ALBERT

Description: Normal human erythrocyte phosphofructokinase (ATP:c D-fructose-6, P-1- phosphotransferase, EC 2.7.1.11; PFK) has recently been shown to consist of a heterogeneous mixture of five tetrameric isozymes: M4, M3L, M2L2, ML3, and L4 (M, muscle type; L, liver type). In the light of these findings, we have investigated the molecular basis of the inherited erythrocyte PFK deficiency associated with myopathy and hemolysis (Tarui disease). The propositus, a 31-yr-old male, suffered from muscle weakness and myoglobinuria on exertion. He showed mild erythrocytosis despite laboratory evidence of hemolysis. In his erythrocytes a metabolic crossover point was found at the level of PFK; 2,3-diphosphoglycerate (2,3-DPG) was also significantly reduced. The PFK from the patient's erythrocytes consisted exclusively of the L4 isozyme, and there was a complete absence of the other four. The leukocyte and platelet PFKs from the patient showed normal activities, chromatographic profiles, and precipitation with anti-M4 antibody. These studies provide direct evidence that in Tarui disease the M-type subunits are absent; but the liver- and platelet-type subunits of PFK are unaffected. The paradox of mild erythrocytosis despite hemolysis reflects the decreased production of 2,3-DPG.

Description: Although alloantibody against the PLA1 platelet antigen is usually found in patients with posttransfusion purpura (PTP), the mechanism of destruction of the patient's own PLA1-negative platelets is unexplained. We used a sensitive immunoblot technique to detect antiplatelet antibodies in a patient with classic PTP. The patient's acute-phase serum contained antibodies against three proteins present in control (PLA1-positive) platelets: an antibody that bound to a previously unrecognized platelet protein of mol wt 120,000 [glycoprotein (GP) 120], antibodies that bound to PLA1 (mol wt 90,000), and an epitope of GP IIb (mol wt 140,000). The antibodies against PLA1 and GP IIb did not react with the patient's own PLA1-negative platelets, control PLA1-negative platelets, or thrombasthenic platelets. In contrast, the antibody against GP 120 recognized this protein in all three platelet preparations, but not in Bernard-Soulier or Leka (Baka)-negative platelets. Antibody against GP 120 was not detected in the patient's recovery serum, although the antibodies against PLA1 and GP IIb persisted. F(ab)2 prepared from the patient's acute-phase serum also bound to GP 120. These results suggest that in PTP, transient autoantibody production may be responsible for autologous (PLA1-negative) platelet destruction. In addition, alloantibodies against more than one platelet alloantigen may be found in this disease. The nature of the GP 120 autoantigen and the GP IIb- related alloantigen defined by our patient's serum remains to be determined.

Description: Transmission of viral diseases through blood products remains an unsolved problem in transfusion medicine. We have developed a psoralen photochemical system for decontamination of platelet concentrates in which platelets are treated with long wavelength ultraviolet radiation (UVA, 320–400 nm) in the presence of 8-methoxypsoralen (8-MOP). Bacteria, RNA viruses, and DNA viruses ranging in genome size from 1.2 x 10(6) daltons, encompassing the size range of human pathogens, were inoculated into platelet concentrates and subjected to treatment. This system inactivated 25 to 30 logs/h of bacteria Escherichia coli or Staphylococcus aureus, 6 logs/h of bacteriophage fd, 0.9 log/h of bacteriophage R17 and 1.1 logs/h of feline leukemia virus (FeLV) in platelet concentrates maintained in standard storage bags. Platelet integrity and in vitro function before, immediately following photochemical treatment, and during prolonged storage after treatment, were evaluated by measuring: (1) extracellular pH; (2) platelet yields; (3) extracellular lactate dehydrogenase (LDH) levels; (4) platelet morphology; (5) platelet aggregation responsiveness; (6) thromboxane beta-2 (TXB-2) production; (7) dense body secretion; and (8) alpha granule secretion. These assays demonstrated that this photochemical inactivation system inactivated bacteria and viruses in platelet concentrates with minimal adverse effects on the in vitro function of platelets in comparison to untreated control concentrates maintained under current, standard blood bank conditions.

Description: Introduction: INTERCEPT Plasma (I-FFP) is prepared as FFP for transfusion using photochemical treatment (PCT) with amotosalen HCl (S-59, 150 μM) and UVA light (3J/cm2) to inactivate a broad spectrum of blood-borne pathogens. Phase 3 clinical trials, supported with an inventory of 〉 19,000 I-FFP units processed in 200 mL/unit with a prototype set, demonstrated retention of coagulation factor activities and hemostatic function in I-FFP for support of patients with congenital and acquired coagulopathies or TTP. For commercial introduction, the clinical prototype I-FFP system has been improved to treat up to 650 mL of plasma in a single, less time-consuming, PCT process yielding up to three 200 mL I-FFP doses per PCT process. The effects of this modified process on coagulation factor activity and yield were evaluated in two European blood centers under routine operating conditions. Methods: A total of 60 plasma units (approximately 600 mL/unit) were collected using the Autopheresis C device (Baxter-Fenwall) at Haukeland University Hospital and the Institute of Immunology and Transfusion Medicine, University of Lübeck. I-FFP units were prepared at each center by blood bank personnel using the improved set. Baseline and I-FFP samples were collected, frozen (

Description: Normal human erythrocyte phosphofructokinase (ATP:c D-fructose-6, P-1- phosphotransferase, EC 2.7.1.11; PFK) has recently been shown to consist of a heterogeneous mixture of five tetrameric isozymes: M4, M3L, M2L2, ML3, and L4 (M, muscle type; L, liver type). In the light of these findings, we have investigated the molecular basis of the inherited erythrocyte PFK deficiency associated with myopathy and hemolysis (Tarui disease). The propositus, a 31-yr-old male, suffered from muscle weakness and myoglobinuria on exertion. He showed mild erythrocytosis despite laboratory evidence of hemolysis. In his erythrocytes a metabolic crossover point was found at the level of PFK; 2,3-diphosphoglycerate (2,3-DPG) was also significantly reduced. The PFK from the patient's erythrocytes consisted exclusively of the L4 isozyme, and there was a complete absence of the other four. The leukocyte and platelet PFKs from the patient showed normal activities, chromatographic profiles, and precipitation with anti-M4 antibody. These studies provide direct evidence that in Tarui disease the M-type subunits are absent; but the liver- and platelet-type subunits of PFK are unaffected. The paradox of mild erythrocytosis despite hemolysis reflects the decreased production of 2,3-DPG.

Description: We have established a murine model and techniques with which to serially study thrombocytopoiesis after induction of experimental immune thrombocytopenia of variable severity and duration. Bone marrow megakaryocyte ploidy distribution was determined by using unfractionated bone marrow, a polyclonal megakaryocyte-specific probe, and two-color, fluorescence-activated flow cytometry. With these techniques, the modal megakaryocyte ploidy class in normal murine bone marrow was 16N. Serial studies of bone marrow megakaryocyte ploidy after the induction of acute, severe thrombocytopenia (platelet count, less than 0.05 X 10(6) microL) demonstrated no detectable change in the ploidy distribution at 12, 24, and 36 hours after the onset of thrombocytopenia. At 48 hours, the modal ploidy class shifted from 16N to 32N, and the 64N class increased significantly (P less than .001). The ploidy distribution returned to normal 120 hours after the onset of thrombocytopenia. A lesser degree of thrombocytopenia (platelet count reduction to 0.100 to 0.200 X 10(6)/microL) delayed the modal ploidy class shift from 16N to 32N until 72 hours after the onset of thrombocytopenia. Chronic, severe thrombocytopenia (platelet count, less than 0.05 X 10(6)/microL for seven days) resulted in a modal ploidy class shift from 16N to 32N during the thrombocytopenic phase and an enhanced increase in the 64N megakaryocyte class during the recovery phase. Mean platelet volume (MPV) was simultaneously measured on isolated total platelet populations after induction of thrombocytopenia. MPV was significantly increased (P less than .001) as early as eight hours after the onset of acute, severe thrombocytopenia, 40 hours before a shift in the ploidy distribution. Mild thrombocytopenia (platelet count reduction to 0.400 X 10(6)/microL) was not associated with a ploidy shift but did result in a significantly increased MPV (P less than .001). These studies demonstrate that the temporal relationship and magnitude of the effects of thrombocytopenia upon megakaryocyte ploidy distribution are dependent upon the degree and the duration of the thrombocytopenic stimulus and that the effects of experimental thrombocytopenia on platelet volume and megakaryocyte ploidy are dissociated.

Description: Although alloantibody against the PLA1 platelet antigen is usually found in patients with posttransfusion purpura (PTP), the mechanism of destruction of the patient's own PLA1-negative platelets is unexplained. We used a sensitive immunoblot technique to detect antiplatelet antibodies in a patient with classic PTP. The patient's acute-phase serum contained antibodies against three proteins present in control (PLA1-positive) platelets: an antibody that bound to a previously unrecognized platelet protein of mol wt 120,000 [glycoprotein (GP) 120], antibodies that bound to PLA1 (mol wt 90,000), and an epitope of GP IIb (mol wt 140,000). The antibodies against PLA1 and GP IIb did not react with the patient's own PLA1-negative platelets, control PLA1-negative platelets, or thrombasthenic platelets. In contrast, the antibody against GP 120 recognized this protein in all three platelet preparations, but not in Bernard-Soulier or Leka (Baka)-negative platelets. Antibody against GP 120 was not detected in the patient's recovery serum, although the antibodies against PLA1 and GP IIb persisted. F(ab)2 prepared from the patient's acute-phase serum also bound to GP 120. These results suggest that in PTP, transient autoantibody production may be responsible for autologous (PLA1-negative) platelet destruction. In addition, alloantibodies against more than one platelet alloantigen may be found in this disease. The nature of the GP 120 autoantigen and the GP IIb- related alloantigen defined by our patient's serum remains to be determined.

Description: A quantitative high yield method utilizing isosmotic arabino-galactan (Stractan) solutions and isopycnic centrifugation was developed to isolate and to fractionate total human platelet populations into density-dependent subpopulations. Isolated platelets were free of factor VIII/von Willebrand factor and other plasma proteins. They responded to ADP, epinephrine, and collagen with a sensitivity equal to platelet-rich plasma platelets. The correlation of platelet density with volume and ultrastructure was examined for normal subjects. Recovery of total platelet populations averaged 92.76%+/-3.64% (SD). Normal individuals exhibited a narrow range of platelet buoyant density distribution. Computerized probability plot analysis of platelet volume distribution for 15 normal subjects' total platelet populations showed a mean volume of 5.17+/-0.46 cu mum (SD). Platelets with buoyant density less than or equal to 1.062 g/ml had a mean volume of 4.50+/- 0.48 cu mum, while platelets with buoyant density greater than 1.071 g/ml, but less than or equal to 1.084 g/ml, had a mean volume of 5.32+/- 0.63 cu mum (SD). The volume difference by paired t test was significant, p less than 0.001. Thin-section electron microscopy demonstrated a significant reduction of granule content in light platelets, as compared to heavy platelets, but an equal number of mitochondria for both groups. These differences of platelet volume and structure between light and heavy platelets suggested that aging may be a determinant of platelet heterogeneity.

Description: Transfusion-induced hemochromatosis is a major complication in the therapy of severe chronic anemia. Improvement of transfused cell survival with a reduction in transfusion frequency is one possible approach to this problem. Using continuous-flow centrifugation (CFC), young red blood cells (YRBC) with enhanced in vivo survival have been isolated, but the expense and donor time required with this technique prohibit its widespread use for patient support. We studied the use of the IBM 2991 cell washer (CW) to isolate YRBC ex vivo from previously collected donor blood. Age-dependent red cell separation could be achieved using this instrumentation. Autologous mean red cell half-life (RBC-T50) (n = 9) for the younger cell fractions was 43.9 +/- 7.8 days compared to 34.7 +/- 5.8 days for the older cell fractions (n = 6, p less than 0.05). Paired measurement of RBC-T50 for young and old fractions in three donors showed an average survival increase of 41% for the YRBC. Adequate quantities of YRBC with enhanced survival can be obtained with less cost and less donor stress using the CW system compared to CFC. This approach could improve the management of patients with chronic transfusion requirements and merits further examination.