T lymphocytes among HIV-infected and -uninfected infants: CD4/CD8 ratio as a potential tool in diagnosis of infection in infants under the age of 2 years.

BACKGROUND: Serologic tests for HIV infection in infants less than 18 months do not differentiate exposure and infection since maternally acquired IgG antibodies may be detected in infants. Thus, the gold standard for diagnosis of HIV-1 infection in infants under the age of 2 years is DNA or reverse transcriptase polymerase chain reaction. There is an urgent need to evaluate alternative and cost effective laboratory methods for early diagnosis of infant HIV-1 infection as well as identifying infected infants who may benefit from cotrimoxazole prophylaxis and/or initiation of highly active antiretroviral therapy. METHODS: Whole blood was collected in EDTA from 137 infants aged 0 to 18 months. DNA polymerase chain reaction was used as the reference standard for diagnosis of HIV-1 infection. T-cell subset profiles were determined by flow cytometry. RESULTS: Seventy-six infants were DNA PCR positive while 61 were negative. The median CD4 counts of PCR negative infants were significantly higher than those of the PCR positive infants, p < 0.001. The median CD4/CD8 ratio and the %CD4 of the PCR positive infants were both significantly lower than those of the negative infants, p < 0.001. The CD4/CD8 ratio had a >98% sensitivity for diagnosis of HIV-1 infection and a specificity of >98%. CONCLUSION: The CD4/CD8 ratio appears useful in identifying HIV-infected infants. The development of lower cost and more robust flow cytometric methods that provide both CD4/CD8 ratio and %CD4 may be cost-effective for HIV-1 diagnosis and identification of infants for cotrimoxazole prophylaxis and/or highly active antiretroviral therapy.

Affordable CD4(+)-T-cell counting by flow cytometry: CD45 gating for volumetric analysis.

The flow cytometers that are currently supported by industry provide accurate CD4(+)-T-cell counts for monitoring human immunodeficiency virus disease but remain unaffordable for routine service work under resource-poor conditions. We therefore combined volumetric flow cytometry (measuring absolute lymphocyte counts in unit volumes of blood) and simpler protocols with generic monoclonal antibodies (MAbs) to increase cost efficiency. Volumetric absolute counts were generated using CD45/CD4 and CD45/CD8 MAb combinations in two parallel tubes. The percentage values for the various subsets were also determined within the leukocyte and lymphocyte populations utilizing a fully automated protocol. The levels of agreement between the newly developed method and the present industry standards, including both volumetric and bead-based systems using a full MAb panel for subset analysis, were tested by Bland-Altman analyses. The limits of agreement for CD4 counts generated by the volumetric methods using either CD45/CD4 (in a single tube) or the full Trio MAb panel (in three tubes) on the CytoronAbsolute flow cytometer were between -29 and +46 cells/mm(3) with very little bias for CD4 counts (in favor of the Trio method: +8 CD4(+) lymphocytes/mm(3); 0.38% of lymphocytes). The limits of agreement for absolute CD4 counts yielded by the volumetric CD45/CD4 method and the bead-based method were between -118 and +98 cells/mm(3), again with a negligible bias (-10 CD4(+) lymphocytes/mm(3)). In the volumetric method using CD45/CD8, the strongly CD8(+) cells were gated and the levels of agreement with the full Trio showed a minor bias (in favor of the Trio; +40 CD8(+) cells/mm(3); 5.2% of lymphocytes) without a significant influence on CD4/CD8 ratios. One trained flow cytometrist was able to process 300 to 400 stained tubes per day. This workload extrapolates to a throughput of >30,000 samples per year if both CD45/CD4 and CD45/CD8 stainings are performed for each patient or a throughput of >60,000 samples if only CD45/CD4 counts are tested in a single tube. Thus, on the basis of the high efficiency and excellent agreement with the present industry standards, volumetric flow cytometers with automated gating protocols and autobiosamplers, complemented by generic CD45, CD4, and CD8 MAbs used in two-color immunofluorescence, represent the most suitable arrangements for large regional laboratories in resource-poor settings.

Stabilised cellular immuno-fluorescence assay: CD45 expression as a calibration standard for human leukocytes.

We first confirmed the precision of the quantitative indirect immunofluorescence (QIFI) test by demonstrating that blood lymphocytes from different individuals expressed CD45 leucocyte antigens at a very similar level (mean: 201 x 10(3) antibody binding capacity (ABC)/lymphocyte) with only minimal variation (CV% 2.5%). These values were maintained for 4 days in blood samples when kept at 20 degrees C, and for up to 14 days in samples fixed with TransFix. Using long-term stabilisation, after an initial drop of 10-15% the CD45 ABC/lymphocyte values remained stable at an 85-90% level for >1 year. These biological standards were used to check other quantitative IF techniques. The quantum simply cellular (QSC) method showed variable results (85-240%), and the QuantiBRITE method gave values as low as 30-40% of the expected values, indicating that biological standards such as CD45 ABC/lymphocytes are absolutely essential to check the performance of methods that claim to quantify immunofluorescence (IF). Next, these standards were used to establish the stabilised cellular immuno-fluorescence assay (SCIFA) as follows. The ABC x 10(3)/cell values established by QIFI on leucocyte populations such as lympho-, mono- and granulocytes were used to create calibration curves for the CD45 antigen and its isoforms CD45RA, -R0 and -RB. The same cell populations were then stained with monoclonal antibodies (MAbs) directly conjugated to different fluorochromes in order to translate the mean fluorescence intensity (MFI) values seen with the directly labelled reagents to values of ABCx10(3)/cells. Using SCIFA with a triple-colour direct IF, the display of CD45 and its isoforms were quantitated on the 'virgin' or 'unprimed' (CD45RA+) and 'primed' (CD45R0+) subsets in both the CD4+ and CD8+ T cell lineages. We also observed that the CD4+ and CD8+ T cells in transit between the 'virgin' and 'primed' subsets frequently displayed different levels of the CD45RA and -R0 molecules, pointing to the physiological variability of the CD45RA-R0 switching process. In conclusion, internal biological standards, with known stable expression of ABC/cell, should be used to evaluate IF staining patterns in a quantitative manner during routine investigations.