CD4 Cell Count Threshold for Cryptococcal Antigen Screening of HIV-Infected Individuals: A Systematic Review and Meta-analysis.

Background: Current guidelines recommend screening all people living with human immunodeficiency virus (PLHIV) who have a CD4 count ≤100 cells/µL for cryptococcal antigen (CrAg) to identify those patients who could benefit from preemptive fluconazole treatment prior to the onset of meningitis. We conducted a systematic review to assess the prevalence of CrAg positivity at different CD4 cell counts. Methods: We searched 4 databases and abstracts from 3 conferences up to 1 September 2017 for studies reporting prevalence of CrAg positivity according to CD4 cell count strata. Prevalence estimates were pooled using random effects models. Results: Sixty studies met our inclusion criteria. The pooled prevalence of cryptococcal antigenemia was 6.5% (95% confidence interval [CI], 5.7%-7.3%; 54 studies) among patients with CD4 count ≤100 cells/µL and 2.0% (95% CI, 1.2%-2.7%; 21 studies) among patients with CD4 count 101-200 cells/µL. Twenty-one studies provided sufficient information to compare CrAg prevalence per strata; overall, 18.6% (95% CI, 15.4%-22.2%) of the CrAg-positive cases identified at ≤200 cells/µL (n = 11823) were identified among individuals with a CD4 count 101-200 cells/µL. CrAg prevalence was higher among inpatients (9.8% [95% CI, 4.0%-15.5%]) compared with outpatients (6.3% [95% CI, 5.3%-7.4%]). Conclusions: The findings of this review support current recommendations to screen all PLHIV who have a CD4 count ≤100 cells/µL for CrAg and suggest that screening may be considered at CD4 cell count ≤200 cells/µL.

Haematological and immunological reference intervals for adult population in the state of Amhara, Ethiopia.

OBJECTIVE: Reference intervals (RIs) currently being used in Ethiopia are derived from western populations. Thus, this study aimed to establish locally derived haematological and immunological RIs. METHOD: The study was conducted in Amhara State, Ethiopia with a total of 967 (55.2% males) participants. 56.9% of males and 43.1% of females were eligible for haematological and immunological RI determination. A non-parametric test was used for the determination of upper (97.5th percentile) and lower (2.5th percentile) reference interval limits with 95% CI. The Harris and Boyd Rule was used to determine the need of partitioning of reference intervals based on gender. RESULT: The established 95% reference intervals (2.5th-97.5th percentile) were: for WBC: 3-11.2 × 109 /l; for platelet: 90-399 × 109 /l; for RBC: 4-6 × 1012 /l for males and 3.5-5.6 × 1012 /l for females; for haemoglobin: (Hgb) 12-18.9 g/dl for males and 10.7-17.5 g/dl for females; for PCV: 35.7-55.3% for males and 32.2-50.1% for females; for CD4: 400-1430 × 109 /l for males and 466-1523 × 109 /l for females; for CD4 percentage: 18-49.1% for males and 21.3-52.9% for females; for MCV: 81-100 fl; for MCH: 25.3-34.6 pg; MCHC: 28.8-36.9%; for RDW: 11.6-15.4% and for MPV: 8-12.3 fl. Males had significantly higher RBC, Hgb and PCV than females. CD4 counts and CD4 percentage were significantly higher in females. CONCLUSION: The reference intervals established in this study differ from others and thus should be used for the interpretation of laboratory results in diagnosis and safety monitoring in clinical trials in Amhara.

Cox regression with dependent error in covariates.

Many survival studies have error-contaminated covariates due to the lack of a gold standard of measurement. Furthermore, the error distribution can depend on the true covariates but the structure may be difficult to characterize; heteroscedasticity is a common manifestation. We suggest a novel dependent measurement error model with minimal assumptions on the dependence structure, and propose a new functional modeling method for Cox regression when an instrumental variable is available. This proposal accommodates much more general error contamination than existing approaches including nonparametric correction methods of Huang and Wang (2000, Journal of the American Statistical Association 95, 1209-1219; 2006, Statistica Sinica 16, 861-881). The estimated regression coefficients are consistent and asymptotically normal, and a consistent variance estimate is provided for inference. Simulations demonstrate that the procedure performs well even under substantial error contamination. Illustration with a clinical study is provided.

Quantification of cells with specific phenotypes I: determination of CD4+ cell count per microliter in reconstituted lyophilized human PBMC prelabeled with anti-CD4 FITC antibody.

A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties. Twelve different flow cytometer platforms were evaluated using a standard protocol that included calibration beads used to obtain quantitative measurements of CD4+ T cell counts. There was good overall cross-platform and counting method agreement with a grand mean of the laboratory calculated means of (301.7 ± 4.9) µL(-1) CD4+ cells. Excluding outliers, greater than 90% of participant data agreed within ±15%. A major contribution to variation of sLL CD4+ cell counts was tube to tube variation of the calibration beads, amounting to an uncertainty of 3.6%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalyzed. Remaining variation was attributed to instrument specific differences. CD4+ cell counts obtained in CCQM-P102 are in excellent agreement and show the robustness of both the measurements and the data analysis and hence the suitability of sLL as a reference material for interlaboratory comparisons and external quality assessment.

Quantification of cells with specific phenotypes II: determination of CD4 expression level on reconstituted lyophilized human PBMC labelled with anti-CD4 FITC antibody.

This report focuses on the characterization of CD4 expression level in terms of equivalent number of reference fluorophores (ERF). Twelve different flow cytometer platforms across sixteen laboratories were utilized in this study. As a first step the participants were asked to calibrate the fluorescein isothiocyanate (FITC) channel of each flow cytometer using commercially available calibration standard consisting of five populations of microspheres. Each population had an assigned value of equivalent fluorescein fluorophores (EFF denotes a special case of the generic term ERF with FITC as the reference fluorophore). The EFF values were assigned at the National Institute of Standards and Technology (NIST). A surface-labelled lyophilized cell preparation was provided by the National Institute of Biological Standards and Control (NIBSC), using human peripheral blood mononuclear cells (PBMC) pre-labeled with a FITC conjugated anti-CD4 monoclonal antibody. Three PBMC sample vials, provided to each participant, were used for the CD4 expression analysis. The PBMC are purported to have a fixed number of surface CD4 receptors. On the basis of the microsphere calibration, the EFF value of the PBMC samples was measured to characterize the population average CD4 expression level of the PBMC preparations. Both the results of data analysis performed by each participant and the results of centralized analysis of all participants' raw data are reported. Centralized analysis gave a mean EFF value of 22,300 and an uncertainty of 750, corresponding to 3.3% (level of confidence 68%) of the mean EFF value. The next step will entail the measurement of the ERF values of the lyophilized PBMC stained with labels for other fluorescence channels. The ultimate goal is to show that lyophilized PBMC is a suitable biological reference cell material for multicolor flow cytometry and that it can be used to present multicolor flow cytometry measurements in terms of ABC (antibodies bound per cell) units.

Opportunities to improve storage and transportation of blood specimens for CD4 testing in a rural district in Zimbabwe using BD vacutainer CD4 stabilization tubes: a stability and diagnostic accuracy study.

BACKGROUND: CD4+ T-cell testing of blood specimens collected in standard EDTA Vacutainer tubes and transported at ambient temperature, must be completed within 48 hours with the BD FACSCount™ flow cytometer, restricting specimen collection in remote clinics with no on-site testing and limited specimen transport services. We conducted a study in Buhera District, Zimbabwe, to assess the stability and accuracy of CD4+ T-cell results of samples collected in Stabilization Tubes (ST) and stored at ambient temperature for varying time periods. METHODS: Paired EDTA and ST samples were collected from 51 HIV-positive patients aged 18 years and older. CD4+ T-cell testing was done on arrival in the laboratory (Day 0). ST samples were retested on Days 3, 5, and 7. Nineteen ST samples were stored for an additional week and retested on Day 14. RESULTS: There was a strong correlation between absolute CD4+ T-cell counts measured in the EDTA Day 0 reference sample and Day 7 ST sample (Spearman's rho: 0.9778; mean difference: -4.9 cells/µL and limits of agreement (LOA): 98.5 and 88.7 cells/µL); and the reference sample and Day 14 ST sample (Spearman's rho: 0.9632; mean difference 5.1 cells/µL and LOA: -99.6 and 109.8 cells/µL. Using a 350 cells/µL threshold, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were all 100% on Day 7, and 83.3%, 100%, 100% and 92.9% on Day 14. Using a 500 cells/µL threshold, the sensitivity, specificity, PPV and NVP were 100%, 88.5%, 88.5% and 100% on Day 7 and 88.9%, 80.0%, 80.0% and 88.9% on Day 14. CONCLUSIONS: CD4 ST can be used and stored up to 7 days as a reliable alternative to standard EDTA tubes in settings where CD4+ T-cell testing within 48 hours is not feasible. Despite the small sample size, results suggest that ST may be stored up to 14 days at room temperature for CD4 testing, without compromising accuracy. However, further studies with larger sample sizes are needed to confirm this preliminary finding.

Operational challenges in delivering CD4 diagnostics in sub-Saharan Africa.

Access to reliable and low cost CD4 T-cell enumeration to stage illness and monitor anti-retroviral therapy remains elusive in resource-limited settings. We report challenges in delivering CD4 testing using the microcapillary Fluorescence-Activated Cell Sorter (FACS) methodology (Guava EasyCD4 instrument Guava Technologies, Hayward) in Burkina Faso and Zimbabwe. Resources, instruments, reagents, and training were provided to local laboratories within the existing infrastructure and data on CD4 were collected from routine laboratory testing. Challenges encountered included frequent instrument breakdown; poor manufacturer maintenance; difficulties in managing reagent stocks; high technician turnover; reliance on antiquated data management systems; redundant service provision; and lack of repeat testing in male HIV+ patients and in patients with higher CD4 counts after initial staging. While adopting newer, less expensive technologies such as fluorescent platforms and point of care tests can facilitate access to lower cost CD4 testing, our experience suggests that supply chain, corporate commitment to implementation, and community factors also require consideration.

Low-cost assays for monitoring HIV infected individuals in resource-limited settings.

Use of a combination of CD4 counts and HIV viral load testing in the management of antiretroviral therapy (ART) provides higher prognostic estimation of the risk of disease progression than does the use of either test alone. The standard methods to monitor HIV infection are flow cytometry based for CD4+ T cell count and molecular assays to quantify plasma viral load of HIV. Commercial assays have been routinely used in developed countries to monitor ART. However, these assays require expensive equipment and reagents, well trained operators, and established laboratory infrastructure. These requirements restrict their use in resource-limited settings where people are most afflicted with the HIV-1 epidemic. With the advent of low-cost and/or low-tech alternatives, the possibility of implementing CD4 count and viral load testing in the management of ART in resource-limited settings is increasing. However, an appropriate validation should have been done before putting them to use for patient testing.

Clinical laboratory reference values in adults in Kisumu County, Western Kenya; hematology, chemistry and CD4.

BACKGROUND: Clinical laboratory reference intervals (RIs) are essential for diagnosing and managing patients in routine clinical care as well as establishing eligibility criteria and defining adverse events in clinical trials, but may vary by age, gender, genetics, nutrition and geographic location. It is, therefore, critical to establish region-specific reference values in order to inform clinical decision-making. METHODS: We analyzed data from a prospective observational HIV incidence cohort study in Kombewa, Kenya. Study participants were healthy males and females, aged 18-35 years, without HIV. Median and 95% reference values (2.5th percentile to 97.5th percentile) were calculated for laboratory parameters including hematology, chemistry studies, and CD4 T cell count. Standard Deviation Ratios (SDR) and Bias Ratios (BR) are presented as measures of effect magnitude. Findings were compared with those from the United States and other Kenyan studies. RESULTS: A total of 299 participants were analyzed with a median age of 24 years (interquartile range: 21-28). Ratio of males to females was 0.9:1. Hemoglobin range (2.5th-97.5th percentiles) was 12.0-17.9 g/dL and 9.5-15.3 g/dL in men and women respectively. In the cohort, MCV range was 59-95fL, WBC 3.7-9.2×103/µL, and platelet 154-401×103/µL. Chemistry values were higher in males; the creatinine RI was 59-103 µmol/L in males vs. 46-76 µmol/L in females (BRUL>.3); and the alanine transferase range was 8.8-45.3 U/L in males vs. 7.5-36.8 U/L in females (SDR>.3). The overall CD4 T cell count RI was 491-1381 cells/µL. Some parameters including hemoglobin, neutrophil, creatinine and ALT varied with that from prior studies in Kenya and the US. CONCLUSION: This study not only provides clinical reference intervals for a population in Kisumu County but also highlights the variations in comparable settings, accentuating the requirement for region-specific reference values to improve patient care, scientific validity, and quality of clinical trials in Africa.

Nevirapine/zidovudine/lamivudine has superior immunological and virological responses not reflected in clinical outcomes in a 48-week randomized comparison with abacavir/zidovudine/lamivudine in HIV-infected Ugandan adults with low CD4 cell counts.

BACKGROUND: Triple nucleoside reverse transcriptase inhibitor regimens have advantages as first-line antiretroviral therapy (ART), avoiding hepatotoxicity and interactions with anti-tuberculosis therapy, and sparing two drug classes for second-line ART. Concerns exist about virological potency; efficacy has not been assessed in Africa. METHODS: A safety trial comparing nevirapine with abacavir was conducted in two Ugandan Development of Antiretroviral Therapy in Africa (DART) centres: 600 symptomatic antiretroviral-naïve HIV-infected adults with CD4 counts <200 cells/microL were randomized to zidovudine/lamivudine plus abacavir or nevirapine (placebo-controlled to 24-week primary toxicity endpoint, and then open-label). Documented World Health Organization (WHO) stage 4 events were independently reviewed and plasma HIV-1 RNA assayed retrospectively. Exploratory efficacy analyses are intention-to-treat. RESULTS: The median pre-ART CD4 count was 99 cells/microL, and the median pre-ART viral load was 284 600 HIV-1 RNA copies/mL. A total of 563 participants (94%) completed 48 weeks of follow-up, 25 (4%) died and 12 (2%) were lost to follow-up. The randomized drug was substituted in 21 participants (7%) receiving abacavir vs. 34 (11%) receiving nevirapine (P=0.09). At 48 weeks, 62% of participants receiving abacavir vs. 77% of those receiving nevirapine had viral loads <50 copies/mL (P<0.001), and mean CD4 count increases from baseline were +147 vs. +173 cells/microL, respectively (P=0.006). Nine participants (3%) receiving abacavir vs. 16 (5%) receiving nevirapine died [hazard ratio (HR) 0.55; 95% confidence interval (CI) 0.24-1.25; P=0.15]; 20 receiving abacavir vs. 32 receiving nevirapine developed new or recurrent WHO 4 events or died (HR=0.60; 95% CI 0.34-1.05; P=0.07) and 48 receiving abacavir vs. 68 receiving nevirapine developed new or recurrent WHO 3 or 4 events or died (HR=0.67; 95% CI 0.46-0.96; P=0.03). Seventy-one participants (24%) receiving abacavir experienced 91 grade 4 adverse events compared with 130 events in 109 participants (36%) on nevirapine (P<0.001). CONCLUSIONS: The clear virological/immunological superiority of nevirapine over abacavir was not reflected in clinical outcomes over 48 weeks. The inability of CD4 cell count/viral load to predict initial clinical treatment efficacy is unexplained and requires further evaluation.

Establishment of reference range of CD4 T-lymphocyte in healthy Nepalese adults.

OBJECTIVE: CD4 T lymphocytes are the most widely used cellular markers to assess the course of HIV infection, clinical staging and, monitoring the effect of antiretroviral therapy. The regional reference range for Eastern, Central and Western development region of Nepal had already been established whereas the same was still lacking in Mid-western and Far-western development region. The objective of this study was to establish reference range of CD4 T lymphocyte in the remaining two development regions and finally the national reference range using data from previous study. RESULTS: The average values (mean ± SD) of CD4 and CD3 T cell in present study was (819 ± 294) cells/µl and (1546 ± 532) cells/µl, respectively. The absolute CD4 T cell (914 ± 303) and CD3 T cell (1671 ± 560) count in female were significantly higher than those from male, CD4 (757 ± 270) and CD3 (1465 ± 499) (p value-0.000). National reference value of CD4 was determined to be (798 ± 335) cells/µl for healthy Nepalese adults.

Accuracy of WHO CD4 cell count criteria for virological failure of antiretroviral therapy.

OBJECTIVES: To examine the accuracy of the World Health Organization immunological criteria for virological failure of antiretroviral treatment. METHODS: Analysis of 10 treatment programmes in Africa and South America that monitor both CD4 cell counts and HIV-1 viral load. Adult patients with at least two CD4 counts and viral load measurements between month 6 and 18 after starting a non-nucleoside reverse transcriptase inhibitor-based regimen were included. WHO immunological criteria include CD4 counts persistently <100 cells/microl, a fall below the baseline CD4 count, or a fall of >50% from the peak value. Virological failure was defined as two measurements > or =10 0000 copies/ml (higher threshold) or > or =500 copies/ml (lower threshold). Measures of accuracy with exact binomial 95% confidence intervals (CI) were calculated. RESULTS: A total of 2009 patients were included. During 1856 person-years of follow up 63 patients met the immunological criteria and 35 patients (higher threshold) and 95 patients (lower threshold) met the virological criteria. Sensitivity [95% confidence interval (CI)] was 17.1% (6.6-33.6%) for the higher and 12.6% (6.7-21.0%) for the lower threshold. Corresponding results for specificity were 97.1% (96.3-97.8%) and 97.3% (96.5-98.0%), for positive predictive value 9.5% (3.6-19.6%) and 19.0% (10.2-30.9%) and for negative predictive value 98.5% (97.9-99.0%) and 95.7% (94.7-96.6%). CONCLUSIONS: The positive predictive value of the WHO immunological criteria for virological failure of antiretroviral treatment in resource-limited settings is poor, but the negative predictive value is high. Immunological criteria are more appropriate for ruling out than for ruling in virological failure in resource-limited settings.

External Quality Assessment on CD4+ T-Cell Count Using in-House Proficiency Testing Panels for CD4 Count Laboratories in Addis Ababa, Ethiopia.

BACKGROUND: CD4+ T-cell count External Quality Assessment program is important for the evaluation of performance of CD4 count laboratories. The aim of this study was to assess the quality of CD4count laboratory performance using in-house Proficiency testing panels that perform routine CD4 counts in Addis Ababa, Ethiopia, 2013/14. METHODS: Participating laboratories were 20, 23 and 25 in trials 1, 2 and 3, respectively. In-house prepared fresh whole blood samples both with "normal" and "low" CD4 values were sent to participating laboratories. Percentage and absolute counts of CD4+ T-lymphocytes were done using their routine procedures. Data were analyzed for each trial including trimmed mean, standard deviation (SD), percent coefficient of variation (%CV), residual, and standard deviation index (SDI) values for both absolute counts and percentages of CD4+ lymphocytes (%CD4). RESULTS: Most participating laboratories produced results that were within 2SD of the mean. Average inter-laboratory precision (trimmed %CV) was 10.87% and 5.14% for CD4 absolute counts and %CD4, respectively. For normal material, the trimmed mean %CV was 9.59% and3.23% for CD4 absolute counts and %CD4, respectively. For low material, the trimmed mean % CV was 12.15% and 7.05% for CD4 absolute counts and %CD4 respectively. BDFACSCount™ users showed the best accuracy and precision as evidenced by longitudinal analysis. CONCLUSION: This study was found to help facilities in early identifying their gaps with regard to their CD4 count performance and in avoiding the challenges encountered during participation in external EQA providers like the high cost, transportation problem, feedback delay and CD4laboratory coverage.

Field Performance of PIMA Point-of-Care Machine for CD4 Enumeration Under a Mobile HIV Counseling and Testing Program in Remote Fishing Communities of Lake Victoria, Uganda.

Uganda is among the most HIV/AIDS-afflicted countries, and many HIV-infected persons live in remote areas with poor access to health care. The success of HIV care programs relies in part on patient monitoring using CD4 T cell counts. We conducted an evaluation of the point-of-care PIMA test using BD FACSCount as a gold standard. One hundred fifty-one participants were enrolled, provided venous blood and samples tested at the point of care with the Alere PIMA™ CD4 Analyzer and the BD FACSCount in the UVRI-IAVI main laboratory. Correlation between the methods was assessed, as was the ability of the Pima Analyzer to predict values <200, <350, and ≥500 CD4 cells/mm3 when compared with BD FACSCount as the gold standard. A near-perfect positive Pearson correlation coefficient (r = 0.948; p < .0001) between the two methods was observed. The Alere PIMA Analyzer had a mean bias of -32.5 cells/mm3. The sensitivity and specificity, for PIMA to predict CD4 lymphocyte count less than 200 cells/mm3, were 71.4% and 100%, respectively; less than 350 cells/mm3 were 84.6% and 94.6%, respectively; and at CD4 count less than 500 cells/mm3 were 94.4% and 100%. The Alere Pima Analyzer provides reliable CD4 cell count measurement and is suitable for monitoring and screening eligible HIV patients in hard-to-reach settings.

Delayed diagnosis of HIV infection and late initiation of antiretroviral therapy in the Swiss HIV Cohort Study.

OBJECTIVES: To investigate delayed HIV diagnosis and late initiation of antiretroviral therapy (ART) in the Swiss HIV Cohort Study. METHODS: Two sub-populations were included: 1915 patients with HIV diagnosis from 1998 to 2007 and within 3 months of cohort registration (group A), and 1730 treatment-naïve patients with CD4>or=200 cells/microL before their second cohort visit (group B). In group A, predictors for low initial CD4 cell counts were examined with a median regression. In group B, we studied predictors for CD4<200 cells/microL without ART despite cohort follow-up. RESULTS: Median initial CD4 cell count in group A was 331 cells/microL; 31% and 10% were <200 and <50 cells/microL, respectively. Risk factors for low CD4 count were age and non-White race. Homosexual transmission, intravenous drug use and living alone were protective. In group B, 30% initiated ART with CD4>or=200 cells/microL; 18% and 2% dropped to CD4 <200 and <50 cells/microL without ART, respectively. Sub-Saharan origin was associated with lower probability of CD4 <200 cells/microL without ART during follow-up. Median CD4 count at ART initiation was 207 and 253 cells/microL in groups A and B, respectively. CONCLUSIONS: CD4<200 cells/microL and, particularly, CD4<50 cells/microL before starting ART are predominantly caused by late presentation. Earlier HIV diagnosis is paramount.

Corrected Lymphocyte Percentages Reduce the Differences in Absolute CD4+ T Lymphocyte Counts between Dual-Platform and Single-Platform Flow Cytometric Approaches.

OBJECTIVE: To determine whether a corrected lymphocyte percentage could reduce bias in the absolute cluster of differentiation (CD)4+ T lymphocyte counts obtained via dual-platform (DP) vs standard single-platform (SP) flow cytometry. METHODS: The correction factor (CF) for the lymphocyte percentages was calculated at 6 laboratories. The absolute CD4+ T lymphocyte counts in 300 blood specimens infected with human immunodeficiency virus (HIV) were determined using the DP and SP methods. RESULTS: Applying the CFs revealed that 4 sites showed a decrease in the mean bias of absolute CD4+ T lymphocyte counts determined via DP vs standard SP (-109 vs -84 cells/µL, -80 vs -58 cells/µL, -52 vs -45 cells/µL, and -32 vs 1 cells/µL). However, 2 participating laboratories revealed an increase in the difference of the mean bias (-42 vs -49 cells/µL and -20 vs -69 cells/µL). CONCLUSIONS: Use of the corrected lymphocyte percentage shows potential for decreasing the difference in CD4 counts between DP and the standard SP method.

All-printed cell counting chambers with on-chip sample preparation for point-of-care CD4 counting.

We demonstrate the fabrication of fully printed microfluidic CD4 counting chips with complete on-chip sample preparation and their applicability as a CD4 counting assay using samples from healthy donors and HIV-infected patients. CD4 counting in low-income and resource-limited point-of-care settings is only practical and affordable, if disposable tests can be fabricated at very low cost and all manual sample preparation is avoided, while operation as well as quantification is fully automated and independent of the skills of the operator. Here, we show the successful use of (inkjet) printing methods both to fabricate microfluidic cell counting chambers with controlled heights, and to deposit hydrogel layers with embedded fluorophore-labeled antibodies for on-chip sample preparation and reagent storage. The maturation process of gelatin after deposition prevents antibody wash-off during blood inflow very well, while temperature-controlled dissolution of the matrix ensures complete antibody release for immunostaining after the inflow has stopped. The prevention of antibody wash-off together with the subsequent complete antibody release guarantees a homogeneous fluorescence background, making rapid and accurate CD4 counting possible. We show the successful application of our fully printed CD4 counting chips on samples from healthy donors as well as from HIV-infected patients and find an excellent agreement between results from our method and from the gold standard, flow cytometry, in both cases.

External quality assessment for CD4 + T-lymphocyte count test: Performance of the Brazilian public health laboratories network.

The National Network for CD4+ T-lymphocyte counting of Brazil comprises 93 laboratories. This study reports the laboratory performances achieved in external quality assessment (EQA) rounds provides by Ministry of Health to evaluate the quality of the kits used and the performance of test by the technicians.Ten EQA rounds were analyzed according the EQA criteria aimed to evaluate individual laboratory performance on the basis of the accuracy of their results compared to the general mean obtained by all participating laboratories and the reproducibility of the results obtained between 2 samples from the same donor.The percentage of approved and failed laboratories in the EQAs tends to follow a uniform pattern. Since 2011, approval has remained above 80% and the failure rate has never exceeded 15%.EQA is very important to evaluate the performance of the laboratories, to identify monitor, and to resolve errors as quickly as possible.

Estimating Normal Values of Rare T-Lymphocyte Populations in Peripheral Blood of Healthy Cuban Adults.

INTRODUCTION Flow cytometry allows immunophenotypic characterization of important lymphocyte subpopulations for diagnosis of diseases such as cancer, autoimmune diseases, immunodeficiencies and some infections. Normal values of rare lymphoid cells in blood, quantified by cytometry, vary among different populations; so it is indispensable to obtain normal national values that can be used in clinical practice. OBJECTIVE Characterize distribution of rare T-lymphocyte populations in peripheral blood, specifically double-positive T, natural killer T and activated T lymphocytes, as well as their relationship to sex and age. METHODS A cross-sectional study was carried out in 129 adults (68 women, 61 men) aged >18 years, without chronic diseases or unhealthy habits, who signed informed consent. Peripheral blood was collected for immunophenotyping of lymphocyte subpopulations with monoclonal antibodies specific for CD4+CD8+ double-positive T cells, CD3+CD56+ natural killer T cells, and CD3+CD25+HLA-DR+ activated T cells. An eight-color flow cytometer (Beckman Coulter Gallios) was used. The analytic strategy was modified, associating variables of interest in a single graphic, using conventional monoclonal labeling antibodies. Medians and minimum and maximum percentiles (2.5 and 97.5, respectively) were used as descriptive statistics, stratified by sex, for cell counts and percentages. A linear regression model was applied to assess age effects and a two-tailed Mann-Whitney U test for independent samples was used to assess sex differences. The significance threshold was set as p ≤0.05. RESULTS Median percentages of total lymphocytes: natural killer T cells 6.3% (1.4%-23%) in men and 4.7% (0.8%-11.3%) in women (p = 0.003); activated T cells 1.0% (0.2%-2.2%) in men and 1.2% (0.4%-3.1%) in women, without statistical significance; and double positives 0.8% (0.1%-4.2%) in men and 0.9% (0.3-5.1) in women, also without statistical significance. Median cell counts (cells/mL) were: natural killer T cells, 126 (27-580) in men and 105 (20-279) in women (p = 0.023); activated T cells: 20 (4-46) in men and 25 (7-75) in women, (p = 0.013) and double-positive T cells: 17 (2-85) in men and 21 (7-154) in women, without statistical significance. Sex influenced natural killer T cells, but age did not. CONCLUSIONS Age does not affect counts and percentages of rare T lymphocyte subpopulations in the blood of healthy Cuban adults. Sex differences found for some phenotypes suggest the need for different reference values for women and men.