Paolo Perutelli

Processing of Human Cord Blood by Three Different Procedures for Red Blood Cell Depletion and Mononuclear Cell Recovery

Background and Objectives: Human cord blood (CB) is an important source of stem cells which may be used for hematopoietic reconstitution as an alternative to bone marrow transplantation. Banking of CB would be accomplished by removing red blood cells (RBC) and plasma from CB collections. Our aim was to compare three different procedures for CB processing. Materials and Methods: Poligeline, hydroxyethyl starch gel (HES) and gelatin were used as separation media in processing 79 CB units for RBC depletion and mononuclear cell (MNC) recovery. Results: The best MNC recoveries were obtained performing the HES– and the gelatin–based procedures (80.9 and 84.7%, respectively), but the gelatin procedure allowed us to obtain the highest RBC depletion (96.4%); CD34+ cell recovery was higher using HES or gelatin as separation media (85.6 and 85.9%, respectively). Conclusion: The best results, as far as RBC removal and MNC recovery are concerned, were obtained by using gelatin as RBC sedimentation medium. Gelatin is a low–cost, animal–derived reagent, which has been successfully used for CB transplantation; the procedure is simple to perform and appears to be suitable for large–scale banking in view of CB transplantation.

von Willebrand factor: biological function and molecular defects.

The human von Willebrand factor (vWF) plays a pivotal role in the mechanisms of blood clotting and platelet thrombus formation; it also binds and stabilizes factor VIII procoagulant protein. The biological functions of vWF are dependent on distinct molecular domains responsible for the specificity and affinity for ligands. The multimeric structure of vWF provides an array of binding sites that allow multivalent interactions, thus supporting the formation of stable platelet aggregates at the site of vascular injury, particularly under flow conditions characterized by high shear stress. Quantitative and qualitative abnormalities of vWF cause the most common congenital bleeding disorder in humans, the von Willebrand disease (vWD). This review will provide an update on the recent advances toward the elucidation of structure-function relationships and the detection of molecular defects leading to vWD and will highlight the revised classification of vWD.

Platelet Antibody Detection in Pediatric Immune Thrombocytopenic Purpura: Evaluation of Three Screening Methods

Background and objectives: Immune thrombocytopenic purpura (ITP) is a common hematologic disorder, two forms of which occur in children. The detection of circulating platelet antibodies is helpful in diagnosis. Materials and methods: We evaluated three different immunological methods for detecting platelet antibodies in the serum of children with ITP. These were: a solid‐phase red‐cell adherence test (SPRCA), an enzyme immunoassay (EIA), and an immunofluorescence test (PSIF). Results: The sensitivity of the methods in detecting IgG antibodies ranged from 28.1 (EIA) to 39.4% (SPRCA). We also looked for IgM antibodies by PSIF, thus raising the sensitivity of this test from 32.0 to 40.0%. A combination of two tests (SPRCA and EIA) allowed us to detect 61.8% positive samples. By doing all three tests, we obtained 71.3% positive samples. Finally, we reached 73.5% by adding PSIF for IgM. We found a higher frequency of circulating antibodies in both acute and chronic ITP at onset than in clinical remission. There were a few positive sera in chronic ITP, but not in the acute form in remission. Conclusion: The individual tests each have a relatively low sensitivity, but the combination of all three increases the diagnostic effectiveness. The finding of platelet antibodies during remission may predict evoluation toward a systemic autoimmune state.

Diagnosis of Glanzmann’s Thrombasthenia and Carrier Detection using Monoclonal Antibodies to Platelet Glycoprotein lIb and IIIa in Immunoblotting

Glanzmanns thrombasthenia is a rare hemorrhagic syndrome, characterized by a quantitative or functional defect of the platelet glycoprotein GPIIb-IIIa complex. The authors describe a method to diagnose thrombasthenic patients and identify carrier subjects by using monoclonal antibodies specific for GPIIb and GPIIIa in an immunoblotting technique. The immunoreaction patterns of two thrombasthenic patients lacking GPIIb or GPIIIa, respectively, are shown. The described method produces further evidence concerning the biochemical heterogeneity of Glanzmanns thrombasthenia.

A simple, rapid method for screening hybridomas producing monoclonal antibodies to platelet proteins

Several parameters influence the outcome of somatic cell fusions based on the Köhler and Milstein technology, and a number of steps are of critical importance, including the screening strategy. The procedure chosen, appropriate for the type of antibody required, should be rapid and sensitive, in order to clone the relevant hybrids as quickly as possible. A simple and quick dot blot-based method is reported, suitable for screening hybridoma culture supernatants in order to identify clones producing monoclonal antibodies to platelet constituents.

Reply: About the Von Willebrand Factor

I thank Prof. Ozsoylu for his helpful criticism about the paper, “von Willebrand Factor: Biological Function and Molecular Defects.” The von Willebrand factor (vWF) serves two distinct functions: as a cofactor for platelet adhesion to subendothelial matrix molecules, and as the circulating protein that binds and stabilizes procoagulant factor VIII. In the latter function, vWF takes role in blood clotting.