Levels of cytokines and immune activation markers in plasma in human immunodeficiency virus infection: quality control procedures

Abstract

Procedures for quality control (QC) in a laboratory that concentrates on cytokine and soluble marker measurements in biological fluids are outlined. Intra-assay, interassay, and interlaboratory experiences are presented. Plasma and serum beta2-microglobulin (beta2M) and neopterin test data are presented in greatest detail, along with substantial tumor necrosis factor alpha (TNF-alpha), gamma interferon, soluble interleukin-2 receptor-alpha (sIL-2Ralpha), sTNF-RII, IL-4, and IL-6 data. Recommended QC procedures for cytokine and soluble-marker testing include replicate testing of two or more reference samples provided by the kit manufacturer, replicate testing of in-house frozen reference QC samples that represent normal and abnormal analyte contents, retesting 15 to 20% of randomly selected samples, and comparing normal reference ranges each year. Also, eight cytokines and soluble markers were evaluated in human immunodeficiency virus (HIV)-seronegative and HIV-seropositive individuals stratified on the basis of CD4 T-cell numbers. Levels of some but not all cytokines in serum increased in HIV infection. There was a tendency for cytokines to increase with more advanced disease, defined by reduced CD4 T-cell numbers. Cytokine changes did not relate closely to CD4 level, indicating that separate information was provided by the measurements of TNF-alpha, sTNF-RII, sIL-2Ralpha, beta2M, and neopterin. Serum IL-4 and TNF-alpha levels were not increased. The quality of laboratory data can impact on clinical relevance. Interlaboratory comparisons revealed substantial differences at some sites and documented the need for external proficiency-testing quality assurance programs.

FIG. 1

Interassay variations in concentrations of neopterin (NPT) (A) and sTNF-RII (B) in normal (filled circles) and abnormal high (open circles) in-house QC preparations. Each point represents the mean value of triplicate testing.

FIG. 2

Levels in plasma of elevated (A and C) and normal (B and D) external QA samples for β2M (A and B) and neopterin (C and D) assayed in laboratories 1 (•), 2 (○), 3 (▾), and 4 (▿), participating in an external QA program from 1989 to 1992. To avoid a change in the presentation of the other data, the outlier value of test 11 in panel A is reported without being plotted in the scale.

FIG. 3

Cytokine and soluble marker levels in serum (means ± standard error) of 15 HIV-seronegative (○) and 56 HIV-seropositive subjects stratified by CD4⁺-lymphocyte levels: >500 (A), 350 to 499 (B), 200 to 349 (C), and <200 (D) lymphocytes per μl.