HLA class I depletion by citric acid, and irradiation of apheresis platelets for transfusion of refractory patients.

BACKGROUND: Patients can form antibodies to foreign human leukocyte antigen (HLA) Class I antigens after exposure to allogeneic cells. These anti-HLA class I antibodies can bind transfused platelets (PLTs) and mediate their destruction, thus leading to PLT refractoriness. Patients with PLT refractoriness need HLA-matched PLTs, which require expensive HLA typing of donors, antibody analyses of patient sera and/or crossmatching. An alternative approach is to reduce PLT HLA Class I expression using a brief incubation in citric acid on ice at low pH. METHODS AND MATERIALS: Apheresis PLT concentrates were depleted of HLA Class I complexes by 5 minutes incubation in ice-cold citric acid, at pH 3.0. Surface expression of HLA Class I complexes, CD62P, CD63, phosphatidylserine, and complement factor C3c was analyzed by flow cytometry. PLT functionality was tested by thromboelastography (TEG). RESULTS: Acid treatment reduced the expression of HLA Class I complexes by 71% and potential for C3c binding by 11.5-fold compared to untreated PLTs. Acid-treated PLTs were significantly more activated than untreated PLTs, but irrespective of this increase in steady-state activation, CD62P and CD63 were strongly upregulated on both acid-treated and untreated PLTs after stimulation with thrombin receptor agonist peptide. Acid treatment did not induce apoptosis over time. X-ray irradiation did not significantly influence the expression of HLA Class I complexes, CD62P, CD63, and TEG variables on acid treated PLTs. CONCLUSION: The relatively simple acid stripping method can be used with irradiated apheresis PLTs and may prevent transfusion-associated HLA sensitization and overcome PLT refractoriness.

Fast and low-cost direct ELISA for high-throughput serological HPA-1a typing.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloantibodies against fetal human platelet antigens (HPAs), mostly caused by anti-HPA-1a. Population-based screening for FNAIT is still a topic of debate. Logistically and financially, the major challenge for implementation is the typing of pregnant women to recognize the 2% HPA-1a-negative women. Therefore, there is need for a high-throughput and low-cost HPA-1a-typing assay. STUDY DESIGN AND METHODS: A sandwich ELISA was developed, using a monoclonal anti-GPIIIa as coating antibody and horseradish-peroxidase-conjugated recombinant anti-HPA-1a, as detecting antibody. The ELISA results were compared to an allelic discrimination PCR-assay. In phase I, samples from unselected consecutive pregnant women were tested with both assays. Phase II was part of a prospective screening study in pregnancy and genotyping was restricted to samples with an arbitrary set, OD < 0.500. RESULTS: The ELISA was optimized to require no additional handling (swirling or spinning) of stored tubes. During phase I, 506 samples were tested. In phase II, another 62,171 consecutive samples were phenotyped, with supportive genotyping in 1,902. In total 1,585 HPA-1a negative and 823 HPA-1a positive women were genotyped. The assay reached 100% sensitivity with a cut-off OD from 0.160, corresponding with a 99.9% specificity and a false-HPA-1a negative rate of 0.03. CONCLUSION: A high-throughput, low-cost, and reliable HPA-1a phenotyping assay was developed which can be used in population-based screening to select samples for testing of presence of anti-HPA-1a. Because plasma from tubes of 3- to 6-days-old samples can be used, this assay is applicable to settings with suboptimal conditions.

Buccal swab genomic DNA fragmentation predicts likelihood of successful HLA genotyping by next-generation sequencing.

Many clinical human leukocyte antigen (HLA) laboratories are adopting next-generation sequencing (NGS) technology for HLA genotyping. There have been several reports of the cost-benefit and reduction in turn-around-time provided by NGS. Ninety-six percent of buccal swabs and peripheral blood samples had reportable HLA genotyping by NGS. The HLA loci most likely to fail genotyping from buccal swabs were DQB1, DPB1, and DPA1. Successful buccal swab samples had significantly less genomic DNA fragmentation compared to buccal swab samples that were unsuccessful. Increasing sequencing depth of coverage for heavily fragmented samples rescued HLA genotyping. This information provides laboratories with a quality assurance parameter that reduces the amount of repeat NGS needed to achieve high-resolution HLA genotyping. This information should further reduce laboratory and patient costs for HLA genotyping.

The Incremental Cost of Incompatible Living Donor Kidney Transplantation: A National Cohort Analysis.

Incompatible living donor kidney transplantation (ILDKT) has been established as an effective option for end-stage renal disease patients with willing but HLA-incompatible living donors, reducing mortality and improving quality of life. Depending on antibody titer, ILDKT can require highly resource-intensive procedures, including intravenous immunoglobulin, plasma exchange, and/or cell-depleting antibody treatment, as well as protocol biopsies and donor-specific antibody testing. This study sought to compare the cost and Medicare reimbursement, exclusive of organ acquisition payment, for ILDKT (n = 926) with varying antibody titers to matched compatible transplants (n = 2762) performed between 2002 and 2011. Data were assembled from a national cohort study of ILDKT and a unique data set linking hospital cost accounting data and Medicare claims. ILDKT was more expensive than matched compatible transplantation, ranging from 20% higher adjusted costs for positive on Luminex assay but negative flow cytometric crossmatch, 26% higher for positive flow cytometric crossmatch but negative cytotoxic crossmatch, and 39% higher for positive cytotoxic crossmatch (p < 0.0001 for all). ILDKT was associated with longer median length of stay (12.9 vs. 7.8 days), higher Medicare payments ($91 330 vs. $63 782 p < 0.0001), and greater outlier payments. In conclusion, ILDKT increases the cost of and payments for kidney transplantation.

Performance Characteristics and Validation of Next-Generation Sequencing for Human Leucocyte Antigen Typing.

High-resolution human leukocyte antigen (HLA) matching reduces graft-versus-host disease and improves overall patient survival after hematopoietic stem cell transplant. Sanger sequencing has been the gold standard for HLA typing since 1996. However, given the increasing number of new HLA alleles identified and the complexity of the HLA genes, clinical HLA typing by Sanger sequencing requires several rounds of additional testing to provide allele-level resolution. Although next-generation sequencing (NGS) is routinely used in molecular genetics, few clinical HLA laboratories use the technology. The performance characteristics of NGS HLA typing using TruSight HLA were determined using Sanger sequencing as the reference method. In total, 211 samples were analyzed with an overall accuracy of 99.8% (2954/2961) and 46 samples were analyzed for precision with 100% (368/368) reproducibility. Most discordant alleles were because of technical error rather than assay performance. More important, the ambiguity rate was 3.5% (103/2961). Seventy-four percentage of the ambiguities were within the DRB1 and DRB4 loci. HLA typing by NGS saves approximately $6000 per run when compared to Sanger sequencing. Thus, TruSight HLA assay enables high-throughput HLA typing with an accuracy, precision, ambiguity rate, and cost savings that should facilitate adoption of NGS technology in clinical HLA laboratories.

Coeliac screening in a Scottish cohort of children with type 1 diabetes mellitus: is DQ typing the way forward?

BACKGROUND: Children with type 1 diabetes mellitus (T1DM) are at increased risk of coeliac disease (CD). Recent guidelines indicate coeliac screening should include HLA typing for CD predisposing (DQ2/DQ8) alleles and those negative for these alleles require no further coeliac screening. METHODS: Children (n=176) with T1DM attending clinics across two Scottish regions were screened for HLA DQ2/DQ8 as part of routine screening. Data collected included the frequency of DQ2/DQ8 genotypes and the additional cost of HLA screening. RESULTS: Overall, DQ2/DQ8 alleles were identified in 94% of patients. The additional cost of HLA typing was £3699.52 (£21.02 per patient). All patients with known CD (11/176) were positive for DQ2/DQ8 and all were diagnosed with CD within 5 years of T1DM diagnosis. CONCLUSIONS: The vast majority of children with T1DM have CD-predisposing HLA genotypes limiting the number of patients that can be excluded from further screening. We conclude that HLA genotyping is not currently indicated for CD screening in this population.

Cost-effectiveness of pediatric heart transplantation across a positive crossmatch for high waitlist urgency candidates.

Allosensitized children listed with a requirement for a negative prospective crossmatch have high mortality. Previously, we found that listing with the intent to accept the first suitable organ offer, regardless of the possibility of a positive crossmatch (TAKE strategy), results in a survival advantage from the time of listing compared to awaiting transplantation across a negative crossmatch (WAIT). The cost-effectiveness of these strategies is unknown. We used Markov modeling to compare cost-effectiveness between these waitlist strategies for allosensitized children listed urgently for heart transplantation. We used registry data to estimate costs and waitlist/posttransplant outcomes. We assumed patients remained in hospital after listing, no positive crossmatches for WAIT, and a base-case probability of a positive crossmatch of 47% for TAKE. Accepting the first suitable organ offer cost less ($405 904 vs. $534 035) and gained more quality-adjusted life years (3.71 vs. 2.79). In sensitivity analyses, including substitution of waitlist data from children with unacceptable antigens specified during listing, TAKE remained cost-saving or cost-effective. Our findings suggest acceptance of the first suitable organ offer for urgently listed allosensitized pediatric heart transplant candidates is cost-effective and transplantation should not be denied because of allosensitization status alone.

Do human leukocyte antigen-typed cellular therapeutics based on induced pluripotent stem cells make commercial sense?

The promise of off-the-shelf cellular therapeutics (CTPs) based on allogeneic induced pluripotent stem cells (iPSCs) may be hindered by alloimmunity, leading many to suggest that such products could be based on a series of human leukocyte antigen (HLA)-typed iPSC lines allowing at least some degree of tissue matching. While based on sound scientific principles, this suggestion presupposes that other immune responses will not be limiting. Technically this approach would present a number of major challenges, the first being the development of a suitably reliable reprogramming method amenable to validation that results in highly consistent iPSC lines. Further, the resulting array of HLA-typed iPSCs would need to be shown to be capable of being manufactured into the same CTP and exhibit comparable quality, safety, and efficacy. When the enormities of these challenges are laid out, it becomes apparent that the manufacturing and product development challenges would be unprecedented. Given the uncertainties and lack of clinical experience with iPSC-based CTPs at this time, the financial costs and commercial risks do not appear to be acceptable.

Impact of three Illumina library construction methods on GC bias and HLA genotype calling.

Next-generation sequencing (NGS) is increasingly recognized for its ability to overcome allele ambiguity and deliver high-resolution typing in the HLA system. Using this technology, non-uniform read distribution can impede the reliability of variant detection, which renders high-confidence genotype calling particularly difficult to achieve in the polymorphic HLA complex. Recently, library construction has been implicated as the dominant factor in instigating coverage bias. To study the impact of this phenomenon on HLA genotyping, we performed long-range PCR on 12 samples to amplify HLA-A, -B, -C, -DRB1, and -DQB1, and compared the relative contribution of three Illumina library construction methods (TruSeq Nano, Nextera, Nextera XT) in generating downstream bias. Here, we show high GC% to be a good predictor of low sequencing depth. Compared to standard TruSeq Nano, GC bias was more prominent in transposase-based protocols, particularly Nextera XT, likely through a combination of transposase insertion bias being coupled with a high number of PCR enrichment cycles. Importantly, our findings demonstrate non-uniform read depth can have a direct and negative impact on the robustness of HLA genotyping, which has clinical implications for users when choosing a library construction strategy that aims to balance cost and throughput with data quality.

Can a combined screening/treatment programme prevent premature failure of renal transplants due to chronic rejection in patients with HLA antibodies: study protocol for the multicentre randomised controlled OuTSMART trial.

BACKGROUND: Renal transplantation is the best treatment for kidney failure, in terms of length and quality of life and cost-effectiveness. However, most transplants fail after 10 to 12 years, consigning patients back onto dialysis. Damage by the immune system accounts for approximately 50% of failing transplants and it is possible to identify patients at risk by screening for the presence of antibodies against human leukocyte antigens. However, it is not clear how best to treat patients with antibodies. This trial will test a combined screening and treatment protocol in renal transplant recipients. METHODS/DESIGN: Recipients>1 year post-transplantation, aged 18 to 70 with an estimated glomerular filtration rate>30 mL/min will be randomly allocated to blinded or unblinded screening arms, before being screened for the presence of antibodies. In the unblinded arm, test results will be revealed. Those with antibodies will have biomarker-led care, consisting of a change in their anti-rejection drugs to prednisone, tacrolimus and mycophenolate mofetil. In the blinded arm, screening results will be double blinded and all recruits will remain on current therapy (standard care). In both arms, those without antibodies will be retested every 8 months for 3 years. The primary outcome is the 3-year kidney failure rate for the antibody-positive recruits, as measured by initiation of long-term dialysis or re-transplantation, predicted to be approximately 20% in the standard care group but <10% in biomarker-led care. The secondary outcomes include the rate of transplant dysfunction, incidence of infection, cancer and diabetes mellitus, an analysis of adherence with medication and a health economic analysis of the combined screening and treatment protocol. Blood samples will be collected and stored every 4 months and will form the basis of separately funded studies to identify new biomarkers associated with the outcomes. DISCUSSION: We have evidence that the biomarker-led care regime will be effective at preventing graft dysfunction and expect this to feed through to graft survival. This trial will confirm the benefit of routine screening and lead to a greater understanding of how to keep kidney transplants working longer. TRIAL REGISTRATION: Current Controlled Trials ISRCTN46157828.

Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing.

BACKGROUND: A close match of the HLA alleles between donor and recipient is an important prerequisite for successful unrelated hematopoietic stem cell transplantation. To increase the chances of finding an unrelated donor, registries recruit many hundred thousands of volunteers each year. Many registries with limited resources have had to find a trade-off between cost and resolution and extent of typing for newly recruited donors in the past. Therefore, we have taken advantage of recent improvements in NGS to develop a workflow for low-cost, high-resolution HLA typing. RESULTS: We have established a straightforward three-step workflow for high-throughput HLA typing: Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 are amplified by PCR on Fluidigm Access Array microfluidic chips. Illumina sequencing adapters and sample specific tags are directly incorporated during PCR. Upon pooling and cleanup, 384 samples are sequenced in a single Illumina MiSeq run. We developed "neXtype" for streamlined data analysis and HLA allele assignment. The workflow was validated with 1140 samples typed at 6 loci. All neXtype results were concordant with the Sanger sequences, demonstrating error-free typing of more than 6000 HLA loci. Current capacity in routine operation is 12,000 samples per week. CONCLUSIONS: The workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing. Despite the focus on cost efficiency, resolution exceeds the current standards of Sanger typing for donor registration.

'It's a regional thing': financial impact of renal transplantation on live donors.

INTRODUCTION: There has been no research exploring the financial impact on the live renal donor in terms of testing, hospitalisation and surgery for kidney removal (known as nephrectomy). The only mention of financial issues in relation to live renal transplantation is the recipients' concerns in relation to monetary payment for the gift of a kidney and the recipients' desire to pay for the costs associated with the nephrectomy. The discussion in this article posits a new direction in live renal donor research; that of understanding the financial impact of live renal donation on the donor to inform health policy and supportive care service delivery. The findings have specific relevance for live renal donors living in rural and remote locations of Australia. METHODS: The findings are presented from the first interview (time 1: T1) of a set of four times (time 1 to time 4: T1-T4) from a longitudinal study that explored the experience of live renal donors who were undergoing kidney removal (nephrectomy) at the Renal Transplantation Unit at the Princess Alexandra Hospital, Brisbane, Australia. A qualitative methodological approach was used that involved semi-structured interviews with prospective living kidney donors (n=20). The resulting data were analysed using the qualitative research methods of coding and thematic analysis. RESULTS: The findings indicate that live renal donors in non-metropolitan areas report significant financial concerns in relation to testing, hospitalisation and surgery for nephrectomy. These include the fact that bulk billing (no cost to the patient for practitioner's service) is not always available, that individuals have to pay up-front and that free testing at local public hospitals is not available in some areas. In addition, non-metropolitan donors have to fund the extra cost of travel and accommodation when relocating for the nephrectomy to the specialist metropolitan hospital. CONCLUSION: Live renal transplantation is an important new direction in medical care that has excellent long-term results for individuals diagnosed with end-stage renal disease. An essential element of the transplantation procedure is the voluntary donation of a healthy kidney by the live renal donor. Such an altruistic gift, which has no personal health benefit for the donor, is to be applauded and supported. The present research demonstrates that for some donors, particularly those living outside the metropolitan area, the gift may also include a range of financial costs to the donor. There is no prior research available on the financial impact of live renal donation for individuals living in non-metropolitan areas. Thus, this article is a seminal work in the area. The findings affirm 'rural disadvantage' by demonstrating that it is the live renal donors in non-metropolitan areas who are reporting financial concerns in relation to testing, hospitalisation and surgery for nephrectomy. It is the hope and expectation that the reporting on these costs will encourage further work in this area and the findings will be used for health policy and service delivery considerations.

New law of renal transplantation in Portugal associated with more acute rejection episodes and higher costs.

The new law implemented in August 2007 changed the criteria to select renal transplantation (RT) candidates in Portugal, favoring hyperimmunized subjects and those on the waiting list for a longer time, making human leukocyte antigen (HLA) compatibilities less important. The authors compared patients who received a deceased donor kidney between 2005 and 2010. Patients were divided in group A who underwent transplantation before August 2007 (n = 132) and group B (n = 125) after that date. We considered a value of P < .05. Overall mean age at RT was 46.6 ± 13.9 years with 58.8% men, 88% on hemodialysis (HD), with a mean dialysis time of 82.8 ± 119 months. Also, 10.5% of patients underwent a previous transplantation. The mean follow-up was 35 ± 17.1 months. Group B showed significant adverse differences, including dialysis time (50.9 vs. 117 months), length of hospitalization (14.4 vs. 23.2 days), need for HD (1 vs. 3.4 days), HLA match (3.3 vs. 1.4 compatibilities), previous sensitization (4.4% vs. 21.7%), acute rejection episodes in the 1st year (23% vs. 37%), greater use of immunosuppressive drugs, higher costs of induction therapy (2790 vs 4360ϵ), and greater costs of drugs during first hospitalization (3456 vs. 7144ϵ). Among the 16 subjects who lost their grafts, 7 were in group A (3 in the first year) and 9 in group B all in the first year. There was a 5.1% decrease in graft survival at 12 months (P = .07). Univariate analysis showed an association of acute rejection episodes with HLA mismatches, hyperimmunized patients, absence of immediate graft function, hospitalization time, longer HD need, and higher creatinine level at months 1, 2, 3, and 6. Multivariate analysis revealed acute rejection episodes to be associated with a lower number of HLA compatibilities (odds ratio = 0.65; 95% confidence interval, [0.46-0.9]). Application of the law has led to a greater number of acute rejection episodes in the first year and increased costs.

Multiplexed, ligation-dependent probe amplification for rapid and inexpensive HLA-DQB1 allelotyping.

Many effective options exist to accurately type DNA for human leukocyte antigen (HLA) alleles. However, most of the existing methods are excessively costly in terms of overall monetary costs, DNA requirements, and proprietary software. We present a novel assay capable of resolving heterozygous HLA-DQB1 allelotypes at two digits, with even greater specificity for the HLA-DQB1*06 allele family, by using the multiplexed ligation-dependent probe amplification technology. This assay provides more specific allele data than genome-wide analysis and is more affordable than sequencing, making it a useful intermediate for researchers seeking to accurately allelotype human DNA samples.

A novel single cDNA amplicon pyrosequencing method for high-throughput, cost-effective sequence-based HLA class I genotyping.

Human leukocyte antigen (HLA) genotype influences the immune response to pathogens and transplanted tissues; accurate HLA genotyping is critical for clinical and research applications. Sequence-based HLA typing is limited by the cost of Sanger sequencing genomic DNA (gDNA) and resolving cis/trans ambiguities, hindering both studies correlating high-resolution genotype with clinical outcomes, and population-specific allele frequency surveys. We present an assay for sequence-based HLA genotyping by titanium read length clonal Roche/454 pyrosequencing of a single, universally diagnostic polymerase chain reaction (PCR) amplicon from HLA class I cDNA that captures most of exons 2, 3, and 4 used for traditional sequence-based typing. The amplicon is predicted to unambiguously resolve 85% of known alleles. A panel of 48 previously HLA-typed samples was assayed with this method, demonstrating 100% non-null allele typing concordance. We show that this technique can multiplex at least 768 patients per sequencing run with multiplex identifier sequence bar-coding. Unprecedented typing throughput results from a novel single cDNA-PCR amplicon strategy requiring only 1 PCR amplification per sample. This method dramatically reduces cost for genotyping of large cohorts.

Prediction of duration and success rate of unrelated hematopoietic stem cell donor searches based on the patient's HLA-DRB1 allele and DRB1-DQB1 haplotype frequencies.

Rapid identification of a matched unrelated donor is essential for patients in need of hematopoietic SCT. We carried out a retrospective evaluation of 549 unrelated donor searches (UDSs), which were completed in 2005 for 23 German transplant centers. On the basis of the patient's HLA-DRB1 allele and DRB1-DQB1 haplotype frequencies, UDSs were divided into four groups with different search success probability predictions. For 90.5% of the patients, an acceptable HLA-matched, and for 61.6% an HLA-A-B-Cw-DRB1-DQB1-identical (10/10 matched) unrelated donor was found. The median search duration was 22 days. In the groups with high (n = 318), medium (n = 157), low (n = 56) and very low (n = 18) UDS success probability, an acceptable donor was found for 99.1, 86.6, 75.0 and 22.2% of the patients, and a 10/10-matched donor was found for 78.3, 49.7, 17.9 and 4.5% of the patients, respectively. The median search duration was 20, 27, 45 and 477 days in the groups with high, medium, low and very low probability, respectively. The search success rate and duration can be predicted on the basis of the patient's HLA-DRB1 allele and HLA-DRB1-DQB1 haplotype frequencies. An unrelated donor can be found for most of the patients, even if the indication for transplantation is urgent.

[Economic evaluation of the organization of a registry of haematopoietic stem cell donors]. / Evaluation économique de l'organisation d'un registre de donneurs de cellules souches hématopoïétiques.

BACKGROUND: Availability of a healthy, human-leukocyte-antigen-matched hematopoietic stem cell source is a prerequisite for successful allogenic hematopoietic stem cell transplantation. In 70% of cases, the search of hematopoietic stem cells shifts from siblings to unrelated donor registries. Given that the Human Leucocytes Antigens (HLA) system is highly polymorphic and that the cost of HLA typing remains high, the adequacy between registry content and patient needs must be assessed. Registries should be optimally organized to increase the probability for any given patient to find a donor. METHODS: A welfare function associated with the existence of an HLA registry was defined as was a measure of the advantage for laboratories having performed HLA typing. We hypothesized a way to formalize registry efficiency and applied it to the French Hematopoietic Stem Cell donors Registry. RESULTS: The model determined an implicit value for the stem cell graft and showed that efficiency increased very slowly with increasing number of potential donors in registries. The optimal size of a registry was found to be sensitive to model parameters. CONCLUSION: Increased registry size, in terms of number of donors foreseeable in the French registry, would have a limited impact on registry efficiency and thus social effectiveness. Nevertheless, the calibration of the model justifies the goal of recruiting 100000 new volunteer donors over the next 10 years as proposed by the French government in the "Graft Plan". The policy of the regulatory agency should be oriented towards improving the probability a compatible potential donor identified during a preliminary search would become an actual fully compatible donor and towards reducing the cost of typing.

The co$t of mi$matching.

We have shown that the number of HLA mismatched antigens correlates with the development of new or changes in existing HLA-specific antibodies. We have further shown that the magnitude of the effect varies among groups defined by whether or not HLA-specific antibody was present prior to transplant, by the transplant number, by recipient race, and by donor type. The increases in antibody, which increase with increasing degree of mismatch, result in differences in waiting times reflective of the number of previous mismatches. For many patients, increased waiting time represents not only reduced quality of life but deteriorating health and shortened life expectancy. Globally, increased waiting times translate into increased costs for dialysis, antibody testing, and health care. These factors suggest that HLA matching should not be abandoned but should be given consideration for those patients most affected by mismatches.

Prospective epidemiologic study of the outcome and cost-effectiveness of antenatal screening to detect neonatal alloimmune thrombocytopenia due to anti-HPA-1a.

BACKGROUND: To assess the value of antenatal screening to detect neonatal alloimmune thrombocytopenia (NAIT) due to anti-HPA-1a, a prospective study was carried out to quantify the potential clinical benefits and determine whether screening would be cost-effective. STUDY DESIGN AND METHODS: An observational prospective controlled study was carried out on 26,506 pregnant women over 2 years. HPA-1a phenotyping was performed in the first trimester and women confirmed HPA-1a-negative were tested for anti-HPA-1a during pregnancy, at delivery, and 10 to 14 days after birth. Babies of HPA-1a-negative women were tested at delivery for thrombocytopenia and examined for signs of bleeding. Economic evaluation was undertaken on the basis of the data collected during the study. RESULTS: Twenty-five of 318 women (7.9%) had anti-HPA-1a detected for the first time. Eight women (43 per 100,000) gave birth to babies with NAIT, and 5 (27 per 100,000) had severe thrombocytopenia. Three babies had mild signs of bleeding, and no cases of intracranial hemorrhage (ICH) or fetal loss were detected. It is estimated that it would cost 60,596 pounds (98,771 US dollars) to detect a case of severe NAIT, where anti-HPA-1a has been identified for the first time, and 1,151,323 pounds (1,876,656 US dollars) to prevent a case of ICH, assuming that detection allowed successful intervention. CONCLUSIONS: Our data suggest that severe HPA-1a NAIT is underdiagnosed in the absence of routine antenatal screening. Serious bleeding complications and ICH, however, occur less frequently in first cases of NAIT than suspected from the literature, and the costs of screening and possible intervention must be balanced against the procedural risks.