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This study prospectively compared the effect of secondary prophylaxis to episodic treatment on target joint (TJ) range of motion (ROM), number of joint haemorrhages and new TJ development in patients with moderate or severe haemophilia. Two-hundred and eighty-six males, 17% in prophylaxis, 83% in episodic treatment group, participating in the Centers for Disease Control and Prevention's Universal Data Collection project, fulfilled inclusion criteria: age >2 years at enrollment, free of TJs at enrollment, developed at least one TJ after enrollment, and received either prophylaxis or episodic treatment continuously for two follow-up visits after TJ development. The outcomes of interest - percentage change in TJ ROM, number of joint haemorrhages and new TJ development, were modelled using multivariate linear, Poisson and logistic regression techniques respectively. Individuals who received secondary prophylaxis in comparison to episodic treatment were younger at TJ development (P < 0.01); there was no difference in the decrease in TJ ROM between the two groups (P = 0.9). Factors significantly associated with a higher rate of haemarthroses included episodic treatment, severe haemophilia, age >5 years at TJ development, obesity and inhibitor negative status. Secondary prophylaxis significantly decreased haemarthroses but was not associated with a significant improvement in TJ ROM or with new TJ development., (© 2013 Blackwell Publishing Ltd.)

Alloantibody tests demonstrate immunological causes of insufficient increments in random platelet transfusions. The value of a positive or negative test result in predicting the outcome of human leucocyte antigen (HLA)-matched transfusions in patients refractory to leucodepleted random platelet transfusions has not been assessed. We retrospectively evaluated the outcome of the first HLA-matched platelet transfusion in 72 patients with haematological diseases in two ways: first, the strategy according to which the patient was selected for HLA-matched platelet transfusions was analysed. The strategies were: (i) results of alloantibody tests were not available, (ii) a positive alloantibody test, (iii) a negative alloantibody test. Secondly, the outcome of the first HLA-matched transfusion was investigated relative to the results of alloantibody tests, irrespective of the decision strategy. No significant association was found between the decision strategy and the outcome of the first HLA-matched platelet transfusion. Positive alloantibody tests, however, predicted a better outcome of the first HLA-matched platelet transfusion (P = 0.04 and P = 0.03 after 1 and 16 h respectively). In patients refractory to random platelet transfusions, positive alloantibody tests predicted a better outcome of HLA-matched platelet transfusions. Patients with negative alloantibody tests, however, may benefit from HLA-matched platelet transfusions.

The detection of platelet-specific antibodies is of high clinical interest in diseases with immune thrombocytopenia. The glycoprotein-specific platelet-antigen capture (PAC)-assay developed in this study is especially suited to the differentiation of platelet-specific immunoglobulin (Ig) G subclasses and the determination of platelet-specific IgM in serum or on platelets. The problems with unspecific signals or low sensitivity usually seen with the detector antibodies available are effectively overcome, as unbound detector antibodies are removed at an early stage in the assay. We investigated 14 maternal alloantisera from cases of neonatal alloimmune thrombocytopenia (NAITP) and six sera from patients with autoimmune thrombocytopenic purpura (AITP). In NAITP sera, we found IgG1 alone in 57%, IgG1 + IgG3 in 21% and IgG1 + IgG2 in 14% of cases. One serum contained IgG1 + IgG2 + IgG3. In AITP, three out of the six sera contained IgG1 alone. One serum contained IgG1 + IgG2. One patient, with highly refractory AITP, had platelet-specific IgG1 + IgG2 + IgG3 in his serum. A patient with AITP in remission and normal platelet counts only showed platelet-bound IgG2. The detection of platelet-specific 'whole IgG' is possible too. However, at this time the commonly used monoclonal antibody-specific immobilization of platelet antigens (MAIPA) method should not be replaced for this purpose, as it is well standardized and used with similar results in many laboratories. The PAC assay sensitively detects the subclasses of platelet-specific IgG and human leucocyte antigen (HLA)-antibodies independently. It is easy to perform and takes less time than other platelet glycoprotein-specific methods.

The glycosylphosphatidylinositol-linked platelet protein CD109 carries the biallelic alloantigen system Gov. There is limited information on the incidence of Gov alloantibodies in neonatal alloimmune thrombocytopenia (NAITP), post-transfusion purpura (PTP) and platelet refractoriness. We adapted the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay to the detection of Gov antibodies and determined their incidence in 605 archived samples (112 with HPA antibodies) referred for the aforementioned conditions. Here, we show that CD109 expression was reduced upon platelet storage in saline or by cryopreservation, but was stable when stored as whole blood or therapeutic platelet concentrate. Fourteen of the 605 samples contained Gov alloantibodies (anti-Gova, n = 10; anti-Govb, n = 4), with the majority in platelet refractoriness (n = 9) and, of the remaining five, four in NAITP and one in PTP. In seven cases, no other HPA antibodies were detected, three being NAITP cases. The incidence of Gov antibodies was significantly lower than HPA-1 system antibodies (n = 87), but equalled the number of HPA-5 system antibodies (n = 14) and outnumbered HPA-2 and -3 system antibodies (10 altogether).

Severe combined immune deficient (SCID) mice were engrafted with human (Hu) peripheral blood lymphocytes (PBL) from a previously alloimmunized donor and transfused with HLA-mismatched platelets. We have previously shown this to be a useful model for platelet transfusion. These engrafted mice (Hu-PBL-SCID mice) produced high levels of alloantibody in response to standard platelet preparations. However, when the first platelet challenge was presensitized with anti-HLA antibody and then transfused there was a marked reduction in the amount of alloantibody produced to five subsequent untreated platelet transfusions. Platelets pretreated with platelet-specific anti-HPA-1a (PL(A1)) sera did not induce a decrease in the anti-HLA alloantibody response. This demonstrated that platelet-induced HLA alloimmunization can be blocked by anti-HLA antibody-sensitized cells.

The formation of erythrocyte autoantibodies following transfusion therapy has been described in case reports and small series. To determine the frequency, serological characteristics, and clinical significance of this phenomenon in paediatric patients with sickle cell disease, we analysed the patient database at the Duke University Pediatric Hematology Clinic. We identified children who received multiple erythrocyte transfusions, then reviewed clinical records to identify children who developed erythrocyte autoantibodies in association with transfusions. Among 184 paediatric patients who received multiple erythrocyte transfusions, 14 children (7.6%) developed warm (IgG) erythrocyte autoantibodies. Median transfusion exposure at the time of autoantibody formation was 24 erythrocyte units, range 3-341 units. The autoantibody reacted as a panagglutinin in 11 cases but had anti-e specificity in three patients. Surface complement also was detected in five patients. Clinically significant haemolysis was documented in four patients, each of whom had both surface IgG and C3 detected. The development of erythrocyte autoantibodies was associated with the presence of erythrocyte alloantibodies. Formation of warm erythrocyte autoantibodies in association with transfusions is not rare in paediatric patients with sickle cell disease. Clinicians should be aware of this complication and recognize that the presence of surface C3 is often associated with significant haemolysis.

We report a case of a patient with Glanzmann's thrombasthenia and anti-GPIIb/IIIa alloantibodies who developed life-threatening and intractable bleeding from gastrointestinal telangiectatic lesions. After a period of transfusion-dependent gastrointestinal bleeding despite tranexamic acid, oral iron, omeprazole and platelet transfusions, the use of oral norethisterone produced a significant improvement with a marked reduction in her transfusion requirements.

We describe the first human monoclonal anti-D (LOR-15C9) which reacts with a D-specific motif exposed either on a native form on intact D-positive red cells or on a denatured form of the RhD protein (33 kD), and detected by immunoblotting. LOR-15C9 was able to precipitate RhD but not RhcE proteins produced by in vitro transcription-translation assays. The reactivity of the antibody, using panels of red cells with various partial D phenotypes known to lack some D epitopes and corresponding in RHD gene variants, suggested that LOR-15C9 reactivity depends on the portion of the RhD polypeptide encoded by the exon 7 (amino acids 314-358). These findings correlate well with the reactivity of LOR-15C9 with erythrocytes of some nonhuman primates (D(gor)-positive gorillas), but not of chimpanzee and Old or New World monkeys. In membrane proteins from partial D(VI) red cells, LOR-15C9 detected two proteins of molecular weight 33 and 21 kD: the presence of the latter was specific for category D(VI) and presumably represented the product of an alternatively spliced RHD(VI) transcript in these cells. This is consistent with the finding that LOR-15C9 can precipitate a shortened D protein mutant resulting from in vitro transcription-translation and lacking amino-acids 163-313 encoded by exons 4-6. In addition, a 21 kD band polypeptide was detected by immunoblot in all red cell samples but D--, using a rabbit anti-Rh polypeptide antibody (MPC8) raised against the C-terminal domain of Rh proteins. This 21 kD polypeptide most probably results from the translation of an alternatively spliced RHCE gene transcript. This study demonstrates that LOR-15C9 detects an epitope on the RhD protein that is independent of the membrane environment, and therefore could be a useful tool for the study of RhD polypeptides.

Immune thrombocytopenic purpura (ITP) is a disorder caused by anti-platelet autoantibodies (Ab), most of which are directed against epitopes on platelet membrane glycoprotein complexes GPIIb/IIIa and GPIb/IX. To detect platelet Ab, reliable techniques, such as MAIPA or immunobead assay, have been developed. They all achieve their selective specificity by the use of monoclonal antibodies (MoAb) against defined glycoproteins of the platelet membrane. In order to determine the most frequent Ab-specificities, a novel enzyme-linked immunosorbent assay, named platelet-glycoprotein-ELISA (P-GP-ELISA), has been developed. It uses purified GPIIb/IIIa and GPIb/IX complexes, respectively, as antigens and enables determination of platelet-associated as well as circulating Ab (IgG, IgM). MoAbs are not required and therefore there is no risk of competition between MoAb and Ab. Levels of Ab in patients with the clinical diagnosis of an idiopathic thrombocytopenic purpura were analysed. 92.7% (76/82) platelet eluates with significantly increased levels of Ab against at least one of the glycoproteins were found, whereas no sample from healthy volunteers (0/37) gave a positive result, pointing to a high sensitivity and specificity of the test system. Since its application is also easy and quick, P-GP-ELISA should facilitate detection of Ab against platelet membrane proteins in routine determinations.

A total of 1997 pregnant women were screened during their first antenatal visit for irregular antibodies for the prevention of haemolytic disease of the newborn. Patient sera were tested against a panel of group O screen cells including one with the expression of Miltenberger determinants GP.Mur. 17 women (0.85%) had irregular antibodies of which four were of potential clinical significance, including one with anti-D, two with anti-E and one with anti-D, anti-E and anti-G. Although antenatal antibody screening is mandatory in Western populations, our results suggest that this may not be necessary in the Chinese population except for those who are Rh D-negative or who have a history of haemolytic disease of the newborn.

This report describes the results of eight workshop exercises which were designed to test the proficiency of laboratories in the detection of antibodies to human platelet antigens (HPA). Detection of the most clinically significant alloantibody, anti-HPA-1a, is adequate. However, despite improvements in consistency of test results between laboratories over the last 3 years, there is still a high probability that clinically significant antibodies against other HPA alloantigens will not be detected.

Neonatal alloimmune thrombocytopenia (NAIT) is induced by maternal alloantibodies to fetal platelet antigens. This prospective study was carried out to evaluate the incidence of anti-platelet antibodies in 933 mother-child pairs where the mother and child were typed for the human platelet antigens (HPA)-1, -2, -3, -5. Sera from mismatched mother-child pairs were screened for anti-platelet antibodies, anti-HLA class I and blood group ABO IgG antibodies. Platelet-specific antibodies were anti-HPA-3a in one and anti-HPA-5b in 17 neonates, respectively. All these neonates had normal platelet counts. One woman had autoreactive antibodies. Anti-HLA class I and anti-blood group A IgG antibodies were detected in five and four neonates, respectively, born with a platelet count < 150 x 10(9)/l. None of the 11 homozygous HPA-1b mothers became immunized against their heterozygous offspring. The maternal HLA-allotypes HLA-DR52 and -DR6, typically found in individuals immunized against HPA-1a and -5b, respectively, were found in three of 11 HPA-b/b nonresponders and eight of the anti-HPA-5b responders. The results indicate that a risk for NAIT due to HPA-2 and -3 alloimmunization is low. The HLA allotypes do not predict the risk for NAIT due to HPA-1 or -5 alloimmunization. Maternal anti-HPA-5b antibodies do not correlate with the platelet count in the neonate.

We report a family with a neonate who was severely damaged by intracranial haemorrhages. These probably occurred before the 20th week of gestation. The neonate had a moderate thrombocytopenia. In the maternal serum anti-HPA-1b and anti-HPA-2a alloantibodies were detected. Third-generation assays were applied to identify the alloantibodies. No other cause for the bleeding was found. Probably the combination of anti-HPA-1b and anti-HPA-2a alloantibodies, directed against the platelet fibrinogen receptor and the von Willebrand receptor, respectively, induced a thrombocytopenia and a thrombocytopathy.

Fifty polytransfused patients were prospectively studied to determine the frequency of post-transfusion alloimmunization and its influence on the response to platelet transfusion. Platelet- and HLA-specific antibodies were detected by means of the standard and antiglobulin-augmented lymphocytotoxicity techniques (LCT), the platelet suspension indirect immunofluorescence test (PSIIFT), and monoclonal antibody immobilization of platelet antigens (MAIPA). HLA antibodies were detected in 13 patients (26%) (IgM = 6; IgG = 6; IgM + IgG = 1). The standard LCT was positive in 12 of these 13 patients. Complement-independent HLA antibodies, only detectable in the PSIIFT and the antiglobulin-augmented LCT, were documented in two patients and were associated with poor post-transfusion platelet recovery in the patient who could be evaluated. All the HLA antibodies were detected in the PSIIFT, while only four were detected in the MAIPA. Platelet-specific alloantibodies were found in two patients by means of PSIIFT or MAIPA and may have led to poor post-transfusion platelet recovery in one patient. Platelet autoantibodies were detected in two patients but were not associated with platelet refractoriness. Paraformaldehyde-dependent platelet antibodies were detected in 11 patients but were not associated with platelet refractoriness.

In 40 cases of haemolytic disease of the fetus due to RhD immunization where the fetus required intrauterine transfusion, fetal packed cell volume was compared with the following parameters of maternal anti-D: (a) concentration, (b) IgG subclass, (c) activity in a macrophage binding assay, and (d) activity in a monocyte antibody-dependent cell mediated cytotoxicity (ADCC) assay. The anti-D concentration exceeded 4 iu/ml in all cases, correctly indicating the risk of haemolytic disease. A relationship between IgG subclass composition of the anti-D and severity of anaemia was not observed; IgG1, IgG3 and IgG1 + 3 antibodies were all detected. The ADCC assay gave the best correlation between assay results and fetal packed cell volume; high results correctly indicated fetal anaemia in 95% of cases. Macrophage binding assay results were only considered as high in 70% of cases. Overall, these results indicate that serological tests and bioassays in highly immunized mothers may not generate any information that proves more useful than ultrasonography and their previous obstetric history.

Maternal alloimmunization against fetal platelets can cause fetal and neonatal thrombocytopenia (NAIT). The HPA-1a (PIA1, Zwa) antigen is by far the most common antigen implicated in NAIT. However, today another antigen often linked with that affection is HPA-5b (Br(a)). This is a report of 39 cases of NAIT involving the HPA-5b antigen. Thrombocytopenia may be of grave consequence. Three infants developed intracerebral haemorrhages (ICH). Of these, one died presumably as a consequence of ICH. Central nervous system (CNS) sequelae in the neonatal period was observed in two children. The potential hazards of death or disabling neurologic sequelae following intracerebral haemorrhage call for rapid and reliable diagnosis and effective therapy. Because there is high risk that subsequent pregnancies might be also affected by NAIT, the mothers of a previously affected child should be managed similarly to the HPA-1b mothers (PIA2, Zwb). The antenatal diagnosis of thrombocytopenia should be made and if necessary the in utero therapy instituted.

Serum samples containing IgG red blood cell (RBC) antibodies were collected without reference to clinical information from 131 pregnant alloimmunized women. Anti-D and anti-K were present in sera from 75 and 20 patients respectively. Antibody titres were determined by indirect antiglobulin test (IAGT), anti-D levels were measured by AutoAnalyzer, RBC-binding IgG was quantified using an enzyme-linked immunosorbent assay (SOL-ELISA), and functional activities were measured using the monocyte chemiluminescence (CL) test, antibody-dependent monocyte-mediated and K cell-mediated cytotoxicity (ADCC) assays, and rosette formation with U937 cells. Details of clinical outcomes were obtained retrospectively from 104 pregnancies. Forty-one babies were 'antigen-negative', and of the remainder, four required top-up transfusions, 12 required exchange transfusions, three received intrauterine transfusions, and two died in utero. A comparison of test results with severity of haemolytic disease of the newborn (HDN) showed that, provided sera tested were collected within 8 weeks of the expected delivery date, the CL test and the monocyte-mediated ADCC assay differentiated those D-positive babies which required exchange transfusions from those unaffected or only mildly affected. The usefulness of results from the AutoAnalyzer and IAGT in predicting disease severity was compromised by the wide range of results from mothers of unaffected babies. This variability was less apparent in the SOL-ELISA which predicted severe HDN with greater precision. Results from the U937 rosette assay and the K cell-mediated ADCC assay failed to correlate with disease severity.

Quantification of platelet-bound immunoglobulins (PBIg) with radiolabelled murine monoclonal antibodies (mAbs) has been described only for IgG so far. Here we describe some modifications of this mAb radioimmunoassay (MARIA) and show that by using a panel of radiolabelled specific mAbs it is possible to quantify not only PBIgG but also PBIgG subclasses and PBIgM. Analysis by gel filtration showed that all anti-IgG and anti-IgG-subclass mAbs bound to their respective antigens in a ratio of about 1:1. However, the binding ratio for the anti-IgM Mab could not be established. There was a good correlation between the antibody-density per platelet as determined with the anti-IgG mAb and determined as the sum of the IgG molecules of different subclass per platelet (r = 0.90). Platelet fragments did not interfere in the assay. 89 normal healthy controls had 140 IgG molecules per platelet and bound 269 anti-IgM molecules per platelet (geometric means). In a study on the detection of PBIg in 147 thrombocytopenic patients, it appeared that the MARIA had a sensitivity of 61% and a specificity of 45% for the diagnosis of idiopathic thrombocytopenic purpura (ITP). Both in ITP and in secondary thrombocytopenia (STP), PBIgG1 and PBIgG3 were found more frequently (60% and 61%, respectively) than PBIgG2 and PBIgG4 (13% and 9%, respectively). There was no relation between the amount of total PBIgG or PBIgM and the platelet count in either ITP or STP. Also, if IgG antibodies of only one subclass were found, there was no relation between the severity of the thrombocytopenia and the amount of PBIgG. By applying the MARIA, it is possible to quantify PBIgG, all four PBIgG-subclasses and PBIgM in ITP and STP in a reliable way.

Reported here is the first example of a partial D antigen stimulating the production of anti-D: stimulation was of fetal origin. During her second pregnancy, anti-D developed in the serum of a D-negative mother who had received Rh immunoglobulin after the birth of her first D-positive child. Her second baby had moderate neonatal jaundice and was successfully treated by phototherapy. Subsequently the red cells of the father and of the first child were shown to carry a partial D antigen of category DVa type. Six available batches of Rh immunoglobulin reacted with DVa cells.

The natural history of alloimmunization to the PlA1 platelet antigen is uncertain. We followed 50 PlA1-negative pregnant women during pregnancy and for 6 months post-partum in order to determine this natural history. The cohort of PlA1-negative women was obtained by PlA1 typing 5000 women. Three PlA1-negative women formed anti-PlA1 antibodies during this prospective study, two in pregnancy and one in the immediate post-partum period. All three PlA1 antibody producers were HLA-DR3 positive, a histocompatibility phenotype that is strongly associated with alloimmunization to the PlA1 antigen. One of the three infants delivered to these mothers was thrombocytopenic (platelet count 9 x 10(9)/l). The remaining two infants had normal platelet counts at birth (160 and 174 x 10(9)/l). The HLA-A1, -B8, -DR3 and -DRw52 phenotype frequencies in the group of PlA1-negative women who did not form PlA1 antibodies (n = 47) was similar to that found in their husbands, and that expected in a normal Caucasian population. From our data we estimate that alloimmunization to the PlA1 antigen occurs in approximately one out of every 1000 pregnancies in a Caucasian population. It is important to recognize that not all pregnancies in which a mother has formed PlA1 alloantibodies will result in the delivery of a thrombocytopenic infant. These findings are relevant to programs designed to either prevent alloimmunization to the PlA1 antigen (through passive administration of anti-PlA1 immunoglobulin to at-risk PlA1-negative mothers), or to identify women at risk of delivery of thrombocytopenic infants (by antenatal screening to detect women alloimmunized to the PlA1 antigen).

A simple technique to separate erythrocytes from bone marrow by sedimentation under gravity after addition of hydroxyethyl starch is described. This method of removing red blood cells from the donor marrow was evaluated in 23 consecutive allogeneic bone marrow transplants between ABH-incompatible individuals. Infusion of the separated marrow was well tolerated, i.e. graft-rejection did not occur, and sustained engraftment was documented in all evaluable cases. This technique obviates the need for cumbersome and potentially hazardous large volume plasma exchange that is commonly utilized to deplete recipient isohaemagglutinins prior to transplantation.

By applying the platelet suspension immunofluorescence test (PSIFT), platelet-specific alloantibodies responsible for post-transfusion purpura were detected in eight patients within a period of 2 years. All patients were female and had previously received blood or had been pregnant. The platelet-specific alloantibodies had the specificity anti-Zwa in all the patients, who were all Aw(a-). In two patients the platelets were tested in the acute phase of the disease and found to be coated with IgG. In one patient an ether eluate was prepared from the platelets that reacted strongly with Zw(a+) platelets, but weakly with platelets from Zwb-homozygous individuals. The sera of these two patients, and of two others whose platelets were not directly tested, taken in the acute phase of the purpura, reacted strongly with Zwa-positive platelets. The four sera also reacted, however weakly, with Zwa-negative platelets, with autologous platelets taken during remission and with platelets from patients with Glanzmann's disease. It is postulated that Zwa-anti-Zwa complexes, present in the eluate and the sera, caused the reaction with Zwa-negative platelets and the patients' own platelets. Immunochemical characterization of the post-transfusion purpura antibodies showed that in all patients these were IgG, in two combined with IgM antibodies. Antibodies of the sub-class IgG1 were found in all patients, sometimes together with IgG3. In the indirect immunofluorescence test with anti-complement serum, the PTP antibodies in only four sera were able to fix complement. In only two of these sera were these complement-binding antibodies detectable in the 51 Cr-lysis technique and then in a much lower titre than in the immunofluorescence technique.

An international collaborative study of anti-D assays has been carried out by 21 laboratories in 11 countries. Samples of anti-D immunoglobulin assayed in this study included two dilutions of a preparation used in clinical trials to determine a dose-protection relation, a national standard, commercial clinical preparations and the proposed international reference preparation in coded ampoules. Manual, automated haemagglutination and isotope labelling methods all gave similar relative potencies. Several of these estimates were significantly (P=0.95) heterogeneous and some modifications to improve assay design and procedure are suggested. The coded preparation was shown to be stable and suitable for comparative assays. It was estimated to contain 60 microgram of of anti-D IgG immunoglobulin per ampoule, and 150 i.u./ampoule when assayed against the International Standard for Incomplete Anti-D Blood Typing Serum. Thus for this preparation 1 microgram of IgG anti-D immunoglobulin identical to 2.5 i.u. anti-D antibody. At its 28th meeting the Expert Committee on Biological Standardization of WHO established the preparation 68/419 as the International Reference Preparation of Anti-D Immunoglobulin and assigned to it a potency of 150 i.u. per ampoule. The Preparation has been widely used (with a nominal content of 60 microgram of IgG anti-D immunoglobulin) for control of clinical preparations.

After a single antigenic stimulus, not greater than 5.0 ml R2R2 red cells, anti-D was detected in 79% of D-negative volunteers increasing to 88% after subsequent spaced stimuli. The use of repeated antigenic stimuli at 2- or 4-weekly intervals to induce the immune response did not appear to increase the frequency of responders. The results obtained are compared with those of other workers and evidence is presented to suggest that the immunogenicity of red cells from different donors may have a role in determining the frequency of responders with detectable anti-D and is possibly associated with the R2 antigenic complex.

Antibodies against factor VIII collected from six patients were studied for their effect on factor-VIII coagulant activity, Willebrand factor activity (WF) and factor-VIII-related antigen. The results demonstrate differences between individual antibodies; some acting primarily as anti factor VIII, others inhibiting both after VIII and WF. Differences in effect between plasma and serum indicate that the process of coagulation alters the interaction of these antibodies.

The role of humoral immune mechanisms in the pathogenesis of the neutropenia in five patients with chronic killer (K)-cell lymphocytosis was studied. For the detection of neutrophil antibodies in the patient's blood, immunofluorescence, cytotoxicity and agglutination tests and a sensitive antibody-dependent cellular cytotoxicity (ADCC) assay were applied. An assay for bone-marrow colony growth (CFU-GM, BFU-E) inhibition was applied as well. A C1q-binding test was used to detect immune complexes. The lymphocytes of all five patients had the capacity to lyse alloantibody sensitized neutrophils. Neutrophil-bound IgG was found only in one patient. Two patients had neutrophil-reactive antibodies in their serum; in one patient these antibodies were only detectable in the ADCC using the patient's serum to sensitize target cells. However, these antibodies reacted also with lymphocytes, were absorbable with platelets and thus probably were HLA antibodies. The serum of one of these two patients showed complement-dependent CFU-GM and BFU-E inhibition, but the observed inhibition was probably also due to the anti-HLA antibodies present in his serum, as the inhibiting effect disappeared after absorption of the serum with platelets. The serum of only one patient contained low amounts of immune complexes, as measured in the C1q-binding test. Our data suggest that humoral autoimmune mechanisms, such as autoantibodies against neutrophils or neutrophil precursors or circulating immune complexes, do not seem to play an important role in the K-cell lymphocytosis/neutropenia syndrome. The possible role of the expanded K-cell population, and its humoral products (tumour necrosis factor, interferons), in the syndrome is discussed.

Incubation of factor IX with non-precipitating alloantibodies to factor IX gives rise to soluble complexes between factor IX and the alloantibodies. These complexes appear as a factor IX molecule with a reduced mobility in crossed immunoelectrophoresis against a rabbit antiserum to factor IX. This factor IX immunoprecipitate was used for the study of alloantibodies to factor IX from five patients with severe haemophilia B (antibody titres 0.1--800 units/ml plasma). Incorporation of antiserum to k light chains or lambda light chains of human immunoglobulin G in an intermediate gel in crossed immunoelectrophoresis gave a reduction of the factor IX precipitin arc, indicating the presence of immunoglobulin G alloantibodies containing both k and lambda light chains in complex with factor IX. Incubation of the factor IX immunoprecipitate with peroxidase-conjugated antisera to the same immunoglobulins, and staining for peroxidase activity, confirmed the presence of immunoglobulin G containing both types of light chains in the factor immunoprecipitate. It is concluded that all five alloantibodies were polyclonal immunoglobulin G antibodies. The technique had the advantage that the light chain types of low-titre antibodies could be determined, and it may be suitable for further characterization of alloantibodies to factor IX if antibodies to immunoglobulin subclasses are available.

Alloimmunization by platelet transfusions was studied in 154 patients with acute leukaemia. 17 patients had HLA antibodies at initial presentation induced by previous transfusions or pregnancies; one of these also had platelet-specific antibodies and one other patient had platelet-specific antibodies alone. A further 38 patients developed HLA antibodies during therapy; three also had platelet-specific antibodies and two patients developed platelet-specific antibodies alone. Of these, 37 patients with HLA antibodies including three with platelet-specific antibodies and one patient with platelet-specific antibodies alone survived their initial therapy and formed the basis of this study. Antibodies once detected persisted throughout the study in seven of the 37 patients with HLA antibodies including one patient with platelet-specific antibodies and in the patient with platelet-specific antibodies alone. HLA antibodies disappeared after discontinuation of transfusions in six patients, and after switching to HLA matched platelet transfusions and leucocyte-poor blood in eight patients; two of the latter patients also had platelet-specific antibodies which disappeared. The other 16 patients with HLA antibodies lost their antibodies despite continued transfusions.

Red cells coated with as little as 0.3-0.5 mug anti-A/ML cells were found to agglutinate if mixed with plasma and rocked on a tile, whereas the minimum amount of IgG anti-Rh required for agglutination under these conditions was about 50 mug antibody/ml cells. Complement-binding by red cells coated with IgG anti-A could be demonstrated only when the amount of antibody on the cells was at least 14 mug antibody/ml cells. Since in ABO haemolytic disease the amount of antibody on the cells is frequently less than 0.6 mug/ml cells, it seems that red cell destruction in this syndrome is not due to the activation of complement but may be due to the sequestration of agglutinated cells. A few experiments are described in which the survival in vivo of small amounts of agglutinated cells was studied. Reasons are given for considering that such experiments fail to mimic the conditions prevailing in ABO haemolytic disease.

An agarose plate method for detecting human alloantibodies (inhibitors) to coagulant factor IX has been developed. The assay is based on the ability of such antisera to inhibit the coagulation of a mixture of haemophilia B plasma, normal plasma and platelet subsitute in an agarose matrix. The agarose plate method was also adopted to measure levels of FIX antigen (IX:Ag) in plasma. Using this technique, 12 of 17 obligate carriers of haemophilia B demonstrated reduced levels of IX:Ag. Three of the five carriers with normal IX:Ag levels were members of kindred in which affected individuals had normal or near normal levels of IX:Ag.

An indirect immunofluorescence technique for the detection of alloantibodies against granulocytes was developed. A major problem was the unspecific fluorescence of granulocytes, due to unspecific adherence of immunoglobulins to the cell membrane. This could be suppressed by the prefixation of the granulocytes with paraformaldehyde. The developed test proved to be specific and sensitive and to have a high reproducibility. Strongly positive reactions were obtained with antisera containing granulocyte-specific agglutinins or granulocyte-cytotoxins, but also with some sera that did not react in either the agglutination or the cytotoxicity test. HLA antisera also gave positive reactions, but strong anti-A, anti-B or anti-D sera did not react. With this technique leucocyte antibodies can be detected in a higher percentage of patients with febrile transfusion reactions.

One hundred and forty-eight red cell alloantibodies, of specificities generally considered to be of clinical significance, were studied in vitro for their ability to induce phagocytosis of sensitized red cells by allogeneic mononuclear phagocytes. Results indicate that only 53% of the alloantibodies studied mediated significant phagocytosis in vitro. The percentages for each blood group system were as follows: Kell, 73%; Jka, 32%; Jkb, 67%; D, 75%; E, 60%; Fya, 62%; Yta, 25%; Ge, 22%; and Vel, 25%. Significant phagocytosis was independent of the strength of the indirect antiglobulin test. The percentage of anti-Jka and anti-Fya mediating significant phagocytosis was increased when fresh complement was added during the sensitization procedure and/or red cells homozygous for the antigen in question were used. The in vivo clinical significance or lack of significance was documented for nine alloantibodies; five caused haemolysis and four did not. Those causing in vivo haemolysis mediated in vitro phagocytosis by monocyte-macrophages whereas the antibodies that did not result in haemolysis showed no increased in vitro phagocytosis. Autologous monocytes were more reliable than random allogeneic monocytes in that phagocytosis was increased over that obtained using allogeneic monocyte-macrophages with two of four alloantibodies having documented clinical significance. The use of target red cells homozygous for the antigen in question, the addition of fresh complement in the antibody sensitization procedure, and use of autologous and allogeneic monocyte-macrophages appear necessary for optimal results. Since 47% of those alloantibodies generally considered to be clinically significant failed to mediate phagocytosis in vitro, the monocyte-macrophage assay should not be considered a predictive assay of a given alloantibody's in vivo significance or lack of significance until more extensive correlation of these assays with in vivo red blood cell survival is obtained.

Erythrocyte membranes from an anaemic patient receiving gold therapy for rheumatoid arthritis had reduced beta-sialoglycoprotein (beta-SGP) content but normal expression of sialoglycoproteins alpha, delta and gamma. Elliptocytes were present in the peripheral blood. The serum of the patient contained anti-beta-SGP which did not appear to bind to her own cells. It reacted with all erythrocytes apart from beta-SGP deficient Leach phenotype cells. The antibody was inhibited by purified beta-SGP from normal red cells, bound to beta-SGP on immunoblots and also reacted with the abnormal beta-related-SGP in erythrocyte membranes of both the Gerbich type and Yus type of Gerbich negative. Two years later the patient was no longer anaemic, no elliptocytes were seen in her peripheral blood film and her erythrocyte membranes had normal beta-SGP content. Antibody was no longer present in her serum and antibody from the earlier sample now reacted with the patient's erythrocytes. Erythrocyte membrane beta-SGP is known to be important in the maintenance of normal cell shape. It is likely that the transient occurrence of elliptocytes in the patient resulted from the concurrent temporary reduction in beta-SGP content of her erythrocyte membranes.

Twenty-nine Rho(D) negative male volunteers previously immunized with D-positive red cells have received series of antigenic stimuli repeated at intervals of 2 or 4 weeks. Although there is an individual variation in the magnitude of the anti-D response, the levels of anti-D achieved are, in general, higher than those obtained after stimuli spaced at intervals of several months. Moreover, certain volunteers whose plasma was not suitable for inclusion into pools for the preparation of anti-D immunoglobulin after spaced stimuli could donate regularly following the repeated stimuli. Whilst untoward clinical reactions did not occur as a result of the repeated stimuli five volunteers developed unwanted antibodies outside the Rh system and this may be related to the total dose of red cells injected.

Investigations on six males with naturally occurring Rh antibodies are described. In two subjects in whom the antibody (one anti-E and one anti-D) could be detected only by a two-stage papain technique, the survival of incompatible red cells was normal. In the remaining four subjects, the antibodies (two anti-E and two anti-D) could be detected by the indirect antiglobulin test and, in these, incompatible red cells were destroyed at an accelerated rate; in two of the subjects, 75-99% of the cells were cleared within 24 h; in the other two, 50% of the cells were cleared within 24 h and the remaining cells were cleared far more slowly. All six antibodies were mainly or wholly IgG; a clear-cut immune response was observed in only one case.

Delayed haemolytic transfusion reactions (DHTRs) are a recognized sequel of blood transfusion. The true incidence and importance of this complication have been difficult to estimate due to the lack of any prospective studies. We have carried out such a study by testing 530 patients who were transfused during cardiac surgery. 2% of the patients had new red cell alloantibodies detectable 1 week following transfusion. Despite this finding, and the fact that at the time the study was performed pre-transfusion antibody screening of recipients was not routine practice, no DHTRs were diagnosed on clinical or laboratory criteria. These results indicate that the reported incidences, based on retrospective recognition of DHTRs, are not a serious underestimate of the frequency of the complication.

Fourteen high-risk cases of Rh alloimmunized women were treated by intensive plasma exchange on the cell separator throughout their pregnancies. The mean volume of plasma exchanged per week was 3.21 with a total volume of 10-123 1. The replacement fluid was mainly plasma protein fraction (PPF) supplemented with fresh frozen plasma (FFP). The mean duration of treatment was 131/2 weeks, ranging from 2 to 22 weeks, commencing at 12-30 weeks gestation. The complications encountered and the selection of the optimal time for delivery are discussed. The expected stillbirth rate in this series as determined by their past obstetric histories and anit-D levels was 62%. Intrauterine transfusion was given to only two of the infants and both were later stillborn. It was possible to reduce and maintain a lower level of anti-D in the serum of most of the patients and a successful outcome was achieved in nine of the 12 cases included for analysis (75%). Plasma exchange commenced early in pregnancy is recommended as a non-hazardous form of treatment in the management of severe Rh haemolytic disease. However, if the mean level of anit-D cannot be maintained at less than 35 iu/ml then the outcome is more likely to be fatal. Amniocentesis should be delayed where possible until 28 weeks gestation and intrauterine transfusion reserved for those cases where the anti-D level becomes uncontrollably high and the fetal death is imminent.