Human neutrophil antigen-1, -3, -4, and -5 allele and genotype frequencies in the Croatian blood donor population and their clinical significance.

OBJECTIVES: Human neutrophil antigens (HNAs) and antibodies play an important role in allo- and autoimmunity associated with immune neutropenia and transfusion reactions. The aim of this study was to determine the HNA-1, -3, -4 and -5 allele and genotype frequencies in the Croatian blood donor population to assess the role of HNA-1, -3, -4, and -5 alleles in the development of neonatal alloimmune neutropenia and antibody-mediated transfusion-related acute lung injury. MATERIAL AND METHODS: A total of 371 blood samples from unselected healthy blood donors were analyzed. Samples from all 371 donors were genotyped for HNA-1, samples from 160 donors were genotyped for HNA-3, and samples from 142 donors were genotyped for HNA-4 and HNA-5 using the polymerase chain reaction with sequence-specific primers (PCR-SSP) method. RESULTS: The frequencies of the FCGR3B*01, FCGR3B*02 and FCGR3B*03 HNA-1 alleles were 0.393, 0.607 and 0.022, and of the SLC44A2*01 and SLC44A2*02 HNA-3 alleles 0.781 and 0.219, respectively. The frequencies of the ITGAM*01 and ITGAM*02 HNA-4 alleles were 0.796 and 0.204, and of the ITGAL*01 and ITGAL*02 HNA-5 alleles 0.718 and 0.282, respectively. CONCLUSION: These are the first results on the HNA allele and genotype frequencies in the Croatian blood donor population. We observed no deviations from previous reports on Caucasian populations. Determination of the HNA antigen frequencies in the population is important to estimate the risk of alloimmunization to HNA, especially the risk of fetal-maternal incompatibility and alloantibody production by transfusion of the HNA incompatible blood components.

Transient pseudothrombocytopenia (PTCP) in the neonate due to the mother.

Thrombocytopenia with platelet count <50×109/L is common laboratory finding in a severely ill newborn in neonatal intensive care units (ICU). Neonates with severe thrombocytopenia are at risk of bleeding. Most dangerous is intracerebral hemorrhage (ICH) frequently leading to death or lifelong neurological sequels. Pseudothrombocytopenia (PTCP) is a rare in vitro phenomenon of falsely low platelet count determined on hematology analyzers due to platelet clumping in ethylenediaminetetraacetic acid (EDTA) anticoagulated blood. PTCP was also reported in pregnant women with isolated thrombocytopenia. EDTA-PTCP in the neonate due to the transplacental transmission of maternal antibodies has been reported only in a few cases. Although PTCP is rare phenomenon, it should always be excluded in newborns with isolated thrombocytopenia to avoid erroneous interpretation of platelet and leukocyte count, unnecessary laboratory investigation of false positive antiplatelet antibodies and needless platelet transfusions. We report on two cases of transient PTCP in a neonate due to transplacental transfer of maternal EDTA-dependent autoantibodies of IgG class from the same mother.

Evaluation of reference genes for RT-qPCR gene expression analysis in Arabidopsis thaliana exposed to elevated temperatures.

Increases in environmental temperature are directly linked to the issue of climate change and are known to significantly disrupt plant growth and development. Studies of gene expression in plants commonly include RT-qPCR but the reliability of the method depends on the use of suitable reference genes for data normalization. Despite this, no reference genes have been validated specifically for experiments in Arabidopsis thaliana employing treatments with elevated temperature. Here, ten genes were selected for expression stability analysis based on the screening of available literature and microarray data from temperature-treated A. thaliana. Expression levels of candidate reference genes were measured in 12-day-old seedlings, rosette leaves and flower buds of 5-week-old A. thaliana plants exposed to five different temperatures (22°C, 27°C, 32°C, 37°C and 42°C) and their expression stabilities were assessed using four statistical algorithms (BestKeeper, geNorm, NormFinder and comparative ΔCq method). This study provides reliable reference genes for use in A. thaliana RT-qPCR expression analyses employing elevated temperature treatments, namely OGIO and PUX7 in seedlings, UBC21 and PUX7 in leaves, TIP41 and UBC21 in buds, and TIP41 and UBC21 in all three tissues combined. Orthologues of these genes can be of potential use in less studied plants, especially agricultural species heavily affected by climate change.

COVID-19 convalescent plasma as long-term therapy in immunodeficient patients?

OBJECTIVES: The patients with hematological malignancies are a vulnerable group to COVID-19, due to the immunodeficiency resulting from the underlying disease and oncological treatment that significantly impair cellular and humoral immunity. Here we report on a beneficial impact of a passive immunotherapy with convalescent plasma to treat a prolonged, active COVID-19 infection in a patient with a history of nasopharyngeal diffuse large B-cell lymphoma treated with the therapy inducing substantial impairment of particularly humoral arm of immune system. The specific aim was to quantify SARS-CoV2 neutralizing antibodies in a patient plasma during the course of therapy. MATERIALS AND METHODS: Besides the standard of care treatment and monitoring, neutralizing antibody titers in patient's serum samples, calibrated according to the First WHO International Standard for anti-SARS-CoV-2 immunoglobulin (human), were quantified in a time-dependent manner. During the immunotherapy period peripheral blood flow cytometry immunophenotyping was conducted to characterize lymphocyte subpopulations. RESULTS: The waves of clinical improvements and worsening coincided with transfused neutralizing antibodies rises and drops in the patient's systemic circulation, proving their contribution in controlling the disease progress. Besides the patient's lack of own humoral immune system, immunophenotyping analysis revealed also the reduced level of helper T-lymphocytes and immune exhaustion of monocytes. CONCLUSION: Therapeutic approach based on convalescent plasma transfusion transformed a prolonged, active COVID-19 infection into a manageable chronic disease.

Review of indications for immunoglobulin (IG) use: Narrowing the gap between supply and demand.

Cellular blood components and plasma-derived medicinal products (PDMPs) are obtained from blood donated by volunteers. In a growing number of countries, in line with World Health Organization advice issued since the mid-1970s, donors are not remunerated. In recent decades, considerable efforts have been made to restrict the indications for labile blood components to those based on evidence, to ensure efficacy and safety. By contrast, the producers of PDMPs have developed pathogen reduction techniques for inactivating the microorganisms in (pooled) plasma, but little attention has been paid to the pertinence of the clinical indications for these products. The use of blood, and of erythrocytes in particular, has declined by almost 40%, but the use of immunoglobulins (IG) tripled between 2004 and 2018, making it necessary to pay donors to obtain sufficient blood to meet the market demand for these products. We analyzed the reasons for this increase to unsustainable levels of use, by investigating (practice) guidelines, recommendations, Cochrane data analyses and systematic reviews for clinical indications for IG over time. We found no new evidence explaining the huge increase up to 2018 or the predicted 5-7% yearly annual increase until 2024. For some former evidence-based indications, biologics have largely replaced IG, but the administration of IG for doubtful indications (up to 40%) has not decreased in recent decades. The main development since 2004 is that IG use in Europe has become market-driven rather than evidence-guided. As transfusion specialists and blood therapists, we must raise the alarm that this situation is likely to continue in the absence of good clinical studies determining the place of IG alongside other treatments, and for as long as market profitability remains the dominant driving force. We discuss here approaches for reversing this trend and moving towards European self-sufficiency through non-remunerated voluntary blood donation.

Transfusion medicine: Overtime paradigm changes and emerging paradoxes.

This essay aims to discuss some aspects of blood transfusion in the perspective of the changes that occurred over time as well as modifications of the paradigms that transformed the activities and the organization of blood transfusion services. Without specific knowledge, pioneers envisioned precision and personalized medicine, rendering transfusion medicine operational. Transfusion medicine is like The Picture of Dorian Grey: always young despite being old and sometimes appearing old-fashioned. Over the years, the transfusion medicine discipline has evolved, and major progress has been achieved, despite some troublesome periods (for example, the tainted blood scandal, and-at the time being-the offending plasma market and the selling of human parts). Transfusion medicine has at all times implemented the rapidly developing biomedical technologies to secure blood components. The safety of blood components has now reached an exceptional level in economically wealthy countries, especially compared to other health care disciplines. Strengthening of the safety has mandated that blood donors and recipients are unrelated, an issue which has eased preservation and fractionation practices; blood is no longer arm-to-arm transfused and neither is whole blood, the commonest component. However, it is interesting to note that a revival is occurring as whole blood is back on stage for certain specific indications, which is one among the many paradoxes encountered while studying this discipline.

Non-conformities in tissue typing laboratory: Croatian experience.

OBJECTIVES: Our study aimed to determine the frequency and type of non-conformities that occurred in the tissue typing laboratory (TTL), Rijeka, Croatia, in order to evaluate the quality of testing and compliance with the requirements of the quality management system (QMS). BACKGROUND: The QMS in a TTL should lead to improvements in the accuracy and timeliness of results. One of its essential elements is non-conformity management. METHODS: A retrospective analysis of non-conformities recorded in the TTL Rijeka from 2006 to 2017 revealed the overall frequency of 0·43%. Classification was performed according to the examination phase, employee characteristics, relation to the field of work, part of laboratory and priority of testing. RESULTS: The most reported non-conformities occurred during the examination phase (54·0%), followed by the pre- and post-examination phase (31·2 and 14·8%, respectively). Inadequate competency and less experience were associated with the number of reported non-conformities while no difference was observed in relation to the profession or position in the laboratory. Non-conformities were equally distributed between organ transplantation and HLA-associated disease diagnostic processes, mostly referring to the entire laboratory work. CONCLUSION: Intensifying training and education, implementation of information technology into laboratory processes, more rigorous error detection and reporting are fundamental steps in improving quality of laboratory services.

Estimation of the hepatitis E assay-dependent seroprevalence among Croatian blood donors.

BACKGROUND: Seroprevalence of hepatitis E virus (HEV) in blood donors presenting to the Croatian Institute of Transfusion Medicine was assessed with 4 available tests (3 ELISA tests and 1 immunoblot (IB) test). MATERIALS AND METHODS: In October and November 2014, a total of 1,036 serum samples of blood donors were collected for the study. Samples were primarily tested for total HEV antibodies by Dia.Pro HEV Ab test (a). All reactive samples were tested by ELISA tests: Dia.Pro HEV IgG (b) and IgM (c), Mikrogen recomWell HEV IgG_old (d) and IgM_old (e), recomWell HEV IgG_new (f) and IgM_new (g), and IB Mikrogen recomLine HEV IgG (h) and IgM (i). HEV IgM reactive samples also positive by the IB were further tested for HEV RNA. RESULTS: There were 21.5% of samples reactive for total HEV antibodies (a). Seroprevalence of HEV IgG according to the b, d, f and h tests was 20.2%, 9.6%, 18.1% and 17.8%, respectively. Seroprevalence of HEV IgM according to the c, e, g and i tests was 4.4%, 1.5%, 2.0% and 1.7%, respectively. Out of 46 HEV IgM (Dia.Pro HEV IgM) positive samples, 18 (39.1%) were also positive by IB. HEV RNA was not detected in any of those samples. There was a significant association between age and HEV seroprevalence (P<0.001). CONCLUSION: Different HEV antibody detection assays showed a high HEV IgG seroprevalence in Croatian blood donors. Among HEV IgG and HEV IgM positive samples HEV RNA was not detected.

Combined cell index in assessing blood donor iron stores.

OBJECTIVES: The aim of this study was to assess the appropriateness of using combined cell index (CCI) in the assessment of iron stores in blood donors. This index is calculated by the formula: red blood cell distribution width (RDW) × 104 × mean corpuscular volume (MCV)-1 × mean corpuscular haemoglobin (MCH)-1 . BACKGROUND: Ferritin measurement is a reliable method for estimating iron stores in blood donors. The sensitivity of red blood cell (RBC) parameters of complete blood count in detecting non-anaemic iron deficiency is significantly lower. Consequently, there were several attempts to increase the detection sensitivity by combining these parameters in different indices. METHODS: This study included 1084 male and 792 female whole blood donors accepted for blood donation. For six RBC parameters with the highest level of correlation relative to ferritin [Hgb, MCV, MCH, mean corpuscular haemoglobin concentration (MCHC), RDW and CCI], diagnostic efficacy in the detection of iron depletion (ferritin <12 µg L-1 ) was assessed using receiver operating characteristic (ROC) analysis. RESULTS: CCI showed the highest degree of correlation with ferritin (r = -0·373 for men and r = -0·590 for women) and the highest area under the curve (0·961 for men and 0·864 for women). Using the cut-off value of 52·6 for men and 50·6 for women, the corresponding Youden index was the highest for CCI in both genders (0·851 for men and 0·612 for women). The sensitivity and specificity of CCI in the population of male donors were higher in comparison to female donors (0·941 and 0·910 vs 0·851 and 0·761, respectively). CONCLUSIONS: Study results confirmed the satisfactory diagnostic value of CCI in detecting depleted iron stores in blood donors.

Quality control of buffy coat removed red cell concentrates--a Croatian experience.

OBJECTIVES: Results are presented of the statistical quality control of red cell concentrate buffy coat removed in additive solution (RCC/BC/AS) and red cell concentrate buffy coat removed and leucoreduced in additive solution (RCC/BC/LR/AS) produced at the Croatian Institute of Transfusion Medicine during an 8-year period (2005-2012). The aim was to assess quality conformity of these products with specified requirements, as well as the suitability and justification of current regulations on the minimal quality requirements. METHODS: The measurements of all the study parameters of the products analysed are expressed using descriptive statistics and graphs showing the distributions of observed parameters. RESULTS: In RCC/BC/AS, the mean (± SD) volume was 279 ± 17 mL; haematocrit, 0.60 ± 0.03 L L(-1); haemoglobin content, 55 ± 5 g; leucocyte count, 0.65 ± 0.41 × 10(9); and haemolysis at expiry date, 0.16 ± 0.13%. In RCC/BC/LR/AS (post-production filtration), the mean (± SD) volume was 255 ± 14 mL; haematocrit, 0.60 ± 0.02 L L(-1); haemoglobin content, 51 ± 4 g; leucocyte count, 0.11 ± 0.16 × 10(6); and haemolysis at expiry date, 0.11 ± 0.07%. In RCC/BC/LR/AS (inline filtration), the mean (± SD) volume was 254 ± 15 mL; haematocrit, 0.61 ± 0.02 L L(-1); haemoglobin content, 51 ± 5 g; leucocyte count, 0.04 ± 0.06 × 10(6); and haemolysis at expiry date, 0.16 ± 0.10%. The standards were just met for leucocyte count in RCC/BC/AS (90%), whereas for all other parameters satisfactory results were obtained in at least 99% of products analysed. Total incidence of bacterial contamination was 0.23% for all products. CONCLUSION: Results of the RCC/BC/AS and RCC/BC/LR/AS quality control showed very high conformity with the specified requirements in the majority of study parameters, suggesting that the current requirements could be redefined and improved at the institutional level.

Quality control of leucocyte-depleted platelet concentrates obtained by buffy-coat method.

OBJECTIVES: Quality control results on leukoreduced buffy-coat platelet pools during the 7-year period (2005-2011) are presented with the aim to assess their overall quality and trends recorded during the study period. METHODS: Data of the Quality Assurance Department, Croatian Institute of Transfusion Medicine were used in the study. Measurement results of all study parameters are presented for the entire study period, while the rate of controlled platelet pools consistent with the specified quality requirements is additionally presented for each study year. RESULTS: The mean component volume was 50 ± 9 mL per 6 × 10(10) platelets (91.5% of conformable results), mean platelet count 7.81 ± 1.26 × 10(10) per single unit equivalent (97.5% of conformable results), mean leucocyte count 0.01 ± 0.08 × 10(6) per single unit equivalent (99.6% of conformable results), and mean pH 7.45 ± 0.16 (99.4% of conformable results). Bacteriologic testing showed negative result in 99.8% of tested components. CONCLUSION: Study results indicated the results of platelet pool quality control to be highly conformable with the specified quality requirements. It is illustrated how simple interventions in the preparation process can have significant impact on product quality improvement and thus on redefining quality requirements.

Bacterial contamination of blood products at the Croatian Institute of Transfusion Medicine: results of eleven-year monitoring.

OBJECTIVES: The aim of this study is to present the results and experiences of the Croatian Institute of Transfusion Medicine (CITM) in blood product testing for the presence of bacterial contamination. This is the first study analysing the results of bacterial testing of blood products in Croatia. METHODS: Results of monitoring blood products for the presence of bacterial contamination during an 11-year period (2000-2010) were retrospectively analysed. As universal screening of platelet concentrates for bacterial contamination is not mandatory in Croatia, the results presented refer to the products tested within the frame of statistical process control. RESULTS: A total of 23,130 blood products were tested during the study period. There were 122 (0·53%) initially positive and 41 (0·18%) confirmed positive blood products, whereas suspicion of bacterial contamination could be neither confirmed nor ruled out in 8 (0·03%) blood products. While the frequency of bacterial contamination of plasma products was very low (0·03%), there was no statistically significant difference between bacterial contamination of platelet concentrates (0·26%) and RBC concentrates (0·20%). There were 73 (0·32%) false-positive blood products, with nearly equal proportion of causes related to laboratory contamination (n = 34; 0·15%) and those related to the testing system (n = 39; 0·17%). CONCLUSION: The results obtained in the study did not differ significantly from literature data. A number of measures to reduce the risk of bacterial contamination of blood products have been implemented at CITM. The introduction of universal screening of platelet concentrates for the presence of bacterial contamination should be taken into consideration.

Inventory management.

A critical aspect of blood transfusion is the timely provision of high quality blood products. This task remains a significant challenge for many blood services and blood systems reflecting the difficulty of balancing the recruitment of sufficient donors, the optimal utilization of the donor's gift, the increasing safety related restrictions on blood donation, a growing menu of specialized blood products and an ever-growing imperative to increase the efficiency of blood product provision from a cost perspective. As our industry now faces questions about our standard practices including whether or not the age of blood has a negative impact on recipients, it is timely to take a look at our collective inventory management practices. This International Forum represents an effort to get a snap shot of inventory management practices around the world, and to understand the range of different products provided for patients. In addition to sharing current inventory management practices, this Forum is intended to foster an exchange of ideas around where we see our field moving with respect to various issues including specialty products, new technologies, and reducing recipient risk from blood transfusion products.