THE USE OF ISOTYPIC CONTROL ANTIBODIES IN THE ANALYSIS OF CD3-CYTOMETRY - ARE THEY REALLY NECESSARY( AND CD3+, CD4+ LYMPHOCYTE SUBSETS BY FLOW)

Objective.-Isotypic control reagents are defined as irrelevant antibod ies of the same immunoglobulin class as the relevant reagent antibody in a flow cytometry panel. The use of the isotypic control antibody ha s been advocated as a necessary quality control measure in analysis of flow cytometry. The purpose of this study was to determine the necess ity of an isotypic control antibody in the analysis of CD3+ and CD3+, CD4+ lymphocyte subsets. Materials and Methods.-We performed a prospec tive study of 46 consecutive patient samples received for lymphocyte s ubset analysis to determine the need for the isotypic control. For eac h sample, a sham buffer (autocontrol) and isotypic control reagent wer e stained for three-color immunofluorescence, processed, and identical ly analyzed with Attractors software. The Attractors software allowed independent, multiparametric, simultaneous gating; was able to identic ally and reproducibly process each list mode file; and yielded populat ion data in spreadsheet form. Results.-Statistical analysis (Fisher's z test) revealed no difference between the CD3+ autocontrol and CD3+ i sotypic control (correlation = 1, P < .0001) or between the CD3+, CD4 autocontrol and the CD3+, CD4+ isotypic control (correlation = 1, P < .0001). The elimination of the isotypic control reagent resulted in a total cost savings of $3.36 per test. Additionally, the subtraction o f isotypic background can artifactually depress population enumeration . Conclusions.-The use of an isotypic control antibody is not necessar y to analyze flow cytometric data that result in discrete cell populat ions, such as CD3+ and CD3+, CD4+ lymphocyte subsets. The elimination of this unnecessary quality control measure results in substantial cos t savings.