Feasibility of performing multiple point of care testing for HIV anti-retroviral treatment initiation and monitoring from multiple or single fingersticks.

BACKGROUND: Point of Care testing (POCT) provides on-site, rapid, accessible results. With current South African anti-retroviral treatment guidelines, up to 4 fingersticks /patient/clinic visit could be required if utilizing POC. We determined the feasibility and accuracy of a nurse performing multiple POCT on multiple fingersticks followed by simplification of the process by performance of multiple POC on a single fingerstick. METHOD AND FINDINGS: Random HIV positive adult patients presenting at a HIV treatment clinic in South Africa, for ART initiation/ monitoring, were approached to participate in the study between April-June 2012. Phase I: n=150 patients approached for multiple POCT on multiple fingersticks. Phase II: n=150 patients approached for multiple POCT on a single fingerstick. The following POC tests were performed by a dedicated nurse: PIMA (CD4), HemoCue (hemoglobin), Reflotron (alanine aminotransferase, creatinine). A venepuncture specimen was taken for predicate laboratory methodology. Normal laboratory ranges and Royal College of Pathologists Australasia (RCPA) allowable differences were used as guidelines for comparison. In 67% of participants, ≥3 tests were requested per visit. All POCT were accurate but ranged in variability. Phase I: Hemoglobin was accurate (3.2%CV) while CD4, alanine aminotransferase and creatinine showed increased variability (16.3%CV; 9.3%CV; 12.9%CV respectively). PIMA generated a misclassification of 12.4%. Phase II: Hemoglobin, alanine aminotransferase and creatinine showed good accuracy (3.2%CV, 8.7%CV, 6.4%CV respectively) with increased variability on CD4 (12.4%CV) but low clinical misclassification (4.1%). No trends were observed for the sequence in which POC was performed on a single fingerstick. Overall, PIMA CD4 generated the highest error rate (16-19%). CONCLUSIONS: Multiple POCT for ART initiation and/or monitoring can be performed practically by a dedicated nurse on multiple fingersticks. The process is as accurate as predicate methodology and can be simplified using a single fingerstick.

Quality control of the total lymphocyte count parameter obtained from routine haematology analyzers, and its relevance in HIV management.

Lylmphocyte subsets/CD4 T Helper cell enumeration in HIV care and treatment in resource constrained settings can be difficult to ascertain as a result of the lack of the necessary instrumentation, capacity and infrastructure. However. it is imperative to gain such information for patient monitoring in HIV. The Total Lymphocyte Count (TLC) is useful as a surrogate marker for CD4 count as recommended by the World Health Organisation (WHO) and to calculate CD4% for pacdiatric use. This study therefore sets out to determine and compare the accuracy of the total lymphocyte counts obtained from three haematology analysers designated A. B and C. that are in regular use for routine haemnatological parameters at the main referral hospital in Barbados. West Indies. The TLC of 263 HIV treatment naive individuals attending the HIV Reference Unit in Barbados were enumnerated on the three haematology analysers. The lymphosumn (Sum of lymphocyte subsets: T-helper cell. T-cytotoxic cells. B lymphocytes and Natural killer cells) should be equal to the TLC. and is derived by immunophenotypic analysis on a 4-colour flowcytometer. Machine C had the highest positive correlation between the TLC and the lymphosumn with and R' of 0.9031 compared to machine A with an R values of 0.7119 and Machine B with R(2) values of 0.637. These results show that there can be dramatic inaccuracies when using routine haematology analysers for both routine use. as a surrogate marker of CD4 or for derivation of CD4% in HIV management. It further demonstrates that all haematology analyzers require some form of Quality control. The possible lack of accuracy of the TLC by haematology analysers should be taken into consideration when following the recommendations of the WHO in resource poor settings or using it as a denominator for calculating CD4%.

Assessment of selected adhesion molecules and lymphocyte subpopulations in children with IgA nephropathy.

PURPOSE: The aim of the study was to assess the expression of selected adhesion molecules on mononuclear cells of peripheral blood and lymphocyte subpopulations in children with IgA nephropathy (IgAN). MATERIAL AND METHODS: 14 children with IgAN and 20 healthy controls were included in the study. Flow cytometry was used to determine the expression of such adhesion molecules as L selectin (CD62L), VLA-4 integrin (CD49d), intracellular molecule ICAM-1 (CD54) and cytotoxic lymphocyte molecule CTLA-4 (CD152), as well as the lymphocyte antigens: CD3, CD4, CD8, CD19, CD1656 (NK), CD4 and CD8 RO+ and RA+. RESULTS: The findings revealed that the expression of the adhesion molecules VLA-4 and CTLA-4 did not differ from that of the healthy controls (p > 0.05). However, the expression of CD62L (L-selectin) was increased (p < 0.05). The expression of ICAM-1 was reduced, but not significantly, compared to the control group (p > 0.05). We found a decrease in the expression of NK cells (CD1656) and CD4/CD8 ratio, and an increase in CD8 cells (p < 0.05). In the group of 9/14 children, with proteinuria over 1.0 g/24 hours, a decreased expression of CD4 was additionally found (p < 0.05). CONCLUSIONS: The children with IgAN show: 1. Changes in peripheral lymphocyte subpopulations involving an increase in CD8 cells and a decrease in CD1656(NK) cells, a reduction in the CD4/CD8 ratio, and additionally in cases with proteinuria a reduction in CD4 cell count, 2. Increased expression of L-selectin (CD62L) on peripheral blood mononuclear cells.

An assessment of concentrations of soluble CD4 and CD8 receptors in serum before and after surgical treatment in patients with chronic maxillary sinusitis.

BACKGROUND: The aim of this study was to assess the concentrations of soluble CD4 (sCD4) and sCD8 receptors in serum of patients before and after surgical treatment of chronic maxillary sinusitis. METHODS: We examined 57 patients, aged 20-63 years (mean age, 41 +/- 0.5 years), and divided them into four groups: group I, 14 patients with chronic maxillary sinusitis without allergy; group II, 15 patients with chronic maxillary sinusitis with allergy; group III, 16 patients with cyst of maxillary sinuses without allergy (control); and group IV, 12 patients with cyst of maxillary sinuses with allergy (control). The assay of sCD4 and sCD8 receptor concentrations was performed by means of enzyme-linked immunosorbent assay method. The concentrations of sCD4 and sCD8 receptors before and after 30 days of surgical treatment of maxillary sinuses were examined. RESULTS: In our studies the increase of concentration of sCD4 in groups I and II in comparison with the concentration in control groups were statistically significant. The differences between mean concentrations of sCD8 in groups I and II and in the control groups were not statistically significant. After surgical treatment of chronic maxillary sinusitis, a significant decrease in values of sCD4 and sCD8 in comparison with the results before surgical treatment suggest that the measurement of cell suppression product concentration can be used to assess the extirpation of the inflammatory process and the effectiveness of the operation method. CONCLUSION: Changes in concentration of sCD4 and sCD8 manifest activation or suppression of cells with particular receptor expression.

T-cell subset counting and the fight against AIDS: reflections over a 20-year struggle.

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.

Evaluation of stabilized blood cell products as candidate preparations for quality assessment programs for CD4 T-cell counting.

BACKGROUND: Exceptionally robust cell preparations are needed for quality assessment programs (QAPs) such as the International Program for Quality Assessment and Standardization for Immunological Measures (QASI) relevant to HIV/AIDS. A suitable product must withstand environmental stress related to transportation for a minimum of 6 days. The two objectives of this study are (1) to evaluate the performance of various commercial preparations with multicenter participation and (2) to evaluate the robustness of stabilized blood cell products. METHODS: Phase 1: The performance of stabilized blood cell products was evaluated in a multicenter QAP utilizing various staining procedures and flow cytometers. Absolute cell enumeration was achieved using single-platform T-cell subset methodology. Phase 2: The robustness of stabilized blood cell products was evaluated by monitoring T-cell subset values from samples stored at 4 degrees C, 22 degrees C, and 37 degrees C for up to 10 days. RESULTS: The largest interlaboratory variation in both absolute and relative T-cell values was 16% in samples with CD4 levels > or =400 cells per microliter and 21% in samples with CD4 levels <400 cells per microliter. Six preparations retained their phenotypic expression for 7 days at 4 degrees C and 22 degrees C. However, only two preparations remained stable for 4 days at 37 degrees C. CONCLUSION: Some stabilized cell preparations are more robust and therefore more suitable for quality assessment purposes.

Quality control of CD4+ T-lymphocyte enumeration: results from the last 9 years of the United Kingdom National External Quality Assessment Scheme for Immune Monitoring (1993-2001).

The human immunodeficiency virus (HIV) global epidemic has necessitated the routine enumeration of T-lymphocyte subsets, which has created a need for external quality assurance (EQA). The United Kingdom National External Quality Assessment Scheme (UK NEQAS) for Immune Monitoring provides EQA for 296 laboratories in 40 countries. In 1993, UK NEQAS developed and incorporated into its program stabilized whole blood that enables the accurate monitoring of laboratory performance. Overall, the mean interlaboratory coefficient of variation (CV) for percentage CD4(+) T-lymphocyte subset enumeration has fallen from 15% to less than 5%, as a direct result of the increased use of CD45/ side scatter (SSC) gating. Laboratories using alternative gating strategies (i.e., CD45/CD14 or forward scatter [FSC]/SSC) were about 7.4 times more likely to fail an EQA exercise. Furthermore, the adoption of single-platform technology resulted in a reduction of the overall mean interlaboratory CV for absolute CD4(+) T lymphocytes from 56% (prior to the widespread use of single-platform technology) to 9.7%. Individual laboratory deficiencies were also identified using a performance monitoring system and, through re-education by collaboration with the coordinating center, satisfactorily resolved. In conclusion, during the last 9 years, the UK NEQAS for Immune Monitoring program has highlighted the significant technological advances made by laboratories worldwide that undertake lymphocyte subset enumeration.

BD Biosciences contributions in CD4 counting and immune status for HIV/AIDS.

BD Biosciences is a leader in the use of flow cytometry for determining immune system status and for counting CD4 cells in patients with human immunodeficiency virus (HIV) infection. The company has gained this position through many years of basic research and product development in immunology and cell biology, dye chemistry, immunoassays, instrumentation, and software. Some of the highlights of these developments and their historical perspective are described in this review.

Assessment of hematological and immunological function during long-term follow-up after peripheral blood stem cell transplantation.

BACKGROUND AND OBJECTIVE: Long-term hemopoietic and immunological profile after autologous peripheral blood progenitor cells transplantation (PBPCT), in patients affected by hematological malignancies is largely unknown. The aim of this work was to detect the impact of high dose chemotherapy and PBPCT on hemopoietic and immunological function compared to conventional chemotherapy. DESIGN AND METHODS: Patients had to fulfill the following criteria: continuous complete remission after PBPCT, follow-up longer than 12 months, no chemo or radiotherapy or biological response modifiers after PBPCT. Twenty-five patients were considered eligible for this analysis. Stable and complete hemopoietic reconstitution (Hb > 12 g/dL, WB > 4.0 x 10(9)/L, ANC > 1.5 x 10(9)/L and Pits count > 150 x 10(9)/L), morphological examination of peripheral blood and bone marrow, cytogenetic analysis and immunological profile were evaluated at 12 months and yearly thereafter. RESULTS: Immunological reconstitution showed a persistent reduction of CD4/CD8 ratio up to five years after PBPCT. This reduction was related to a persistent increase of CD8+ lymphocytes and a constant reduction of CD4+ lymphocytes. INTERPRETATION AND CONCLUSIONS: Defects observed in PBPCT patients are induced by the procedure itself, by the conditioning regimen or both. The different behavior in the immune reconstitution of CD8+ subset after PBPCT may be favored by an extrathymic origin of these cells while CD4+ subset recovery which is thymus-dependent is impaired after PBPCT in adult population. Long-term hemopoietic reconstitution after PBPCT is rapidly obtained and is stable over the years, long-term immunological function seems to be abnormal in these patients and these abnormalities are long-lasting.