD4+CD45RO+ and CD8+CD45RO+. These results suggest that the reduced incidence of GVHD in CB transplantation may partly due to the altered cytokine profile seen in CBL.

The need for a rapid and reliable marker for the engraftment potential of hematopoietic stem and progenitor cell (HPC) transplants has led to the development of flow cytometric assays to quantitate such cells on the basis of their expression of CD34. The variability associated with enumeration of low-frequency cells (i.e., as low as 0.1% or 5 cells/microl) is exceedingly large, but recent developments have improved the accuracy and precision of the assay. Here, we review and compare the major techniques. Based on the current state of the art, we recommend 1) bright fluorochrome conjugates of class II or III monoclonal antibodies (mAbs) that detect all glycoforms of CD34, 2) use of a vital nucleic acid dye to exclude platelets, unlysed red cells, and debris or use of 7-amino actinomycin D to exclude dead cells during data acquisition, 3) counterstaining with CD45 mAb to be included in the definition of HPC, 4) during list mode data analysis, Boolean gating to resolve the CD34+ HPCs from irrelevant cell populations on the basis of the low levels of CD45 expression and low sideward light-scatter signals of HPCs, 5) inclusion of CD34dim and CD34bright populations in the CD34+ cell count, 6) omission of the negative control staining, and 7) for apheresis products, enumeration of at least 100 CD34+ cells to ensure a 10% precision. Unresolved technical questions are 1) the replacement of conventional dual-platform by single-platform assay formats, i.e., derivation of absolute CD34+ cell counts from a single flow cytometric assessment instead of from combined flow cytometer (percent CD34+) and hematology analyzer (absolute leukocyte count) data, 2) the cross-calibration of the available single-platform assays, and 3) the optimal method for sample preparation. An important clinical question to be addressed is the definition of the precise phenotypes and required numbers of HPCs responsible for short- and long-term recovery to optimize HPC transplant strategies.

Forty-seven HIV-infected adults had broncho-alveolar lavage stimulated with purified protein derivate of Mycobacterium tuberculosis. Eighteen of 19 (95%) with tuberculosis co-infection had interferon-gamma synthetic CD4 lymphocyte responses > 1% versus three of 28 (11%) without (P < 0.0001). Lung response was unrelated to blood CD4 cell count. BAL HIV tuberculosis responses were similar in 25 HIV-uninfected tuberculosis patients. Responses in matched blood samples were often undetectable. Therefore, immunological tuberculosis assays seem less affected by HIV co-infection when lung-based.

Simple models of CD4 lymphocyte interactions with human immunodeficiency virus (HIV) lead to the hypothesis that progression of HIV infection involves an increase in viral replicative capacity, due either to changes in the virus or in the host environment, or both. In order to consider how changes in plasma virus load after transient, potent antiretroviral therapy can be used to test the above hypothesis--a simple mathematical model that encompasses the processes of (1) arrival of new CD4 lymphocytes, (2) death/removal of these cells by HIV-independent mechanisms, (3) infection of susceptible CD4 lymphocytes by HIV, and (4) death/removal of infected cells was investigated. This showed that the in vivo rate of increase in plasma virus load immediately after transient therapy provides a measure of the viral replicative capacity. Thus, the hypothesis that progression of HIV infection involves an increase in viral replicative capacity can be tested by measuring this viral growth rate in patients with high CD4 counts and in patients with low CD4 counts. Studies should thus investigate dynamics of changes in virus levels after stopping antiretroviral therapy and, in particular, measure rates of increase in virus in patients at high and low CD4 counts. In practice, such data may assist in therapeutic management of patients with HIV infection.

Objective: To describe survival after a CD4+ T-cell count of less than 5 〈 10 6 /l and to identify possible baseline factors associated with outcome. Design: A prospective cohort study. Setting: A large teaching hospital in North London. Patients and participants: Patients treated at the Royal Free Hospital, London, who had at least one reported CD4+ T-cell count of less than 5 〈 10 6 /l and were being followed up for clinical care prior to the date of this cell count. Main outcome measure: Death. Methods: Proportional hazards models, Kaplan‐Meier analysis. Results: One-hundred and sixty-nine patients were included in the study. The median survival after a very low CD4+ T-cell count was 0.95 years (95% confidence interval, 0.78‐1.19), although 20% survived for over 2 years. Older age and a previous AIDS diagnosis were related to poorer outcome. A higher CD8+ T-cell count at baseline was also associated with a better prognosis. Conclusions: A CD4+ T-cell count of less than 5 〈 10 6 /l did not necessarily mean imminent death, with a median survival after this count of just under 1 year. These results will enable clinicians to provide appropriate counselling for patients at this late stage and to plan terminal care. AIDS 1997, 11:1123‐1127

The International Society of Analytical Cytology (ISAC) Biohazard Working Group presents guidelines for sorting of unfixed cells, including known biohazardous samples, using jet-in-air, deflected-droplet cell sorters. There is a risk that personnel operating these instruments could become exposed to droplets and aerosols containing biological agents present in the samples. The following guidelines can aid in the prevention of exposures of laboratory personnel to pathogens contained in the sort samples. The document provides biosafety recommendations for sample handling, operator training and protection, laboratory facility design, and instrument setup and maintenance. In addition, it describes in detail methods for assessment of instrument aerosol containment. Recommendations provided here may also help laboratories to obtain institutional and/or regulatory agency approval for sorting of unfixed and known biohazardous samples.

Activated interleukin-2 (IL-2)–dependent T cells express high levels of Bcl-2 protein. On cytokine withdrawal, Bcl-2 expression decreases and the cells die rapidly by apoptosis. We have previously shown that the survival of IL-2–deprived T cells can be promoted by factor(s) secreted by fibroblasts. Here we report that reduced glutathione (GSH), but not its oxidized counterpart GSSG, also enhances the in vitro survival of these cells. Exogenous GSH mediates its effect intracellularly, as (1) endogenous glutathione concentrations are increased up to fivefold in the presence of GSH, and (2) acivicin, an inhibitor of transmembrane GSH transport, abrogates GSH-dependent survival. The GSH-rescued T cells do not proliferate and express only low levels of Bcl-2, resembling WI38 fibroblast-rescued T cells. We, therefore, investigated a role for GSH in fibroblast-promoted T-cell survival. We show that WI38-promoted survival results in elevated GSH levels in surviving T cells and is abrogated by buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. Furthermore, both WI38-promoted T-cell survival and GSH upregulation are associated with large molecular weight molecules (

In order to examine the relevance of early immune activation to the long-term course of HIV infection, we evaluated the ability of the serum beta 2-microglobulin level measured in 63 haemophiliacs on average 4.9 months from HIV seroconversion to predict the rate of development of server immunodeficiency (CD4) lymphocyte count 50/mm3) or AIDS over the following 10 years. Patients with higher beta 2-microglobulin values tended to develop severe immunodeficiency/AIDS more rapidly than those with lower levels (relative risk 1.68 per 1 mg/L increase; 95% CI 1.26-2.26; p = 0.0004). Older patients also progressed more rapidly, and these two factors acted independently (relative risk 1.65 per 1 mg/L increase; 95% CI 1.21-2.72; p = 0.002 for beta 2-microglobulin and 1.22 per 10 years; 95% CI 1.01-1.48; p = 0.04 for age). These results provide further evidence that the long-term course of HIV infection can, to some extent, be predicted soon after infection. Older patients with high beta 2-microglobulin levels warrant close monitoring and consideration for early antiretroviral therapy.

During the immunodiagnosis of 517 cases of acute myelogenous leukemia (AML) entered into the Medical Research Council (MRC) AML10 trials, we have observed the CD34 precursor cell antigen more frequently in AML of M2 morphology, especially in the 84% of cases with the t(8;21) chromosomal translocation, than in any other French-American-British classification group. CD34 expression was then quantified (using QIFI and Quantum Simply Cellular beads [Flow Cytometry Standards, Research Triangle Park, NC] and CD34(+) standard cells). When CD34 antibody-binding capacity (ABC) of normal bone marrow (BM) precursors and leukemic blasts was compared, it was shown that AML M2 cases with t(8;21) not only had the highest percentages of CD34(+) blasts, but in >80% of CD34(+) cases the individual blasts expressed higher than normal levels of CD34 antigen (>60 x 10(3) ABC per cell). In addition, in 73% of this group CD34 antigen was overexpressed in an asynchronous combination with cytoplasmic myeloperoxidase (MPO). Other signs of asynchrony included high CD34 expression with CD15 and/or CD56, as well as aberrant combinations of CD13 with terminal deoxynucleotidyl transferase (TdT) and CD19. These findings demonstrate that asynchrony is identifiable in virtually every case of AML with t(8;21), although it does not always involve the same antigens, M2 cases with t(8;21), mostly CD34(+), had a 100% remission rate and 71% 5-year survival rate; other patients with CD34(+) or CD34(-) AML showed 69% and 84% remission rates and 31% and 36% 5-year survival rates, respectively. Consequently, individual markers such as CD34 should be interpreted in relation to other features such as chromosomal changes. These simple methods, which are well suited to quantify the expression of ligands, are a useful contribution to diagnosis: 60% to 65% of M2 cases with t(8;21) are rapidly identified by CD34 overexpression alone. This aberration, together with the other signs of asynchrony seen at presentation, can be used to search for residual leukemia after therapy. (C) 1996 by The American Society of Hematology.

Although there are wide differences in prognosis between patients with AIDS they are often thought of as a single homogeneous group. We think a simple staging system that accounts for important prognostic factors including type and number of AIDS diseases and the CD4 lymphocyte count is required. We followed 363 AIDS patients at the Royal Free Hospital and reported the occurrence of 680 AIDS-defining diseases (ADDs). We measured CD4 counts at approximately monthly intervals. Severity of AIDS diseases was defined a priori on the basis of survival in the AIDS in Europe study of 6578 AIDS patients: mild-oesophageal candidiasis, Kaposis sarcoma (cutaneous), Pneumocystis carinii pneumonia, extrapulmonary tuberculosis; severe-all other ADDs except lymphoma; very severe-lymphoma. The risk of death increased by 15% (p = 0.08) for each mild condition experienced, by 89% (p < 0.0001) for each new severe condition and by 535% (p < 0.0001) when a lymphoma developed. Estimates from the Cox model were used to derive a score reflecting the risk of death. Patient experience was divided into three categories. Patients in AIDS Grade I had an average death rate of one per 10.1 years, compared with one per 2.8 years in AIDS Grade II and one per 1.1 years in AIDS Grade III. Similar rates were seen in an independent validation study on 1230 AIDS patients at different hospital. Our grading system should be useful for patient management, clinical trial design, surveillance, and resource management.

We have compared CD10 antigen expression in normal fetal bone marrow with that of B-lineage acute lymphoblastic leukemia (ALL). Both quantitative indirect immunofluoresence (QIFI) and direct immunofluorescence (IF) tests with Quantum beads were used to convert median fluorescence intensity (MFI) values into numbers of antigen molecules expressed per cell (AgE). Lymphoid precursors in the fetal marrow and liver expressed 3–12.5 × 103 CD10 molecules/cell with an upper limit of 5 × 104/cell (MaxAgE). The median CD10 AgE in the different cases of acute B-lineage ALL were variable and ranged from undetectable to very high values (> 1.8 × 105). In 24 of the 72 cases (33%) tested with QIFI the median CD10 AgE was above the highest values seen in normal samples (> 5 × 104/cell). An additional 23.6% of cases had higher median values than the normal median CD10 AgE. Next, CD10 antigen was quantitated in 78 cases during the routine multiparameter analysis of B-lineage leukemia using CD10/class II/CD34 3-color IF test or CD10/TdT 2-color IF test. The aberrant overexpression was confirmed in 43.6% of ALL cases. The CD10bright display suggested ALL diagnosis even when few cells were available for study, e.g., in early relapse and in ALL masquerading as aplastic anemia. The levels of CD10 expression were maintained in relapse. In addition, different CD10 levels were associated with the various chromosomal alterations: high CD10 levels (> 3 × 104/cell) with hyperdiploidy, low CD10 levels (1.8–4 × 103/cell) with the t(1;19), and undetectable levels (< 1.2 × 103/cell) with the t(4;11) translocations. These findings show that while all these diseases are part of the precursor B-cell spectrum, the CD10 changes are linked to disease-associated alterations instead of truly reflecting the features of subtypes of normal B-cell precursors. The quantitative CD10 assessment should therefore be part of the routine flow cytometric assessment in ALL, and further careful studies are warranted during the regeneration phase following chemotherapy. High CD10 expression alone during strong bone marrow regeneration may not be leukemia specific and both the multiparameter analysis described above and parallel studies for gene rearrangements will be necessary to establish the relative values of these assays in detecting minimal disease amidst regenerating marrow. © 1994 Wiley-Liss, Inc.

During viral infections, CD8+,CD45RO+ T populations expand. These primed cells express abundant levels of cytoplasmic granules that contain perforin and TIA-1. Recent work has suggested that the majority of this CD8+ population downregulates Bcl-2 protein expression and is destined to undergo apoptosis. In this study we have investigated the elimination of these apoptotic CD8+ T cells by both human monocyte-derived and murine bone marrow macrophages. We have found that these phagocytes recognize and ingest both apoptotic CD8+ and CD4+ T cells using an alpha v beta 3 (vitronectin receptor)/CD36/thrombospondin recognition system, with the same receptors being used in the recognition of apoptotic neutrophils. These data provide new evidence for a mechanism that enables the clearance of greatly increased populations of CD8+ effector cells which are found during viral infections. This enables cellular homeostasis to occur in the host upon resolution of viral diseases in vivo.

OBJECTIVES To determine the prevalence of HIV infection among people attending a confidential, non-genitourinary medicine based testing clinic that provides HIV antibody test results on the day of consultation. METHODS Retrospective analysis of data collected on 2635 individuals attending the Same-Day HIV Testing Clinic at the Royal Free Hospital, London between March 1992 and February 1993. RESULTS A total of 1612 men and 1023 women were tested for HIV antibody. The primary risk for HIV infection was heterosexual (71.7%; 1889 out of 2635) and homosexual contact (24.5%; 646 out of 2635). Fifty-four individuals were given positive HIV test results (46 men, median age 34 years; eight women, median age 27 years). Overall HIV seroprevalence was 2.0% (95% confidence interval, 1.5-2.5). HIV seroprevalence was highest among homosexual men (6.5%; 41 out of 635) and injecting drug users (5.7%; four out of 70). The rates for heterosexual men and women were 0.2% (two out of 915) and 0.7% (seven out of 974), respectively. Of the 54 individuals who were HIV-antibody-positive, 44 were Centers for Disease Control and Prevention stage II/III, eight stage IV and one was tested at the time of seroconversion (stage I; data were not available for one patient). Of the total numbers attending this clinic 27% (702 out of 2635) had previously been tested and received a negative result. Of a total of 54 HIV-antibody-positive individuals, 40% (21) had previously received a negative test result. CONCLUSIONS The data suggest that seroprevalence amongst homosexual men attending a designated HIV testing clinic in London is lower than that reported by genitourinary clinic based testing sites. The large number of repeat testers who subsequently became infected with HIV suggests that there is a population requiring specific targetting for HIV risk reduction.

Objectives and design: The expression of the accessory molecule CD28 was compared in various populations of T and natural killer (NK) cells from HIV-1-negative and HIV-1-positive individuals and correlated with activation using mitogens in vitro.Methods: Multiparameter flow cytometric analysis using combinations of CD3 CD28 and other markers was performed together with absolute cell counting in peripheral blood. Blast transformation and proliferative responses were also quantitated using the Cytoron(absolute) after stimulation with phytohaemagglutinin (PHA) and anti-CD3. CD28- cells were also purified to confirm the observations.Results: In HIV-1-negative individuals >90% of CD3+ T cells were CD28+ and responded to stimulation, while CD3- CD16+ CD57+ NK-like cells were CD28- and failed to respond. In HIV-1-positive individuals the expression of CD28 was greatly reduced and the proportion of CD3+CD28- T cells expanded. CD8 lymphocytosis was caused entirely by the accumulation of CD28- T cells and many of these expressed activation markers human lymphocyte antigen-DR, CD38 and CD45RO on their membrane and molecules such as TIA-1 and perforin, associated with cytolytic function, in their cytoplasm. The strong positive correlation (r = 0.66) between the lack of CD28 expression and the poor proliferation from HIV-1-positive individuals was confirmed by demonstrating that only CD28+ cells transformed into lymphoblasts and proliferated. Although the CD28- including CD3+ T cells transiently expressed CD25 (interleukin-2Ralpha), they did not undergo blastogenesis or activation measured by bromodeoxyuridine uptake and died after 3-4 days in culture. These observations were confirmed in costimulation experiments with anti-CD2 and anti-CD28.Conclusion: In HIV-1 infection activated CD3+CD28- T cells accumulate but are unresponsive to mitogens and anti-CD28. These cells appear to represent terminally differentiated effector cells which fail to respond to further stimuli because of the absence of a CD28 second signal.