Characterization of Novel RHD Allele Variants and Their Implications for Routine Blood Group Diagnostics.

The Rh system, including the highly immunogenic D antigen, is one of the clinically most important blood group systems in transfusion medicine. Numerous alleles of the RHD gene are associated with variant RhD phenotypes. In case of Rh incompatibility, some of them can induce hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Thus, accurate blood group diagnostics are critical for safe transfusion therapy. We characterized phenotypes of four individuals revealing weakened D expression during routine pre-transfusion testing. Standard gel card matrix techniques with monoclonal and polyclonal anti-D antibodies were used for serological typing, complemented using D epitope and antigen density analysis. Genotyping employing PCR with sequence-specific primers, genomic and allele-specific Sanger sequencing and in silico protein analysis were performed. Four novel RHD alleles associated with weak D or partial D phenotypes were identified. One of the mutations is predicted to disrupt the terminal stop codon and result in an elongated translation of the mutant D protein that phenotypically exhibits a loss of D epitopes. Furthermore, a hybrid gene formed with the homologue RHCE gene is described. The presented data enhances the understanding of the Rh system and may contribute to continued advances in blood group diagnostics.

Cumulative incidence and risk factors for cutaneous squamous cell carcinoma metastases in organ transplant recipients: The Skin Care in Organ Transplant Patients in Europe-International Transplant Skin Cancer Collaborative metastases study, a prospective multicenter study.

INTRODUCTION: Solid organ transplant recipients (SOTRs) are believed to have an increased risk of metastatic cutaneous squamous cell carcinoma (cSCC), but reliable data are lacking regarding the precise incidence and associated risk factors. METHODS: In a prospective cohort study, including 19 specialist dermatology outpatient clinics in 15 countries, patient and tumor characteristics were collected using standardized questionnaires when SOTRs presented with a new cSCC. After a minimum of 2 years of follow-up, relevant data for all SOTRs were collected. Cumulative incidence of metastases was calculated by the Aalen-Johansen estimator. Fine and Gray models were used to assess multiple risk factors for metastases. RESULTS: Of 514 SOTRs who presented with 623 primary cSCCs, metastases developed in 37 with a 2-year patient-based cumulative incidence of 6.2%. Risk factors for metastases included location in the head and neck area, local recurrence, size > 2 cm, clinical ulceration, poor differentiation grade, perineural invasion, and deep invasion. A high-stage tumor that is also ulcerated showed the highest risk of metastasis, with a 2-year cumulative incidence of 46.2% (31.9%-68.4%). CONCLUSIONS: SOTRs have a high risk of cSCC metastases and well-established clinical and histologic risk factors have been confirmed. High-stage, ulcerated cSCCs have the highest risk of metastasis.

High Rate of Passenger Lymphocyte Syndrome after ABO Minor Incompatible Lung Transplantation.

Rationale: Passenger lymphocyte syndrome (PLS) may complicate minor ABO mismatched lung transplantation (LuTX) via donor-derived red cell antibody-induced hemolysis.Objectives: To ascertain the incidence and specificity of PLS-relevant antibodies among the study population as well as the dynamics of hemolysis parameters and the transfusion requirement of patients with or without PLS.Methods: In this cohort study, 1,011 patients who received LuTX between January 2010 and June 2019 were studied retrospectively. Prospectively, 87 LuTX (July 2019 to June 2021) were analyzed. Postoperative ABO antibody and hemolytic marker determinations, transfusion requirement, and duration of postoperative hospital care were analyzed. Retrospectively, blood group A recipients of O grafts with PLS were compared with those without.Measurements and Main Results: PLS affected 18.18% (retrospective) and 30.77% (prospective) of A recipients receiving O grafts, 5.13% of B recipients of O grafts, and 20% of AB patients receiving O transplants. Anti-A and anti-A1 were the predominant PLS-inducing antibodies, followed by anti-B and anti-A,B. Significantly lower hemoglobin values (median, 7.4 vs. 8.3 g/dl; P = 0.0063) and an approximately twice as high percentage of patients requiring blood transfusions were seen in PLS. No significant differences in other laboratory markers, duration of hospital stay, or other complications after LuTX were registered.Conclusions: Minor ABO incompatible LuTX recipients are at considerable risk of developing clinically significant PLS. Post-transplant monitoring combining red cell serology and hemolysis marker determination appears advisable so as not to overlook hemolytic episodes that necessitate antigen-negative transfusion therapy.

Immunoglobulin Class Profiles of ABO Antibodies in Saliva and Serum of Healthy Individuals.

Introduction: The coronavirus disease (COVID-19) pandemic gave rise to studies investigating the association of ABO blood group with COVID-19 susceptibility. It is hypothesized that ABO antibodies might play a role in neutralizing SARS-CoV-2. However, ABO antibodies were exclusively analyzed in blood samples. Investigation of ABO antibodies in saliva, an easy-to-obtain surrogate for respiratory secretions, may provide novel insights into mucosal immunity crucial in early defense against respiratory pathogens. Methods: In this study, saliva and serum samples from healthy individuals with known blood groups were investigated using a flow cytometric method for separate anti-A/anti-B IgA, IgM, and IgG class antibody detection. Saliva samples were additionally tested using hemagglutination-based neutral and indirect anti-human globulin test gel cards. This method comparison was complemented by dilution experiments with a high-titer anti-A/anti-B WHO standard. Results: In saliva, IgA was the most abundant ABO antibody class, followed by IgM; IgG was detected only in low levels in all non-AB blood types. In serum, IgM was the predominant ABO antibody class in all non-AB blood types, followed by IgA and IgG, the latter mainly detected in group O individuals. Saliva and serum samples of group O individuals yielded the highest variability of ABO-specific antibody levels. Regardless of sample material and blood type, major interindividual differences in ABO antibody reactivities were recorded. Antibody levels correlated moderately between these two body fluids. There were no significant sex and age-group differences in ABO antibody levels in both serum and saliva. WHO standard dilution experiments yielded technique-specific limits of detection, illustrating the inherent differences of immunofluorescence versus agglutination. Conclusion: For the first time, salivary ABO antibodies were investigated by separate detection of the three most relevant antibody classes IgA, IgM, and IgG in a healthy cohort. This study opens new perspectives regarding mucosal ABO antibody class profiles and their potential influence on respiratory infections.

Tailored positioning and number of hand rub dispensers: the fundamentals for optimized hand hygiene compliance.

BACKGROUND: Availability of alcohol-based hand rub (ABHR) dispensers at positions adapted to the work flow of healthcare workers (HCWs) is decisive in order to carry out indication-based hand rubbing. Although requirements and guidelines regarding the positioning of ABHR dispensers are in place, scientific evidence is often lacking. METHODS: In order to analyse the impact of the location and number of ABHR dispensers on hand hygiene performance, additional dispensers were systematically placed in patient rooms in a surgical 38-bed ward at Marburg University Hospital, Germany to complement the existing dispenser locations. ABHR use was monitored continuously before and after complementation using the NosoEx hand hygiene monitoring system. The ward had 53 dispensers before the intervention and 82 dispensers after the intervention. RESULTS: The addition of dispensers increased ABHR consumption across the entire ward. Before the intervention, mean consumption was 20.6 mL/patient-day, whereas mean consumption after the intervention was 25.3 mL/patient-day. Depending on the combination of dispenser locations, consumption increased through targeted supplementation in patient rooms. The presence of two or three dispensers per patient room resulted in significantly greater ABHR consumption compared with one dispenser per patient room. The preferred location combinations were entrance-front bed-back bed and entrance-foot end. CONCLUSION: ABHR consumption can be increased significantly by optimizing the position and number of dispensers. The outstanding factors are visibility and integrability of dispenser use into the work flow; in particular, a dispenser should be positioned in the entrance area. Recommendations should be optimized with regard to the required number and location of dispensers in patient rooms.

World human neutrophil antigens investigation survey.

BACKGROUND AND OBJECTIVES: Human neutrophil antigens (HNAs) are categorized into five systems: HNA-1 to HNA-5. Given the importance of neutrophils in immunity, we sought to create awareness of the role of HNA diagnostic services in managing immune neutropenia and transfusion-related acute lung injury. To provide health communities all around the world with access to these services, we conducted a survey to create a directory of these HNA diagnostic services. MATERIALS AND METHODS: An Excel table-based survey was created to capture information on the laboratory's location and was emailed to 55 individuals with known or possible HNA investigation activity. The collected data were then summarized and analysed. RESULTS: Of contacted laboratories, the surveys were returned from 23 (38.2%) laboratories; 17 have already established HNA diagnostic (of them 12 were regular participants of the International Granulocyte Immunobiology Workshop [ISBT-IGIW]), 4 laboratories were in the process of establishing their HNA investigation and the remaining 2 responder laboratories, did not conduct HNA investigations. In established laboratories, investigation for autoimmune neutropenia (infancies and adults) was the most frequently requested, and antibodies against HNA-1a and HNA-1b were the most commonly detected. CONCLUSION: The directory of survey respondents provides a resource for health professionals wanting to access HNA diagnostic services. The present study offers a comprehensive picture of HNA diagnostics (typing and serology), identifying weak points and areas for improvement for the first time. Identifying more laboratories involved in HNA diagnostics with limited access to international societies in the field will globally improve HNA diagnostics.

Lower Levels of ABO Anti-A and Anti-B of IgM, IgG and IgA Isotypes in the Serum but Not the Saliva of COVID-19 Convalescents.

Individuals with ABO type O, naturally possessing anti-A and anti-B antibodies in their serum, are underrepresented among patients infected with SARS-CoV-2 compared with healthy controls. The ABO antibodies might play a role in the viral transmission. Therefore, we aimed to quantify anti-A/anti-B, including their subclasses IgM, IgG and IgA, in the serum and saliva of Caucasians (n = 187) after mild COVID-19 to compare them with individuals who had never been infected with SARS-CoV-2. Two samples were collected within two months after the diagnosis (median days: 44) and two months later. ABO antibodies were determined by flow cytometry. Additionally, total IgA in saliva and antibodies specific to SARS-CoV-2 were tested by ELISA. COVID-19 convalescents had significantly lower levels of anti-A/anti-B IgM, IgG and IgA in their serum than control subjects (p < 0.001). Interestingly, no significant differences were observed in saliva. ABO antibody levels remained stable over the period considered. No relation of ABO to the level of SARS-CoV-2-specific antibodies was observed. Total IgA was lower in convalescents than in controls (p = 0.038). Whereas ABO antibodies in the saliva may not contribute to the pathogenesis of COVID-19, individual pre-existing high serum concentrations of anti-A/anti-B may have a protective effect against SARS-CoV-2 infection.

HautTief Multidisciplinary Educational Program for Patients with Psoriasis or Atopic Dermatitis: A Randomized Controlled Study.

BACKGROUND: Improving health-related quality of life (HRQoL), disease severity, and treatment adherence through patient education is an increasingly important, yet relatively new area in dermatology. This randomized controlled trial aims to contribute to this growing area of research by exploring the effects of a 9-week educational program for patients with chronic skin diseases. OBJECTIVE: The aim of the study was to evaluate the effect of a multidisciplinary educational program on HRQoL and disease severity in patients with psoriasis or atopic dermatitis (AD). METHODS: Sixty-four patients with diagnosed psoriasis or AD were recruited from University Hospital Zurich and randomized (1:1) to the intervention or control group. To assess HRQoL, the following self-reported questionnaires were used: Dermatology Life Quality Index (DLQI), Skindex-29, EuroQol-5D (EQ-5D), RAND 36-Item Short Form Survey (SF-36), and Beck Depression Inventory (BDI) to measure depression symptoms. Psoriasis Area and Severity Index (PASI) and the Eczema Area and Severity Index (EASI) were used to capture disease extent. These scores were assessed at four study visits, which were performed at baseline and 3, 6, and 9 months after the start of the program. RESULTS: At month 6, an improvement of at least 25% in BDI was recorded in 15 (68.2%) of 22 patients in the intervention group and 6 (27.3%) of 22 patients in the control group (difference 40.9%, p = 0.016). 53.3% (16 of 30) of patients achieved an improvement in one subdomain of the SF-36 score (role limitations due to emotional problems) at 6-month follow-up, compared with 23.1% (6 of 26) of those not attending the educational program (difference 30.2%; p = 0.042). No significant differences in DLQI, Skindex-29, EQ-5D, PASI, and EASI between both groups at the three time points were found. CONCLUSION: An educational program may improve HRQoL and depression status of patients with psoriasis or AD.

Maternal bleeding complications in pregnancies affected by red blood cell alloimmunization.

OBJECTIVE: To investigate whether women with red blood cell (RBC)1 alloimmunization are more likely to experience bleeding complications during pregnancy or delivery than women without RBC alloimmunization. STUDY DESIGN: Retrospective study involving all singleton pregnancies affected by RBC alloimmunization and without pre-existing maternal bleeding disorders or placenta previa, from 1 July 1999 to 30 June 2019 ("cases"). Only bleedings not related to invasive procedures (amnio- or cordocenteses) were included. Patients who were already at increased risk of pre- or perinatal bleeding due to their medical history (pre-existing bleeding disorders, antithrombotic therapy), or known obstetrics parameters (placental abnormalities etc.) were not included a priori. Cases were compared to controls without RBC alloimmunization, matched for maternal age and body mass index, from the same tertiary referral center in Austria. RESULTS: 130 cases were compared to 130 controls. Cases had significantly more previous pregnancies and miscarriages and their newborns had lower birthweight and were more often transferred to the intensive care unit than newborns of controls. 18/130 (13.8%) cases, compared to 8/130 (6.2%) controls experienced any bleeding during pregnancy or delivery (p = 0.061). Bleeding most often happened during the third trimester (cases: 4.6% vs. controls 0.8%, p = 0.12) and during or after delivery (cases: 7.7% vs. controls: 4.6%, p = 0.168). Binary logistic regression for the prediction of any bleeding complication during pregnancy, delivery or postpartum revealed immunization against RBC antigens as the only independent contributor (p = 0.04). Age, smoking, or previous obstetric history had no influence on the likelihood of maternal bleeding complications. Neither RBC antibody specificity nor titers were predictive of maternal bleeding during pregnancy or delivery. CONCLUSION: Pregnancies affected by RBC alloimmunization might be at increased risk of maternal bleeding complications during pregnancy and delivery.

External quality assessment providers' services appear to more impact the immunohaematology performance of laboratories than national regulatory and economic conditions.

OBJECTIVES: Medical laboratories may, at their own discretion, exceed but not undercut regulatory quality requirements. Available economic resources, however, may drive or hinder eagerness to exceed minimum requirements. Depending on the respective scopes of regulatory and economic framework conditions, differing levels of quality efforts to safeguard laboratory performance can be anticipated. However, this has not yet been investigated. METHODS: Immunohaematology external quality assessment (EQA) results collected by 26 EQA providers from their participant laboratories in 73 countries from 2004 to 2019 were evaluated. Error rates were aggregated in groups according to the respective national regulatory and economic framework conditions, to whether or not expert advice was provided in case of incorrect results, and the frequency of EQA samples. RESULTS: These representative data indicate no association between national regulatory (mandatory participation in EQA, monitoring of performance of individual laboratories by authorities, financial consequences of incorrect results) and economic (level of national income, share of national health expenditure) conditions to the quality performance of medical laboratories in immunohaematology. However, EQA providers' support for laboratories in the event of incorrect results appear to be associated with lower error rates, but a high EQA sample frequency with higher error rates. CONCLUSIONS: Further research into the impact of introducing or changing services of EQA providers is needed to confirm the results found in this first of its kind study.

Increased Serum Levels of soluble ST2 as a Predictor of Disease Progression in Systemic Sclerosis.

OBJECTIVE: Interleukin-33 (IL-33) has been investigated as a mediator in the pathogenesis of fibrosis in lung, liver, and heart. There is accumulating evidence for the involvement of the IL-33/IL-33 receptor ST2L signalling pathway in systemic sclerosis (SSc). Little is known about the role of serum sST2 in SSc, which is the subject of the present investigation. METHOD: Serum levels of sST2 were measured in 49 patients with SSc, recruited prospectively between November 2017 and March 2019. Patients were divided into those with progressive and those with stable disease. Receiver operating characteristics (ROC) curve analysis was applied to study sST2 as a marker for identifying patients with progressive disease. We used multivariate logistic regression analysis to evaluate the predictive value of sST2 for progressive disease after adjustment for potential confounding factors. RESULTS: Serum sST2 levels in patients with progressive disease were significantly elevated compared with patients with stable disease (mean ± sem: 50.4 ± 4.7 ng/mL vs 29.2 ± 2.97 ng/mL, p < 0.001). ROC curve analysis identified an sST2 cut-off value of 37.8 ng/mL as optimal for discriminating patients with progressive disease from those with stable disease (sensitivity 80.0%, specificity 79.3%, area under the curve 0.80). After controlling for potential confounding factors (age, gender, C-reactive protein, pro-brain natriuretic peptide, and sum of internal medicine comorbidities), sST2 remained predictive of progressive disease (odds ratio 1.070, 95% confidence interval 1.017-1.126, p < 0.009). CONCLUSION: In the present study, sST2 serum levels were predictive of disease progression in patients with SSc.

Consensus-Based Recommendations on the Prevention of Squamous Cell Carcinoma in Solid Organ Transplant Recipients: A Delphi Consensus Statement.

IMPORTANCE: There is a paucity of evidence to guide physicians regarding prevention strategies for cutaneous squamous cell carcinoma (CSCC) in solid organ transplant recipients (SOTRs). OBJECTIVE: To examine the development and results of a Delphi process initiated to identify consensus-based medical management recommendations for prevention of CSCC in SOTRs. EVIDENCE REVIEW: Dermatologists with more than 5 years' experience treating SOTRs were invited to participate. A novel actinic damage and skin cancer index (AD-SCI), consisting of 6 ordinal stages corresponding to an increasing burden of actinic damage and CSCC, was used to guide survey design. Three sequential web-based surveys were administered from January 1, 2019, to December 31, 2020. Pursuant to Delphi principles, respondents thoroughly reviewed all peer responses between rounds. Supplemental questions were also asked to better understand panelists' rationale for their responses. FINDINGS: The Delphi panel comprised 48 dermatologists. Respondents represented 13 countries, with 27 (56%) from the US. Twenty-nine respondents (60%) were Mohs surgeons. Consensus was reached with 80% or higher concordance among respondents when presented with a statement, question, or management strategy pertaining to prevention of CSCC in SOTRs. A near-consensus category of 70% to less than 80% concordance was also defined. The AD-SCI stage-based recommendations were established if consensus or near-consensus was achieved. The panel was able to make recommendations for 5 of 6 AD-SCI stages. Key recommendations include the following: cryotherapy for scattered actinic keratosis (AK); field therapy for AK when grouped in 1 anatomical area, unless AKs are thick in which case field therapy and cryotherapy were recommended; combination lesion directed and field therapy with fluorouracil for field cancerized skin; and initiation of acitretin therapy and discussion of immunosuppression reduction or modification for patients who develop multiple skin cancers at a high rate (10 CSCCs per year) or develop high-risk CSCC (defined by a tumor with approximately ≥20% risk of nodal metastasis). No consensus recommendation was achieved for SOTRs with a first low risk CSCC. CONCLUSIONS AND RELEVANCE: Physicians may consider implementation of panel recommendations for prevention of CSCC in SOTRs while awaiting high-level-of-evidence data. Additional clinical trials are needed in areas where consensus was not reached.

Ethical Issues and Management of Fetal Hemolytic Anemia Caused by Anti-Rh17 in a Multipara with Rare -D- Phenotype.

BACKGROUND: The development of allo-anti-Rh17 (anti-Hr0) in a -D- phenotype whose red blood cells (RBCs) lack CcEe antigens is most likely triggered by transfusion, transplantation, or pregnancy. Gene conversion is the predominating factor in generating RHD-CE-D and RHCE-D-CE hybrids like -D-. METHODS: We report here immunohematological and obstetrical data from 2 of the 5 pregnancies of a 24-year-old woman presenting with the -D- phenotype with anti-Rh17. Blood group typing, antibody screening, antibody differentiation, direct antiglobulin test (DAT), and antibody titers were performed by routine gel technology and tube testing. Additionally, molecular genetic analysis was performed. Fetal surveillance was done by sonographic evaluation of the fetal middle cerebral artery peak systolic velocity (MCA-PSV). RESULTS: Blood group typing showed O, C-c-D+E-e- and the DAT was negative. DNA sequencing revealed homozygosity for an RHCE-D(3-9)-CE null allele. Anti-Rh17 titers in the fourth pregnancy remained between 1:8 and 1:128, and no signs for a fetal anemia were observed. However, in the fifth pregnancy, the antibody titers increased up to 1:4,096. Signs of moderate fetal anemia were detected and cesarean section was performed at 34 + 6 weeks of gestation. The newborn presented with hemolytic anemia (cord blood hemoglobin [Hb] = 8.5 mg/dL). She received 2 compatible (small) packed RBC concentrates, phototherapy, and intravenous immunoglobulins. CONCLUSION: Our case shows that the risk for hemolytic complications increases with the number of pregnancies of sensitized women. Only people who also lack CcEe antigens are compatible as donors. The role of such rare donors as lifesavers, their freedom, and voluntariness conflict with the urgent need for compatible blood.

Increased extracellular water measured by bioimpedance analysis and increased serum levels of atrial natriuretic peptide in polymyalgia rheumatica patients : Signs of volume overload.

BACKGROUND: Water retention is a typical feature of acute inflammatory episodes, chiefly implemented by the sympathetic nervous system (SNS) and the hypothalamic-pituitary-adrenal (HPA) axis. This is an important compensatory mechanism counteracting expected water loss, e.g., due to sweating. Both the SNS and HPA axis are activated in polymyalgia rheumatica (PMR). As retention mechanisms may similarly apply in this disease, we hypothesized increased water retention in PMR. METHODS: Using bioimpedance analysis body composition was investigated in 64 healthy controls and 32 treatment-naive PMR patients. All PMR patients satisfied the 2012 EULAR/ACR classification criteria for PMR. 32 PMR patients were tested before and after 7 days of glucocorticoid-based therapy. Serum levels of pro-atrial natriuretic peptide (proANP) were investigated in all PMR patients and 15 healthy controls. RESULTS: Extracellular water (ECW) was markedly higher in PMR patients than in controls (mean ± SD: 49.1 ± 6.0% versus 36.3 ± 2.5% of total body water, p < 0.001). Patients with PMR demonstrated significantly higher serum levels of proANP compared to controls. Even before glucocorticoid treatment was initiated, systolic and diastolic blood pressure were higher in PMR patients compared to controls. Extracellular water levels did not change in PMR patients upon 7 days of intensified treatment. CONCLUSION: This study demonstrated increased extracellular water and elevated serum levels of proANP as signs of fluid overload in patients with PMR. Volume changes are imprinted as long-lasting mechanisms as water distribution is not affected by short-term anti-inflammatory therapy.

Skin Cancer Development in Solid Organ Transplant Recipients in Switzerland (Swiss Transplant Cohort Study).

IMPORTANCE: Skin cancer, in particular squamous cell carcinoma, is the most frequent malignancy among solid organ transplant recipients with a higher incidence compared to the general population. OBJECTIVE: To determine the skin cancer incidence in organ transplant recipients in Switzerland and to assess the impact of immunosuppressants and other risk factors. DESIGN: Prospective cohort study of solid organ transplant recipients in Switzerland enrolled in the Swiss Transplant Cohort Study from 2008 to 2013. PARTICIPANTS: 2,192 solid organ transplant recipients. MATERIALS AND METHODS: Occurrence of first and subsequent squamous cell carcinoma, basal cell carcinoma, melanoma and other skin cancers after transplantation extracted from the Swiss Transplant Cohort Study database and validated by medical record review. Incidence rates were calculated for skin cancer overall and subgroups. The effect of risk factors on the occurrence of first skin cancer and recurrent skin cancer was calculated by the Cox proportional hazard model. RESULTS: In 2,192 organ transplant recipients, 136 (6.2%) developed 335 cases of skin cancer during a median follow-up of 32.4 months, with squamous cell carcinoma as the most frequent one. 79.4% of skin cancer patients were male. Risk factors for first and recurrent skin cancer were age at transplantation, male sex, skin cancer before transplantation and previous transplantation. For a first skin cancer, the number of immunosuppressive drugs was a risk factor as well. CONCLUSIONS AND RELEVANCE: Skin cancer following solid organ transplantation in Switzerland is greatly increased with risk factors: age at transplantation, male sex, skin cancer before transplantation, previous transplantation and number of immunosuppressive drugs.

Single molecule distribution of RhD binding epitopes on ultraflat erythrocyte ghosts.

The Rh blood group system plays a key role in transfusion and organ transplant medicine. The complex transmembrane Rh polypeptides RhD and RhCE carry numerous antigens, including the extremely immunogenic D antigen. The Rh polypeptides form multimolecular Rh complexes with certain transmembrane and skeletal proteins, with so far only incompletely understood physiological functions. Determination of the energy landscape of individual Rh binding epitopes towards their specific interaction partners as well as their localization across the red blood cell (RBC) membrane requires single molecule approaches including large area high resolution recognition imaging. Atomic force microscopy based molecular recognition force spectroscopy in combination with single molecule recognition imaging fulfills these requirements. For unbiased single molecule results, nano-mechanical influences due to cell elasticity have to be eliminated. This is realized by generation of ultra flat erythrocyte ghosts on a solid support. We developed a protocol for the preparation of complete ultraflat erythrocyte ghosts and determined the molecular binding behaviour of different anti-D antibodies towards their binding epitopes on RhD positive and negative erythrocytes. Performing optimized topography and recognition imaging at 16 Mpixel resolution allowed localisation of individual RhD molecules at the single molecule level across an entire RBC. A map of Rh antigens across integer ultraflat RBC ghosts was generated with nanometer resolution. Here we show a homogeneous distribution on rim and dimple regions with comparable receptor densities. Furthermore, differences in the energy landscape between specific monoclonal antibodies were determined at the single molecule level.

To Win the Battle, First Know Your Enemy: Error Rates in Immunohematology External Quality Assessment Results.

BACKGROUND: As some errors in pretransfusion testing remain unrecognized, error rates and the resulting need for corrective measures are probably underestimated. External quality assessment (EQA) schemes could provide valuable input for identifying error-prone laboratory tests because they are designed to monitor test performance and errors. So far, however, there are only limited published data on error rates in such schemes. METHODS: The types and incidence of incorrect results in an EQA scheme for red cell immunohematology with 187 participating laboratories were examined. The results of 58 distributions between 1999 and 2017 were evaluated, considering also the employed determination methods. RESULTS: Out of a total of 58,726 results, 563 (0.96%) were incorrect. Error rates were 5.45% for antibody identification, 1.39% for Rh phenotyping, 0.83% for serologic cross-match, 0.60% for direct antiglobulin test, 0.20% for Kell phenotyping, 0.16% for antibody screening, and 0.14% for ABO phenotyping. During the observation period, 53 participants reported error-free results, while 37 reported one incorrect result and 97 repeatedly reported incorrect results for one or more analytes. Error rates obtained by manual methods significantly surpassed those obtained by automated methods (1.04 vs. 0.42%). The introduction of double testing with two different systems reduced error rates in Rh phenotyping from 1.55 to 0.50%. CONCLUSION: Risk assessment should consider that error rates in pretransfusion test results vary. These data delineate the error risk potential of individual laboratory tests and thus should aid in tailoring appropriate improvement measures.