Immunological diagnostics

Philip Cunningham : NSW State Reference Laboratory for HIV, St Vincent’s Hospital Sydney NSW
Mark Kelly : AIDS Medical Unit, Brisbane QLD
John Ray : St Vincent’s Hospital, Sydney NSW
Andrew McLean-Tooke : Sir Charles Gairdner Hospital, WA
In addition to clinical and virological staging of HIV infection patients need to be staged immunologically. This is currently based on the use of flow cytometry assays using fluorescent cell surface markers (usually labelled antibodies) to identify lymphocyte subsets including T cells. T cells express CD3 (part of the T cell receptor) and can be divided according to expression of CD4 and CD8 molecules with further populations identified on the basis of expression of markers of activation or immunological memory. In normal healthy individuals the proportions and numbers of these populations remain relatively fixed, but these may be altered in disease states. In HIV the number and proportion of CD4 T cells relates to the stage of infection and risk of infection. Additional populations may also provide additional information about disease progression e.g. increased activated cells are a strong predictor of disease progression independent of CD4 count [1] and may predict CD4 T cell recovery on ART [2] [Add reference PMID: 20182359] although this information is not typically necessary to make treatment decisions for patients. In addition to T cells subsets, specific T cell immune responses to HIV can be measured. Whilst these tests are not used in the routine clinical setting, they have utility in research settings in understanding immunological response to HIV and particularly in assessment of HIV vaccine candidates. Tables 1 and 2 summarise the assays used to identify T-cell subsets (these are not human immunodeficiency virus (HIV) specific) and assays used to detect HIV-specific responses, respectively.
Table 1.  Assays used to identify T-cell subsets (not HIV specific)
Characteristic Markers Cell type Method





Helper T cells

Cytotoxic T cells

Naïve or Memory1 

Flow cytometry
Activation status CD38+ HLA-DR+ Activated Flow cytometry
Origin TRECs2 Recent thymic emigrants PCR

1. Memory T cells can be further classified into central and effector memory on the basis of expression and/or downregulation of CD45RA, CCR7, CD28 and CD27.

2. Interpretation of TREC is most accurate with simultaneous measurement of peripheral lymphocyte proliferation.

TREC: T cell receptor excision circle; PCR: polymerase chain reaction; HLA-DR: human leukocyte antigen- antigen D Related.

Table 2.    Assays used to detect cellular HIV-specific responses 
 Table 2.    Assays used to detect HIV-specific responses     
Assay CD4 CD8 Quantitative Functional Interpretation
Tetramer 1  √2  √  √   Number of T cells recognising specific HIV epitopes
ELISPOT 3  √  √  √  √ Number of T cells capable of producing cytokines following recognition of HIV antigens
Intracellular cytokine production 4  √  √  √  √ Number of T cells capable of producing cytokines following recognition of HIV antigens
51Cr release assay 5    √  √  √ Number of (CTLs able to kill cells expressing HIV antigens)
Degranulation assay6.   Number of T cells able to degranulate following recognition of HIV antigens
Lymphoproliferative assay 7  √      √ Number of CD4 cells able to proliferate following recognition of HIV antigens
1. Tetrameric HLA-peptide complexes detect T cells that express a T cell receptor complex which specifically recognises a given peptide presented by a given human leukocyte antigen molecule. This assay uses flow cytometric methods.

2. Less developed for CD4 cells as tetramers harder to create, specific CD4 T cell frequency lower and reduced MHC class II and T cell receptor affinity.

3. T cells are exposed to HIV antigens. Cells recognising these antigens produce cytokines such as interferon-gamma (IFNgamma). Secreted IFNgamma is captured by specific antibodies coating the well. Cells are then removed by washing and bound IFNgamma is detected by colorimetric techniques.

4. T cells are exposed to HIV antigens. Cells recognising these antigens produce cytokines such as IFNgamma. Cell membranes are then made permeable to permit fluorescent monoclonal antibodies to enter cells, which bind to the cytokine of interest. The cytokine-containing cells are then enumerated by flow cytometry.

5. Autologous B cells are transformed to express HIV antigens on their cell surface. These cells are pulsed with 51Chromium (51Cr) which remains intracellular. CD8 cells are then mixed with autologous B cell targets. HIV-specific cytotoxic T lymphocytes lyse target cells and thus release 51Cr which can then be measured.

6. T cells are exposed to HIV antigens. CD8 T cells recognising these antigens degranulate which can be detected by CD107a expression on cell surface by flow cytometry.

7. Lymphoproliferative assay – CD4 cells are mixed with HIV antigen and 3H-thymidine. CD4 cells which recognise HIV antigens proliferate and incorporate 3H-thymidine. The stimulation index reported compares the amount of 3H-thymidine incorporated into cells exposed to HIV antigens compared with those exposed to control antigens.

ELISPOT: enzyme-linked immunospot assay; CTL = cytotoxic T lymphocyte.

Therapeutic drug monitoring

Using therapeutic drug monitoring (TDM) to monitor patient plasma drug levels and to adjust doses for toxicity or treatment of drug-related toxicity is described elsewhere.

Sample analysis

A whole blood sample collected in a tube with ethylenediaminetetraacetic acid (EDTA) or citrate anticoagulant is recommended for most drug analyses. The use of gel separator tubes for drug analysis is not recommended as drugs have been shown to bind to the gel. Whole blood samples can be stored overnight at 4oC. TDM of the non-nucleoside reverse transcriptase inhibitor (NNRTI) and protease inhibitors (PIs) is performed using high-performance liquid chromatography (HPLC). Samples are treated with the organic solvent chlorobutane to extract the drug from the plasma. The extract is analysed on a high-performance liquid chromatography to separate the drug of interest from endogenous interference. The drug is detected by light absorption in the ultraviolet region and quantitated using known standards. Figure 1 shows chromatograms from patients with and without antiretroviral therapy. Peaks are compared with control panels of purified antiretroviral compounds to determine area under the curve and subsequent blood plasma drug concentrations.

Figure 1 Chromatograms of drug concentrations in blood plasma



Note: Chromatograms of blank human plasma (left panel) and a standard extracted from human plasma (right panel), showing specific peaks for known antiviral medications.
(1) = indinavir; (2) = ritonavir; (3) = lopinavir; (4) = internal standard; mAbs = milliabsorbant units.
Source: John Ray, St Vincent’s Hospital, Sydney. Used with permission.

Chromatographic assay availability

Currently in Australia, there is limited availability of pharmacokinetic chromatographic assays for NNRTIs and PIs; many specialist hospital-based referral centres have developed in-house assays. Generally, in-house assays vary in three areas:

  • Extraction procedure
  • Chromatographic process (column type and mobile phase)
  • Sensitivity (sensitivity depends on the purpose of the assay – TDM or single-dose pharmacokinetics).

Acceptable criteria for assay variability is less than 10% (co- efficient of variation) and ±15% at the limit of quantitation (lowest standard). Assay specificity (the ability to separate the drug of interest from other drugs, metabolites and endogenous compounds) is a significant problem. Chromatographic analysis solves this problem by selective extraction procedures, varying the chromatographic process (changing columns and mobile phase) and selective detection mechanisms, such as diode array ultraviolet detection. This technique involves comparing an ultraviolet fingerprint of the chromatographic peak with a library of pure standards (controls). This produces a match with a purity assessment. The gold standard chromatographic system for specificity is the liquid chromatographic mass spectrometer, used by only a few laboratories because of the purchase cost.

Quality assurance system

A mandatory requirement of laboratory accreditation is enrolment in an external quality assurance system. An international program has been established and measures the performance of a particular laboratory against a panel of 30 laboratories[3] . This program is new and only three cycles of assessment have been completed. Has this been updated? Preliminary data for the PIs have shown large variability in the performance between laboratories. Mean inaccuracies for all PIs were 25.3% for low concentrations and 17.1% for high concentrations. Enrolment in the international program should alert poorly performing laboratories to their weaknesses. Improvement in performance is expected as the program continues.

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Hunt PW, Cao HL, Muzoora C, Ssewanyana I, Bennett J, Emenyonu N, Kembabazi A, Neilands TB, Bangsberg DR, Deeks SG, Martin JN. Impact of CD8+ T-cell activation on CD4+ T-cell recovery and mortality in HIV-infected Ugandans initiating antiretroviral therapy. AIDS. 2011 Nov 13;25(17):2123-31
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