Flow cytometric detection of intracellular myeloperoxidase, CD3 and CD79a interaction between monoclonal antibody clones, fluorochromes and sample preparation protocols

Detection of intracellular myeloperoxidase (MPO), CD79a and CD3 has become the most specific tool for the assignment of myeloid, B- and T-lymphoid lin eages in acute leukemias. In order to establish the best combination of mon oclonal antibody reagent and sample preparation technique for the intracell ular detection of these three markers, we compared six different cell fixat ion-permeabilization kits (Cytofix/Cytoperm(TM), Fix and Perm(TM), Intrapre p(TM), Intrastain(TM), Permeacyte(TM) and Permeafix(TM)) using 12 fluorochr ome conjugates derived from seven monoclonal antibody (mAb) clones. A total of 21 samples corresponding to normal peripheral blood (n=4), normal bone marrow (n=3), acute myeloblastic leukemia (AML, n=6), precursor B-acute lym phoblastic leukemia (ALL, n=6) and T-ALL (n=2) cases, were analysed in two centers. All fixation/permeabilization methods resulted in decreased side s catter and mostly increased forward scatter as compared to erythrocyte-lyse -washed and 1% paraformaldehyde fixed samples, The autofluorescence levels of the leukocyte populations was only significantly increased with use of t he Cytofix/Cytoperm(TM) kit and mildly with the other techniques. In additi on, non-specific staining increased significantly for combinations of any a nti-MPO mAb with the Cytofix/Cytoperm(TM) kit and for the CD3 clone S4.1 co mbined with any intracellular method. Anti-MPO antibodies gave a stronger f luorescence signal when conjugated to PE than when coupled to FITC. In conc lusion, MPO-7-PE, UCHT-1-PE (CD3) and any HM57-PE conjugate (CD79a) in comb ination with Fix and Perm(TM), Intraprep(TM), Intrastain(TM) or Permeafix(T M), provided specific staining of the respective markers in sufficient inte nsities. Thus, combined selection of fixation/permeabilization kits and mon oclonal antibody reagents against CD3, CD79a and MPO is required for obtain ing optimal cytoplasmic detection of these antigens. (C) 2000 Elsevier Scie nce BN, All rights reserved.