What's happening--probing of HPA-1a antigen-antibody interaction by surface plasmon resonance technology.

This brief report summarizes the use of surface plasmon resonance technology (SPRT) in probing HPA-1a antigen-antibody interactions, based on a poster presented at the 60th meeting of the American Association of Blood Banks. It was concluded that the GP purification method could affect the performance of antigen in SPRT. It also highlighted that chips immobilised with Monoclonal antibody (Mab)-purified GP-IIb/IIIa work satisfactorily with both monoclonal and recombinant Abs with the appropriate concentration and binding affinity, while determination of the avidity and concentration of maternal polyclonal antibodies in respect to clinical severity on NAIT warrants further development.

Repeat ABO-incompatible platelet transfusions leading to haemolytic transfusion reaction.

A 65-year-old woman, blood group A RhD positive, who had completed her first course of induction chemotherapy for acute myeloid leukaemia was transfused with apheresis platelets over a number of days. On three occasions she received group O RhD positive units, which had been screened and found not to contain high-titre anti-A,B isoagglutinins. Following the third unit, she developed a haemolytic transfusion reaction and died soon thereafter. This has led to change in policy of the supplying centre in testing for high-titre anti-A,B isoagglutinins. Blood group O apheresis platelets and fresh-frozen plasma units are now labelled as high titre with a cut-off of 1/50 as compared to the previous cut-off of 1/100 for anti-A,B isoagglutinins. A universal approach to testing donations for high-titre anti-A,B isoagglutinins, better compliance of guidelines and monitoring of patients is necessary.

The analysis and quantification of a clonal B cell response in a hyperimmunized anti-D donor.

Healthy volunteers are hyperimmunized with RhD-positive red cells in order to obtain plasma containing high titres of anti-D immunoglobulin, which is used for the prevention of haemolytic disease of the fetus and newborn. We analysed the anti-D immune response in a donor who had been hyperimmunized for 7 years and who showed declining anti-D titres despite re-immunization. A phage display library representing the complete immunorepertoire and a second library representing the IGHV3 superspecies family genes (IGHV3s) repertoire in the donor were constructed and analysed. A clonal Ig-gene rearrangement was quantified in the peripheral blood by limiting dilution polymerase chain reaction (PCR) All RhD-binding phages from both libraries, except one, had heavy chains with IGH-VDJ rearrangements of the same clonal origin, but with different patterns of somatic mutations and joined with different light chains. Limiting dilution PCR performed on mRNA and genomic DNA showed a frequency of 1 clonal B cell in 2000 IgG1/3-positive B cells. We show the presence of clonally related RhD-specific B cells in a hyperimmunized anti-D donor who had declining anti-D titres and who was unresponsive to re-immunization. Furthermore, we found a high frequency of clonal B cells. These results contribute to the understanding of the immune response against RhD in hyperimmunized anti-D donors.

Alloimmunity to RhD in humans.

The RhD protein is expressed only on human red blood cells (RBC), and is one of the most immunogenic of the blood groups. It is of clinical importance since the alloantibody (anti-D) can hemolyse D positive RBC after blood transfusion, or cause hemolytic disease of the newborn. The immunogenicity of D is better understood with the knowledge of the genetic basis of the protein(s) involved, the molecular orientation in the RBC membrane, and the nature of the cellular immune response to proteins. The adaptive humoral response consists of antigen presenting cells, T-cells and B-cells, which interact cooperatively to result in antibody against the antigen in question. The anti-D that B-cells produce is targeted against surface membrane determinants (B-cell epitopes) and are conformational i.e. non-contiguous amino acids. The antigen specific T-cells recognize short linear peptides in the context of MHC class II, and these T-cell epitopes can reside anywhere in the protein. Since the RhD protein in D-positive individuals differs by some 35 amino acids from the RhCcEe protein in D-negative individuals, the opportunity for generating immunogenic T-cell epitopes is much greater than that for alleles characterized by a single amino acid difference e.g. E and e. Multiple conformational B-cell epitopes are also created by the presence of several D-specific amino acids in extracellular loops on the red cells surface, which may stimulate several B-cell clones and develop a strong polyclonal antibody response. With greater understanding comes the possibility of manipulating the immune response to D in clinical situations.

Is there a relationship between anti-HPA-1a concentration and severity of neonatal alloimmune thrombocytopenia?

There is uncertainty about the relationship between anti-HPA-1a levels and severity of neonatal alloimmune thrombocytopenia (NAIT). To investigate this relationship further,the concentration of anti-HPA-1a in HPA-1b homozygous women was determined, using a newly developed quantitative ELISA that uses purified anti-HPA-1a to obtain a standard curve. Seventy-eight samples collected from 22 HPA-1b homozygous pregnant women at various stages of pregnancy were tested. These included five women who had delivered babies with severe NAIT. A national HPA-1a antibody standard (NIBSC 93/710), designated as 1 arbitrary unit/mL (AU/mL),was used in each ELISA to calibrate the purified anti-HPA- 1a, enabling the presentation of results as AU/mL. Moreover, selected samples were also assayed by PAK 12 and their reactivity compared with quantity of antibody. The use of the purified HPA- 1a antibody yielded consistent sigmoid curves, enabling the measurement of HPA-1a antibody concentration in the test samples. The antibody concentration was significantly correlated with the antibody titer in the 78 samples studied (R = 0.54, p < 0.001). Furthermore, there was a significant correlation between PAK 12 and the quantitative ELISA in a selected number of cases, with or without NAIT (R = 0.71, n = 10; p < 0.02). On the other hand, there was no correlation of antibody concentration with NAIT incidence (R = -0.046). This study indicates that there is no relationship between anti-HPA-1a concentration and severity of NAIT when ELISA is used, although the correlation between ELISA and other methods, such as monoclonal antibody immobilization of platelet antigens (MAIPA) assay, remains to be determined.

Report on the 12th International Society of Blood Transfusion platelet immunology workshop.

BACKGROUND AND OBJECTIVES: The aims of the 12th International Society of Blood Transfusion (ISBT) Platelet Immunology Workshop were to evaluate the proficiency of molecular human platelet antigen (HPA) genotyping and detection of platelet antibodies of unusual specificity or reactivity, to assess whether quantification of anti-HPA-1a is practicable, and to determine the variability of reagents and components used in the monoclonal antibody immobilization of platelet antigens assay (MAIPA). MATERIALS AND METHODS: Forty participants from 23 countries were sent 10 samples for DNA typing, five samples for antibody detection, a freeze-dried anti-HPA-1a standard, three samples for anti-HPA-1a quantification and a MAIPA method questionnaire. RESULTS: The detection and identification of HPA antibodies varied from 2.7 to 95% of participants. The number of HPA genotyping errors per sample ranged from 0 to 3.96% per HPA loci. The majority of laboratories were able to assign an arbitrary number of units/ml of anti-HPA-1a activity to the unknown samples. The MAIPA questionnaire indicated a wide variation among participants, both in method and in reagents used. CONCLUSIONS: The results obtained from this workshop highlighted deficiencies in testing regimes and identified a need for internationally available reference materials.

Donor exposure rate to transfusion ratio: a better discriminator of improvement in neonatal transfusion practice.

This study identifies the benefit of using donor exposure rate (DER) to transfusion rate (TR) ratio as a discriminative index for assessing improvement in practice pattern in multiple-transfused neonates. It provides a methodology to demonstrate reduction in donor exposure that is not evident from the use of DER alone. Two time points, one 12-month period (1996-1997) before and one 12-month period (1999-2000) following the introduction of a paedipack system, were reviewed. Blood issued and wasted was quantified. The 1994 BSCH guidelines to define transfusion were used for both time periods, and recombinant erythropoietin (EPO) was not used. Following implementation of paedipack system, 186 donor units were made into satellite bags and kept for 35 days. A dramatic decrease in DER : TR ratio was noted for 79 transfused infants. The DER : TR ratio was 1 : 1 before and 1 : 3.2 after introduction of paedipacks, giving a 70.5% reduction in donor exposure risk. This was not evident from the use of DER alone, which remained the same (2.4) in the historical and study groups. High transfusions per donor unit (TPDU) correlated with the reduction in DER : TR ratio. Red cell wastage per transfusion was 190 +/- 30 mL before and 24.5 +/- 10 mL after intervention.

Genetic polymorphism of HLA DR in a Scottish population of patients with pars planitis.

PURPOSE: Human leucocyte antigen (HLA) class II influences the immunological susceptibility for a variety of diseases including many types of non-infectious intraocular inflammation. Previous studies on North American patients with pars planitis, a subtype of intermediate uveitis, reported an increased prevalence of HLA DR15 in this population. In contrast, two European studies could not find an association between HLA DR2 or its allelic subtype DR15 and various forms of intermediate uveitis. We therefore investigated the genotype frequency of HLA DR alleles in a Scottish population of patients with typical pars planitis. METHODS: Twenty patients with pars planitis were identified from the uveitis database of Grampian University Hospitals. Only patients with bilateral vitritis and snowbanks in at least one eye in the absence of systemic disease were included in the study. Fifteen patients and 34 healthy controls underwent HLA DR genotyping for all DRB genes using PCR sequence specific primers. RESULTS: HLA DR15 was found in 13% of patients with pars planitis and in 24% of controls. There was no statistically significant difference between these two groups. Furthermore, the frequencies of HLA DR 1, 3-14, and 16 did not differ significantly between patients and controls. CONCLUSIONS: There appears to be no association between the occurrence of pars planitis and the HLA DR15 or other known HLA DR genotypes in Scottish patients. However, the small sample size limits the power of this study.

Semiautomated data analysis of flow cytometric estimation of fetomaternal hemorrhage in D- women.

BACKGROUND: Accurate and reliable measurement of the volume of fetal D+ cells in D- women is required for adequate anti-D prophylaxis. A semiautomated flow cytometry assay based on a standardized calibration curve that was created with simulated fetomatemal hemorrhage (FMH) mixtures was developed. STUDY DESIGN AND METHODS: A calibration range of 0.083- to 2-percent D+ cells in the D-RBC mixtures (2-44 mL calculated FMH) was analyzed by use of a flow cytometer (XL-MCL, Coulter Electronics Ltd). Linear regression analysis of the calibration curve data with computer software (Excel, Microsoft) allowed semiautomated determination of the FMH volume. To optimize the assay, fresh versus frozen and thawed RBCs, RBCs from adults who are heterozygous for D or cord RBCs, and indirect- or direct-labeling techniques were evaluated by use of MoAbs. RESULTS: Fresh RBCs from adults heterozygous for D were chosen for routine use, although equivalent calibration curves were obtained with all cells tested (n = 12 calibration assays; r2 = 0.999; mean SD, 14%). A monoclonal anti-D reagent (Therad 10, Diagnostics Scotland) worked well in both indirect-(anti-IgG F(ab)-FITC) and direct-(anti-D-FITC) labeling methods compared to the use of BRAD-3 FITC. In routine practice, the FMH volumes obtained were mainly lower than those obtained in the Kleihauer Betke test when there was less than 4 mL of FMH. CONCLUSION: Semiautomated data acquisition and calibration curve analysis represents a further step toward standardization of flow cytometry for accurate FMH quantification and facilitates evaluation and control of day-to-day variations between laboratories, flow cytometers, and operators.

Limited efficacy of universal leucodepletion in reducing the incidence of febrile nonhaemolytic reactions in red cell transfusions.

This article demonstrates a 62% reduction in the number of febrile nonhaemolytic transfusion reactions (FNHTRs) and 50% reduction in febrile reaction rate associated with red cell transfusions following graded introduction of universal leucodepletion. Though this is a statistically significant reduction (P = 0.009), it shows limited efficacy in abrogating this complication. We also found a reduction in the proportion of cases of FNHTRs with lymphocytotoxic antibodies over the period studied from 54% in 1998, 28% in 1999 to 23% in 2000. This corresponds to a relative increase in the number of febrile reactions without human leucocyte antigen (HLA) antibodies following full implementation of universal leucodepletion, as the total number of reported reactions actually fell considerably during the period. The increase in the number of cases without HLA antibodies was directly proportional to the increase in the number of leucodepleted units used.

In vivo platelet activation in atherothrombotic stroke is not determined by polymorphisms of human platelet glycoprotein IIIa or Ib.

Platelet membrane glycoprotein polymorphisms are candidate risk factors for thrombosis, but epidemiological data are conflicting. Thus, demonstration of a genotype-dependent alteration in function is desirable to resolve these inconsistencies. We investigated in vivo platelet activation in acute thrombosis and related this to platelet genotype. Frequencies of the 1b and 2b alleles of the HPA 1a/1b and HPA 2a/2b platelet glycoprotein polymorphisms were determined in 150 (52 men/98 women, mean age 58.3 years) patients with atherothrombotic stroke, and the influence of genotype on markers of platelet activation was assessed. Platelet P-selectin (CD62P) expression and fibrinogen binding was measured using whole blood flow cytometry within 24 h of stroke and 3 months later in 77 patients who provided a repeat blood sample. Results were compared with matched controls. Neither the 1b allele [allele frequency 0.11 vs. 0.13, odds ratio (OR) confidence interval (CI) 0.8 (0.5-1.3)] nor the 2b allele [0.09 vs. 0.07, OR (CI) 1.4 (0.8-2.4)] was significantly over-represented in patients. Increased numbers of activated platelets were found following stroke (acute mean P-selectin expression 0.64% vs. control 0.35%, P < 0.001; acute mean fibrinogen binding 1.6% vs. control 0.9%, P < 0.001). Activation persisted in the convalescent phase (P < 0.001 and P = 0.005 vs. controls for P-selectin and fibrinogen respectively). Expression of P-selectin and fibrinogen was not influenced by either the HPA 1a/1b genotype (P > 0.95 for each marker, Scheffe's test) or the 2a/2b genotype (P > 0.95 for each). Although persisting platelet activation is seen in atherothrombotic stroke, it is independent of HPA 1a/1b and 2a/2b genotypes. These data suggest an underlying prothrombotic state, but do not support the polymorphisms studied as risk factors for thrombotic stroke in this population.

Immunogenetics and clinical phenotype of sympathetic ophthalmia in British and Irish patients.

BACKGROUND/AIMS: Sympathetic ophthalmia (SO) is a classic example of autoimmune disease where human leucocyte antigen (HLA) genomic associations could provide further understanding of mechanisms of disease. This study sought to assess HLA genetic polymorphism in British and Irish patients with SO, and to assess whether HLA gene variants are associated with clinical phenotype or disease severity. METHODS: High resolution DNA based HLA typing using polymerase chain reaction sequence specific primers was performed in 27 patients with SO and 51 matched healthy controls. Clinical phenotype and markers of disease severity were determined prospectively in 17 newly diagnosed patients and from medical record review and repeat clinical examination in 10 previously diagnosed patients. RESULTS: HLA-Cw*03 (p=0.008), DRB1*04 (p=0.017), and DQA1*03 (p=0.014) were significantly associated with SO. For class II alleles at higher resolution, only HLA-DRB1*0404 (relative risk (RR) = 5.6, p = 0.045) was significantly associated with SO. The highest relative risk for any of the associated haplotypes was with HLA-DRB1*0404-DQA1*0301 (RR=10.9, p=0.019). Patients with the DRB1*04-DQA1*03 associated haplotype were significantly more likely to develop SO earlier, with fewer inciting ocular trauma events, and to require more systemic steroid therapy to control inflammatory activity. CONCLUSIONS: Sympathetic ophthalmia is associated with HLA-DRB1*04 and DQA1*03 genotypes in white patients, similar to Japanese patients. Differences in DRB1*04 gene variant associations (-0404 in Britain and Ireland and -0405 in Japan) may have implications for HLA peptide binding in disease initiation. The DRB1*04-DQA1*03 haplotype is a marker of increased SO susceptibility and severity, as in Vogt-Koyanagi-Harada disease, which also has similar clinicopathological and HLA associations.

Fetomaternal alloimmune thrombocytopenia.

Thrombocytopenia is the second commonest haematological abnormality in the neonatal period after anaemia due to iatrogenic blood letting. One to four percent of all newborn babies have a platelet count < 150 x 10(9)/l at birth and approximately 20-40% of neonates in intensive care units are affected by neonatal thrombocytopenia. The most common cause of severe neonatal thrombocytopenia is fetomaternal platelet incompatibility and subsequent alloimmunisation. During the last decade recent advances in molecular techniques have led to rapid and efficient methods for diagnosis. Progress in fetal medicine has enabled accurate determination of fetal status, allowing improvements in fetal diagnosis and therapy. Human platelet antigen (HPA)-1a is by far the most frequently involved platelet antigen system in Caucasians accounting for 90% of cases, followed at a much lower frequency by HPA-5b (5-15%) and HPA-3a. The incidence is estimated to be 1 per 2000 to 1 per 5000 live births, but this is low in comparison to the incidence of fetomaternal platelet antigen incompatibility especially for the HPA-1 alloantigen system in the Caucasian population in whom the estimated frequency of HPA-1b1b individuals is 2%. Retrospective and prospective studies have reported that the immunogenetic background is important, and the chance of HPA-1a alloimmunisation is strongly associated with maternal HLA class II DRB3*0101 (DR52a) type. A significant association (p = 0.004) between severe thrombocytopenia and a third trimester antiHPA titre >1:32 has been observed. It is now possible to genotype the fetus or neonate and the parents, which provides confirmation as to which HPA systems are incompatible between the mother and father. Simultaneous genotyping of HPA-1, 2, 3 and 5 can be carried out using the polymerase chain reaction-sequence specific primers (PCR-SSP) protocol, which has been widely used for HLA class II determination. The platelet count may continue to fall during the first 48 h after birth and the risk of intracranial bleeding is highest during this period. The best option is transfusion of specially selected antigen negative compatible donor platelets or if unavailable, maternal washed platelets. Antenatal screening for the most common form of fetomaternal alloimmune thrombocytopenia (FMAIT), due to antiHPA-1a is under consideration, but there is no established method at present. The Scottish National Blood Transfusion Service started a study in August 1999 on 25,000 pregnancies to carry out a cost benefit analysis of routine antenatal screening. The aims of the study are to determine the frequency of HPA-1b homozygosity; monitor antibody titres during pregnancy and confirm correlation of antibody emergence with HLA-DRB3*0101, and finally to access cost effectiveness of routine screening across Scotland. Of 26,509 women screened in three Scottish regions 501 (1.9%) are HPA-1b homozygous and about 9%, of the consented women are antibody positive.