Serological and molecular characterisation of the most prevalent weak D variants in Croatian population.

INTRODUCTION: Existence of hundreds of RHD gene variants contributes to variable D antigen expression and inconsistencies in reporting the RHD results. The aim of the study was to determine the serological and molecular characteristics of the most prevalent RHD alleles encoding serologically weak D variants. MATERIAL AND METHODS: Blood donors (n = 145 924) were typed for D antigen using the direct serologic micromethod. Nonreactive samples were analysed in IAT method with the IgM/IgG anti-D monoclonal blend, and 0,2% (n = 263) confirmed weak D antigen expression. After genomic DNA extraction (Qiaqen, Germany), RHD genotyping was performed using in house reagents and PCR-SSP kits (Inno-Train, Germany). RESULTS: The prevalence of serologically weak D in blood donor population was 0.2% (n = 263). RHD genotyping confirmed weak D allele in 92.4% and partial D allele in 7.6%. The most common was weak D type 1 (49.7%) followed by weak D type 3 (24.7%) and type 2 (9.5%). Relatively high frequency was detected for weak D type 14 (4.6%) and type 64 (2.3%). In the category of partial D phenotypes, only DVI variant was found. Direct typing has shown great variability in the strength of reactions with different clones of anti-D reagents. CONCLUSION: Weak D type 1 is the most common weak D variant in Croatian blood donor population. The frequency of D variants and distribution of Rh phenotypes in our study was in concordance with other studies. It has been shown that serological methods and the combination of clones used, cannot distinguish variant D types, which justifies the use of molecular methods.

RhD alloimmunization by DEL variant missed in donor testing.

INTRODUCTION: Exposure to normal or variably expressed RhD antigens in an antigen-negative individual can elicit an immune response and lead to the formation of clinically significant anti-D alloantibodies. We present the case of anti-D alloimmunization by DEL variant missed in routine blood donor screening. MATERIAL AND METHODS: Blood donors were typed for D antigen using the direct serologic micromethod. Nonreactive samples were confirmed in the indirect antiglobulin method with an IgM/IgG anti-D monoclonal reagent. Genomic DNA was extracted using a commercial QIAamp DNA Blood Mini kit on the QIAcube device (Qiaqen, Germany). RHD genotyping was performed using the PCR-SSP genotyping kits- Ready Gene D weak, Ready Gene D weak screen, Ready Gene CDE, and Ready Gene D AddOn (Inno-Train, Germany). Unidentified alleles were sent for DNA genome sequencing. RESULTS: After identifying DEL positive blood units in RhD negative blood donor pool, a look-back study was performed to determine if their previous donations caused alloimmunization in recipients. Out of 40 D negative recipients, one developed anti-D alloantibody after 45 days. The patient did not receive other RhD positive blood products. Blood donor typed D negative in direct and indirect agglutination method. RHD screening was positive, but RHD genotyping and DNA sequencing showed no mutation indicating the normal genotype. CONCLUSION: Currently used methods in RHD genotyping are insufficient to identify many variant alleles, especially intronic variations. We suggest additional gene investigation including yet unexplored regions of regulation and intron regions to justify our serological finding.

D variants in the population of D-negative blood donors in the north-eastern region of Croatia.

OBJECTIVES: The aim of this study was to determine RHESUS D GENE (RHD) allelic variants among Croatian D-negative blood donors and compare our results with respective data from other European countries. BACKGROUND: Altered or reduced D antigen expression can result in D variants, which can be mistyped and can lead to the alloimmunisation of the blood recipient. RHD genotyping can distinguish D variants: weak D, partial D and DEL, thus preventing alloimmunisation. MATERIAL/METHODS: A total of 6523 samples obtained from D-negative Croatian donors were screened for the presence of RHD using the real-time polymerase chain reaction (PCR) method. PCR-SSP was performed for D variant genotyping by using commercial genotyping kits (Inno-Train, Kronberg, Germany). Genomic DNA sequencing for all 10 exons of the RHD was performed when the genotyping kits failed to assign a D variant. RESULTS: RHD molecular screening revealed 23 (0.35%) RHD-PCR positive samples, all C/E positive, in decreasing frequency: 11 hybrid RHD-CE (2-9) D-CE variants, 4 weak partial D type 11 and 2 weak D type 2. Six samples remained unresolved and were sequenced. For 12 of 23 samples (excluding large hybrids), an adsorption/elution of anti-D serum was performed, confirming that all 12 were RhD+. The calculated frequency of clinically significant D alleles in RhD-negative blood donors was 1:543 (0.18%) or 1:53 (1.89%) in C/E blood donors. CONCLUSION: Data on the significant frequency of D variants among serologically D-negative blood donors in the north-eastern region of Croatia could help in introducing RHD molecular screening of blood donors in a routine workflow.

Anti-HBc prevalence among Croatian blood donors in a 14-year period (2004-2017): Assessment of trends, risks and need for implementing routine testing.

OBJECTIVES: The anti-HBc prevalence over a 14-years period (2004-2017), trends, infectivity, residual risk, and need for testing in blood donors (BD) of the Croatian Institute of Transfusion Medicine were assessed. MATERIAL AND METHODS: Anti-HBc was tested in 19,969 BD serum samples collected in 2004 (N=7561), 2013 (N=7318) and 2017 (N=5090). All serums were initially screened for HBsAg, anti-HCV, HIV Ag/Ab, and anti-TP. 2013 and 2017 samples were also tested by ID-NAT. RESULTS: Over a 14-years period, the anti-HBc prevalence significantly decreased among Croatian BD (5.24% in 2004, 2.56% in 2013, and 1.32% in 2017). Similarly, the prevalence of anti-HBc-only profiles decreased from 0.62% in 2004, 0.25% in 2013, and 0.21% in 2017. The 4-time decreasing trend was observed in all age groups of BD from 2017 but mostly among repeat donors (5.90% to 1.38%). First-time donors showed no significant difference in anti-HBc prevalence probably due to their younger age (<29 years) and HBV vaccine status. However, similar anti-HBs carriage rates (80.56%, 87.57%, and 82.09%) were reported in anti-HBc positive donors over the study period. HBsAg and HBV DNA were not detected. No OBI infection was found in the study despite an OBI frequency of 1:10,900 donations previously reported in Croatia. A HBV decreasing residual risks of 68, 88, and 12 per million donations were estimated for years 2004, 2013, and 2017, respectively. CONCLUSION: Anti-HBc testing is an additional measure of preventing HBV infection by transfusion. Implementation of anti-HBc testing will result in the deferral of 1.3% BD and should be supported by cost-benefit analyses.

Anti-D reagents should be chosen accordingly to the prevalence of D variants in the obstetric population.

BACKGROUND: Resolving ambiguous results of D antigen typing is crucial for appropriate and rational administration of anti-D immunoprophylaxis and transfusion practice in obstetric population. The aim of the study was to establish selection criteria of anti-D reagents for our population. METHODS: A total of 12 689 samples from primiparous women in Split-Dalmatia County, Croatia, were typed for RhD antigen during the period of 5 years. Ambiguous results were submitted to additional serologic investigation and genotyping. RHD genotyping was performed by commercial genotyping kits (Ready Gene weak D ® and Ready gene CDE, Inno-Train, Kronberg, Germany). Relative frequencies and accompanying 95% confidence intervals were used to estimate the prevalence of variants. RESULTS: The prevalence of D variants was 0.42% (95% CI 0.31; 0.53). The most common partial D variant was D Va (RHD*05.05), with the prevalence of 0.08% (95% CI 0.03; 0.13). All weak D variants were weak D types 1, 2 and 3 (RHD*weak D type 1, RHD*weak D type 2, RHD*weak D type 3). Weak D samples were distinguishable from partial D in routine typing due to the difference in reactivity of partial D samples with clones D7B8 and RUM-1. Cell line RUM-1 gives weak or negative reactions with partial DVa category. CONCLUSION: The most common partial D variant in our population is DVa. It is recommended to use cell lines which do not strongly agglutinate DVa variant in routine RhD typing. The appropriate choice of reagents will enable the serology methods to recognize the cases in which RHD genotyping is required.

Association of Plasminogen Activator Inhibitor-1 Gene Polymorphisms and Methylene Tetrahydrofolate Reductase Polymorphisms with Spontaneous Miscarriages.

AIM: The objective of this study was to investigate a possible correlation between the plasminogen activator inhibitor-1 (PAI-1) and methylene tetrahydrofolate reductase (MTHFR) polymorphisms and unexplained spontaneous miscarriages (SM). MATERIALS AND METHODS: PAI-1 polymorphisms were evaluated in 150 women with pregnancy in their history. One hundred women with a history of SM formed the study group and 50 women with normal pregnancies served as the control group. Also, the combination of PAI-1 and MTHFR polymorphisms were evaluated in 138 women out of a total of 150, which included 92 women with SM in their history compared to 46 women in the control group. For statistical analysis, χ2 test, Phi, and Cramer V tests were used; p < 0.05 was taken as a statistically significant result. RESULTS: Our findings show: (a) the correlation between SM and PAI-1 mutations reaches statistical significance (p = 0.026); (b) there was a statistically significant difference between heterozygous PAI-1 in women with only 1 SM compared to the control group (p = 0.047); (c) the comparison of combinations of both mutations, PAI-1 and MTHFR, with the control group demonstrates statistical significance in favor of women with SM and both mutations (p = 0.022). CONCLUSION: PAI-1 and MTHFR polymorphisms may play an important role in pregnancy complications because heterozygous PAI-1 mutations and a combination of both PAI-1 and MTHFR mutations might contribute to SM.

Three-Year Experience in NAT Screening of Blood Donors for Transfusion Transmitted Viruses in Croatia.

BACKGROUND: Croatia implemented individual donation (ID)-NAT testing of blood donors in 2013 for three viruses HBV, HCV, and HIV-1 as a mandatory test for all blood donors. This study assessed the impact of NAT screening 3 years after its implementation. METHODS: A total of 545,463 donations were collected and screened for HBV, HCV, and HIV-1 using the Procleix Ultrio Plus Assay. All initially reactive (IR) NAT samples were retested in triplicate and, if repeatedly reactive (RR), NAT discriminatory assay (dNAT) was performed. ID-NAT positive donations were confirmed by RT-PCR on the COBAS AmpliPrep/TaqMan platform. RESULTS: Out of 545,463 samples tested, 108 (0.02%) were RR in NAT. There were 82 (75,9%) HBV reactive, 16 (14.8%) HCV reactive, and 10 (9.3%) HIV-1 reactive samples. 51 (47.2%) samples were ID-NAT positive only. Out of these 51 NAT yield cases, 1 window period HIV-1 and 50 occult HBV infections (OBI) were determined. There were only two potential HBV DNA transmissions from OBI donors. CONCLUSION: The implementation of NAT screening for three viruses has improved blood safety in Croatia. During the 3-year period, 1 window period HIV-1 and a number of occult HBV donations were identified.

Anti-D Antibodies in Pregnant D Variant Antigen Carriers Initially Typed as RhD.

BACKGROUND: To evaluate the incidence, the consequences, and the prevention strategy of anti-D alloimmunizations of D variant carriers in the obstetric population of Split-Dalmatia County, Croatia. METHODS: RhD immunization events were evaluated retrospectively for the period between 1993 and 2012. Women were tested for RhD antigen and irregular antibodies. Those with anti-D antibody who were not serologically D- were genotyped for RHD. They were evaluated for their obstetric and transfusion history and their titer of anti-D. The neonates were evaluated for RhD status, direct antiglobulin test (DAT), hemoglobin and bilirubin levels, transfusion therapy as well as phototherapy and outcome. RESULTS: Out of 104,884 live births 102,982 women were tested for RhD antigen. Anti-D immunization occurred in 184 women which accounts for 0.9% of individuals at risk of anti-D formation. 181 cases occurred in women serologically typed as D-. Three women were partial D carriers (DVa n = 2, DNB n = 1), initially typed RhD+, and recognized as D variant carriers after the immunization occurred. Anti-D titer varied from 1:1 to 1:16. Six children were RhD+, four had positive DAT, and two underwent phototherapy. CONCLUSION: Anti-D immunization occurred in pregnant partial D carriers (DVa, DNB). RhD+ children had serologic markers of hemolytic disease of the fetus and newborn (HDFN), with no cases of severe HDFN.

Characteristics and frequency of DEL phenotype detected by indirect antiglobulin test in Dalmatia county of Croatia.

D variants, collectively called DEL, express trace amounts of D antigen which is considered to be serologically detectable only by adsorption-elution techniques. We detected six cases of DEL phenotype by indirect antiglobulin test, in Dalmatia County of Croatia by routine serological testing of D antigen of new blood donors. RH genotyping found that all six donors carry allele RHD(M295I), RH genotype CcDdee. D antigen densities of D variants were very low, between 26 and 44 D antigens per red blood cell. The frequency of D variants detected by IAT allele RHD(M295I) was 1:272 in D negative donors. Obviously, DEL phenotype is more common in some parts of European population than initially thought. In conclusion, our routine serological testing of D antigen can detect extremely weak D antigen, even RBCs with DEL phenotype and antigen density as low as 26 D antigens per RBC.

Management of complaints in blood establishments: thirteen years of experience at the Croatian Institute of Transfusion Medicine.

BACKGROUND: The aim of the study is to present the results and experience in the management of complaints in a transfusion service in order to draw attention to the importance of this segment of quality management and to stimulate publication of other studies on the topic. MATERIALS AND METHODS: This study is based on data from the Croatian Institute of Transfusion Medicine obtained by analysis of complaints recorded during a 13-year period (1998-2010). The distribution of the types and frequencies of complaints is presented, along with the level of their justifiability and criticality. The dynamics of the complaints is analysed overall and within particular categories. In addition, corrective actions and other factors that may have influenced the trends observed are discussed. RESULTS: During the study period, 817 complaints were received, most of which (40.9%) referred to the positive direct antiglobulin test in red cell concentrates, followed by blood product issuing and distribution (12.9%) and blood product quality (9.4%). Of the 817 complaints, 177 (21.7%) were assessed as serious and 645 (78.9%) as justified based on the testing performed. CONCLUSION: Data collected by systematic recording and analysis of complaints provide a basis for problem identification, implementation of corrective and preventive actions, and improvement of product and service quality, and, thereby, customer satisfaction.

ABO blood groups and genetic risk factors for thrombosis in Croatian population.

AIM: To assess the association between ABO blood group genotypes and genetic risk factors for thrombosis (FV Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T mutations) in the Croatian population and to determine whether genetic predisposition to thrombotic risk is higher in non-OO blood group genotypes than in OO blood group genotypes. METHODS: The study included 154 patients with thrombosis and 200 asymptomatic blood donors as a control group. Genotyping to 5 common alleles of ABO blood groups was performed by polymerase chain reaction with sequence specific primers (PCR-SSP). FV Leiden was determined by PCR-SSP, while prothrombin and methylenetetrahydrofolate reductase were determined by PCR and restriction fragment length polymorphism (PCR-RFLP). RESULTS: There was an association between non-OO blood group genotypes and the risk of thrombosis (odds ratio [OR] 2.08, 95% confidence interval [CI], 1.32-3.27). The strongest association with thrombotic risk was recorded for A1B/A2B blood group genotypes (OR, 2.73; 95% CI, 1.10-6.74), followed by BB/O1B/O2B (OR, 2.29; 95% CI, 1.25-4.21) and O1A1/O2A1 (OR, 1.95; 95% CI, 1.15-3.31). FV Leiden increased the risk of thrombosis 31-fold in the group of OO carriers and fourfold in the group of non-OO carriers. There was no significant difference in the risk of thrombosis between OO and non-OO blood groups associated with prothrombin mutation. Non-OO carriers positive for methylenetetrahydrofolate reductase had a 5.7 times greater risk of thrombosis than that recorded in OO carriers negative for methylenetetrahydrofolate reductase. CONCLUSION: Study results confirmed the association of non-OO blood group genotypes with an increased risk of thrombosis in Croatia.

[Neonatal alloimmune thrombocytopenia in Croatia in 1997-2007]. / Neonatalna aloimuna trombocitopenija u Hrvatskoj od 1997. do 2007. godine.

Neonatal alloimmune thrombocytopenia (NATP) is caused by maternal sensitization to paternal alloantigens on fetal platelets during pregnancy. Although the disease is rare, the severity of clinical picture and its sequels associated with central nervous system hemorrhage impose the need of an early diagnosis, and timely and specific treatment of the disease. Based on these and literature data on the prevalence of NATP in Caucasians of 1-2 cases per 1000-5000 live births, it is estimated that 10 to 50 serologically verified cases of NATP and approximately a twofold number of requests for serologic testing for suspected NATP could be expected in Croatia per year. In the present study, results of serology testing and clinical laboratory data of twenty five cases of NATP in Croatia during the 1997-2007 period are evaluated. In 20/25 cases platelet antibody screening was positive. Specific platelet antibodies were confirmed in 14/20 (70%) cases with positive screening (anti-HPA-la in 7/14, anti-HPA-5b in 5/14, and anti GP Ib-IX in 2/14 cases). Only fourteen serologically confirmed cases of NATP per -45,000 live births per year in Croatia indicate the prevalence of the disease to be considerably lower than expected.