Ivana Babić

Distribution of weak D types in the Croatian population

Dear Sir, The Rh blood group system is determined by the highly homologous RHD and RHCE genes located on the first chromosome, which encode for the RhD and RhCE polypeptides. D antigen is of special clinical relevance in the fields of transfusion medicine and obstetrics. Owing to its high immunogenicity, D antigen can induce the production of alloantibodies and thus cause posttransfusion haemolytic reaction and haemolytic disease of the newborn (Wagner et al., 2000). About 0·2–1% of the European population are carriers of structurally altered RHD alleles encoding for various types of weak D proteins. At the molecular level, point mutations resulting in amino acid substitutions in the intracellular or transmembranous segments of RhD protein are causing weak D phenotypes. More than 170 different RHD alleles closely related to the expression of the respective D phenotype, including more than 70 weak D types, have been discovered to date (Flegel, 2007). Some weak D types (types 1, 2 and 3) are not associated with the development of alloantibodies; however, alloimmunisation in weak D types 4·2, 11 and 15 carriers have been reported (Flegel, 2006). Owing to the extremely small phenotypic variation, particular weak D types are very difficult to differentiate by serology and can only be identified by molecular methods, thus enabling definitive decision on the mode of transfusion treatment and the need of anti-D prophylaxis in pregnant women. The individuals who are carriers of weak D types 1, 2 and 3 can receive transfusion of D+ red blood cell (RBC) units, although such pregnant women do not require anti-D prophylaxis. Thus, the unnecessary utilisation of D− RBC units and RhIg is avoided (Flegel and Wagner, 2002). Particular segments of the RHD gene sequence are multiplied by RHD genotyping using primers specific for the known mutations characterising particular weak D types by use of the polymerase chain reaction with sequence-specific priming (PCR-SSP). This procedure is employed to determine polymorphism of the weak D types.

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.

Detection of three blood donors with multiple myeloma by routine viral individual-donor nucleic acid testing screening: LETTERS TO THE EDITOR

Individual-donor nucleic acid testing (ID-NAT) of all donations collected from voluntary nonremunerated blood donors (VNRDs) in Croatia was introduced in 2013. By the end of August 2016, a total of 639,688 whole blood donations from 148,703 VNRDs had been screened by using the Procleix Ultrio Plus assay on the Tigris devices (Grifols) for simultaneous detection of HBV DNA, HCV RNA, and HIV-1 RNA. During that period three VNRDs gave donations that created strong interference in routine NAT. Their samples were retested two to five times. Procleix Panther System software declared all test results as invalid without any specific information about the cause of hardware error. When the first VNRD created massive hardware failure, in the collaboration with Grifols technical support personnel we revealed protein clot blockage of aspiration system as a main reason for the interference. According to the test manufacturer, a higher rate of invalid results may be observed in serious clinical conditions associated with high plasma protein content (diseases with positive rheumatoid factor or positive antinuclear antibodies, systemic lupus erythematosus, multiple myeloma, multiple sclerosis, rheumatoid arthritis, hyperglobulinemia, alcoholic cirrhosis). We were interested in investigating the health status of three VNRDs with presumably high plasma protein content causing invalid testing results. Donors were invited to the Croatian Institute of Transfusion Medicine (CITM) for an interview and additional testing: basic coagulation tests, total plasma protein (TP) and serum protein electrophoresis (SPE). Interestingly, for the first VNRD, the donation given 4 months before the incriminating one showed inhibition of NAT reaction (low signal-to-cutoff ratio for internal control) that was not resolved by retesting. Donation was rejected but the donor was not informed. All VNRDs were male, 48 to 58 years old, who considered themselves as healthy individuals. During the interview, the second and third donors reported increased sweating several months before the donation, while the third also reported mild anemia established in a clinical laboratory 2.5 months before the donation with invalid NAT results. He was also treated by a physiatrist because of pain in the arm and neck. At the time of control visit to CITM all VNRDs had normal coagulation test results and increased amount of TP (89.9, 99.4, and 94.5 g/L, respectively). The first VNRD presented only with a low albumin-to-globulin ratio while the other two also had M-protein on SPE. They were all advised to visit a hematologist and later diagnosed as myeloma multiplex (MM) IgG lambda. The first two VNRDs were classified as International Staging System (ISS) III and the third as ISS II. For the first VNRD, the time from invalid results to diagnosis was 8 months. The second had a MM diagnosis within 2 weeks and the third within 2 months. The first VNRD was presented by hyperviscosity syndrome, increased calcium level, renal dysfunction, and destructive bone lesions. The second had 40% plasma cells in the marrow, without bone lesions but with anemia and rapid growth of serum IgG. The third donor had smoldering MM and no indication for treatment according to the guidelines of International Myeloma Working Group. All three VNRDs were whole blood donors and in Croatia SPE is performed annually only for apheresis donors. La Raja and colleagues found 104 cases of monoclonal gammopathy including two cases of MM by performing annual SPE for 8197 VNRDs in their study. We did not try to establish and the manufacturer does not precisely cite threshold in TP level, which can cause interference; in that sense the sensitivity of the described accidental finding remains unknown. We assume that high robustness of the Procleix Ultrio Plus assay used for ID-NAT screening allows us to detect only donations with higher protein level, although the TP level measured for two of three VNRDs with MM would be acceptable according to criteria of Canadian Blood Services along with normal results of SPE. This could explain the very low frequency (0.002%) of monoclonal gammopathy coincidentally found and later diagnosed as MM in the population of Croatian whole blood VNRDs. Nevertheless, based on three cases we could assume that finding a protein clot blockage as an interference in NAT has extremely high positive predictive value in the diagnosis of monoclonal gammopathy or even multiple myeloma. Also, extremely rare occasions of inability to solve inhibition by ID-NAT retesting should be investigated as a precautionary measure to examine VNRD health status. In our opinion, the cause of every persistent interference in ID-NAT routine screening should be explored in detail as a sign of possible serious disease. The described cases enabled the transfusion service professionals to take additional care and helped in providing earlier diagnosis of MM. We believe that our experience will be helpful to other colleagues working on NAT in drawing attention to this important finding.