Development and Properties of a Monoclonal Antibody Specific for Human Ecto-5'-Nucleotidase

After immunization with purified human placental ecto-5'-nucleotidase and fusion, 18 different hybrids were obtained which produce an antibody inhibitory for enzyme activity. These antibodies show complete cross-reactivity with the enzyme in a membrane-bound form or on the surface of intact cells. After cloning and ascites production, the antibody of one clone (IFH-5N1) was studied in greater detail. The IFH-5N1 antibody is of the IgG 1 subclass. Optimal enzyme inhibition is 90% for purified 5'-nucleotidase and 80% for the enzyme on lymphoblasts. The specificity of this antibody is further demonstrated by enzyme inhibition assays and fluorescence labeling using various 5'-nucleotidase-positive and -negative human cells such as peripheral blood lymphocytes, leukemic cells, lymphoblastoid B- and T-cell-lines and fibroblasts. The antibody should provide a useful tool for the diagnosis of certain forms of acute leukemias and for the study of normal human lymphocyte subpopulations.     

TRANSPLANTATION AND CELLULAR ENGINEERING: Increasing the economic efficacy of peripheral blood progenitor cell collections by monitoring peripheral blood CD34+ concentrations

BACKGROUND: After mobilization, the collection of peripheral blood progenitor cells (PBPCs) can either be started a fixed number of days after having passed the white blood cell nadir (fixed‐day scheme) or be based on monitoring of CD34+ cells. This study was conducted to compare both approaches and to assess possible financial consequences. STUDY DESIGN AND METHODS: For 29 patients daily enumeration of CD34+ cells was used to guide leukapheresis timing. In a retrospective analysis for the same group of patients, application of a fixed‐day scheme was assumed. For scenarios of beginning apheresis 2, 3, 4, or 5 days after WBC nadir, the number of apheresis days and granulocyte–colony‐stimulating factor (G‐CSF) application days that could be saved was calculated. RESULTS: A total of 44 apheresis procedures were performed resulting in a mean CD34+ cell content per apheresis product of 10.4 × 10⁶ (range, 0.1 × 10⁶‐49.5 × 10⁶)/kg of body weight. The smallest number of deviation days compared to a fixed‐day scheme was found for beginning an apheresis on Day 3. In comparison to this, CD34+ monitoring reduced the number of G‐CSF days by 9 and the number of apheresis procedures by 11 overall, resulting in savings of €19,965 (US$28,788) in comparison to expenses of €826 (US$1191) for CD34+ monitoring. CONCLUSIONS: Measurement of CD34+ cells has reached a precision enabling a prediction of the harvest success. In comparison to a fixed‐day scheme, daily CD34+ monitoring reduces the donor's exposition to G‐CSF, enables collection of a sufficient number of PBPCs in the least possible number of apheresis sessions, and improves the economic efficacy of the institution.     

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.     

Binding of activated platelets to WBCs in vivo after transfusion

BACKGROUND: During preparation and storage of apheresis concentrates, platelets are being activated. One of the alterations that occur during this process is an increased expression of P-selectin (CD62p) on the cytoplasmic surface of platelets. This neoepitope represents a ligand for the binding of platelets to WBCs. It has been suggested that the activation of platelets is associated with the sequestration of platelets after transfusion. In this in vivo study, the binding of platelets to WBCs was analyzed following transfusion of platelet concentrates (PCs). STUDY DESIGN AND METHODS: Double apheresis concentrates were prepared with two different cell separators. One of the split products was stored for 1 to 2 days and the other one for 3 to 5 days. Flow cytometry was applied to analyze the degree of platelet activation in vitro, and also to measure the extent of platelet binding to WBC subclasses in vivo after transfusion into patients. RESULTS: The results of this study show that platelet activation occurs during apheresis and storage of PCs. After transfusion of the PCs, no significant binding of platelets to T or B-cells could be detected. However, a significant binding of platelets to monocytes and neutrophil granulocytes occurs. While in Baxter PCs stored for 1-2 days the amount of platelet-leukocyte aggregates in vivo was higher compared to COBE PCs, no such difference could be detected anymore for the PCs stored for 3-5 days. CONCLUSION: This study demonstrates that binding of activated platelets occurs to monocytes and neutrophil granulocytes but not to T- and B-cells in the circulation after transfusion. In addition, the interaction of platelets and WBCs is dependent on the degree of P-selectin expression. Platelets showing a higher degree of activation adhere to WBCs to a higher degree than nonactivated platelets.     

Cytomegalovirus cultured from different major leukocyte subpopulations: association with clinical features in CMV immunoglobulin G-positive renal allograft recipients

Cytomegalovirus (CMV) cultured from peripheral blood mononuclear cells (PBMCs) was shown to be associated more closely with clinical manifestations than infectious CMV in polymorphonuclear leukocytes (PMNLs) of renal allograft recipients with secondary CMV infection. Shell vial culture was carried out with ficoll-purified PBMCs and PMNLs of 71 CMV IgG-positive patients after kidney transplantation. Thirty-six patients experienced active CMV infections. Of these, 17 developed clinical symptoms. The diagnostic value of PMNLs and PBMCs viremia was determined in comparison to pp65 antigenemia, leukoDNAemia, plasma DNAemia, and detection of cytomegalic endothelial cells. In both PMNLs and PBMCs (with or without detectable endothelial cells), frequencies and levels of viremia were significantly higher among symptomatic patients. Regarding the occurrence of clinical CMV manifestations, the sensitivity of culture from PMNLs and from PBMCs fractions was 100%. Viremia in PBMCs, however, was far more specific (94%) than in PMNLs (74%). Cutoff values established previously for pp65 antigenemia and leukoDNAemia, standard markers in the laboratory, had similar specificity (96% each) to PBMCs viremia, but were less sensitive (88% each). Plasma DNA-emia was both less sensitive (82%) and less specific (69%) than PBMCs viremia. Detection of endothelemia showed maximal specificity (100%), but inferior sensitivity (47%). All patients had PBMCs viremia before the onset of symptoms. In conclusion, infectious CMV present in PBMCs may prove to be a determinant of clinical CMV manifestations in seropositive immunocompromised individuals. Factors involved in PBMCs tropism may help to understand the pathogenetic mechanisms of CMV dissemination in this group of patients.     

Standardization of the PFA-100® platelet function test in 105 mmol/l buffered citrate: effect of gender, smoking, and oral contraceptives

The PFA-100(R) (PFA) diagnostic system for the detection of platelet dysfunction was evaluated to determine reference ranges in a normal population. The PFA determines the primary haemostasis capacity (PHC) of anticoagulated whole blood, expressed by the system's closure time (CT). In this study the CT reference ranges were determined for blood samples collected in 105 mmol/l (3.2%) buffered citrate and the effect of gender, smoking, and use of oral contraceptives on reference ranges was assessed. Each of the 309 healthy blood donors from five blood centres was confirmed to have normal platelet function before inclusion in the study. Blood samples were tested in duplicate with both the collagen/epinephrine (Col/Epi) and collagen/ADP (Col/ADP) test cartridges. PFA reference ranges (90% central intervals of measured closure times) for both cartridge types were similar for all groups. Subgroup analysis showed that neither gender nor oral contraceptive usage had any effect on PHC. The 95% cut-off value for the Col/Epi CT was slightly higher for smokers than for non-smokers, an effect more pronounced in female than in male donors. However, the small difference did not justify establishment of specific reference ranges for smokers. Data from all included subjects were pooled to calculate the CT reference ranges for blood samples collected in 105 mmol/l buffered citrate (Col/Epi 82-150 s; Col/ADP 62-100 s). Normal levels of fibrinogen, as well as normal platelet counts and normal haematocrit levels, appeared not to influence the PHC. Because slight but significant differences of the reference ranges were observed between some of the participating sites, in-house confirmation of these reference range guidelines is recommended.     

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...