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The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

Neonatal alloimmune thrombocytopenia (NAIT) results from maternal immunization against fetal platelet antigens and can occur during the first pregnancy. The most common complications of NAIT are neonatal thrombocytopenia, intracerebral hemorrhage, and fetal death. Most cases of NAIT in Caucasians are caused by anti-HPA-1a (PlA1). Anti-HPA-5b (Bra) accounts for only 4.3 percent of all NAIT cases. NAIT due to anti-HPA-5b is thought to be milder and have fewer complications than NAIT caused by anti-HPA-1a because of the lower number of HPA-5b antigenic sites per platelet. This report describes a severe case of NAIT due to anti-HPA-5b that was treated by intrauterine platelet transfusion.

Acute cellular rejection in cardiac allografts is a major cause of graft loss, and is associated with activation of the coagulation system. We investigated whether plasma markers of coagulation predict the presence of allograft rejection.A total of 132 blood specimens and endomyocardial biopsies were collected from 35 patients, between February of 1997 and May of 1998. We measured plasma prothrombin fragment 1.2 (PF1.2) and p-selectin, fibrinogen, thrombomodulin, and d-dimer. Biopsies were graded according to the International Society of Heart and Lung Transplantation system, with a range of 0 to 4. Grades 0 and 1A were grouped as "no rejection," and the higher grades as "rejection." Linear and logistic regression, accounting for longitudinal data, were the principal analytic tools.p-Selectin level increased progressively with increasing rejection grade (P0.001). With multivariate analysis, both p-selectin and prothrombin fragment levels significantly predicted rejection. p-Selectin levels were predictive of prothrombin fragment levels (P0.0001) but not of d-dimer, fibrinogen, or thrombomodulin levels. This model allowed correct prediction of rejection, based on p-selectin and prothrombin fragment values, up to 85% of the time. Dichotomizing patients by a p-selectin level of 65 ng/ml resulted in an odds of rejection of 21.4 [95% C.I. 7.1-64.7] for the patients in the high- compared with the lower risk group.In heart transplant recipients, p-selectin levels and PF 1.2 levels are highly predictive of organ rejection. The elevation of PF 1.2 suggests that there is systemic generation of thrombin generation. These markers may be useful for noninvasively monitoring patients for organ rejection or for after response to treatment.

Point-of-care testing (POCT) can provide rapid test results, but its impact on patient care is not well documented. We investigated the ability of POCT to decrease inpatient and outpatient waiting times for cardiovascular procedures.We prospectively studied, over a 7-month period, 216 patients requiring diagnostic laboratory testing for coagulation (prothrombin time/activated partial thromboplastin time) and/or renal function (urea nitrogen, creatinine, sodium, and potassium) before elective invasive cardiac and radiologic procedures. Overall patient management and workflow were examined in the initial phase. In phase 2, we implemented POCT but utilized central laboratory results for patient management. In phase 3, therapeutic decisions were based on POCT results. The final phase, phase 4, sought to optimize workflow around the availability of POCT. Patient wait and timing of phlebotomy, availability of laboratory results, and therapeutic action were monitored. Split sampling allowed comparability of POCT and central laboratory results throughout the study.In phase 1, 44% of central laboratory results were not available before the scheduled time for procedure (n = 135). Mean waiting times (arrival to procedure) were 188 +/- 54 min for patients who needed renal testing (phase 2; n = 14) and 171 +/- 76 min for those needing coagulation testing (n = 24). For patients needing renal testing, POCT decreased patient wait times (phases 3 and 4 combined, 141 +/- 52 min; n = 18; P = 0.02). For patients needing coagulation testing, wait times improved only when systematic changes were made in workflow (phase 4, 109 +/- 41 min; n = 12; P = 0.01).Although POCT has the potential to provide beneficial patient outcomes, merely moving testing from a central laboratory to the medical unit does not guarantee improved outcomes. Systematic changes in patient management may be required.

High circulating levels of the procoagulant molecule tissue factor (TF) are associated with thrombosis in a variety of diseases including unstable angina, cancer, and sepsis. Currently, there are no clinical assays to measure the level of TF activity in whole blood. We present an assay called Tissue Factor Clotting Time ("TiFaCT") that detects fibrin formation in human blood. The mean baseline clotting time in a healthy population was 472 +/- 94 s (mean +/- SD, n = 150). Bacterial lipopolysaccharide (LPS or endotoxin) shortened the clotting time in a time-dependent manner. Inhibitory anti-TF antibodies prolonged the clotting time of LPS-stimulated blood, indicating that the shortened clotting time was due to induction of TF expression. Patients with unstable angina had shortened mean baseline clotting time (284 +/- 86, n = 13) compared with healthy volunteers (474 +/- 98, n = 30), suggesting that these patients had elevated levels of circulating TF. The TiFaCT assay should prove clinically useful in quantifying the levels of circulating TF in patients at risk of thrombosis.

The human platelet alloantigen 1 system (HPA-1) is determined by a polymorphism at position 33 in the N-terminus of human glycoprotein IIIa (GPIIIa). This naturally occurring substitution creates a conformation in the HPA-1a allelic form that can be antigenic when presented to an individual expressing the HPA-1b form. Anti-HPA-1a antibodies generated by this immune response can lead to the destruction of platelets, as seen in the clinical disorders, neonatal alloimmune thrombocytopenia (NAIT) and posttransfusion purpura (PTP). To understand better the structural requirements for recognition by these pathogenic antibodies, we investigated the N-terminal 66 amino acids from the HPA-1a form of human GPIIIa and the analogous amino acids from the nonimmunogenic murine homolog. Our objectives were to define further the boundaries of the HPA-1a epitope(s) in the N-terminus of human GPIIIa, to isolate the murine 5' nucleotide sequence and compare the deduced murine N-terminal sequence to that of human, and to mutate the murine sequence systematically to include an HPA-1a epitope(s). Murine amino acids that differed from human were changed by site-directed mutagenesis to the analogous residues in the HPA-1a form of human GPIIIa, starting and radiating from murine position 33 (site of human polymorphism). This systematic approach allowed us to pinpoint amino acids critical to a conformation recognized by anti-HPA-1a antibodies. Our results show that an HPA-1a epitope can be created within the N-terminus of murine GPIIIa and raise the possibility that murine models of HPA-1a sensitization can be developed.

The human platelet alloantigen HPA-1a (PlA1) is responsible for most cases of post-transfusion purpura and neonatal alloimmune thrombocytopenia in the Caucasian population. HPA-1a and HPA-1b are two allelic forms of the platelet membrane glycoprotein IIIa (GPIIIa) gene that differ by a single amino acid. In this report, we describe the development of a recombinant heavy chain antibody fragment capable of distinguishing between the homozygous forms of HPA-1a and HPA-1b. This antibody fragment was isolated from the lymphocytes of an immunized individual through the use of a phage display library system. The recombinant antibody fragment reacted with human platelet lysates from HPA-1a homozygous donors, the HPA-1a form of recombinant N-terminal GPIIIa and intact HPA-1a platelets, but did not react with platelet lysate from HPA-1b homozygous donors, reduced HPA-1a form of platelet GPIIIa or other platelet glycoproteins. This HPA-1a specific human antibody fragment works well in common laboratory assays such as ELISA and flow cytometry, which can assist in identifying HPA-1b homozygous individuals who are known to have a higher risk for developing neonatal alloimmmune thrombocytopenia and post-transfusion purpura. Thus, selection of recombinant antibody fragment using phage display offers a promising alternative to hybridoma technology for the production of human antibodies against human alloantigens and holds potential as a technique in therapeutic applications.

In alloimmune thrombocytopenia, maternal sensitization to a fetal platelet alloantigen results in fetal thrombocytopenia. Even in primipara, 20% of offspring can suffer intracranial hemorrhage, half of which occur in utero. Ninety percent of subsequent pregnancies will be equally or more severely affected. We describe two patients whose previous pregnancies were complicated by neonatal alloimmune thrombocytopenia (NAIT). As expected, NAIT recurred, with devastating sequelae in both cases. One case presented with hydrops fetalis, a previously unreported association; the other fetus developed extensive intracranial hemorrhages in utero. Because both previous obstetrical histories had gone unrecognized, no preventative strategies had been undertaken.Better recognition of this disease through a positive obstetrical history is needed in order to properly counsel patients and institute appropriate prenatal treatment regimens.

Autoimmune hemolytic anemia (AIHA) presents a difficult challenge to clinicians and blood bankers alike. Autoantibodies in the serum significantly complicate serologic evaluation, and necessitate performing procedures such as adsorptions to eliminate the possibility of underlying alloantibodies. In many instances the blood that is issued may be phenotypically similar but remains crossmatch incompatible, generating a considerable degree of anxiety among the clinical staff who are responsible for transfusing the patient. We report a case of warm autoimmune hemolytic anemia (WAIHA) in which the transfusion of red cells was complicated by a febrile transfusion reaction. Evaluation of the reaction resulted in a significant delay in transfusion therapy. Subsequent administration of leukocyte-poor red cells resulted in uneventful transfusions with a good therapeutic response. Retrospective analysis of the pretransfusion sample demonstrated significant levels of anti-neutrophil antibodies. This case resulted in the establishment of our policy to administer all red cell transfusions to patients with autoantibodies (warm or cold) as leukocyte- poor red cells.

Neonatal alloimmune thrombocytopenia (NATP) and post-transfusion purpura (PTP) are acquired bleeding disorders caused by alloimmune thrombocytopenia. In most cases, the thrombocytopenia is due to an alloantibody directed against the platelet glycoprotein IIb-IIIa (GPIIb-IIIa) complex. During the course of routine studies on the role of GPIIb-IIIa in inherited and acquired bleeding and thrombotic disorders, we unexpectedly identified an individual whose platelets reacted by non-reduced Western blot analysis with anti-GPIIIa polyclonal antisera, but did not react with a commercially available monoclonal antibody (SZ21) specific for GPIIIa. We screened all 14 GPIIIa exons for possible nucleotide changes which might alter amino acids and found variations in only exons 3 and 10. Nucleotide sequencing revealed that only the exon 3 alteration changed the predicted amino acid sequence. This variation was caused by homozygosity for the uncommon P1A2 allele of the GPIIIa gene. Platelets from two additional unrelated normal individuals known to be homozygous for P1A2 also lacked reactivity with SZ21 by Western blot. Using flow cytometry with intact platelets, we observed a markedly reduced binding of SZ21 to platelets with the P1A2 genotype. Scatchard analyses indicated that SZ21 bound to P1A1/A1 platelets with a Kd of approximately 8.26 x 10(-10) M, and to P1A2/A2 platelets with a Kd of approximately 5.58 x 10(-9) M. Thus, we have characterized a readily available monoclonal antibody able to distinguish between the two P1A alleles of the GPIIIa gene. Because incompatibility for this platelet polymorphism is the most common cause of neonatal alloimmune thrombocytopenia and posttransfusion purpura, and because platelet immunophenotyping reagents lack specificity and are not easily available, this monoclonal antibody could facilitate the management of patients with these disorders.

To assess the efficacy of immune globulin in preventing CMV infection, interstitial pneumonia, GVHD and death after BMT, we reviewed and synthesized data from 12 published studies (with 1282 patients) in which immune globulin was used prophylactically in BMT patients, controls were included and clinical outcomes were assessed. Data synthesis indicates that immune globulin significantly reduces fatal CMV infection (odds ratio (OR) 0.47, 95% confidence interval (CI) 0.23-0.99), CMV pneumonia (OR 0.61, CI 0.42-0.89), non-CMV interstitial pneumonia (OR 0.57, CI 0.35-0.95) and total mortality (OR 0.74, CI 0.55-0.99). The reduction in acute GVHD was not quite significant (OR CI 0.45-1.02). Complications decrease with both hyperimmune and conventional immune globulin. For CMV-negative transplant recipients, immune globulin decreases symptomatic CMV infection (OR 0.55, CI 0.31-0.94) and interstitial pneumonia (OR 0.34, CI 0.15-0.77). For CMV-positive recipients, immune globulin prevents interstitial pneumonia (OR 0.45, CI 0.26-0.80) but not symptomatic CMV infection (CI 0.41-2.80). We conclude that immune globulin is efficacious in preventing major complications of BMT in both CMV-negative and CMV-positive recipients.

Plasma converted to serum by the addition of bovine thrombin prior to compatibility testing agglutinated all donor red cells. This finding prompted an investigation of bovine thrombin- associated incompatibility that showed that (1) thrombin derived from bovine plasma contains IgG antibodies directed against all human red cells, and (2) excess (50 units/mL) bovine thrombin used for conversion of plasma to serum may cause hemagglutination and erroneous serologic test results because of the presence of heteroagglutinins.

Neonatal alloimmune thrombocytopenia is caused by the transplacental passage of maternal antibodies directed against platelet-specific antigens. Because complications from bleeding may result, prompt institution of therapy may be necessary. Accurate diagnosis by serologic techniques permits appropriate therapy to be administered both prenatally and postnatally.

Immune hemolytic anemia due to minor ABO incompatibility between recipient and donor is a well-recognized occurrence in kidney and liver transplantation. In some cases, the responsible antibodies have been shown to be derived from the donor passenger lymphocytes using Gm allotyping. We report a case of acute, transient hemolysis following heart-lung transplantation in which serologic and Gm allotype studies confirmed the etiology of hemolysis.

To determine the medical and laboratory characteristics of bacteremia secondary to transfusion of microbiologically contaminated platelet concentrates.Febrile transfusion reactions were prospectively monitored over 42 months. Units involved in reactions were evaluated with Gram's stain and culture tests.Comprehensive cancer center.Patients receiving platelet transfusions for thrombocytopenia secondary to bone marrow failure.Seven cases of transfusion-associated sepsis were observed. Multidonor platelet products stored for 5 days resulted in an incidence of sepsis five times higher than those stored for 4 days or less (P less than .01). Investigation indicates that contamination most likely occurred at the time of blood collection. Clinically, septic reactions were associated with greater temperature elevations (average increase, 2.0 degrees C) than febrile reactions to sterile products.Contamination of platelet concentrates remains a significant clinical problem. Septic episodes may be reduced by transfusion of platelets with shorter storage intervals.

Baka is a platelet alloantigen whose putative allele, Bakb, has not been identified previously. By using a serum, “Har,” obtained from a patient with posttransfusion purpura, we describe the platelet alloantigen Bakb. The Har serum reacted with an NP-40-extractable platelet membrane protein of 142 kd with mobility similar to platelet glycoprotein IIb alpha. We found that the antigen recognized by the Har serum is inherited in an autosomal dominant mode with an apparent gene frequency of .39. Chi-square analysis of observed and expected phenotype frequencies indicated that serum Har recognizes Bakb, the anticipated allele of Baka. Our findings provide new evidence for polymorphism of glycoprotein IIb and for the association of posttransfusion purpura with alloimmunization to determinants on this glycoprotein.

Posttransfusion purpura typically occurs in PLA1 negative blood recipients who have been previously immunized to the PLA1 antigen. Following transfusion, severe thrombocytopenia develops with the formation of anti-PLA1. Since the patients' platelets lack the PLA1 antigen, one would not expect this antibody to destroy autologous platelets. In this study we show that PLA1 antigen exists in stored blood and can absorb to PLA1 negative platelets making them PLA1 reactive. Incubating PLA1 (-) platelets with ultracentrifuged plasma from PLA1 (+) blood donors allowed anti-PLA1 to bind to PLA1 (-) platelets. Control plasma from PLA1 (-) blood donors did not lead to anti-PLA1 binding. Using an inhibition assay, we showed that stored blood contains PLA1 material that was not removed by ultracentrifugation. The material absorbing to PLA1 (-) platelets represented the PLA1 antigen, which was confirmed by Western blotting. After incubating plasma containing PLA1 antigen with PLA1 (-) platelets, reactivity at 95,000 D was observed. Native PLA1 (+) platelets showed a similar band. When PLA1 (-) platelets were incubated with plasma from a PLA1 (-) donor, this band was not present. These studies show that a soluble form of PLA1 antigen exists in stored blood that can absorb to PLA1 (-) platelets. Consequently, anti-PLA1 can bind to these platelets leading to thrombocytopenia. These observations may explain the autologous destruction of platelets in posttransfusion purpura.

We describe a method for determining haptoglobin with a centrifugal analyzer that is based on haptoglobin combining stoichiometrically with hemoglobin to form a complex that has peroxidase-like activity proportional to the quantity of haptoglobin present. Under assay conditions, unbound hemoglobin exhibits only a small fraction of the total peroxidase activity. Activity is measured colorimetrically at 405 nm after reaction with o-dianisdine and ethyl hydrogen peroxide. The procedure is standardized by saturating aknown amount of hemoglobin with a serum whose hemoglobin binding capacity exceeds the amount of hemoglobin in the assay system. The mean and mean within-run precision of our method, determined by performing 17 replicate assays of both a pooled normal serum and a 10-fold dilution of the serum, was 1.13 g/liter (CV, 2.9%), and 106 mg/liter (cv, 5.8%), respectively. The 95 percentile estimate of the normal range by our method is 0.45-1.85 g/liter hemoglobin binding capacity. When results by our automated method were compared to those by a manual method [Scand. J. Clin. Lab. 2nvest. 18, 80 (1965)], the slope of the unweighted linear least-squares regression line was .970 the y-intercept 26 mg/liter, and the correlation coefficient .995.

This case report describes a patient who presented with Stage IV B Hodgkin's disease and autoimmune thrombocytopenia. Prior to the institution of therapy the presence of platelet-associated IgG was documented. When the patient was treated with steroids and chemotherapy, the thrombocytopenia resolved and platelet-associated IgG disappeared. Splenectomy alone did not correct the thrombocytopenia. The literature on Hodgkin's disease and autoimmune thrombocytopenia is reviewed.

We report a patient who, while receiving probenecid and colchicine for acute gouty arthritis, developed severe hemolytic anemia in association with a generalized rash. The hemolysis was immune mediated as shown by a positive direct Coombs' test. In vitro hematologic studies showed that a probenecid dependent antibody was present.

We report a patient who developed arthritis and pancytopenia without an underlying systemic disease. Autoantibodies to all lines were demonstrated using an 125I-anti human IgG assay. Elution studies documented cross reactivity of an antibody between erythrocytes and platelets, a pathogenetic variation not previously reported.

The effect of repeated phlebotomy on serum immunoreactive erythropoietin levels was studied prospectively in 69 autologous blood donors. At the time of the initial phlebotomy, 11 men (33%) and two women (6%) were anemic; during the course of blood donations, anemia (defined as a hematocrit less than 0.41 for men and less than 0.36 for women) developed in an additional 17 men (71%) and 14 women (45%). Although there was an increase in the level of serum immunoreactive erythropoietin with successive phlebotomies, the increase was not substantially out of the normal range. The lack of an erythropoietic response to repeated phlebotomies in association with the small increment in the serum erythropoietin level was not due to iron deficiency, since the level of red-cell free protoporphyrin did not increase in these patients. We conclude that within the hematocrit range permissible for autologous blood donation, the degree of anemia experienced is insufficient to initiate an adequate increase in erythropoietin production; as a consequence, mild anemia develops in a majority of donors, and the volume of blood donated is inadequate to meet their operative needs.

It has been proposed that the blood group antibody, anti-P, produced by p or Pk individuals may cause abortion early in pregnancy. The authors have studied and successfully treated a Pk woman with anti-P who had 13 consecutive first-trimester miscarriages. Anti-P was implicated as the cause of repeated pregnancy loss after extensive clinical, endocrinologic, immunologic, and chromosomal evaluations. To remove P blood group antibodies, plasmapheresis was begun at five weeks' gestation during the 14th pregnancy with one plasma volume exchange two to three times per week. This therapy resulted in a reduction in the titer of anti-P, and the patient was delivered of a viable female infant after 33 weeks' gestation. The management and outcome indicate that habitual abortion presumably due to anti-P can be successfully treated with plasmapheresis. This case provides additional evidence that anti-P is responsible for abortions in p or Pk women, and that these abortions are immunologically mediated.