Quantification of apoptosis by the Abbott CD4000 hematology analyzer.

We propose a simple and fast method of detecting apoptosis using an automated hematology analyzer. Detection is based on cellular optical light scatter properties and demonstration of the membrane fragility which characterizes cells undergoing the process of apoptosis. As part of it's routine leucocyte differential analysis, the Abbott Cell-Dyn 4000 collects multi-angle cellular light scatter data. In addition red fluorescence (FL3) emitted by cells following propidium iodide labeling is collected. This provides quantitation of both the erythroblast count and a leukocyte viability index (WVF). Fresh or cryopreserved peripheral blood cells from 17 B-chronic lymphocytic leukemia (B-CLL) patients were incubated in presence of theophylline, fludarabine or in medium alone. After 36-hrs of culture the percentage of apoptotic cells of the sample was determined from the parameters of the CD 4000 described above and thereafter this was compared with reference methods for estimation of apoptosis. The reference methods used were in situ detection of cell death on slides (TUNEL test) and also flow cytometry (Annexin V). Results showed an excellent correlation between the 3 techniques. This rapid, easy and reliable method of quantifying apoptosis may be very useful means of routinely predicting the response to chemotherapy.

VH gene expression in hairy cell leukaemia.

Hairy cells are characterized by their typical morphology and expression of specific surface antigens. Although their B-cell origin is now confirmed, their exact position in B-cell development remains unclear. To better define the origin of hairy cells, we analysed the immunophenotype and the Ig VH nucleotide sequence of seven cases of hairy cell leukaemia (HCL). Six of them were typical HCL and the remaining case corresponded to a variant HCL. Analysis of sequenced VH genes revealed that the VH1 family was used in one case, VH2 in one, VH3 in two, VH4 in two and VH5 in one. No preferential usage of VH genes was observed in this small series. In five cases high rates of somatic mutations were observed, with a predominance of mutations and replacements in CDR regions for three. indicating that these cells originate from cells that have been exposed to the hypermutation mechanism. The distribution of mutations in our small series provides some evidence of a selective mutational process.

Performance evaluation of the haematological analyser Cell-Dyn 3000 (Abbott).

The Abbott Cell Dyn (CD) 3000 is an automatic analyser, designed to give a complete blood count (CBC) and white blood cell differential (WBCD) by 4 angle diffraction analysis. This instrument was evaluated by comparison of results obtained with those obtained from a Technicon H1 analyser and by microscopic examination. Technical performances with regard to reproducibility, linearity and carryover was acceptable and in normal samples there was close correlation with the optical method (R > 0.9) for neutrophils (NE), lymphocytes (LY) and eosinophils (EO). Correlation for monocytes (MO) and basophils (BA) was poorer but without clinical consequences. Significant thresholds for immature granulocyte (IG) and variant lymphocyte (VL) flags were determined and using these thresholds the false positive rate was reduced to 7%. In haematological diseases, no false negatives were observed as all samples were flagged. However, since no case of acute lymphoblastic leukaemia (ALL) was studied, the detection of lymphoblasts which is known to present difficulties for analysers remains to be evaluated. Blasts in acute myeloblastic leukaemia (AML) and hairy cells were recognised, while in chronic lymphocytic leukaemia (CLL) it was possible to define 3 groups according to the flags released. The CD appears to be a satisfactory analyser for use in general or haematological laboratories performing a large number of WBCD per day.

Specific polypeptide markers in chronic B cell malignancies detected by two-dimensional gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of silver stained polypeptides was used to discriminate the cell protein profiles in chronic B cell malignancies. We identified 5 distinct polypeptides (H1 to H5) present in hairy cell leukemia (HCL) (14 cases) and absent in most chronic lymphocytic leukemia (CLL) and normal B lymphocytes. Two polypeptides, H2 and H5, were absolutely specific of HCL. The 2-D PAGE profiles found in 3 splenic lymphoma with villous lymphocytes (SLVL) and 2 HCL variants (HCL-V) were similar to HCL with a unique additional peptide present in 3/3 SLVL and 1/2 HCL-V. When HCL and SLVL were assessed for the CLL stage-specific 2-D PAGE patterns, HCL was related to stage A/B while SLVL was related to stage C. In conclusion, 2-D PAGE analysis of the molecular pattern of B lymphocytes provides a new means to recognize the leukemic origin of the sample and distinguish HCL from CLL and SLVL.

Macrophage activation syndromes.

The clinical and laboratory features of 47 cases of macrophage activation syndrome (MAS) were reviewed in a workshop within the Groupe Français d'Hématologie cellulaire. There was no predilection for a particular age group, while common symptoms at presentation included fever, hepatic and splenic enlargement and profound depression of blood count. Examination of bone marrow aspirates allowed diagnosis to be established in almost all cases. The most characteristic sign of MAS was the presence of well differentiated macrophages without notable cytologic abnormalities but shown to be actively ingesting haematopoietic elements. Haemophagocytic syndromes generally occur in patients who develop infections in the context of preexisting immunologic abnormalities or neoplasms. In the majority of patients evolution of the disease was regressive, once spontaneously but often after antibiotic, antiparasitic and/or antiviral treatment accompanied or not by corticotherapy and/or chemotherapy. Some regressive phases were followed by more or less long term relapse, especially in the case of associated systemic lupus erythematosus. There exists at present no explanation for the occurrence of MAS, although one may remark its association with other pathologies, in particular congenital or acquired immune deficiencies and haemopathies. Several hypotheses have been proposed to explain the appearance and evolution of the disease and at present two pathways of investigation of MAS seen to merit attention: exploration of macrophages themselves and their secretion products and exploration of lymphocytes and NK cells. The current possibilities for these investigations should lead to a greater understanding of the physiopathology of MAS and it is to be hoped that a better application of appropriate therapy will enable control of its evolution.