Study of lymphocyte subpopulations in peripheral blood and secondary lymphoid organs in the goat using monoclonal antibodies to surface markers of bovine lymphocytes.

Monoclonal antibodies (mAbs) to surface markers of bovine lymphocytes MHC I, MHC II, B-cells, T-cells (CD2, CD4, CD8 and gamma/delta) and interleukin-2 (IL-2) receptor were tested in the goat by flow cytometry and using immunohistochemical methods. Samples from peripheral blood and secondary lymphoid organs (mesenteric lymph nodes, spleen and ileal Peyer's patch) were studied. The percentage of positive cells obtained by flow cytometry and its compartmentalisation in different tissue sections showed that the mAbs against MHC I, MHC II, CD2, CD4, CD8, gamma/delta and IL-2 receptor recognised lymphocyte subpopulations similar to those present in the bovine. However, the mAbs tested on B-cells reacted only partially in the recognition of this subpopulation.

Cryopreservation modifies flow-cytometric analysis of hemopoietic cells.

Although cryopreservation of human bone marrow has become very common in modern medicine, the knowledge on the effects of this procedure on hemopoietic cells is still limited. We herein have investigated whether the process of concentrating of human bone marrow to its buffy coat, the exposure to the cryoprotectant dimethyl sulfoide (DMSO), and/or the rate of controlled freezing/thawing procedures modifies the flow cytometric analysis of human bone marrow cells. We found that both the exposure of marrow cells to DMSO and/or the freezing procedure significantly modifies both the relative proportions of hemopoietic cell subsets and the intensity of expression of certain surface antigens. Thus, percentages of cells expressing CD7, CD13, CD33 and CD34, were found to be lower in both cryopreserved buffy coat and buffy coat merely exposed to DMSO, in comparison to those in untreated coat samples. Moreover, the intensity of the surface expression of CD33 decreased in cells exposed to DMSO, and further in cryopreserved samples. By contrast, the intensity of the CD19 antigen was higher in the last groups of samples than in the buffy coat or the unfractionated human bone marrow. Our present results suggest that flow-cytometric analysis of cryopreserved human bone marrow cells is not fully equivalent to that corresponding to fresh bone marrow or its fractionated buffy coat.

Comparative study of three methods to detect free plasma antiplatelet antibodies.

We have compared three techniques for the detection of plasma circulating antiplatelet antibodies, i.e., the platelet suspension immunofluorescence test (PSIFT), the platelet radioactive antiglobulin test (PRAT), and the monoclonal antibody immobilization of platelet antigens (MAIPA). Frozen plasma samples from patients with idiopathic thrombocytopenic purpura or HIV-associated thrombocytopenia were used in the study. The PSIFT and PRAT showed the appropriate ease of performance necessary for screening purposes. The PSIFT is free of radioactivity hazards, but seemed to be less sensitive than the PRAT. The MAIPA is a useful tool to detect antibodies against glycoproteins (GPs) Ib/IX and IIb/IIIa. However, in comparison to PSIFT and PRAT, MAIPA is more time consuming, requires considerable technical expertise, and the identification of antiplatelet activity is highly dependent on the selection of an appropriate primary anti-GP monoclonal antibody. This could explain the lower prevalence of antiplatelet activity detected by MAIPA, in comparison to the frequency provided by the PSIFT and PRAT.

Stability of glycoproteins Ib/IX and IIb/IIIa during preparation and storage of platelet concentrates: detection by binding assays with epitope-defined monoclonal antibodies and physiological ligands.

Preservation of the glycoprotein (GP) complexes Ib/IX and IIb/IIIa, because of their role as specific platelet receptors for adhesive proteins, is essential for the haemostatic efficacy of transfusions of platelet concentrates (PCs). We have combined binding assays with epitope-defined monoclonal antibodies, and with the physiological ligands von Willebrand factor and fibrinogen (Fo), to investigate the total expression and functional status of these receptors, in PCs stored for up to 9 days. Routine preparation and storage for up to 5 days have no apparent effect on the surface expression and the functional status of GPs Ib/IX and IIb/IIIa. However, after prolonged storage for up to 9 days we found a 50% decrease in the level of the total and functional GP Ib/IX content of platelets. This finding parallelled a significant rise in plasma glycocalicin, a proteolytic fragment of GP Ib. In addition, long-term storage promoted an impairment in the exposure of GP IIb/IIIa to Fo, and a 50% decrease in Fo binding capacity, without affecting the complex quantitatively. Finally, we also noticed a storage-induced increase in the platelet surface expression of GMP 140, and after 9 days of storage there was a rise in mean platelet volume, and a significant reduction in pH levels.

Association of autoantibodies against platelet glycoproteins Ib/IX and IIb/IIIa, and platelet-reactive anti-HIV antibodies in thrombocytopenic narcotic addicts.

The levels of platelet-associated Igs (PAIgs) and plasma circulating antiplatelet antibodies were evaluated by a flow cytometric immunofluorescence assay (FCIFA), an enzyme-linked immunoassay (ELISA), and a platelet radioactive antiglobulin test (PRAT), in a group of 45 human immunodeficiency virus (HIV)-infected intravenous drug users (IVDUs), with or without thrombocytopenia (TCP). Direct tests demonstrated an increased amount of PAIgs in 40% of the patients, irrespective of their platelet count. These PAIgs were mainly of IgG class and could not be eluted with ether. Plasma IgG with antiplatelet activity was found in 70% of the thrombocytopenic individuals, whereas it was detected in only one patient without TCP. The relative frequencies of antibodies against the platelet glycoproteins (GPs) Ib/IX and IIb/IIIa were assessed in plasma from all patients by means of the monoclonal antibody-specific immobilization of platelet antigens assay (MAIPA). Plasmas from all non-thrombocytopenic patients were negative when tested by indirect MAIPA. In contrast, 10/23 plasma from thrombocytopenic patients reacted with either GP IIb/IIIa, GP Ib/IX, or both GPs. Finally, aiming to investigate whether HIV antibodies from these patients are reactive with normal platelets, we performed absorption-elution experiments, followed by evaluation of HIV antibodies in the indirect eluates by ELISA and Western blot. Interestingly, we detected anti-HIV antibodies that bind to normal platelet antigens in 50% of the ether eluates prepared from control platelets sensitized with plasma from patients with TCP, but in only 5% of eluates obtained from platelets sensitized with plasma from non-thrombocytopenic patients. The present study provides direct evidence that specific autoantibodies against platelet membrane GPs Ib/IX and IIb/IIIa are common in HIV positive thrombocytopenic individuals. The finding in these patients of HIV antibodies cross-reactive with normal platelets, suggests that mimicry of human antigens by HIV could play a key role in the pathophysiology of the HIV-related TCP.

Effect of rhG-CSF on the mobilization of CD38 and HLA-DR subfractions of CD34+ peripheral blood progenitor cells.

Several studies have demonstrated that both CD34+/CD38- and CD34+/HLA-DR- human hematopoietic progenitor cells have properties associated with hematopoietic stem cells. However, the kinetics of these two cell populations in human peripheral blood (PB) after priming with granulocyte colony-stimulating factor (rhG-CSF) has not been investigated. By using flow-cytometric analysis we have shown that administration of rhG-CSF to 14 patients eligible for peripheral blood progenitor cell (PBPC) transplantation led to an increment of CD34+/CD38+ and CD34+/HLA-DR+ cells in the PB that paralleled the increase of total CD34+ cells, indicating that such subpopulations are responsible for the major release of CD34+ cells. Furthermore, rhG-CSF priming led to a significant mobilization of fractions of more immature CD34+/CD38- and CD34+/HLA-DR- cells to the PB. In the leukapheresis preparations, the average frequency of CD34+ cells lacking the CD38 or HLA-DR antigens was low (5% and 30%, respectively), with little overlap between the CD38- and HLA-DR- subpopulations. In addition, the yield of each subset of CD34+ cells (CD34+/CD38 +/- and CD34+/HLA-DR +/-) in the PB correlated with the numbers in the collected material. The results of the present study indicate that administration of rhG-CSF causes a significant increase of CD34+/CD38 +/- and CD34+/HLA-DR +/- cells in PB, and that such cells can be then safely harvested by leukapheresis procedures.

Immune recovery after autologous or rhG-CSF primed PBSC transplantation.

We performed a prospective study in 17 consecutive patients following Autologous bone marrow (BM) or rhG-CSF primed peripheral blood item cell (PBSC) transplantation, with the objective of comparing immune recovery between both procedures and to evaluate results in rhG-CSF mobilized peripheral blood stem cell transplantation (PBSCT). Kinetics of immune reconstitution showed differences, with a faster recovery of CD3+ and CD8+ T cells, and a more rapid and sustained recovery of CD8+/-/CD56+ natural killer (NK) cells in the PBCSCT group. Autologous bone marrow transplantation (ABMT) was associated with a improved reconstitution of the CD19+/CD5+/-subpopulation. Moreover, rhG-CSF mobilized PBSCT generated a greater recovery of CD8+/-/CD56+ cells than previous data concerning transplantation with peripheral blood (PB) progenitors collected after myelosuppressive chemotherapy or myelosuppressive therapy plus rhG-CSF. Our results show differences in the rate and pattern of B and T lymphocytes reconstitution after ABMT and PBSCT. Additionally, we state an enhancement of CD56+ cells in patients undergoing PBSCT mobilized solely using rhG-CSF.