Zsuzsanna Hevessy

The emerging value of P-selectin as a disease marker

Abstract Activated platelets are key components in many arterial disorders. P-selectin is an activation-dependent platelet receptor, which is also identified in endothelial cells. Together with E-and L-selectin it constitutes the selectin family. These transmembrane proteins have continued to attract great interest as they support rapid and reversible cell adhesion in flow systems and thus play an essential role in multicellular interactions during thrombosis and inflammation. Similarly to other lectins, selectins bind to different glycoconjugates with varying affinities. Protein ligands, equipped with the appropriate carbohydrate and sulfate moieties for P-selectin binding, have been identified in normal peripheral blood leukocytes and several non-hematopoietic organs, as well as on cancer cells. For diagnostic purposes, P-selectin can readily be detected on the platelet surface by flow cytometry and by ELISA as a soluble ligand in the plasma. Along with other markers, these data can be used in the assessment of platelet activation status. Such results bear clinical significance since P-selectin has been implicated in the pathogenesis of widespread disorders including coronary artery disease, stroke, diabetes and malignancy.

Pathogenicity and virulence of coagulase negative staphylococci in relation to adherence, hydrophobicity, and toxin production in vitro.

AIMS--To study the pathogenicity and virulence characteristics of Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus sapro-phyticus. METHODS--BALB/c mice were challenged intraperitoneally with graded doses of three strains belonging to each species. LD50s were measured for each strain. Haemolysin (alpha- and delta-) and enzyme (DNAase, lipase, and esterase) production in vitro were measured qualitatively and quantitatively. Adhesion to plastic was measured and related to cell surface hydrophobicity among the strains. RESULTS--S saprophyticus proved the most virulent (LD50 = 2.7-2.9 x 10(7) cfu/g body weight) while S epidermidis was the least virulent (LD50 = 6-8 x 10(7) cfu/g body weight). An enlarged spleen was the most common macroscopic pathological feature. Kidney, liver, and more rarely peritoneal abscesses were also seen in the infected animals. No direct correlation was found between adherence in vitro, cell surface hydrophobicity, or toxin/enzyme biosynthesis and virulence in mice. CONCLUSION--The results show that coagulase negative staphylococci are pathogenic in BALB/c mice. It is clear that these bacteria can cause invasive disease. However, the in vitro characteristics of coagulase negative staphylococci are not related to the pathogenicity of the organisms in mice.

Leukemic lymphoblasts, a novel expression site of coagulation factor XIII subunit A

Blood coagulation factor XIII (FXIII) is a protransglutaminase circulating as a tetramer formed by two types of subunits (A2B2). The intracellular dimeric form of FXIII (A2) is present in platelets, megakaryocytes, monocytes and macrophages and has been detected in mono- and megakaryocytic leukemias. The aim of our study was to investigate FXIII-A expression in newly diagnosed B cell acute lymphoblastic leukemia (ALL) samples. We examined 47 de novo ALL cases of B cell origin by triple color labeling with flow cytometry. FXIII-A was detected by a FITC conjugated monoclonal antibody combined with CD34 and CD45 staining. In selected cases FXIII-A was investigated on slides prepared from blasts and visualized with a fluorescent microscope. In addition, blasts were studied by Western blot analysis and FXIII-A was measured by a highly sensitive ELISA method. By flow cytometry 19 samples of the 47 cases were found to be FXIII-A positive. Antigen concentration was 3.11 +/- 1.19 fg/blast, while normal lymphoid precursors and mature lymphocytes from B-CLL did not contain FXIII-A. In the lysate of lymphoblasts that were positive by flow cytometry, a single band (82 kDa) corresponding to FXIII-A was detected on Western blots. Confocal laser scanning microscopic examination revealed the presence of FXIII-A in the cytoplasm of these lymphoblasts. This novel expression site of FXIII-A in leukemic lymphoblasts can be utilized as a diagnostic tool and may also gain functional significance in B-lineage ALL.

Serum Thymidine Kinase Activity: Analytical Performance, Age-Related Reference Ranges and Validation in Chronic Lymphocytic Leukemia

Background To date no age-related reference ranges are available for serum thymidine kinase (TK1) activity. Being a proliferation marker, it may be used as a prognostic marker in malignant diseases, including chronic lymphocytic leukemia (CLL). Our aim was to establish age-specific reference ranges for TK1 and examine its utility as a screening marker in CLL, a disease of the elderly. Methods Serum TK1 activity was measured by a competitive chemiluminescent immunoassay in 369 healthy adults and 115 de novo CLL patients. Results We observed a statistically significant decline in TK1 activity from young (18–35 years) to middle-aged (36–60 years) and further on to elderly (60–86 years) healthy individuals. Age-related reference range was: <30 U/L for young, <25 U/L for middle-aged and <19 U/L for elderly. There was no difference in TK1 activity between the studied healthy men and women. In CLL patients, TK1 activity was the highest in the advanced Rai stages. The area under the receiver operating characteristic curve (ROC-AUC) for TK1 was 0.840 (95% CI: 0.787–0.892), for differentiating CLL patients from age and sex matched healthy controls, with a cut-off value of 10.5 U/L (sensitivity: 80.9%, specificity: 73.4%). TK1 was significantly elevated in CD38+/Zap70+ CLL patients, and showed significant correlation with WBC and absolute B-cell count. Conclusion In the healthy, serum TK1 activity does not differ in the two sexes but declines significantly with age. As such, use of age-related reference ranges is warranted, especially when evaluating CLL patients who generally belong to the elderly age group.

Expression of coagulation factor XIII subunit A in acute promyelocytic leukemia

Leukemic cells often express markers, which are not characteristic of their particular cell lineage. In this study, we identified the “A” subunit of coagulation factor XIII (FXIII‐A) in leukemic promyelocytes in de novo AML M3 cases. The cytoplasmic presence of factor XIII‐A has previously been shown only in platelets/megakaryocytes and monocytes/macrophages. Furthermore, more recently we described the presence of FXIII‐A in leukemic lymphoblasts.

A Coagulation Factor Becomes Useful in the Study of Acute Leukemias: Studies with Blood Coagulation Factor XIII

The intracellular form of the coagulation factor XIII has previously been identified by immunomorphological techniques using polyclonal antibodies. In these studies, only the A subunit (FXIII‐A) was detectable in megakaryocytes/platelets and in monocytes/macrophages. We developed several novel monoclonal antibody clones directed to both subunits (FXIII‐A and FXIII‐B) and investigated their appearance in normal and leukemic cells. By using 3‐ and 4‐color flow cytometry FXIII expression was investigated in normal peripheral blood and bone marrow samples and in acute myeloblastic (AML) and lymphoblastic (ALL) leukemia cases. Samples were studied by Western blotting and confocal laser scanning microscopy. With a previously published ELISA assay applying two monoclonal antibodies directed to different epitopes in FXIII‐A, we were able to measure the intracytoplasmic content of FXIII‐A in normal cells and leukemic blasts. FXIII‐A was detectable in normal peripheral blood monocytes and in large quantities in platelets, but both cell types were negative for FXIII‐B. There was no surface staining for FXIII‐A, it only appeared intracellularly. In samples derived from patients with AML M4 and M5, FXIII‐A sensitively identified blast cells. Although normal lymphocytes do not express FXIII‐A, 40% of ALL cases showed significant FXIII‐A expression as determined by flow cytometry. FXIII‐A positivity of lymphoblasts was verified by Western blotting, ELISA, and confocal laser scanning microscopy cytometry. These data provide evidence that FXIII‐A is a sufficiently sensitive marker in differentiating myeloblasts and monoblasts and is suitable for identifying leukemia‐associated phenotypes in ALL.

Laboratory evaluation of a flow cytometric BCR-ABL immunobead assay

Abstract Background: A new flow cytometric (FC) BCR-ABL immunobead assay has been developed recently. Here we present the laboratory evaluation of the commercially available kit. Methods: Mononuclear cells were isolated, lysed and processed according to the instructions of the manufacturer. Anti-BCR antibodies adsorbed to capture beads bind the BCR-ABL fusion proteins of the lysed cells, a phycoerythrin (PE)-conjugated anti-ABL antibody is the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Detection of t(9;22)(q34;q11) translocation was carried out with a quantitative PCR assay. Results: MFI results of 20 normal peripheral blood samples were 88±8 (mean±SD), CV 9%. K562 cells were used as positive control. Within-batch imprecision was excellent (3.7% in the normal and 10% in the pathological range). Cut-off was chosen at MFI 112, where both sensitivity and specificity were 100%. Altogether 17 chronic myeloid leukemia (CML) and 16 acute leukemia samples were analyzed. All PCR positive samples (n=14) were positive with the FC method and negative results were also concordant (n=15). Frozen cell lysates can be stored up to 4 weeks without significant decrease of MFI signal. Conclusions: The FC BCR-ABL assay is a fast, reproducible and reliable method that may be incorporated into standard flow cytometric protocols to help clinical decision-making.

The prognostic value of smudge cells (Gumprecht shadows) in chronic lymphocytic leukaemia

INTRODUCTION Smudge cells (Gumprecht shadows) are chronic lymphocytic leukaemic cells ruptured during peripheral blood smear preparation. It has been demonstrated to be linked to reduced expression of the cytoskeletal protein vimentin and its inverse correlation with the clinical outcome of the disease. AIMS Investigation of the percentage of smudge cells, CD38-, ZAP-70-positive cells and the time to treatment in patients with chronic lymphocytic leukaemia. METHODS Authors investigated the percentage of smudge cells, CD38- and ZAP-70-positive cells in the peripheral blood of 50 patients with chronic lymphocytic leukaemia and their correlation with the time to treatment. RESULTS 21 patients required treatment in the follow-up period. Their median smudge cell percentage was 9.9%, while it was 26.8% in the non-treated group. The cut-off value of smudge cell positivity was set to 20%. 59.3% of the patients with less than cut-off had to be treated in the follow-up time compared to 21.7% of patients with more smudge cells. These findings were similar to the prognostic value of CD38 and ZAP-70. The necessity of treatment increased to 75-77.8% with the combination of investigated markers. The time to treatment was 19 months when smudge cells were less than 20%, but above 20% it was 36.15 months. In case of low smudge cell percentage and CD38 positivity the time to treatment was 14.14 months and in case of high smudge cell percentage and CD38 negativity it was 32.92 months. In discordant cases the time to treatment was 18.43 months. The authors also present a case report that demonstrates the relationship between the percentage of smudge cells and apoptotic cells with annexin V and 7-AAD staining. CONCLUSIONS Estimation of smudge cells on a blood smear could be a simple and cheap prognostic test in chronic lymphocytic leukaemia with sensitivity similar to CD38 and ZAP-70 estimation. Combination of these tests raised the sensitivity of their prognostic value.

Classical and atypical neuroblastoma— Case reports

Neuroblastoma is the most common extracranial solid tumor in early childhood with a pluripotent neural cell origin. The disease often infiltrates the bone marrow, and these cases are candidates for flow cytometric diagnosis and disease follow‐up, more so in atypical cases that are diagnostically challenging by other methods. Here, we report two neuroblastoma cases, one with characteristic neuroblastoma cells forming Homer–Wright rosettes found in the bone marrow sample by histological examination. This case showed the classical immunophenotype with CD81/CD56/CD117 positive and CD45 negative labeling and 80% bone marrow infiltration. Minimal residual disease measurement was performed in a regular fashion, and flow cytometry results showed good correlation with other disease markers. Analyzing 300,000 events provided a sensitivity level of 0.01%. The second case showed atypical cell morphology on histological examination, after which flow cytometric analysis was initiated. Atypical cells displayed CD81 and bright CD56 but CD117 negative immunophenotype, with a 28% bone marrow infiltration. In most cases the primary tumor can be identified, but in this particular case only bone marrow involvement could be detected using flow cytometry, as such making it a powerful tool for diagnosis and disease monitoring.

The impact of delayed sample handling and type of anticoagulant on the interpretation of dysplastic signs detected by flow cytometry

Introduction A growing body of evidence supports the usefulness of dysplastic signs detected by flow cytometry in the diagnosis of myelodysplastic syndromes (MDS). Our aim was to assess the impact of pre-analytical variables (delayed sample handling, type of anticoagulant, and different clones of antibody) in the interpretation of flow cytometric results. Material and methods Bone marrow samples were labelled and analysed immediately after aspiration and on two consecutive days. The effect of anticoagulant type was evaluated in 16 bone marrow samples. Thirty-seven different immunophenotypic variables were recorded after eight-colour staining. Furthermore, 8 normal peripheral blood samples collected in K3-EDTA and Na-heparin were examined with different clones of CD11b antibodies and four parameters were recorded with both anticoagulants on two consecutive days. Results Fourteen significant differences were detected in the initial immunophenotype of fresh samples collected in K3-EDTA and Na-heparin. Regardless of the anticoagulant type, eleven parameters remained stable despite delayed sample handling. Due to delayed sample processing, more alterations were detected in the samples collected in K3-EDTA than in the samples collected in Na-heparin. The type of CD11b clone influenced the reduction of fluorescence intensity only in samples collected in K3-EDTA, where the alterations were contrary to the changes observed in Na-heparin. Conclusions Delayed sample processing causes considerable immunohenotypic alterations, which can lead to false interpretation of the results. If delayed sample evaluation is unavoidable, markers that remain more stable over time should be considered with more weight in the diagnosis of MDS.