Artifactual staining of monoclonal antibodies in two‐color combinations is due to an immunoglobulin in the serum and plasma

Abstract

Two-color whole blood lysis is the assay of choice for lymphocyte immunophenotyping because of the additional information it provides. Recently, artifactual double-staining of some specimens has been observed with this assay. In these cases, the samples appear to be uncompensated for spectral overlap or to inappropriately coexpress two antigens simultaneously. This artifact can result in the apparent coexpression of CD4 and CD8 (observed in lymphoblastic processes) or of CD5 and CD20 (characteristic of chronic lymphocytic leukemia) in normal persons, leading to an erroneous diagnosis. Using plasma, serum, or immunoglobulin preparations from donors who exhibit this artifact we sought to determine 1) the source of the artifact and 2) ways to overcome it. This staining is apparently due to an immunoglobulin in the donors' serum and plasma which does not have specific reactivity with mouse immunoglobulin. Washing whole blood samples or blocking with mouse immunoglobulin is a convenient way of avoiding this artifact. 0 1994 Wiley-Liss, Inc.Key terms: Flow cytometry, immunophenotyping, monoclonal antibodies, fluorescein, phycoerythrin Lymphocyte immunophenotyping of specimens from human immunodeficiency virus (H1V)-infected persons or lcukemia patients has become a routine procedure in many clinical laboratories. To minimize any artifacts that may result from density gradient centrifugation, a whole blood assay has been recommended for immunophenotyping lymphocytes (1,2). In this assay, whole blood is incubated with monoclonal antibodies that are labeled with fluorochromes (direct immunofluorescence). The red blood cells are lysed, and the remaining cells of interest (leukocytes) are analyzed o n a flow cytometer. The flow cytomcter processes light scatter and fluorescence data from the specimens. Currently, two-color immunofluorescence is the method of choice ( 1,2) because it allows better identification of cell populations that may possess more than one antigen of interest and provides more information than single-color immunofluorescence.Recently, we as well as others ( 3 ) have observed aberrant labeling in some specimens which appears to be uncompensated fluorescence or inappropriate double-laheling. In some cases, erroneous results were reported 0 1994 Wiley-Liss, Inc.when CD4 and CD8 cells were artifactually double-labeled and interpreted to be a population of immature T cells or when artifactual double expression of CD5 and CD20 was interpreted as a lymphocytic malignancy (Marti, personal communication).We report the data from four different laboratories concerning this artifactual two-color staining in specimens, including the frequency of occurrence and the conditions under which it is observed. This artifact is idiosyncratic and is apparently due to an immunoglobulin found in plasma and serum of random donors which is not antimouse immunoglobulin antibodies. The immunoglobulin appears to crosslink mouse monoclonal antibodies, possibly through the Fc portion of the antibodies.

Interactions between mouse IgG2 antibodies are common and mediated by plasma C1q

“…Following vortexing, the mixture was incubated for 15 min at room temperature in the dark, lysed with 1.5 ml of HCO 3 -buffered NH 4 Cl containing 0.25% formaldehyde (Ultrapure, Polysciences) for 15 min and was washed once (550g, 5 min) with phosphate buffered saline containing 0.3% bovine serum albumin and 0.1% sodium azide (PBS-A). The sample was resuspended in 100 ml of PBS-A and 100,000 total events acquired on an LSRII (Becton-Dickinson, San Jose, CA).…”

“…2, right), and CD13 (L138, BectonDickinson) and CD5 (BL1a, Beckman-Coulter). To estimate the frequency with which this artifact occurred, 10 peripheral blood samples were evaluated with each of the above three reagent combinations in a standard NH 4 Cl-based whole blood lysis assay, and the artifact was shown to be present with all 10 specimens, albeit with some minor variation in intensity (data not shown). Consequently, it appeared that we had identified a group of artifactual antibody interactions that were exceedingly common, occurring in all specimens evaluated.…”

“…A variety of manipulations were investigated that failed to eliminate the observed antibody interaction, including (data not shown): preincubation with mouse serum or purified murine IgG2, a change in lysing reagent from NH 4 Cl to FACSlyse (Becton-Dickinson), and washing after antibody incubation. One method that consistently and completely eliminated the interaction was removal of plasma prior to antibody addition, either by washing the whole blood three times in PBS (Fig.…”

“…One method that consistently and completely eliminated the interaction was removal of plasma prior to antibody addition, either by washing the whole blood three times in PBS (Fig. 3) or by prelysis of the sample with NH 4 Cl followed by a single PBS wash. These results suggested that a component of plasma was required for mediating the antibody interaction and that the interaction was not due to a direct interaction between the antibodies themselves.…”

“…This is particularly a concern when specimens are prepared using a whole blood lysing technique, common in a clinical setting, as reagents are added to whole blood, whose plasma contains a complex mixture of proteins including numerous immunoglobulins of varied specificity as well as components of the complement system. Despite this concern, antibody interactions as a result of specimen preparation for flow cytometry are believed to be relatively infrequent, with only few descriptions in the literature (3,4).…”