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.

[Molecular biology examination in chronic lymphocytic leukemia]. / Molekuláris biológiai vizsgálatok krónikus lymphoid leukaemiában.

Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia characterised by the accumulation of monoclonal CD5 + B-lymphocytes. The pathogenesis and the biology of CLL is complex and many details are still unknown. Several molecular biological methods have been used in the investigation of CLL, among them the study of apoptosis appears to be one of the most important. Initial experiences obtained by the spontaneous and fludarabine induced apoptosis, multidrug resistance (MDR)-test and fluorescent in situ hybridization (FISH) are reported by the authors. Apoptosis of CLL cells could be induced by fludarabine, while more studies should be performed to determine the exact role of MDR-test and FISH.

Identification of P-selectin glycoprotein ligand-1 as a useful marker in acute myeloid leukaemias.

Immunophenotyping is considered to be less valuable in the diagnosis of acute myeloid leukaemias (AML) compared with acute lymphoid leukaemias. Here, we present data on the use of quantitative flow cytometry (QFC) of P-selectin glycoprotein ligand 1 (PSGL-1, CD162) and three-colour immunophenotyping including CD162 staining in the identification of myeloid precursors in AML. Analysis of normal peripheral blood (n = 20) and normal bone marrow (n = 5) samples and on 20 samples from de novo M1, M2, M4 and M5 AML patients demonstrated that PSGL-1 is differentially expressed on various mature and immature leucocyte subsets. It was found by QFC that neutrophils expressed 26500 +/- 4500 and monocytes 47200 +/- 9900 copies of PSGL-1 on their surface, whereas AML blasts from M1 and M2 AML patients expressed significantly less PSGL-1 (12 000 +/- 5300) than mature neutrophils (P < 0.001). In M4 and M5 leukaemias, however, the amount of PSGL-1 on monocytic precursors is displayed in a fairly broad range that is not significantly different from that of mature monocytes (P = 0.084). Using three-colour immunophenotyping PSGL-1-dim staining was co-expressed with CD7 and C34 positivity and PSGL-1 staining intensity on immature myeloid cells paralleled with CD45 expression. This would imply a differential expression of PSGL-1 during myeloid haematopoietic development and suggests that quantification of surface PSGL-1 may aid in differentiating myeloblasts from monoblasts by immunophenotyping in different AML subsets.

Inhibition of activation-induced apoptosis of thymocytes by all-trans- and 9-cis-retinoic acid is mediated via retinoic acid receptor alpha.

Thymocytes can be induced to undergo apoptotic cell death by activation through the T-cell receptor (TCR). This process requires macromolecular synthesis and has been shown to be inhibited by retinoic acids (RAs). Two groups of nuclear receptors for RAs have been identified: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). All-trans-RA is the high-affinity ligand for RARs, and 9-cis-RA additionally binds to RXRs with high affinity. Because 9-cis-RA is much more potent in inhibiting TCR-mediated death than all-trans-RA, it was suggested that RXRs participate in the process. In the present study various synthetic retinoid analogues were used to address this question further. The results presented suggest that the inhibitory effect of RAs on activation-induced death of thymocytes is mediated via RARalpha, because (1) it can be reproduced by various RARalpha analogues both in vitro and in vivo, (2) the effect of RAs can be inhibited by the addition of an RARalpha antagonist, (3) CD4+CD8+thymocytes, which die on TCR stimulation, express RARalpha. Stimulation of RARgamma, in contrast, enhances the activation-induced death of thymocytes and inhibits its prevention by RARalpha stimulation. RXR co-stimulation suspends this inhibitory effect of RARgamma and permits the preventive function of RARalpha on activation-induced death. Our results suggest a complex interaction between the various isoforms of retinoid receptors and demonstrate that low (physiological) concentrations of all-trans-RA do not affect the activation-induced death of thymocytes because the RARalpha-mediated inhibitory and the RARgamma-mediated enhancing pathways are in balance, whereas if 9-cis-RA is formed, additional stimulation of RXRs permits the inhibitory action of RARalpha.