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 lineages in acute leukemias. In order to establish the best combination of monoclonal antibody reagent and sample preparation technique for the intracellular detection of these three markers, we compared six different cell fixation-permeabilization kits (Cytofix/Cytoperm, Fix and Perm, Intraprep, Intrastain, Permeacyte and Permeafix) using 12 fluorochrome 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 lymphoblastic leukemia (ALL, n=6) and T-ALL (n=2) cases, were analysed in two centers. All fixation/permeabilization methods resulted in decreased side scatter 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 the Cytofix/Cytoperm kit and mildly with the other techniques. In addition, non-specific staining increased significantly for combinations of any anti-MPO mAb with the Cytofix/Cytoperm kit and for the CD3 clone S4.1 combined with any intracellular method. Anti-MPO antibodies gave a stronger fluorescence signal when conjugated to PE than when coupled to FITC. In conclusion, MPO-7-PE, UCHT-1-PE (CD3) and any HM57-PE conjugate (CD79a) in combination with Fix and Perm, Intraprep, Intrastain or Permeafix, provided specific staining of the respective markers in sufficient intensities. Thus, combined selection of fixation/permeabilization kits and monoclonal antibody reagents against CD3, CD79a and MPO is required for obtaining optimal cytoplasmic detection of these antigens.
Kappelmayer
J
J
Department of Clinical Biochemistry and Molecular Pathology, University Medical School of Debrecen, P.O. Box: 40, Nagyerdei krt. 98, H-4012 Debrecen, Hungary. kappelmayer@jaguar.dote.hu
Gratama
J W
JW
Karászi
E
E
Menéndez
P
P
Ciudad
J
J
Rivas
R
R
Orfao
A
A
D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000032:analysis* / Q000276:immunology; D017252:CD3 Complex / Q000032:analysis* / Q000276:immunology; D051925:CD79 Antigens; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005453:Fluorescence; D005456:Fluorescent Dyes; D007424:Intracellular Fluid / Q000737:chemistry; D015470:Leukemia, Myeloid, Acute / Q000097:blood; D010539:Permeability; D009195:Peroxidase / Q000032:analysis* / Q000276:immunology; D010714:Phosphatidylethanolamines; D011933:Reagent Kits, Diagnostic; D011947:Receptors, Antigen, B-Cell / Q000032:analysis* / Q000276:immunology; D012996:Solutions
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D017252:CD3 Complex; D051925:CD79 Antigens; D005456:Fluorescent Dyes; D010714:Phosphatidylethanolamines; D011933:Reagent Kits, Diagnostic; D011947:Receptors, Antigen, B-Cell; D012996:Solutions; D009195:Peroxidase; D016650:Fluorescein-5-isothiocyanate