Prevalence of HEV RNA in Croatian blood donors.

OBJECTIVES: HEV infection is asymptomatic for immunocompetent blood donors (BD). Transfused HEV-infected blood products may cause potentially hazardous HEV infection in immunocompromised patients. Evaluation of the need for routine BD HEV RNA screening primarily demands the establishment of HEV infection prevalence in Croatian BD. MATERIALS AND METHODS: We tested BD samples in ID-NAT with the Procleix UltrioPlex E screening test for simultaneous detection of HBV DNA, HCV RNA, HIV-1,2 RNA, and HEV RNA (Grifols, Spain). HEV infection was confirmed with HEV RNA quantitative test (Altona Diagnostics, Germany) and HEV IgM and HEV IgG antibody test (DIA.PRO Diagnostic Bioprobes, Italy). We analysed the HEV RNA sequence and performed a phylogenetic analysis. We recorded BD's anamnestic data and dietary habits. BDs gave follow-up samples after two months and did not donate blood for six months. RESULTS: Between December 2021 and March 2022, we tested 8,631 donations and found four HEV RNA-positive donations, which equals to one in 2,158 donations (0.046 %, 95 % confidence interval, 0.018 %-0.119 %). Confirmatory HEV RNA testing gave results from negative to 4.73E + 3 IU/ml HEV RNA. Three donations were in the serological window period. We have genotyped HEV RNA of two infected BD as genotype HEV-3c. Blood donors didn't report any health problems and their diet included pork. Testing on follow-up samples presented seroconversion and no HEV RNA could be detected. CONCLUSION: The incidence of HEV RNA infection in BD in Croatia corresponds with other European data. The decision on implementation of HEV NAT screening in Croatia needs an expert team evaluation of the possible risk of TT-HEV infection.

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.

Oxacillinases and antimicrobial susceptibility of Ralstonia pickettii from pharmaceutical water systems in Croatia.

This study evaluated antibiotic susceptibility and presence of blaOXA22 and blaOXA60 genes in 81 isolates of Ralstonia pickettii obtained from different purified and ultra-pure water systems in two different geographical areas of Croatia. E-test and disc diffusion test were performed to determine antibiotic susceptibility. Polymerase chain reaction was applied to detect genes encoding OXA-22 and OXA-60 oxacillinases previously identified in R. pickettii. The isolates were genotyped by pulsed-field gel electrophoresis. The results revealed variable susceptibility/resistance profiles. Our isolates exhibited high susceptibility rates to ceftriaxone, cefotaxime, piperacillin-tazobactam, ciprofloxacin, imipenem, cefepime and in lesser extent to ceftazidime. High rates of susceptibility were also observed for sulphamethoxazole-trimethoprim and piperacillin. High resistance rates were noticed for ticarcillin-clavulanate, aztreonam and meropenem, as well as for all aminoglycosides tested. Modified Hodge test was positive in 51·9% strains, indicating production of carbapenemases. blaOXA22 and blaOXA60 genes were detected in 37·0 and 80·3% strains, respectively. Pulsed-field gel electrophoresis identified three major clusters containing subclusters. R. pickettii should be taken seriously as a possible cause of nosocomial infections to ensure adequate therapy, to prevent the development of resistant strains and to try to reduce the possibility of R. pickettii surviving in clean and ultra clean water systems.

S2k guidelines (consensus statement) for diagnosis and therapy of dermatitis herpetiformis initiated by the European Academy of Dermatology and Venereology (EADV).

INTRODUCTION: Dermatitis herpetiformis (DH) is a chronic, pruritic, gluten-induced skin disorder characterized by subepidermal granular IgA deposition and a variable degree of enteropathy identical to that seen in coeliac disease. So far, there has been no European consensus about the management of DH. METHODS: The guidelines were created by small subgroups of a guideline committee consisting of 26 specialists from various medical fields and one patients' representative. The members of the committee then discussed the guidelines and voted for the final version at two consensus meetings. The guidelines were developed under the support of the European Academy of Dermatology and Venereology (EADV) and in collaboration with the European Dermatology Forum (EDF). RESULTS: The guidelines summarize evidence-based and expert-based recommendations (S2 level) for the management of DH (see Appendix). CONCLUSION: These guidelines will improve the quality of management of DH and support dermatologists in their diagnostic and therapeutic decisions.

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.

Prenatal RHD genotyping in Croatia: preliminary results.

OBJECTIVES: Croatian Institute of Transfusion Medicine (CITM) implemented non-invasive fetal RHD genotyping as a request for targeted antenatal anti-D prophylaxis. The diagnostic performance of in-house RT-PCR method for fetal RHD genotyping and preliminary results are analyzed. MATERIALS AND METHODS: Evaluation included results of RHD genotyping for 205 RhD negative pregnant women, 12-36th week of gestation, whose samples were received in period between 2015 and 2020. QIAsymphony SP DSP Virus Midi Kit was used for cffDNA extraction on QIAsymphony SP platform (Qiagen, Germany). Fragments of RHD exons 7 and 10 and later exon 5 were RT-PCR amplified. As internal controls, amplification of SRY gene or RASSF1A fragment and ß-actin genes digested with BsTUI were used. RESULTS: We identified 70.72% (145/205) positive and 28.78% (59/205) negative fetal RHD genotypes. We had one inconclusive result (0.50%) due to the interference of maternal DNA with variant genotype RHD*09.02.00/01/*01N.01. When compared to newborns RhD phenotypes, no false negative and three false positive results (3/199, 1.50%) were observed. The test yielded 100% sensitivity and 95.08% specificity, while diagnostic accuracy was 98.48%. We were able to determine one case of fetal variant genotype RHD*04.04/*01N.01 inherited from the father. The negative and positive predictive test values were 100% and 97.86%, respectively. CONCLUSION: Automated cffDNA extraction and RT-PCR amplification of fetal RHD exons 5,7,10 and fragments of SRY, RASSF1A genes represents highly reliable system for determining fetal RHD status which enables targeted antenatal anti-D prophylaxis. To obtain high specificity of cffDNA extraction, strict and thoroughly cleaning procedures are required.

Warm red blood cell autoantibodies and clinical diagnoses in patients with or without autoimmune hemolysis.

OBJECTIVES: Red blood cell autoantibodies (RBC autoAbs) of IgG class are found in the majority of patients with warm autoimmune hemolytic anemia (wAIHA) but sometimes also during the pretransfusion testing of patients with different diagnoses but without hemolysis. The aim of the study was to identify the main differences between these two groups of patients according to age, gender, subclass and titer of IgG RBC autoAbs and diagnosis. MATERIAL AND METHODS: In the 9-year retrospective study, data were collected from records of 291 patients with IgG RBC autoAbs detected by gel technique, from which 111 with wAIHA. RESULTS: More than 85% of patients in both groups were over 40 years old, with male to female ratio 1:1.9 in wAIHA vs 1:1.3 in patients without hemolysis (P=0.0916). The main characteristics of patients with wAIHA vs patients without hemolysis were: IgG only 38% vs 70%, IgG+Complement 62% vs 30%, total IgG1 79% vs 55%, IgG1+IgG3 35% vs 11%, titer of 100 for IgG1+IgG3 17% vs 3% (P<0.0001), respectively, while titer of 100 for IgG1 18% vs 9% (P=0.0241). The underlying diagnosis in wAIHA vs patients without hemolysis: hematologic disorders 41% vs 22% (P=0.0006), autoimmune disorders 12% vs 13% (P=0.8033), solid tumors 5% vs 14% (P=0.0154) and surgery procedures 6% vs 26% (P<0.0001). CONCLUSION: We observed more wAIHA patients with high titer of IgG1 and high prevalence of IgG1+IgG3 and consider that patients without hemolysis having identical results might be interesting to find out how they are protected from damage by RBC autoAbs.

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.

Serological diagnostics in the detection of IgG autoantibodies against human collagen VII in epidermolysis bullosa acquisita: a multicentre analysis.

BACKGROUND: Epidermolysis bullosa acquisita (EBA) is a rare, potentially devastating autoimmune disease of the skin. IgG autoantibodies directed against type VII collagen (Col7), the major component of anchoring fibrils, induce skin fragility leading to cutaneous and mucocutaneous blister formation, which is mostly of a scarring phenotype. Thus, powerful and reproducible diagnostic assays are critical to establish the diagnosis of EBA early to avoid irreversible sequelae. OBJECTIVES: The present international, retrospective multicentre study included a large cohort of patients with EBA and evaluated the diagnostic power of four different diagnostic assays for the detection of anti-Col7 IgG autoantibodies. METHODS: Overall, 95 EBA sera and 200 control sera consisting of 100 bullous pemphigoid sera, 50 pemphigus vulgaris sera and 50 sera of healthy controls were tested for anti-Col7 IgG autoantibodies using indirect immunofluorescence (IIF), two commercial enzyme-linked immunosorbent assay (ELISA) systems and Western blot (WB) analysis. EBA sera were taken from patients with positive direct immunofluorescence and IgG reactivity in at least one of the immunoserological assays (IIF, ELISA, WB). RESULTS: A Col7-NC1/NC2 ELISA (MBL, Nagoya, Japan) showed the highest sensitivity (97·9%), followed by a Col7-NC1 ELISA (Euroimmun, Lübeck, Germany) (89·5%), WB with Col7-NC1 (85·3%), and IIF on saline-split human skin (74·7%). The specificities of both ELISA systems were comparable (NC1 98·7%, NC1/NC2 99·3%). Furthermore, WB was more sensitive than IIF, which was more specific. CONCLUSIONS: The two commercially available ELISA systems allow for a highly sensitive and specific diagnosis of EBA. The sensitivity of the Col7-NC1/NC2 ELISA is significantly higher compared with the ELISA based on the Col7-NC1 domain only.

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.

Lamotrigine in the treatment of pain syndromes and neuropathic pain.

Anti-epileptic drugs are increasingly used in the treatment of pain syndromes and neuropathic pain. Sodium channel blockers can be effective in the treatment of pain. The object of our interest is the efficiency of lamotrigine in treating the pain. A MEDLINE search was conducted to identify pertinent studies, case reports, letters, and reviews in English published from 1986 to May 2007. The search has indicated efficiency in treating a number of painful syndromes and neuropathic pain; central pain, trigeminal neuralgia and trigeminal neuralgia in multiple sclerosis, pain in multiple sclerosis, SUNCT syndrome, cluster headache, glossopharyngeal neuralgia, neuropathic pain, allodynia, neuralgia after nerve section, postherpetic neuralgia, HIV-associated neuropathy. Further researches are required on the role of lamotrigine in treating the spinal cord injury pain, neuralgia after nerve section, postoperative analgesic requirement, and in migraine (Tab. 1, Ref. 46). Full Text (Free, PDF) www.bmj.sk.