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Background and objective Routine antenatal anti-D prophylaxis (RAADP) to RhD-negative women is most often administered in gestational age (GA) 28-30 weeks with the next anti-D dose administered postpartum. The aim of this study was to analyse the proportion of RhD-negative women where RAADP is not detectable at term and in a pilot study to investigate whether RAADP administered in GA 28 and 38 results in detectable levels at term, post-term and post-delivery. Materials and methods In a retrospective analysis, 4280 RhD-negative women carrying an RHD positive fetus were included and the proportion with a negative antibody screen at delivery was determined. In the second part, 39 pregnancies were included prospectively, a second dose of RAADP was administered in GA 38 weeks, and anti-D was quantified before the second dose and then weekly for 5 weeks. Results In the retrospective analysis, 20·5% (856/4280) with RAADP administered in GA 28 were negative in routine antibody screening at delivery. In the small prospective study, 18% (7/39) had a negative antibody screen and 26% (10/39) had levels below 0·005 IU/ml, in the quantification assay, in GA 38. Anti-D prophylaxis administered in GA 38 showed detectable levels of anti-D up to 30 days post-delivery, with concentration at delivery 0·060 ± 0·034 IU/ml (mean ± SD). Conclusion Approximately 20% of the RhD-negative women show non-detectable levels of anti-D at term. A second dose of RAADP at GA 38 results in stable concentrations of anti-D at term, post-term and post-delivery, but with large interindividual variation.

Naturally occurring anti-Kpa antibody is extremely rare and was first reported in 1957, named after the first producer 'Penney'. However, the subsequent anti-Kpa reports presented were all anti-Kpa due to isoimmunization. Individuals with severe bacterial infections particularly Gram-negative bacteria are known to be capable of producing cross-reactive antibodies against Kell blood group system. However, such uncommon antibodies like anti-Kpa can be easily missed in routine pre-transfusion testing unless the panel cells containing low incidence antigen are used for antibody screening. Here, we report a case of naturally occurring anti-Kpa antibody, identified incidentally during pre-transfusion testing of a 12-month-old female infant with the diagnosis of Niemann-Pick disease and recurrent bacterial (Escherichia coli) infection.

ALX148, a novel CD47 blocking agent, is in clinical development for the treatment of advanced solid tumors and lymphoma. Because CD47 is highly expressed on red blood cells (RBCs), its therapeutic blockade can potentially interfere with pretransfusion compatibility testing. This study describes the interference of ALX148 in pretransfusion compatibility testing and evaluates the methods used for mitigating such interference. Study design and methods Routine serologic tests were performed on six samples from four patients treated with ALX148. Antibody screening tests were performed on ALX148-spiked plasma, and RBC testing including antigen typing was performed on ALX148-coated RBCs. Soluble CD47 or high-affinity signal regulatory protein α (SIRPα) monomers were used to remove the false-positive reactivity of ALX148-spiked plasma with or without anti-E. Results ALX148 caused false-positive reactivity in antibody screening using indirect antiglobulin testing (IAT) and two-stage papain testing. However, false-positive reactivity was not observed at the immediate spin (IS), room temperature (RT), and 37°C phases. Direct antiglobulin testing, autologous controls, and eluates showed positive results. ALX148 did not affect blood group antigen typing performed at the IS or RT phases. The use of 50- to 100-fold molar excess of soluble CD47 or 300-fold molar excess of high-affinity SIRPα monomers removed false-positive reactivity in IAT without affecting anti-E detection. Conclusion ALX148 generates false-positive reactivity in IAT, interfering with pretransfusion compatibility testing. The use of soluble CD47 or high-affinity SIRPα monomers can resolve the interference without possibly missing clinically significant alloantibodies.

Antiviral monoclonal antibody (mAb) discovery enables the development of antibody-based antiviral therapeutics. Traditional antiviral mAb discovery relies on affinity between antibody and a viral antigen to discover potent neutralizing antibodies, but these approaches are inefficient because many high affinity mAbs have no neutralizing activity. We sought to determine whether screening for anti-SARS-CoV-2 mAbs at reduced pH could provide more efficient neutralizing antibody discovery. We mined the antibody response of a convalescent COVID-19 patient at both physiological pH (7.4) and reduced pH (4.5), revealing that SARS-CoV-2 neutralizing antibodies were preferentially enriched in pH 4.5 yeast display sorts. Structural analysis revealed that a potent new antibody called LP5 targets the SARS-CoV-2 N-terminal domain supersite via a unique binding recognition mode. Our data combine with evidence from prior studies to support antibody screening at pH 4.5 to accelerate antiviral neutralizing antibody discovery. This article is protected by copyright. All rights reserved.

Protein engineering and synthetic biology stand to benefit immensely from recent advances in silico tools for structural and functional analyses of proteins. In the context of designing novel proteins, current in silico tools inform the user on individual parameters of a query protein, with output scores/metrics unique to each parameter. In reality, proteins feature multiple "parts"/functions and modification of a protein aimed at altering a given part, typically has collateral impact on other protein parts. A system for prediction of the combined effect of design parameters on the overall performance of the final protein does not exist. Function2Form Bridge (F2F-Bridge) attempts to address this by combining the scores of different design parameters pertaining to the protein being analyzed into a single easily interpreted output describing overall performance. The strategy comprises of (a) a mathematical strategy combining data from a myriad of in silico tools into an OP-score (a singular score informing on a user-defined overall performance) and (b) the F2F Plot, a graphical means of informing the wetlab biologist holistically on designed construct suitability in the context of multiple parameters, highlighting scope for improvement. F2F predictive output was compared with wetlab data from a range of synthetic proteins designed, built, and tested for this study. Statistical/machine learning approaches for predicting overall performance, for use alongside the F2F plot, were also examined. Comparisons between wetlab performance and F2F predictions demonstrated close and reliable correlations. This user-friendly strategy represents a pivotal enabler in increasing the accessibility of synthetic protein building and de novo protein design.

BACKGROUND Parvovirus B19 (B19V) can cause severe anemia, hydrops foetalis, and even death in vulnerable patients. To prevent transfusion-transmitted B19V infection of at-risk patients, B19V antibody screening of blood donors was implemented. The cost-effectiveness of this intervention is unclear, as the likelihood of transmission through blood and subsequent complications for recipients are unknown. This study estimates the cost-effectiveness of anti-B19V donor screening in the Netherlands. STUDY DESIGN AND METHODS The estimates needed for the cost-effectiveness model were: the occurrence of B19V in Dutch blood donors, the number of anti-B19V tested products required by hospitals, the likelihood of morbidity and mortality given B19V infection, treatment costs, and screening costs. These estimates were obtained from literature and observational data. When data were unavailable, structured expert judgment elicitation and statistical modeling were applied. RESULTS The costs of preventing one transfusion transmitted B19V infection are estimated at €68,942 (€42,045 - €102,080). On average, 1.25 cases of morbidity and 0.12 cases of mortality are prevented annually. Although the perceived risk of transfusion transmitted B19V infection was low, half of the treating physicians favored anti-B19V screening. CONCLUSION The estimated mortality and morbidity caused by B19V infection was low in the risk groups. The cost-effectiveness ratio is similar to other blood safety screening measures. No guidance exists to evaluate the acceptability of this ratio. The explicit overview of costs and effects may further guide the discussion of the desirability of B19V safe blood products.

BACKGROUND An alloimmune response to red blood cell (RBC) transfusion in neonates is a rare event. Several guidelines recommend limited pretransfusion testing in neonates. The evidence for these recommendations is based on small studies with sample sizes of between 30 and 90 infants. STUDY DESIGN AND METHODS We conducted a retrospective cohort study among consecutive patients who received transfusions at a single university medical center. All non-alloimmunized patients who had received their first RBC transfusion between 1994 and 2013 and who underwent at least one antibody screening follow-up visit between 7 and 365 days after transfusion were included. RESULTS The incidence of alloimmunization in the control group of 17,084 adult patients age 45 years or older who had received a median of 5 RBC units (interquartile range, 2-12 RBC units) was 3.55% (n = 607 alloimmunized patients). After transfusion of 40 RBC units, the cumulative incidence of alloimmunization in adult controls was 10.24% (95% confidence interval, 7.71%-13.17%). In total, 1641 neonates and children up to age 3 years received a median of 4 RBC units (interquartile range, 2-7 RBC units) in a median of two RBC transfusion episodes (interquartile range, one to five RBC transfusion episodes). Two children developed anti-M and anti-E antibodies post-transfusion at the ages of 181 and 611 days, respectively. CONCLUSION To our knowledge, this study presents the largest longitudinal cohort study of RBC alloimmunization in neonates. Antibodies against RBC antigens were not detected within the first 6 months of life. Repeat antibody screening and cross-matching during the first months of life can be safely omitted.

SUMMARYObjectives Enzyme indirect antiglobulin test (EIAT) and polyethylene glycol IAT (PIAT) were evaluated for their potential use as tests to distinguish between prophylactic and alloimmune anti-D in plasma by comparing with a tube variation of the standard low ionic strength solution-IAT (LISS-IAT). Background Laboratories performing the screening of RhD-negative pregnant women are required to provide clinicians with guidance as to the source of detected RhD antibodies. Currently, this is derived from RhIg immunoprophylaxis history, agglutination scores and titration results, where performed. A serological test that can differentiate between prophylactic and alloimmune anti-D would be useful in the diagnosis of RhD alloimmunisation in pregnant women. Materials and Methods Plasma samples (n = 273) [fresh (collected from April 2014 to February 2015) and frozen (up to 2 years)] from antenatal females, preoperative males and females over child-bearing age were used in this study. Samples were identified as containing anti-D by routine column agglutination (CAT) and were tested by tube LISS-IAT, EIAT and PIAT, and a score difference was calculated. Results A total of 32% of alloimmune anti-D samples demonstrated an increase in agglutination score (+2 or +3) when tested by EIAT. A significant increase in agglutination score for alloimmune samples using EIAT compared with LISS-IAT was observed. EIAT had a sensitivity (Sn) of 59%, positive predictive value (PPV) of 100% and specificity (Sp) of 100% for alloimmune anti-D. Conclusion EIAT is capable of confirming but not excluding the presence of alloimmune anti-D in samples where anti-D is detected in routine antibody screening.

BACKGROUND Kidd blood group antibodies are notorious for transient detection and hemolytic transfusion reactions. This report compares the rate of detection of anti-Jka when using gel column agglutination versus solid-phase red blood cell adherence (SPRCA) testing and documents the occurrence of hemolytic transfusion reactions in 17 recently transfused patients who developed anti-Jka that were detectable by SPRCA but were undetectable by gel. STUDY DESIGN AND METHODS Before April 20, 2011, the laboratory used gel column agglutination as the primary method for antibody screening and identification. From April 20, 2011, to August 12, 2013, SPRCA was adopted as the primary method for antibody screen with gel remaining the primary method for identification. SPRCA identification was also performed if sufficient sample was available. Medical records were reviewed for evidence of hemolytic reaction in patients whose anti-Jka was negative or inconclusive by gel, but clearly identifiable by SPRCA at the time the anti-Jka was first identified. RESULTS A total of 105 patients were discovered with anti-Jka from 88,478 SPRCA screens performed. In 32 patients, anti-Jka was initially discovered by SPRCA testing and concurrent gel testing was completely negative (n = 26) or inconclusive (n = 6). Seventeen of the 32 patients were recently transfused and of these six met criteria for delayed hemolytic transfusion reaction (DHTR), three had possible DHTRs, and eight had delayed serologic reactions; 13 of the transfused patients received Jk(a–) RBCs to avoid potential hemolysis. CONCLUSION SPRCA testing significantly increased the discovery of clinically significant anti-Jka and facilitated the earlier use of Jk(a–) RBCs to avoid hemolytic transfusion reactions.

SUMMARY Objectives To explore an alternative way for antibody detection testing, the examination of gold nanoparticle solution for enhancing unexpected antibodies for pre-transfusion testing was investigated. Background Exposure of foreign antigens on red blood cells from transfusion can trigger the immune system to produce unexpected antibodies. This immunological response may cause the complication to future transfusion. For detection of unexpected antibodies, the antibody screening test is performed approximately 30–60 min. To reduce turnaround time, enhancing reagent, low-ionic strength solution (LISS), is widely used. However, cost of enhancing reagent is an issue which has concerned in resource limited countries. Gold nanoparticle solution can increase red blood cells agglutination reaction. To solve this issue, study of gold nanoparticle solution was investigated. Methods Samples were performed comparing between LISS and gold nanoparticle solution at antiglobulin phase. After reading the agglutination reaction, supernatants were collected and measured at the optical density at 760 nm by spectrophotometer. Results The optical density in the tube of gold nanoparticle solution was higher than in the tube of 2–5% cell suspension and monoclonal antibody. It has been observed that gold nanoparticle solution enhanced the reaction of agglutination 98% while LISS enhanced the agglutination only 60·8%. Employing a commercially available enhancing reagent, parallel samples confirmed results providing validation of the assay. It approximately costs $1 US dollars compared to $30 for a commercially available reagent. Conclusions The low cost and yet effective time-consuming test for antibody screening is a practical and viable solution alternative way for performing in antibody screening test in resource limited countries.

Combination of mannan antigen and anti-mannan antibody (Mn/A-Mn) testing has been reported a useful and specific strategy for diagnosis of invasive Candida infections (ICIs). We evaluated Mn/A-Mn as a screening tool in patients with haematological malignancies. This clinical prospective study was performed at the Division of Hematology, Medical University Graz, Austria between July and December 2012. Patients at risk for fungal infection were included into the study and twice weekly screened by Mn/A-Mn testing, yielding 650 samples. Of overall 67 patients 66 had no evidence for ICI. From those, 153/640 serum samples (23.9%) were positive for mannan Ab, and nine (1.4%) for Ag. Most false positive Ab results were observed among 375 samples from patients without haematopoietic stem cell transplantation (34.9% resulted positive). Combined specificity of Mn/A-Mn was 74.8%. Of 10 samples obtained in the single patient with candidemia, five were positive for mannan Ag (from the day of diagnosis up to 40 days after detection of candidemia) and none for Ab. In conclusion, mannan Ab screening yielded a high number of false positive results. While mannan Ag was found to be highly specific and may have potential for diagnostic driven testing, mannan Ab testing cannot be recommended based on our study results.

BACKGROUND RhD negativity is distributed unevenly among different ethnicities. In this study we explored the frequencies of RhD negativity in pregnant women in multiethnic Suriname, along with screening results for red blood cell (RBC) antibodies in these women and their offspring. Results may help identify women at risk for hemolytic disease of the fetus and newborn. STUDY DESIGN AND METHODS A retrospective study was performed in pregnant women who delivered at three major hospitals in Suriname between January 1, 2013, and December 31, 2014. RESULTS The overall prevalences of RhD negativity among 8686 women was 4.3%. The percentages of RhD negativity in Maroons, Creoles, and Hindustani women were 7.2, 5.4, and 3.7%, respectively. Chinese and Javanese women had very low prevalences of 0.8 and 0.5%, respectively, and Amerindians showed no RhD negativity. Antibody screening was positive in six D– (five Maroons and one Creole) women and weakly positive in three women (two Creoles and one Maroon), making overall antibody prevalence 4.4%. In 15 (5.5%; 10 Maroons, four Creoles, one Chinese) newborns from D– mothers antibody screening was positive. CONCLUSION In the multiethnic Surinamese population RhD negativity among pregnant women varied between 0.0 and 7.2% between ethnic groups. RBC antibodies were detected during pregnancy and in newborns in 4.4 and 5.5%, respectively.

Background Many centres provide D-mismatched platelet transfusions. Platelet concentrates may contain enough red cells leading to the formation of anti-D antibody in D-negative recipient. In resource-poor settings, random donor platelet concentrate (RDPC) is used often and carries higher risk of alloimmunization. At our institution, platelets are transfused across the ABO and Rh blood group barrier without any RhIG immunoprophylaxis. In this study, we retrospectively appraised alloimmunization after D-positive platelet transfusion in D-negative patients. Materials and Methods Single-centre 5-year data were collected from Hospital Information System (HIS) for D-negative patients who received D-positive platelet transfusions. Naive patients who had neither pre-existing antibody nor received D-positive platelet transfusion before admission were included. Those who had repeat antibody screening at least 28 days after D-positive platelet transfusion were considered. Results A total of 332 D-negative patients who received 1996 D-positive platelet transfusions were analysed. Median follow-up was 7 weeks (range = 4–90). Anti-D was detected in three (0·9%) D-negative patients at 5, 8 and 24 weeks after receiving D-positive platelet transfusions. Two of these patients received RDPCs and one received both RDPCs and single donor platelet concentrates. Conclusion D-positive platelet transfusion to D-negative patients seems an acceptable practice as only 0·9% patients developed anti-D antibody.

Background and Objectives Red cell antibody screening as a part of pretransfusion testing is not a routine practice in many developing countries. This study, was done to determine the incidence of allo- and auto-antibodies, assess their clinical significance and deliver antigen negative blood where warranted. Materials and Methods Red cell antibody screening was done as a part of pretransfusion testing at a tertiary care multispecialty hospital in India on 60 518 patients between August 2007 and July 2014 using column agglutination technology by gel. Results Red cell antibodies were detected in 0·95% (n = 579). Of these, 0·54% (n = 332) were alloantibodies and 0·4% (n = 247) were auto-antibodies. Rh and Kell antibodies detected were anti-D(n = 140), anti-C(n = 10), anti-c(n = 12), anti-E(n = 21), anti-e(n = 4), anti-K(n = 10), anti-k(n = 2). Anti-M (n = 30), anti-N(n = 1), anti-S(n = 7), anti-Le(a)( n = 23), anti-Le(b)( n = 6) and anti-Fy(a)(n = 7) were found. Dual antibodies anti-D&C(n = 36), anti-C&E(n = 1), anti-c&E(n = 2), anti-K&C(n = 1), anti-Jka&E(n = 1) and anti-Jka&D(n = 1) were also detected. Of the 332 alloantibodies detected only 9 could not be typed. Additionally, our study detected 247 auto-antibodies. Conclusion Implementation of red cell antibody screening routinely helped us understand the prevalence of antibodies in our region and showed that wider implementation at the national level is feasible and adds to blood safety.

Background and Objectives Severe transfusion-related acute lung injury (TRALI) events have been linked to donor-derived neutrophil antibodies. The journey to developing mass donor neutrophil antibody screening platforms is challenged by the fragility of neutrophils and their unique-specific antigenic characteristics. Material and Methods This article critically evaluates the capabilities and potential of five emerging antibody screening platforms designed to detect neutrophil reactive antibodies relevant to TRALI. They are compared with established neutrophil serological methods. Results Data from two recombinant antigen platforms and a method using human neutrophil antigens-expressing KY cells indicated high specificity. Two mixed cellular flow cytometric assays have the advantage of presenting native conformation of the human polymorphonuclear neutrophil antigenic epitopes. Conclusions To date, the number and specificity of test sera applied to each platform is small. This needs to be substantially increased and further rigorous serological evaluation is yet needed to compare the sensitivity and specificity limits of each new platform with classical methods. With a limited world supply of TRALI-relevant test sera, a collaborative effort of laboratories with neutrophil and TRALI investigation expertise is required.

Background: In contrast to other studies evaluating machines by testing a great number of samples, we focused here on the sensitivity and on the influence of possible disturbing factors on automated blood group testing. Materials and Methods: As the gelcards of the DiaMed-ID Micro Typing System are well known, this study examines reading and interpretation of the gelcards by the automated analyser Techno TwinStation. The results of automated reading were compared with visual reading of the gelcards taken out from the machine. Results: The sensitivity, tested by the titers of some complete and incomplete antibodies, was at least the same for automated as for visual reading. Samples with weak antigens were well recognised by the analyser. Mixed field agglutinations were recognised as questionable results by automated reading up to a relation of 1 : 10 for the most part. Antibody screening and reverse typing were not influenced by hyperbilirubinaemia, whereas in lipemic and haemolytic samples doubtful results were reported. Conclusion: Interpretation of the results by the automated analyser Techno TwinStation may be disturbed in some difficult samples, but in most of these cases the analyser reports not a false, but a doubtful result, thus triggering visual reading by the operator.

BACKGROUND: Blood donor screening with enzyme immunoassays (EIAs) for antibodies to human T-lymphotropic virus (HTLV)-I, and later to HTLV-I/II, has led to the unnecessary deferral of tens of thousands of individuals. The licensure of the Abbott PRISM HTLV-I/HTLV-II chemiluminescent immunoassay (ChLIA) may permit the reinstatement of historically deferred donors. STUDY DESIGN AND METHODS: The efficacy of a reentry algorithm involving a follow-up sample from EIA-deferred donors testing HTLV-I/II ChLIA nonreactive was evaluated using 386 serologic confirmed-positive samples archived since the inception of anti-HTLV donor screening. Reactivity of the 386 samples by the ChLIA, when coupled with the package insert sensitivity data, may be used to demonstrate efficacy of the reentry algorithm. Donor incidence was also examined from 2008 through 2009 to evaluate changes to the existing HTLV screening policy. RESULTS: From January 1, 1995, to April 28, 2008, a total of 64,052 donors to the American Red Cross were deferred solely because of HTLV EIA false positivity, representing more than 130,000 US donors. HTLV ChLIA identified 386 confirmed-positive donations from 386 randomly selected donors representing reactivity to both the bioMerieux and the Abbott HTLV-I/II EIAs (95% confidence interval [CI], 99.2%-100%); both EIAs have since been discontinued. This is comparable to the detection of 843 of 843 confirmed-positive samples during the ChLIA clinical trials (95% CI, 99.48%-100%). Incident HTLV infections occurred primarily among female repeat donors during 2008 throughout 2009. CONCLUSIONS: Donors testing falsely positive by historic EIAs since 1988 should be considered for reinstatement if a contemporary sample tests ChLIA nonreactive. Changes to the existing screening algorithm seem unlikely since new HTLV infections were detected among repeat donors.

Background and Objectives In 2005, the Australian Red Cross Blood Service implemented a malaria antibody testing based strategy for donors with a history of travel/residence in a malaria endemic country or a past history of malaria. This report assesses the safety and efficacy of the strategy since inception. Materials and Methods Eligible blood donors were tested using the Newmarket malarial antibody EIA at least 4 months after their last potential exposure. Where EIA non-reactive their quarantined red cells were considered for transfusion and they were re-instated for cellular component manufacture at their next donation. The efficiency and safety of this strategy were evaluated based on the additional number of components recovered for transfusion and the observed incidence of transfusion transmitted malaria (TTM) respectively. Results Of the repeat reactive donors, 2696 (> 99·99%) were PCR negative whilst one was PCR positive with very low level parasitaemia. The average number of RBCs and platelets recovered per annum was 64 967 and 7398 representing 7·9 and 5·5% respectively of their annual production. No new TTM cases were recorded and the observed TTM rate of zero was consistent with the upper 95% CI for the pretesting TTM incidence of 0·9 per million donations. Conclusion The study findings support the efficacy and safety of a targeted screening strategy combining a sensitive antibody screening test with a 4-month cellular component restriction period for donors with a declared malarial risk. The TTM risk in Australia remains low and did not measurably change after implementing the testing strategy.

BACKGROUND: The FREELYS Nano system (Diagast) is a manual workstation for ABO/D grouping, Rh phenotyping, K typing, and antibody screening (ABS) for immunoglobulin G (IgG) antibodies only and works with the erythrocyte-magnetized technology (EMT). The principle of EMT is based on magnetization of red blood cells and avoids centrifugation and washing steps. STUDY DESIGN AND METHODS: A total of 304 samples were tested with our routine blood bank methods, 100 samples for ABO/D grouping, 196 samples (100 at first evaluation, 96 at second evaluation) for Rh phenotyping and K typing (PK7200, Olympus), and 108 samples for ABS (DiaMed). All samples were tested in parallel with the FREELYS Nano. RESULTS: We found a 100% concordance between the observed (FREELYS Nano) and the expected (Olympus PK7200) results for ABO/D grouping in all 100 samples. For Rh phenotyping and K tests, in 24 of 100 samples false-positive reactions were observed in the first evaluation by the FREELYS Nano. After changing the test kit batch for Rh phenotyping by the manufacturer, a complete concordance in Rh phenotyping and K tests was observed in a second evaluation. For ABS, the FREELYS Nano showed in 4 of 108 samples (3.7%) false-negative reactions for IgG antibodies (two anti-K, one anti-E, one anti-Cw), and one (0.9%) false-positive reaction. CONCLUSIONS: The FREELYS Nano is reliably suited to ABO/D grouping, Rh phenotyping, and K testing. The rate of false-negative reactions for IgG antibodies should be reduced.

The blood group and gene frequencies within the ABO, MNS, P, Rhesus, Lutheran, Kell, Lewis and Duffy systems have been determined in samples from 802 Danes aged 50 years and 427 Danes aged 70 years, living in a suburban area of Copenhagen. An antibody screening test was performed on sera from the same persons. When the results of the blood groupings in the two materials were compared, no significant difference could be demonstrated. When the present materials were compared with those reported earlier for Danish populations, no significant difference was observed. The frequency of irregular antibodies against erythrocytes in the two materials was lower than that in materials of patients in Rigshospitalet, but the difference was not significant.

A commercially prepared antibody screening system, Selectogen®, is now available in Denmark. A total of 291 selected sera underwent extended antibody studies by means of the Selectogen procedure and the routine methods of the blood grouping laboratory in Arhus. One hundred and thirty three sera contained irregular antibodies which could be analyzed in detail with respect to sensitivity of the methods used. The comparison showed the value of the two-stage papain technique in studies aimed at revealing the presence of weak irregular antibodies. The Selectogen albumin technique was not found to be a satisfactory substitute for the enzyme technique. Good results were obtained by using the Selectogen albumin indirect Coombs technique, but even that could not replace the enzyme technique, particularly not in the demonstration of weak Rhesus or Lewis antibodies.

A survey is presented of 4168 sera containing irregular blood-group antibodies. The antibodies were found during 309,335 antibody screenings and 135,402 cross matchings of blood samples from about 200,000 individuals. About 50 per cent of the antibodies were anti-D, sometimes together with other antibodies in Rh negatives (D neg), 8 per cent were Rh antibodies in Rh positives (D pos) and many of these antibodies were detectable only by a two-stage enzyme technique and not by AHG-technique. Almost 15 per cent of the antibodies belonged to the Lewis-system. There was a significantly increased number in females of the fertile age, and there was a significant decrease in the number of group O individuals among the Lewis-immunized persons. The low number of group O individuals indicate a hitherto unrecognized relationship between the ABO- and Lewis-systems. Altogether 2.1 per cent of the individuals described possessed one or more irregular antibodies. The importance is discussed of avoiding complications from these antibodies during blood transfusions. It is concluded that a reliable cross-match should include a two-stage enzyme technique as well as the usual saline and AHG tests. It is emphasized that the antibody screening is an efficient prophylactic measure, particularly for smaller hospitals, where complicated cross-matchings are impractical.

BACKGROUND: Non-ABO blood group (BG) alloantibodies can disappear over time, confounding compatibility testing and predisposing patients to delayed hemolytic transfusion reactions. The goal of this study was to analyze BG antibody disappearance after transfusion-related alloimmunization in men. STUDY DESIGN AND METHODS: The transfusion service records of 18,750 military veterans at a Department of Veterans Affairs (VA) medical center were screened to identify male patients with one or more BG alloantibodies and who had at least one type and screen performed after initial antibody detection (n = 304). Antibodies were categorized as to whether they were present at a patient's first antibody screening test ("preexisting") or after initial negative testing ("hospital-acquired"). RESULTS: Overall, the evanescence of hospital-acquired antibodies (108/222; 48.6%) was significantly higher (p < 0.0001) than that of preexisting antibodies (36/185; 19.5%). Half (54/108) of evanescent, hospital-acquired alloantibodies disappeared within 6 months of detection, and all disappeared by 10 years. Evanescence of hospital-acquired antibodies was dependent on duration of follow-up testing, because antibodies tested 5 or more years after initial development demonstrated the highest evanescence rate (35/55; 64%; p < 0.01). Some evanescent antibodies (9/407; 2.2% of total antibodies) disappeared and reappeared one or more times without known antigenic reexposure ("multiple evanescence"). Among commonly observed alloantibodies, disappearance varied with antigenic specificity. CONCLUSIONS: Approximately two-thirds of BG alloantibodies disappeared within 5 years of induction, a rate higher than previously reported in mixed-sex populations. Evanescence did not appear to be a random, first-order decay process, as evidenced by the lower evanescence rate of preexisting antibodies.

B lymphocytes were isolated from fresh blood and frozen-thawed lymphocyte preparations from the same donors. A panel of 55 human antisera, previously found to react with human peripheral blood B lymphocytes in cytotoxicity assays, was screened against these B-cell preparations. There was essentially no difference in the reactions of B-cell antisera with fresh B lymphocytes when compared with frozen-thawed cells. The percentage recovery of B cells from the fresh and frozen preparations was not significantly different.

A simple method of freezing lymphocytes requiring a cryoprotective medium consisting of glucose, MEM, and dimethylsulfoxide (DMSO) and an ultra-low freezer to freeze and store the lymphocytes is described. The thawed lymphocytes following 2 weeks' to 5 months' storage were found to be useful for HL—A antibody screening, HL—A typing, and PHA stimulation.

Summary. This document is an introduction to microplate serology prepared at a time of very active research and development. Antibody detection techniques are expected to improve during the lifetime of this document. Approximately 25% of hospital blood banks are ABO and D grouping by microplate techniques. Antibody screening by microplate techniques is only being carried out by a small percentage of hospital blood banks but there is likely to be a change to antiglobulin tests by microplate procedures as soon as the methods have been thoroughly tested and approved. There are one or two weak links in these procedures which could give rise to serious errors:— (1) Antiglobulin reagents standardized for spin-tube tests may be subject to prozones in liquid phase microplate tests. Each new batch of antiglobulin reagent must be standardized by the microplate procedure in use to show that the reagent is at its optimum anti-IgG dilution for use. However dilutions greater than 1:2 may seriously compromise the anti-complement activity of the reagent. (2) Automated cell washers for microplates are currently under development and should be evaluated by replicate tests with weak anti-D sensitized cells. (3) The combination of diluted anti-IgG and poor washing procedures may lead to neutralization of anti-IgG and cause false negative errors. Future development for improved antiglobulin tests in microplates by a solid phase system is now well advanced and workers proposing to change to antibody screening by microplates are advised to bear this in mind. It is hoped that microplate methodology will move towards standardization based on national guidelines using standard antibody reagents for the validation of new microplate procedures.

BACKGROUND: Chagas disease, caused by the parasite Trypanosoma cruzi, represents a serious blood safety problem due to increasing immigration from Latin America. The Food and Drug Administration recently recommended implementation of Chagas antibody screening for US donors as soon as a suitable assay is licensed. An anonymized preclinical study of a prototype T. cruzi lysate-based enzyme-linked immunosorbent assay (ELISA) developed by Ortho-Clinical Diagnostics was conducted. STUDY DESIGN AND METHODS: Two populations of specimens were evaluated: 1) 10,192 sequential donations from blood donors residing in the El Paso, Texas, area and 2) 178 specimens from South America which were presumptively positive for antibodies to T. cruzi and purchased from commercial vendors. RESULTS: A total of 10,189 (99.97%) of the 10,192 screened donor specimens did not react, whereas 3 (0.03%) tested initially reactive. The 3 initially reactive specimens tested repeat reactive and were confirmed by radioimmunoprecipitation analysis (RIPA). Based on antibody profile analysis, 2 of the 3 Chagas-positive specimens were from the same donor. Observed specificity of the test was therefore 100 percent. Of the specimens from South America, 173 of 178 were reactive by the prototype ELISA. Of the 5 nonreactive specimens, all did not react by indirect fluorescence assay, but 4 were positive by RIPA. Therefore, calculated sensitivity of the ELISA was 97.7 percent (173/177). CONCLUSIONS: These studies indicate that the prototype ELISA has excellent sensitivity and specificity for detection of antibodies to T. cruzi in donors. Moreover, among donations from a geographically selected collection region of the United States, observed seroprevalence was 0.03 percent.

BACKGROUND: A number of automated devices for pretransfusion testing have recently become available. This study evaluated a fully automated device based on column agglutination technology (AutoVue System, Ortho, Raritan, NJ). STUDY DESIGN AND METHODS: Some 6747 tests including forward and reverse ABO group, Rh type and phenotype, antibody screen, autocontrol, and crossmatch were performed on random samples from 1069 blood donors, 2063 patients, and 98 newborns and cord blood. Also tested were samples from 168 immunized patients and 53 donors expressing weak or variant A and D antigens. Test results and technician times required for their performance were compared with those obtained by standard methods (manual column agglutination technology, slide, semiautomatic handler). RESULTS: No erroneous conclusions were found in regard to the 5028 ABO group and Rh type or phenotype determinations carried out with the device. The device rejected 1.53 percent of tests for sample inadequacy. Of the remaining 18 tests with discrepant results found with the device and not confirmed with the standard methods, 6 gave such results because of mixed-field reactions, 10 gave negative results with A2 RBCs in reverse ABO grouping, and 2 gave very weak positive reactions in antibody screening and crossmatching. In the samples from immunized patients, the device missed one weak anti-K, whereas standard methods missed five weak antibodies. In addition, 48, 34, and 31 of the 53 weak or variant antigens were detected by the device, the slide method, and the semiautomated handler, respectively. Technician time with the standard methods was 1.6 to 7 times higher than that with the device. CONCLUSION: The technical performance of the device compared favorably with that of standard methods, with a number of advantages, including in particular the saving of technician time. Sample inadequacy was the most common cause of discrepancy, which suggests that standardization of sample collection can further improve the performance of the device.

Antibody-mediated TRALI reduction strategies include the exclusion of high plasma volume blood components from risk donors or testing of these donors for leucocyte antibodies, i.e. HLA class I, HLA class II, and HNA antibodies. Recommendations for the detection of leucocyte antibodies have been made by the ISBT Working Party on Granulocyte Immunobiology.

Objectives: Faster and less labor-intensive crossmatching procedures are needed, but they must be as safe as the traditional antiglobulin method. We present twelve years' experience with a procedure involving antibody screening, blood group checks, and computerized delivery control (ABCD test). Methods: We use a computer for validation and printing documents and declaring compatibility between patient and blood component, based on screening results and earlier-recorded data about the patient. Results: Of 257,400 units transfused during the period, 90% were declared compatible through the ABCD procedure, and 10% had to be crossmatched. We observed no hemolytic transfusion complications due to a failure of the procedure to detect red cell alloantibodies. Labor in the testing laboratory was reduced by 65% compared to a previous crossmatching period. Fewer blood units were returned unused. Conclusion: The procedure using a computerized system as a guard against human mistakes has been found to be safe and reliable and is now widely used in Sweden.

Combinatorial phage display peptide libraries are routinely used to map epitopes of specific monoclonal antibodies. In this study we illustrate that these libraries can be used in the analysis of protein structure. By screening libraries at low stringency, a collection of phages can be obtained. These are characterized by the fact that they are recognized by a given monoclonal antibody yet with various affinities. Comparing the random peptides of these phages indicates the common essential residues necessary for antibody recognition. Aligning the inserts based on the detected homology has revealed structural motifs that correspond to secondary protein structures. The envelope protein of HIV-1 has been studied using this approach. A combinatorial phage display library containing a 20 mer random peptide in protein III of the filamentous phage fd-tet has been used to analyze two different monoclonal antibodies directed against gp120. Our results provide experimental evidence that indicate that the C1 domain of gp120 contains an alpha helix.

Summary. The object of antibody screening is to detect all clinically relevant antibodies. In order to do this effectively red cells are selected with an appropriate antigen profile. The introduction of column techniques for antibody screening by indirect antiglobulin testing (IAT) and two-stage enzyme testing (ETC) is perceived to lead to an increased sensitivity and an ability to detect red cell antibodies more easily than by traditional tube techniques because reactions in columns are more easily read and are stable. We evaluated the use of a column technology with pooled red cells for routine antenatal screening. The pooled cells used contained at least one cell with homozygous antigen expression for the majority of clinically significant antibodies known to be present, except for Kell. Pooled cell results were not as easy to read in gel columns when compared with single cell results due to weaker reactions which were often diffused throughout the gel in the column. We concluded that the use of pooled cells led to a decreased sensitivity which proved problematic for the interpretation of results. We used a two-cell and a three-cell pool and found that detection of known antibodies was reduced in IAT and ETC methods.

UNLABELLED We have studied a previously described solid-phase test (SPH) for screening and identification of antibodies directed against erythrocyte antigens, in large sample numbers and throughout routine processing. The screening results of all samples from pregnant women, patients and blood donors (n = 36,701) sent to our blood bank from August 1992 to July 1993 were compiled and evaluated. Pregnant women were screened by the SPH-antiglobulin technique (IAT) and the SPH-enzyme-enhanced technique (ENZ), while the patients and blood donors were screened only by the SPH-IAT. Samples with known alloimmunization were excluded from this study. Positive screening reactions were further investigated by the SPH-IAT and SPH-ENZ techniques, the manual hemagglutination tests low ionic-strength solution IAT and 2-stage papain. Pregnant women: A positive reaction in one or both of the SPH tests was found in 1.2% of the samples. The SPH tests identified 99 (98.0%) and the manual methods 79 (78.2%) of a total of 101 antibodies. Of the identified antibodies, 61 were to antigens in the Rh blood group system, including 34 Rh immune globulin. The remainder had specificity directed against antigens K, Jka, M, S, Le(a) and Le(b). PATIENTS A positive reaction in the SPH-IAT test occurred in 0.9% of the samples. The antibodies identified had specificity directed against antigens D, C, c and E in the Rh blood group system. The remainder had specificity directed against antigens Fya, K, Jka, M and Le(a). All antibodies identified by SPH-IAT, except 1 anti-M and 2 anti-Le(a), were also identified by manual methods. Blood donors: A positive reaction in the SPH-IAT test was noted in 0.7% of the samples. The identified antibodies had specificity directed against antigens D, C, E, Fya, K and M. All antibodies identified by the SPH-IAT, except 1 anti-D found in a sample from an RhD(+) male, were also identified by the manual methods. This study indicates that the SPH-IAT test is sensitive for detecting anti-M. This was also indicated for the SPH-ENZ test concerning the Jka antibody. An advantage of using solid-phase antibody screening tests for a larger series of samples is the possibility of automated sample identification, dilution, dispensing and interpretation of results. The SPH-IAT and the SPH-ENZ tests permitted us to select test cells, preferably from our own donor pool. We conclude that the SPH-IAT and SPH-ENZ tests are suitable for routine antibody screening of pregnant women and the SPH-IAT test for screening patients and blood donors.

BACKGROUND: In December 1990, the Food and Drug Administration recommended that all United States blood centers implement a policy of asking prospective donors direct oral questions (DOQs) about human immunodeficiency virus (HIV) risk behaviors to increase the safety of the blood supply. STUDY DESIGN AND METHODS: To evaluate the impact of the DOQ policy, HIV-related deferral and HIV seroprevalence data were analyzed at four American Red Cross blood centers for the year before the policy change and the year after. An epidemiologic analysis with stratification was conducted, including the calculation of odds ratios (OR) and 95-percent CIs. RESULTS: Two of the four blood centers showed an overall significant increase in HIV-related deferral after implementation of the DOQ policy: OR = 4.04, (95% CI = 3.41, 4.76); OR = 2.93, (95% CI = 2.67, 3.21). The increase in HIV-related deferral was higher for women. HIV seroprevalence decreased at all four centers, including the two that did not see an increase in HIV-related deferrals. Seroprevalence declined by 14 percent in the two centers with increases in HIV-related deferral, which was neither significant nor attributable to DOQs. CONCLUSION: Given that HIV antibody screening cannot detect HIV-seronegative (but infectious) “window-period” donations, the deferral of at-risk donors may offer some additional protection to the blood supply. However, evidence was not found of an increase in safety of the blood supply as measured by HIV seroprevalence.

Background: The incidence of the Mill phenotype among Chinese blood donors in Taiwan is 7.3 percent. Anti-“Mia” is the most frequently occurring alloantibody of potential clinical significance encountered among Chinese patients in Taiwan, with a frequency of 0.28 percent. Case Reports: A case of hemolytic disease of the newborn and a transfusion reaction due to anti-“Mia” are described. Conclusion: It may be important to include the Mill phenotype in the antibody screening of sera from Chinese patients.

Summary. This study was conducted to compare a new microtube column agglutination technology (CAT) with a previously described tube saline-indirect antiglobulin test (SIAT) with carefully defined performance in the detection of unexpected antibodies. On testing 117 sera from fresh and frozen stock containing antibodies detectable by SIAT, CAT failed to detect two examples of weak anti-K. All other discrepancies between the two techniques involved antibodies generally regarded as clinically insignificant. Titration studies with anti-D (concentration approximately 10 ng/ml) and 23 other antibodies, and studies with 10 weak antibodies of various blood group systems, showed the two techniques to be of similar sensitivity. Equivocal CAT results requiring repeated testing were found in 4·1% of specimens tested. We conclude that the CAT method adequately meets our requirements in sensitivity of detection of unexpected antibodies in pre-transfusion testing and offers opportunities for savings in technical staff time.

Summary. An antihuman globulin (AHG) gel centrifugation technique (AHG gel test) employing microtubes (DiaMed®) has been compared for sensitivity in detection of unexpected antibodies with a saline indirect antiglobulin test (SIAT) which uses a cell-to-serum ratio between 1:120 and 1:200 and provides positive reactions at a concentration of a standard anti-D of 2 ng/mL (0·01 IU/ml) or less. For most antibodies examined the AHG gel test was slightly less sensitive than the SIAT but both methods were capable of detecting anti-D at concentrations of 2 ng/ml or less. The importance of defining in detail the conventional tube test method used in such evaluations is emphasized since previous reports of sensitivity of the DiaMed AHG gel test indicate considerable variation in technique and comparative sensitivity.

Summary. Three antibody-containing samples (anti-Fya anti-S and anti-Jka), each at two dilutions, were distributed to U.K. hospitals and transfusion centres together with an antibody screening panel comprising red cells homozygous for the corresponding antigens. Participants in the study subjected the samples to their routine antibody screening procedures using both their own antibody screening panels and the screening panel provided. A within-group comparison of those participants using their own screening panels having a heterozygous expression of the antigens, with the same participants when using the screening panel provided, showed for five of the samples a greater detection rate in routine antibody screening procedures when using the panel provided, having homozygous expression of the corresponding antigens. The sixth sample, the most potent, was detected equally using both panels. The difference in overall detection rate is statistically significant (chi-square test, 2p0.001). The study shows that the use of red cells presumptively homozygous for Fya, S and Jka improved the detection of the corresponding antibodies.

Summary: An effective, modular automated blood grouping system has been developed which performs more effectively than the AG16C autogroupers and which uses readily obtainable reagents and disposables. Operating and maintenance costs are low by comparison with currently available equipment of similar capacity. The 99·2% success rate for blood grouping using this image analysis-based system compares well with the 98·3% success rate on the AG16C autogroupers. Staffing requirements are reduced and the flexible modular approach allows this Newcastle system to be used for a range of serological functions, including grouping, antibody screening and screening for antigen donors. The ability to operate with small sample numbers without long start-up times and the relatively low capital cost makes the Newcastle system an option that could be considered by the larger hospitals as well as transfusion centres. The sensitivity of the system could be developed to include compatibility testing as a feature of the system.