The influence of different erythrocyte lysing procedures on flow cytometric determination of CD34+ cells in umbilical cord blood transplants

Since the correct determination of CD34+ cells is of great clinical importance for successful transplantation with haematopoietic progenitor cells (HPCs) from cord blood, we investigated the influence of different erythrocyte lysing techniques on the quantification of CD34+ cells in umbilical cord blood.
Flow cytometric determinations of CD34+ cells were performed from 20 cord blood samples, using three different erythrocyte lysing procedures and two monoclonal CD34 antibodies ( n  = 360).
Flow cytometric analysis showed characteristic patterns of the forward (FSC) and side (SSC) scatter light properties for the leucocyte subsets for each of the investigated erythrocyte lysing procedures, indicating that these reagents cause different morphological changes on leucocytes. Furthermore, significant differences of CD34+ cell counts were obtained for identical samples using different lysing techniques ( P  = 0.001 and P  = 0.002). In some cases, a more than 100% difference was found comparing different erythrocyte lysing procedures. In contrast, the determination of CD34+ cells by two CD34 antibodies showed a good reproducibility without significant differences between both antibodies for each of the erythrocyte lysing techniques.
We conclude that the erythrocyte lysing procedure represents a very critical and important step for accurate determination of CD34+ cells in whole blood samples. Especially for the quantification of HPCs in cord blood transplants, this influence may be of high clinical relevance.     

Evaluation of 5′-nucleotidase as biochemical marker in leukemias and lymphomas

Journal of Molecular Medicine - 5′-Nucleotidase (5′-N) is known as a cellular ectoenzyme of wide tissue distribution. The varying expression of the enzyme in leukocytes is thought to be...     

Annexin V and platelet antigen expression is not altered during storage of platelet concentrates obtained with the AMICUS cell separator.

During storage of platelet concentrate the so-called "storage lesion" occurs. During this time, platelets loose their morphological and functional capacities that are necessary for proper in vivo efficacy following transfusion. Annexin V represents a marker for apoptosis. In this study, Annexin V and additional antigens were analyzed by flow cytometry. Platelet concentrates were obtained with a new cell separator (AMICUS Separator, Fenwal). Following apheresis, platelet units were stored for an experimentally prolonged time of seven days. Daily aliquots of the platelet-rich plasma were obtained to measure Annexin V and platelet antigens CD62p, CD63, CD41a, CD42b, and the binding of fibrinogen. All analyses were performed using flow cytometry. During storage, no significant changes in mean channel fluorescence intensity (MCFI) of CD41a (P = 0.99) and CD42b (P = 0.29), percentage of CD62p+ and CD63+ platelets (P = 0.23 for CD62p; P = 0.52 for CD63), and the binding of fibrinogen to platelets occurred (P = 0.85). Also, the expression of Annexin V remained constant with no significant change (P = 0.36). This study shows that antigens of platelets, obtained with the AMICUS cell separator are well preserved during storage. Regarding Annexin V, no obvious signs of apoptosis can be detected by flow cytometry. These findings demonstrate the high degree of biocompatibility of the apheresis device and storage container.     

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A population-based case-control study of EBV and other viral antibodies among persons with Hodgkin's disease and their siblings

The Epstein-Barr virus (EBV) has been consistently found to be associated with Hodgkin's disease (HD) in two ways: cases generally have elevated titer distributions of antibodies against the viral capsid antigen, and the occurrence of HD among persons with a history of EBV infectious mononucleosis is two or three times higher than expected. We evaluated this association by measuring the prevalence and level of antibodies against EBV and related viruses among 304 cases of HD interviewed in a population-based study in comparison to 285 of their siblings. The most significant finding was that antibody titers to the viral capsid antigen of EBV were elevated (± 1:320) in 39% of the cases and in only 14% of the sibling-controls; the relative risk adjusted for age and sex was 4.1. The geometric mean titer was three-fold higher among cases (175.6vs. 58.1) Subjects who reported a history of IM had a higher distribution of titers than those who did not. Cases also had elevated titers against the early antigen of EBV - the D Component being most prominent. A significantly higher proportion of cases has elevated titers against CMV, relative risk = 3.4, but the prevalence of CMV antibody was relatively low and not consistently higher among cases. The findings support the hypothesis that EBV may play a role in the pathogenesis of HD among persons with elevated titers. The findings neither confirm nor deny a possible role of CMV.     

Epidemiologic association between virus-negative feline leukemia and the horizontally transmitted feline leukemia virus

About two-thirds of the natural cases of feline leukemia-lymphoma are assumed to be caused by the feline leukemia virus (FeLV) because they occur in cats, which harbor this agent, and FeLV will induce the disease under laboratory conditions. Epidemiological evidence is presented which associates cases of naturally occurring 'virus negative' feline leukemia with exposure to FeLV.     

School contact among persons with Hodgkin's disease

The authors evaluated the hypothesis that Hodgkin's disease is transmitted among high school students (in a contagious manner) by determining whether members of a population-based series of young adult cases attended school together more frequently than expected. Cases of Hodgkin's disease were diagnosed from July 1, 1973 through December 31, 1977. Of the 245 cases and 483 matched population controls interviewed, 196 cases and 371 controls attended schools in Massachusetts. School histories of subjects obtained by personal interviews were analyzed to determine the frequency with which subjects had been in the same class or had attended the same school at the same time. In high school, cases were schoolmates more often than expected (relative risk (RR) = 1.2), as well as classmates (RR = 1.9). However, in junior high school, cases were schoolmates less often than expected (RR = 0.8), and none were classmates with four expected. In elementary school, cases were somewhat more likely to be schoolmates (RR = 1.4) with no excess of classmates. The 95% confidence interval for all relative risks includes 1.0.     

Flow cytometric analyses of CD34+ cells with inclusion of internal positive controls

BACKGROUND: Flow cytometric measurement of CD34+ events is used to ensure the quality of human progenitor cell grafts. This study was conducted to evaluate whether the spiking of routine samples from peripheral blood and apheresis products with CD34+ positive controls is feasible. STUDY DESIGN AND METHODS: A total of 42 samples from 32 patients and one healthy donor were stained in duplicate for CD34+ cells. Before flow cytometric analysis, one tube was spiked with stabilized CD34+ cells at a defined concentration. RESULTS: Median numbers of viable CD34+ cells/µL did not differ between unspiked and spiked tubes (median 37, range 0‐714; and median 34, range 0‐719, respectively). The 95% confidence interval (CI) of the mean showed a broad overlap between these samples (41.9‐119.1 and 41.4‐119.3, respectively). In addition, the 95% CI of the mean for CD45+ cells/µL overlapped broadly and median numbers did not differ. Median viability of all CD45+ cells was significantly lower in the spiked tubes (96.75, range 64‐98.8 vs. 99.25, range 97.5‐99.8) with no overlap of the 95% CI of the mean viability. CONCLUSION: The results of this study show that spiking of routine samples with internal positive controls does not affect CD34+ cell analyses, but does support the reliability of important clinical data. The inclusion of positive controls is expedient for laboratories that perform analyses with low CD34+ numbers and laboratories that use different flow cytometric analyzers and may also become a requirement to meet statutory regulations.     

Direct volumetric flow cytometric quantitation of CD34+ stem and progenitor cells

Objectives: In this study, we compared a classic single‐platform (SP) method applying beads for enumeration of CD45+ or CD34+ cells with a new device allowing direct volumetric measurements of stem and progenitor cells. Background: Following apheresis and cyropreservation, the precise enumeration of CD34+ cells as key parameter of graft quality is mandatory for the clinical course after transplantation. Currently, flow cytometry with SP technique represents the ‘gold standard’ for such determinations. Methods/Materials: Fresh samples, 14 from mobilised peripheral blood (PB), 9 from apheresis products (AP) and 13 samples from frozen‐thawed (FT) haematopoietic progenitor cell grafts, were analysed for CD34+ cells, CD45+ cells, and in frozen‐thawed samples for viability by a bead‐based flow cytometric method and in parallel by a direct, volumetric flow cytometric method. Results: Comparison of CD34+ analyses revealed a significant correlation (P < 0·01) for each material between both techniques with r = 0·95 (PB), r = 0·933 (AP) and r = 0·929 (FT). Also, for analysis of CD45+ cells µL^?1, the measured numbers evaluated with the different techniques did not significantly differ for all three materials analysed. In frozen‐thawed samples, the analysis of viability was comparable for both techniques. Conclusions: The results of this study demonstrate that a direct volumetric analysis of CD34+ cells µL^?1 or CD45+ cells µL^?1 is feasible. This technique represents a simple and economical approach for standardisation of progenitor and stem cell analyses.