Effect of fixation on quantification of the expression of leucocyte function‐associated surface antigens

Abstract

The surface expression of adhesion molecules and other function-associated antigens on peripheral blood leucocytes may be measured by flow cytometry. However, quantification of these antigens is difficult, because their expression can be rapidly and artefactually modulated if the cells are activated in vitro. Consequently, it is common, when analyzing these antigens either: (1) to label leucocytes in whole blood at 4"C, to lyse erythrocytes and then fix the leucocytes, or (2) to fix the leucocytes in whole blood, to lyse the erythrocytes, and then label the leucocytes. We have compared the mean fluorescence intensity (MFI) values for CDllb, CD18, and L-selectin (Leu8 and TQ1 epitopes) on human peripheral blood leucocytes, using these two approaches. In addition, we have simultaneously evaluated how anticoagulants (acid citrate, K,EDTA, and heparin) and the presence or absence of divalent metal ions (Ca2+ and Mg2+) affect the expression levels of these antigens. The results for all four epitopes varied markedly depending on the preparation procedure used but were less affected by the choice of anticoagulant and whether divalent cations were or were not present in the media used for cell preparation and labelling. Comparison of the results obtained using these procedures, which involve fixation with formaldehyde, with those obtained by a recently developed procedure in which unfixed leucocytes were labelled with the vital nuclear dye LDS-751 and antibodies together, then analysed in unlysed whole blood at 4"C, showed that formaldehyde-based preparation techniques underestimated the expression (MFI) of CD18, Leu-8, and TQ1. It is recommended that, whenever practicable, measurements are made on unfixed cells stained using the newer procedure. o

Comparison of sample fixation and the use of LDS-751 or anti-CD45 for leukocyte identification in mouse whole blood for flow cytometry

“…In this study, we fixed prior to antibody staining, similar to the methods used in other studies (Hageberg, and Lyberg, 2000;Repo, Jansson, Leirisalo-Repo, 1993). McCarthy, et al, (1994) found an overall decrease in mean fluorescence intensity with fixation before or after antibody staining. Because of this finding, we did not include a group that was fixed after sample staining.…”

“…Thus, its use has been controversial for flow cytometric cell analysis because it could potentially alter monoclonal antibody adherence and allow outward diffusion of intracellular probes. McCarthy, et al, (1994) compared the effects of formaldehyde fixation before and after antibody staining and found that overall, fixation underestimated the mean fluorescence intensity of each antigen stained. Our findings are consistent with theirs, as we found that samples fixed with paraformaldehyde before staining demonstrated lower mean fluorescence than unfixed samples.…”

“…However, studies indicate that erythrocyte lysing reagents cause increased leukocyte expression of glycoprotein CD11b and shedding of granulocyte glycoprotein L-selectin (McCarthy, Macey, Cahill, and Newland, 1994;Macey, et al, 1995;Macey, et al, 1999;Alvarez-Larran, Toll, Rivas, and Estella, 2005). Erythrocyte lysing also causes other blood cells, including leukocytes and platelets, to lyse (Terstappen, Meiners, and Loken, 1989;Alvarez-Larran, et al, 2005), creating cell microparticles and debris that interact with and activate leukocytes (Repo, Jansson, and Leirisalo-Repo, 1993).…”

“…An example of a nuclear stain used historically for flow cytometry to label leukocytes is Laser dye styryl-751 (LDS-751) (Hokama, et al, 2000;McDonagh, Hokama, Copeland, and Reynolds, 1997;McCarthy, Macey, Cahill, and Newland, 1994;Macey, et al, 1999;Simon, Chambers, and Sklar, 1990;Terstappen, et al, 1991;Terstappen, Meiners, and Loken, 1989;Repo, Jansson, and Leirisalo-Repo, 1993;Himmelfarb, Hakim, Holbrook, Leeber, and Ault, 1992). Although LDS-751 has been used extensively to discriminate leukocytes in whole blood samples, it has been reported to indiscriminately stain both live and dead cells (O'Brien, and Bolton, 1995) and has been used to identify platelets and nucleated erythrocytes in whole blood (Terstappen, 1991).…”

“…Studies that have examined fixation on sample preparation have produced conflicting results. A number of studies determined that cell fixation decreases the mean fluorescence of several surface antigens, including leukocyte CD11b and CD18, regardless of erythrocyte lysing agents, anticoagulant, and time of fixation (McCarthy, Macey, Cahill, and Newland, 1994;Macey, McCarthy, Milne, Cavenaugh, and Newland, 1999). Conversely, other groups demonstrate that granulocyte CD11b expression and platelet-leukocyte conjugate formation are increased with sample fixation after antibody staining (Repo, Jansson, and Leirisalo-Repo, 1993).…”