Karen M.K. de Vooght

When blood transfusion medicine becomes complicated due to interference by monoclonal antibody therapy

Monoclonal antibodies (MoAbs) are increasingly integrated in the standard of care. The notion that therapeutic MoAbs can interfere with clinical laboratory tests is an emerging concern that requires immediate recognition and the development of appropriate solutions. Here, we describe that treatment of multiple myeloma patients with daratumumab, a novel anti‐CD38 MoAb, resulted in false‐positive indirect antiglobulin tests (IATs) for all patients for 2 to 6 months after infusion. This precluded the correct identification of irregular blood group antibodies for patients requiring blood transfusion.

Management of Gene Promoter Mutations in Molecular Diagnostics

BACKGROUND Although promoter mutations are known to cause functionally important consequences for gene expression, promoter analysis is not a regular part of DNA diagnostics. CONTENT This review covers different important aspects of promoter mutation analysis and includes a proposed model procedure for studying promoter mutations. Characterization of a promoter sequence variation includes a comprehensive study of the literature and databases of human mutations and transcription factors. Phylogenetic footprinting is also used to evaluate the putative importance of the promoter region of interest. This in silico analysis is, in general, followed by in vitro functional assays, of which transient and stable transfection assays are considered the gold-standard methods. Electrophoretic mobility shift and supershift assays are used to identify trans-acting proteins that putatively interact with the promoter region of interest. Finally, chromatin immunoprecipitation assays are essential to confirm in vivo binding of these proteins to the promoter. SUMMARY Although promoter mutation analysis is complex, often laborious, and difficult to perform, it is an essential part of the diagnosis of disease-causing promoter mutations and improves our understanding of the role of transcriptional regulation in human disease. We recommend that routine laboratories and research groups specialized in gene promoter research cooperate to expand general knowledge and diagnosis of gene-promoter defects.

Immunosuppressants and alloimmunization against red blood cell transfusions

Patients receiving red blood cell (RBC) transfusions are at risk of developing alloantibodies against donor RBC antigens. The risk of alloimmunization is dependent on the number of units administered and patients genetic predisposition, but has also been suggested to be modulated by a patients clinical profile. Our aim was to examine whether immunosuppressants suppress the development of clinically relevant RBC antibodies.

Red-blood-cell alloimmunisation in relation to antigens' exposure and their immunogenicity: a cohort study

BACKGROUND Matching donor red blood cells based on recipient antigens prevents alloimmunisation. Knowledge about the immunogenicity of red-blood-cell antigens can help optimise risk-adapted matching strategies. We set out to assess the immunogenicity of red-blood-cell antigens. METHODS In an incident new-user cohort of previously non-transfused, non-alloimmunised white patients receiving non-extended matched red-blood-cell transfusions in six Dutch hospitals between 2006 and 2013, we determined the cumulative number of mismatched red-blood-cell units per patient. We used multiple imputation to address missing antigen data. Using Kaplan-Meier analysis, we estimated cumulative alloimmunisation incidences per mismatched antigen dose as a measure of immunogenicity. FINDINGS Of 54 347 patients assessed, 21 512 were included in our study. Alloantibodies occurred in 474 (2·2%) of all transfused patients, with cumulative alloimmunisation incidences increasing up to 7·7% (95% CI 4·9-11·2) after 40 units received. The antigens C, c, E, K, and Jk(a) were responsible for 78% of all alloimmunisations in our cohort. K, E, and C(w) were the most immunogenic antigens (cumulative immunisation incidences after 2 mismatched units of 2·3% [95% CI 1·0-4·8] for K, 1·5% [0·6-3·0] for E, and 1·2% [0·0-10·8] for C(w)). These antigens were 8·7 times (for K), 5·4 times (for E), and 4·6 times (for C(w)) as immunogenic as Fy(a). The next most immunogenic antigens were, in order, e (1·9 times as immunogenic as Fy(a)), Jk(a) (1·9 times), and c (1·6 times). INTERPRETATION Red-blood-cell antigens vary in their potency to evoke a humoral immune response. Our findings highlight that donor-recipient red-blood-cell matching strategies will be most efficient when primarily focusing on prevention of C, c, E, K, and Jk(a) alloimmunisation. Matching for Fy(a) is of lower clinical relevance. Variations of antigen frequencies determined by ethnic background prevent extrapolating these conclusions to non-white populations. FUNDING None.

Diagnostic Accuracy and User-Friendliness of 5 Point-of-Care D-Dimer Tests for the Exclusion of Deep Vein Thrombosis

BACKGROUND Point-of-care D-dimer tests have recently been introduced to enable rapid exclusion of deep venous thrombosis (DVT) without the need to refer a patient for conventional laboratory-based D-dimer testing. Before implementation in practice, however, the diagnostic accuracy of each test should be validated. METHODS We analyzed data of 577 prospectively identified consecutive primary care patients suspected to have DVT, who underwent 5 point-of-care D-dimer tests-4 quantitative (Vidas®, Pathfast™, Cardiac®, and Triage®) and 1 qualitative (Clearview Simplify®)-and ultrasonography as the reference method. We evaluated the tests for the accuracy of their measurements and submitted a questionnaire to 20 users to assess the user-friendliness of each test. RESULTS All D-dimer tests showed negative predictive values higher than 98%. Sensitivity was high for all point-of-care tests, with a range of 0.91 (Clearview Simplify) to 0.99 (Vidas). Specificity varied between 0.39 (Pathfast) and 0.64 (Clearview Simplify). The quantitative point-of-care tests showed similar and high discriminative power for DVT, according to calculated areas under the ROC curves (range 0.88-0.89). The quantitative Vidas and Pathfast devices showed limited user-friendliness for primary care, owing to a laborious calibration process and long analyzer warm-up time compared to the Cardiac and Triage. For the qualitative Clearview Simplify assay, no analyzer or calibration was needed, but interpretation of a test result was sometimes difficult because of poor color contrast. CONCLUSIONS Point-of-care D-dimer assays show good and similar diagnostic accuracy. The quantitative Cardiac and Triage and the qualitative Clearview Simplify D-dimer seem most user-friendly for excluding DVT in the doctors office.

Visualizing Veins With Near-Infrared Light to Facilitate Blood Withdrawal in Children

Introduction. This study aims to evaluate for the first time the value of visualizing veins by a prototype of a near-infrared (NIR) vascular imaging system for venipuncture in children. Methods. An observational feasibility study of venipunctures in children (0-6 years) attending the clinical laboratory of a pediatric university hospital during a period of 2 months without (n = 80) and subsequently during a period of 1 month with a prototype of an NIR vascular imaging system (n = 45) was conducted. Failure rate (ie, more than 1 puncture) and time of needle manipulation were determined. Results. With the NIR vascular imaging system, failure rate decreased from 10/80 to 1/45 (P = .05) and time decreased from 2 seconds (1-10) to 1 second (1-4, P = .07). Conclusion . This study showed promising results on the value of an NIR vascular imaging system in facilitating venipunctures.

Intensive red blood cell transfusions and risk of alloimmunization.

Exposure to allogenic red blood cells (RBCs) may lead to formation of antibodies against nonself‐antigens in transfused patients. While alloimmunization rates are known to increase with the number of transfusions, the transfusion course in patients can vary from receiving multiple units during a single transfusion event or getting them dispersed over a long(er) period. In this study we compared the immunization risk between different transfusion intensities.

Effect of storage of red blood cells on alloimmunization

Red blood cells (RBCs) undergo changes during storage. Various studies have suggested a higher risk of adverse and often multifactorial clinical outcomes associated with older‐stored RBCs. Our aim therefore was to examine if storage of transfused RBCs is also associated with the risk of RBC‐specific alloantibody formation.

Dealing with anti-CD38 (daratumumab) interference in blood compatibility testing

to 18.5% after the third HP infusion. Additional MB infusion was then given with mild MetHb improvement (14.5%). As his condition continued to worsen, an additional HP unit was given, resulting in MetHb of 25.5% (Fig. 1). He ultimately went into cardiac arrest during infusion of a sixth HP unit. Postmortem examination demonstrated evidence of multiorgan ischemia, acute myocardial infarction, and cerebellar tonsillar herniation. The findings were consistent with the clinical impression of marked, prolonged tissue hypoxia. Our patient with severe AIHA refractory to medications received in total 6 units of HP and developed significant increase of MetHb. Multiorgan failure, as a result of tissue hypoxia, as well as polypharmaceutical state, may have played roles in his inability to compensate for impaired oxygen delivery due to methemoglobinemia. Further, his ischemic status may have been so advanced that any treatment, including RBC transfusion, could not have stopped functional deterioration. This case points out the difficulties of timing of RBC transfusion and of HP use in severe AIHA.

GATA-1 binding sites in exon 1 direct erythroid-specific transcription of PPOX.

We investigated erythroid-specific expression of the human PPOX gene. This gene encodes protoporphyrinogen oxidase, which is involved in synthesizing heme for red blood cells and heme as a cofactor for the respiratory cytochromes. In vitro luciferase transfection assays in human uninduced and hemin induced erythroleukemic K562 cells showed that the presence of exon 1 increased promoter activity fourfold as compared to reporter constructs lacking this exon. This transcriptional regulation was mediated by two GATA-1 sites in exon 1. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that both GATA sites were able to bind GATA-1 in vitro and in vivo. Exon 1 did not affect promoter activity in human hepatoma HepG2 cells and U937 monocytic cells but its presence decreased promoter activity in HeLa human cervical carcinoma cells. We conclude that the GATA-1 binding sites in exon 1 constitute key regulatory elements in differential expression of PPOX in erythroid and non-erythroid cells.