Lymphocyte transfusions prevent diabetes in the Bio-Breeding/Worcester rat.

The Bio-Breeding/Worcester (BB/W) rat develops spontaneous autoimmune diabetes similar to human insulin-dependent diabetes mellitus. Transfusions of whole blood from the nondiabetic W-line of BB/W rats prevent the syndrome in diabetes-prone recipients. We report three experiments designed to determine which blood component is protective. In all experiments, diabetes-prone BB/W rats 23 to 35 d of age were given four or six weekly intravenous injections. In the first experiment, animals received either saline or transfusions of erythrocytes, white blood cells, or plasma from W-line donors. Diabetes occurred in 7/22 (32%) erythrocyte, 2/27 (7%) white cell, 14/24 (58%) plasma, and 15/27 (56%) saline recipients (P less than 0.001). At 120 d of age, peripheral blood was obtained from nondiabetic rats. Fluorescence-activated cell sorter analysis of OX 19 tagged leucocytes revealed 35% T lymphocytes in white cell recipients (n = 13), compared with 9% in saline recipients (n = 7; P less than 0.001). Responsiveness to concanavalin A was also increased in the white cell group, whereas the frequency of both insulitis and thyroiditis was decreased. In the second experiment, 1/19 (5%) rats transfused with W-line spleen cells developed diabetes, as contrasted with 12/18 (67%) recipients of diabetes-prone spleen cells and 19/31 (61%) noninjected controls (P less than 0.001). In the third experiment, diabetes-prone rats received either W-line blood treated with a cytotoxic anti-T lymphocyte antibody plus complement, untreated blood, or saline. Diabetes occurred in 8/20 (40%), 1/20 (5%), and 13/19 (68%) rats in each group, respectively (P less than 0.001). We conclude that transfusions of W-line T lymphocytes prevent diabetes in the BB/W rat.

The value of intravenous heme-albumin and plasmapheresis in reducing postoperative complications of orthotopic liver transplantation for erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is marked by a deficiency of ferrochelatase, which occurs in all cells and tissues, preventing effective conversion of proto porphyrin IX to heme and thereby blocking effective feedback inhibition of heme synthesis. The major source of the excess protoporphyrin is the bone marrow. Protoporphyrin IX may accumulate, with resultant toxicity chiefly of the marrow, skin, nervous system, and liver. Orthotopic liver transplantation (OLT) is, at present, the only adequate intervention for severe liver compromise secondary to protoporphyrin deposition, but it has been complicated by severe photosensitivity and polyneuropathy. Intravenous heme and plasmapheresis have been proposed but not previously reported as means to reduce the protoporphyrin burden before liver transplantation. We report a man with EPP who underwent preoperative heme-albumin administration and plasmaphereses that led to marked reductions in plasma and erythrocyte protoporphyrin levels. His OLT was uneventful, and he developed neither polyneuropathy nor exacerbation of photosensitivity.

Expression of translation initiation factors elF-4E and elF-2alpha and a potential physiologic role of continuous protein synthesis in human platelets.

It is generally believed that platelets do not have a functionally significant protein synthetic machinery. However, our analysis demonstrated that normal bone marrow megakaryocytes express high levels of translation initiation factors eIF-4E and eIF-2alpha and the expression of these protein synthesis initiation factors is continued in platelets (as determined by immunohistochemistry and Western blot analysis). Both eIF-4E and eIF-2alpha are key regulators of protein synthesis. The eIF-4E is a rate-limiting part of a multisubunit complex, eIF-4F, that binds to the 5' cap structure present in virtually all eukaryotic mRNAs, and carries out transfer of mRNAs to ribosomes for translation. Translation initiation factor eIF-2alpha is also a rate-limiting protein which associates with two other proteins to form an eIF-2 initiation factor complex responsible for the transfer of initiator methionyl-tRNA to the 40S ribosomal subunit. We confirm that expression of eIF-4E and eIF-2alpha is biologically relevant in that platelets continue protein synthesis, albeit at a 16 times lower rate than WBC (as determined by 35S-labeled amino acid incorporation, SDS-PAGE and scintillation counting). Finally, we determined that protein synthesis inhibitors (puromycin and emetine) attenuate the platelet aggregation response to a combination of ADP and epinephrine, but potentiate the response to collagen. Our data are consistent with the existence of different signal transducing pathways mediating the response to ADP/epinephrine and collagen. We suggest that the ADP/epinephrine response is positively affected by continuously synthesized proteins, while the response to collagen is modulated by continuously produced inhibitory proteins. Taken together, our results suggest that continuous protein synthesis is important for platelet function and its role in platelet physiology and pathophysiology deserves further study.

Comparison of the effects of transfusions of cryopreserved and liquid-preserved platelets on hemostasis and blood loss after cardiopulmonary bypass.

OBJECTIVE: The aim of the study was to compare the clinical effects and hemostatic efficiency of transfusions of platelets preserved in the frozen state for as long as 2 years with transfusions of platelets preserved in the conventional manner for as long as 5 days in patients undergoing cardiopulmonary bypass. METHODS: Seventy-three patients were prospectively randomly assigned to receive transfusions of cryopreserved or liquid-preserved platelets. Nonsurgical blood loss was measured during and after the operation. Bleeding time, hematologic variables, and the bleeding time site shed blood were assayed before cardiopulmonary bypass and at 30 minutes and 2, 4, and 24 hours after transfusion. In vitro platelet function tests were conducted on platelets obtained from healthy volunteers. RESULTS: No adverse sequelae of the transfusions were observed. Blood loss and the need for postoperative blood product transfusions were lower in the group receiving cryopreserved platelets. Lower posttransfusion platelet increments and a tendency toward decreased platelet survival were observed in patients receiving cryopreserved platelets. Hematocrit and plasma fibrinogen were significantly higher in this group, and the duration of intubation was shorter. In vitro, cryopreserved platelets demonstrated less aggregation, lower pH, and decreased response to hypotonic stress but generated more procoagulant activity and thromboxane. CONCLUSIONS: (1) Cryopreserved platelet transfusions are superior to liquid-preserved platelets in reducing blood loss and the need for blood product transfusions after cardiopulmonary bypass. (2) The reduction in blood loss in the patients receiving cryopreserved platelet transfusions after cardiopulmonary bypass probably reflects improved in vivo hemostatic function of cryopreserved platelets. (3) Some in vitro measures of platelet quality (aggregation, pH, hypotonic stress) may not reflect in vivo quality of platelet transfusions after cardiopulmonary bypass, whereas other in vitro measures (platelet procoagulant activity and thromboxane) do.

Survival of autotransfused red blood cells recovered from the surgical field during cardiovascular operations.

The survival of autologous red blood cells (RBCs) collected during operation from the surgical field and processed immediately by the Haemonetics Cell Saver was compared to the survival of autologous nonprocessed RBCs obtained by venipuncture in nine patients undergoing reconstructive vascular operations and four patients undergoing coronary artery bypass. A double isotope technique (Cr-51 and In-111) was used to determine the survival of the different cell populations. Seven patients undergoing coronary artery bypass served as controls to characterize the isotopes by labeling the same population of RBCs with each radionuclide. Comparison of the data in all groups failed to show any significant difference in either the immediate or long-term survival between autotransfused (Cell Saver--processed) blood and nonprocessed RBCs. This study indicates that shed blood collected and processed at operation with the Haemonetics Cell Saver can be autotransfused and that the in vivo survival of these cells is not significantly different from the survival of nonprocessed blood.

Hematologic changes during and after cardiopulmonary bypass and their relationship to the bleeding time and nonsurgical blood loss.

The hemostatic dysfunction induced by cardiopulmonary bypass is due, in part, to a platelet dysfunction evidenced by a postoperative extension of the bleeding time; it leads to increased postoperative blood loss and morbidity. This study, which was conducted in 85 patients undergoing cardiopulmonary bypass, was designed to characterize the hematologic changes during and after cardiopulmonary bypass and to elucidate the relationships between these changes, the extension of the bleeding time, and the magnitude of the postoperative nonsurgical blood loss. Variables were measured before, during, and 2, 24, 48, and 72 hours after cardiopulmonary bypass. Univariate and multivariate analyses were performed with either the 2-hour postbypass bleeding time or the 4-hour postbypass blood loss as the dependent variables. The reversal of the extension of the bleeding time in the postoperative period was accompanied by a significant increase in the mean platelet volume and by a significant increase in the level of thromboxane B2 measured in the blood shed from the site of the bleeding time determination. The postoperative bleeding time correlated with the postoperative blood loss, and both parameters were dependent on the duration of cardiopulmonary bypass. In addition, the postoperative bleeding time correlated with the skin temperature and the plasma level of D-dimer, while the postoperative blood loss also correlated with temperature and the plasma levels of C3. These data establish a direct relationship between the postoperative bleeding time, the postoperative blood loss, and temperature. They indicate that the reversal of the postoperative extension of the bleeding time is due in part to rewarming and to the release of larger platelets into the circulation, and they suggest that hyperfibrinolysis and complement activation may play an important role in the cardiopulmonary bypass-induced platelet dysfunction.

A new low ionic strength test for assessment of pretransfusion compatibility. Studies in vitro and in vivo.

A new simple low ionic strength antiglobulin test (LIS-AGT) is presented for use in the antibody screening phase of pretransfusion tests. The ionic strength during the incubation phase of LIS-AGT is held between 15-17% of that of indirect AGT, and 8 mM EDTA is added to serum to inhibit false-positive tests. The prevalence of false-positive LIS-AGT was determined to be approximately two times higher than that observed with the indirect AGT. The new test was superior to the indirect AGT in detecting antibodies specific to Rh, Duffy, Kidd, and MNSs antigens, while the indirect AGT was superior in detecting antibodies specific to K and Lewis antigens. On the basis of three 51Cr red blood cell (RBC) survival studies, it was shown that antibodies reactive with LIS-AGT only decreased the long-term survival of incompatible erythrocytes, although the one-hour recovery was not affected. It appeared that antibodies reactive by LIS-AGT only can cause delayed rather than acute hemolytic transfusion reactions. The data shown indicate that the LIS-AGT is a simple and valuable addition to the pre-transfusion antibody screening test.

Mechanism of enhancement of blood group antibody reactions in low ionic strength.

When the ionic strength of blood samples is reduced, blood group antibodies bind to red blood cells that lack the corresponding antigen. Upon normalization of the ionic strength, the antibodies elute into the supernatant fluid. We compared, in semiquantitative terms, this nonspecific and reversible binding of various blood group antibodies. Studied with anti-CD antibodies showed that the nonspecific binding increased when the ion strength was progressively lowered. In 1/10 normal ionic strength, practically all of a high-titer anti-CD antibody bound to Rh(D)-negative RBC (phenotype ce). The majority of the other antibodies studied (anti-C, -c, -Fya, -Fyb, and -S) behaved similarly in 1/10 normal ionic strength, but the binding of some (anti-E, -s, and -Jka) was, at times, less complete. Most examples of anti-K antibodies, however, demonstrated very poor nonspecific binding to K-negative RBC in low ionic strength (LIS). The authors propose that the augmentation of specific blood group antibody reactions in LIS is the direct result of this nonspecific binding, and that the failure of these conditions to support the nonspecific binding of anti-K antibodies might explain the lack of augmentation (and, at times, even the impairment) of the reactions of anti-K with its antigen.

Electron microscopic and immunochemical studies in a patient with hepatitis C virus infection and mixed cryoglobulinemia type II.

The authors describe patient with hepatitis C virus (HCV) infection and mixed cryoglobulinemia type II in whom multiorgan failure was associated with deposits of typical, electron-microscopically visualized paracrystalline tubules in the organs studied. The patient's plasma cryoprecipitate was comprised of monoclonal IgM rheumatoid factor, polyclonal IgG, HCV RNA, and complement component C3. Of the polyclonal IgG, almost half was anti-HCV. The molar ratio between IgG and IgM was approximately 1.5 to 1. On peripheral blood films the cryoprecipitate formed cloudlike structures, which may be a useful diagnostic clue in mixed cryoglobulinemia type II. The ultrastructure of plasma cryoprecipitate and of deposits in skin, renal glomerular capillaries, and blood monocytes was identical. The cross-sectional diameter of the tubules was 30.7 +/- 1.6 nm (mean +/- 1 SD), and they appeared to be surrounded by eight electron-lucent dots. Deposition in organs of complexes containing HCV antigens and antibodies, rheumatoid factor, and C3 contributed to the multiorgan disease in this patient.

Effect of recombinant human erythropoietin on transfusion risk in coronary bypass patients.

BACKGROUND: Patients having a cardiac operation frequently require allogeneic blood transfusions despite surgical blood-conservation techniques. Recombinant human erythropoietin (Epoetin alfa) may augment this conservation by stimulating erythropoiesis. The safety and efficacy of perioperative use of Epoetin alfa to reduce the need of allogeneic transfusion was studied. METHODS: A multicenter double-blind, placebo-controlled, parallel-group study involved 182 patients having coronary artery bypass grafting and randomized to receive Epoetin alfa (300 or 150 IU/kg) or placebo subcutaneously for 5 days before, on the day of, and for 2 days after operation. RESULTS: Perioperative Epoetin alfa resulted in greater increases in baseline to preoperative hemoglobin levels and hematocrit (300 IU/kg) and in presurgery to postsurgical day 1 reticulocyte counts versus placebo (p < or = 0.05). However, there was no significant difference in transfusion requirements. Incidences of adverse events were similar in all study groups. CONCLUSIONS: Lower incidences of allogeneic blood exposure were observed in both Epoetin alfa-treated groups; however, the differences between all treatment groups were not significant. This was probably due to the relatively short 5-day preoperative course of Epoetin alfa therapy. There were no significant differences between the three groups relative to safety. Epoetin alfa was well tolerated in this population.

In vivo survival of red blood cells processed by a bubble or membrane oxygenator during cardiopulmonary bypass surgery.

A dual radioisotope labeling technique was utilized to assess red cell survival differences between cells processed by either a bubble oxygenator (eight patients) or membrane oxygenator (eight patients) in 16 patients undergoing cardiopulmonary bypass surgery. Cells processed by a bubble oxygenator consistently had a shortened survival. The 30-minute recovery of cells was not significantly different between oxygenators in contradiction to some previous studies using plasma hemoglobin as an indicator of hemolysis. The results of this investigation confirm previous studies that a membrane oxygenator provides a survival advantage to red cells during cardiopulmonary bypass surgery.

A new method to collect granulocytes using a low dose of hydroxyethyl starch.

A new granulocyte collection method employing discontinuous flow centrifugation and low donor dose of hydroxyethyl starch (HES) is presented. Following the procedure, the increase in serum polysaccharide concentration was 100 mg/dl, about 20% of that seen after standard discontinuous flow leukapheresis. The average efficiency of granulocyte collection by this method is about 64%. The average yield of WBC from 2,600 ml of donors' whole blood is 1.64 +/- 0.54 x 10(10) (mean +/- 1 SD) and the average granulocyte yield is 1.33 +/- 0.51 x 10(10) (mean +/- 1 SD). The granulocytes were shown to migrate to the sites of infection in vivo. This approach is an improvement over standard discontinuous flow leukapheresis because of the high efficiency of granulocyte collection and because multiple procedures are possible without exposing donors to more HES than is currently given during a single procedure.

PA-IgG on platelet subpopulations of different buoyant densities: high levels on the low density population is an artifact.

We isolated subpopulations of normal blood platelets from platelet rich plasma (PRP) and, in parallel, from platelet rich saline (PRS) on the basis of their buoyant densities. The MPV, being smallest in the lightest subpopulation, correlated significantly with platelet buoyant density (P < 0.002). In agreement with previous reports, we showed that the least dense platelet subpopulation, separated from PRP, had a high level of PA-IgG (3,486 mol/plt). This value was significantly greater (P < 0.02) than PA-IgG in the lightest subpopulation separated from PRS (693 mol/plt). We believe that the procedure of separating platelets from PRP artificially increased PA-IgG in the least dense subpopulation. Our data showed that when subpopulations were prepared from PRS, PA-IgG correlated with platelet buoyant density (P < 0.002), and that the subpopulation with the highest density had the highest level of PA-IgG.

Studies on the preservation of red blood cells. Attachment of the third component of human complement to erythrocytes during storage at 4 degrees C.

Utilizing an automated antiglobulin test, we have investigated the presence of the third and fourth components of human complement on normal red blood cells (RBCs). Only negligible amounts of the fourth component, C4, could be detected on either freshly collected or stored RBCs. The fragment C3d of the third component, C3, was detectable on both freshly collected and stored normal RBCs. A product derived from C3 and reacting with anti-C3c antibody was only barely detectable on freshly collected normal RBCs. During storage of blood at 4 degrees C, increasing quantities of this material were detected on the RBC membrane. Bromelin treatment rendered stored RBCs completely nonreactive with anit-C3c antibody, whereas only partial loss of reactivity was observed following incubation with heated plasma. In contrast, incubation of EC43 with heated plasma completely abolished their ability to react with anti-C3c antibody. We suggest that the presence of this C3 fragment on stored RBCs may contribute to the development of "preservation injury".

Quantitative studies on the Rh-antigen D. Effect of the C gene.

Using an automated antiglobulin consumption test and Scatchard's analysis, the authors measured the number of D antigen epitopes on each of 46 samples of washed red cells (RBC) that represented six different Rh phenotypes. A clear D dosage effect was observed only when C was missing from the Rh genome, which confirmed the previously recognized suppressive effect of C. Thus, RBC of the Rh phenotype R2R2 had almost twice as many D epitopes (mean, 30,300/cell) as RBC of the Rh phenotypes R2r and R0r (means, 17,200 and 16,500 respectively). The data indicated that C in trans and cis suppressed similar amounts of D epitopes.

Evaluation of a large-scale frozen blood program.

The characteristics of previously frozen red blood cells, prepared in a large-scale frozen blood program using the Red Cross method were evaluated. The use of the method as originally described resulted in approximately 91 per cent freeze-thaw-wash recovery of red blood cells. When the glycerolization step was modified by adding the partially glycerolized erythrocytes into 300 ml of 6.2M glycerol, freeze-thaw-wash recoveries were decreased. However, gradient addition of glycerol to the red blood cells without the use of stylet, resulted in acceptable in vitro recoveries. Thawing frozen units in waterbath, to which no antiseptic was added, could introduce bacteria into units of previously frozen red blood cells. Therefore, it seems advisable to use dry heat thawing procedures. Previously frozen red blood cells prepared in the large scale maintained normal levels of ATP and 2,3 DPG. Therapeutic transfusions had acceptable 24-hour survival in vivo.

Quantitative studies on the D antigen of red cells with the Du phenotype.

Rh-positive red cells (RBCs) that fail to agglutinate with commercial anti-D reagents by the tube method are considered to have the Du phenotype. The quantities of D epitopes on such RBCs have been measured previously in one kindred. The authors report on the number of D epitopes on Du RBCs of 23 unrelated individuals, as calculated by Scatchard's analysis. Cell-bound anti-D was measured by an automated antiglobulin consumption technique. On the average, RBCs of the Rh phenotype CcDue had a mean of 1568 +/-1220 (n = 12) D epitopes per cell. The relatively large range of values in this group implies a heterogeneous genetic background. The lowest number of D epitopes, 285 per cell, was observed on the RBCs of one individual who was apparently homozygous for C. In this case, the D antigen was detected only by adsorption/elution tests. RBCs of the phenotype cDuEe had a mean of 775 +/- 378 epitopes per cell (n = 8), and those from two individuals with phenotype cDue had 2840 and 1560 D epitopes, respectively. Thus, on the average, RBCs with the Du phenotype bear about 10 to 20 times less D antigen than normal Rh-positive RBCs. It is suggested that the low D antigen density of Du red cells may account for their poor immunogenicity.

Measurement of fragments of the third component of human complement on erythrocytes by a new immunochemical method.

This paper describes a new antiglobulin consumption method to quantitate the fragments of the third component of human complement (C3) on red blood cell (RBC) membranes. Zymosan-bound C3, which can be stored frozen at -80 degrees C for years, was used as a standard in these tests. Using anti-C3c antibody, zymosan-bound C3 could be calibrated against soluble converted C3 (beta 1A), but not against soluble, native C3 (beta 1C). Calibration with several commercial serum standards yielded virtually identical values. Approximately 79.8 +/- 28.2 C3d molecules (mean +/- 1 SD, n = 50) were detected on normal, freshly collected RBC by this method, whereas no C3c fragments were noted. EC43, prepared by dilution of blood samples with low ionic strength solution, had between 650 and 3,100 C3 molecules/RBC when measured with anti-C3c and between 1,140 and 6,500 C3 molecules when measured with anti-C3d. These data indicated that part of the C3b molecules on EC43 had cleaved to C3d. EC43 are reported to have up to 200,000 C3 molecules when measured by other techniques. To resolve this discrepancy, EC43 were prepared by dialysis of blood samples against low ionic strength solution. About 97.5% of C3 remained in plasma after dialysis supporting the results of our tests. The new assay is an accurate and sensitive method of quantitating C3 fragments bound to RBC in vivo and in vitro.