Application of a Grey Zone for HCV and HIV Immunoassays for Blood Donor Screening: Is It still Necessary? / 대한수혈학회지

BACKGROUND: When enzyme immunoassays (EIA) were implemented, considering the limited sensitivity of 1st generation EIAs, the Korean Red Cross (KRC) applied grey zones for detection of weak reactive samples that could lead to false negative results. Despite improved performance of assays, grey zone application is still in practice. We examined whether application of a grey zone to HCV and HIV EIAs is still necessary. METHODS: HCV and HIV EIA results, number of grey zone results, results of further testing done on grey zone samples, and NAT results from 2005 to 2012 were analyzed retrospectively using the Blood Information Management System of the KRC. RESULTS: Among 18,736,094 cases tested, 4,817 HCV (0.03%) and 5,108 HIV (0.05%) cases repeatedly had grey zone results. Twenty-eight (0.58%) HCV grey zone cases were positive on the recombinant immunoblot assay, but negative on NAT. For HIV, 3 cases were diagnosed as indeterminate by the Korea Centers for Disease Control and Prevention (KCDC). However these cases did not seroconvert after several years and were also negative on NAT. CONCLUSION: For HCV, since the grey zone led to detection of true anti-HCV positive cases, not detected by NAT, application of the grey zone should be continued. For HIV, since none of the grey zone cases has been diagnosed as HIV positive by the KCDC, further application of the grey zone is thought not to be necessary. Re-evaluation of the grey zone will save costs for testing, and prevent discard of blood components and loss of donors.

Influence of Pathogen Inactivation on the Quality of Platelet Rich Plasma Derived Platelets / 대한수혈학회지

BACKGROUND: Pathogen inactivation (PI) is a proactive approach to overcome the limitations of the current testing system and donor questionnaires. Effect of PI on non-leukoreduced platelet rich plasma derived platelets (PRP-PLTs) suspended in plasma has not yet been evaluated. This study was conducted in order to evaluate the effect of PI on the quality of non-leukoreduced PRP-PLTs suspended in plasma. METHODS: PRP-PLTs treated with the Mirasol PRT System and the Intercept Blood System were tested for PLT count, blood gas, PLT activation, and apoptosis on days 3, 5, and 7 of storage. RESULTS: PLT number showed a decrease after PI. No difference in pH was observed until day 5. At day 7, PLTs treated with Mirasol had a lower pH value (6.5), however, it satisfied the quality control criteria. PLTs treated with Mirasol had a lower pO2 compared to pre-inactivation PLTs. pO2 during storage differed significantly between the two PI groups. pCO2 showed a decrease after inactivation and both groups showed a significant difference, compared with the control. PLTs treated with Mirasol had increased P-selectin expression after inactivation; however, difference of P-selectin during storage was not significant compared to the control. P-selectin of PLTs treated with Intercept was significantly different compared to those treated with Mirasol and control. Annexin V showed an increase after inactivation in Mirasol treated PLTs and difference during storage was significant compared to control and Intercept. CONCLUSION: As both PI systems showed satisfactory pH values, the criteria showing a high correlation with in vivo PLT viability and generally applied to monitor quality of PLTs, quality of PRP-PLTs after PI appears not to be negatively affected.

Analysis for Human Immunodeficiency Virus 1 Subtype in Korean Blood Donors / 대한수혈학회지

BACKGROUND: Genetic variants of virus appear to differ depending on the country, race, infection route, and so on. To characterize the main HIV subtype in infected blood donors and inquire about the route of HIV infection, we analyzed HIV subtype for samples that showed reactive results on the anti-HIV 1/2 and HIV-1 NAT test from September 2007 to February 2010. METHODS: To identify the HIV-1 subtype of the 90 samples that showed reactive results on the anti-HIV test and HIV-1 NAT, we performed HIV 1/2 Western blot assay, HIV RNA quantitative assay, HIV-1 nested PCR, and HIV-1 RNA sequencing. RESULTS: A total of 85 samples (94.4%) were confirmed to be HIV-1 subtypes. Among them, 82 samples (96.5%) were subtype B; and subtype A, C, and G was confirmed for one case each (1.2% for each case). We could not identify the subtype of the other five samples. One of them was amplified by nested PCR, but was not confirmed of the subtype, and four samples were not amplified even by nested PCR. CONCLUSION: The main HIV-1 subtype among the HIV-infected blood donors was confirmed to be subtype B. In addition, we identified one case each of HIV-1 subtype A, C, and G, which was not detected in blood donors in the past. It appeared that the route of HIV infection in Korea had become complicated. Therefore, we concluded that continuous research for HIV subtype analysis and efficient management of blood donors is needed.

Analysis of the Coagulation Factors of Donated Plasma for Effective Utilization / 대한수혈학회지

BACKGROUND: The aim of present study was to assess the effect of different freezing time after phlebotomy on the activity of coagulation factors in frozen plasma and to evaluate which source plasma for clotting factor fractionation is appropriate for use. METHODS: Blood plasma units rejected due to a high level of ALT were divided into four groups depending on freezing time after phlebotomy, and each unit of the four groups was assayed for six different clotting factors and blood type. SAS 9.2 was used for statistical analysis of data. RESULTS: A decrease was observed in the activities of FVIII of the plasmas, in the following order: PL-A>FFP>FP(8-24)approximatelyFP(24-72). Results of the assay also showed that the levels of FVIII were significantly higher in the AB type plasmas than in the O type plasmas. PL-A and FFP units met the current quality requirements of the Korean Red Cross, in which the FVIII activity should have more than 0.7 IU/mL in more than 75% of the source plasma, as 85.0% and 82.5%, respectively. On the other hand, FP24 met the Canadian (Quebec) requirements for the source plasma, in which the FVIII activity should have more than 0.52 IU/mL in more than 75% of the source plasma, as 82.6%. CONCLUSION: For use of plasma frozen within 24 hours after phlebotomy (FP24) and plasma of specific blood type, European Pharmacopeia and WHO guidelines on quality control should be adopted for production of plasma-derived coagulation factors in Korea.

Establishment of Plasma Working Standards for the Performance and Quality Assurance of NAT Screening Tests for HIV, HCV and HBV / 대한수혈학회지

BACKGROUND: Since Jan. 2012, for performance evaluation of viral reagents, analysis of domestic samples has been recommended in order to obtain approval from the KFDA when they are first introduced to Korea. This regulation requires the standard domestic materials driven from locally infected samples. We tried manufacturing the plasma working standards of HBV, HCV, and HIV NAT using a mixed titer of viral loads. METHODS: Forty three HBV DNA positive plasmas, 25 HCV RNA positive plasmas, and 26 HIV RNA positive plasmas were evaluated according to viral load and genotype. Several plasma units, which had high-titer viral loads and the common viral genotypes in Korea, were selected as the source materials for each viral standard. To adjust the appropriate concentration based on the detectable range of variable viral reagents, the source plasma was diluted to several concentrations, divided into small vials, and analyzed for quantification. RESULTS: The 13 plasma working standards, which had variable viral loads for the mixed titer performance panel of HIV, HCV, and HBV NAT, were produced. CONCLUSION: These national standard materials were first produced in order to supply the mixed titer performance panel for the viral NAT reagent of the level IV transfusion related high-risk group in Korea.

Establishment of a Korean Hepatitis B Surface Antigen Low Titer Performance Panel for Performance Validation of Hepatitis B Surface Antigen Immunoassays / 대한수혈학회지

BACKGROUND: A range of well characterized materials are needed for validating the performance of hepatitis B surface antigen (HBsAg) immunoassays. These materials are purchased currently from overseas manufacturers at a high cost and with limited quantity. This study was conducted to establish an HBsAg low titer performance panel for use as a national standard for validation of HBsAg immunoassays in Korea. METHODS: 476 plasma units reactive on blood donor screening were collected HBsAg was tested using 3 enzyme immunoassays (EIA) and 1 chemiluminescence immunoassay (CIA). Units reactive on the CIA assay or on 2 or more immunoassays were subjected to hepatitis B virus (HBV) DNA quantification, HBV genotyping and subtyping. Units reactive on HBV DNA quantification were confirmed for HBsAg by neutralization. Candidates for the panel were subjected to a collaborative study performed at 7 laboratories using 7 immunoassays. RESULTS: Eleven HBsAg positive units were selected for the low titer performance panel based on HBsAg immunoassay, HBV DNA quantification, HBV genotyping and subtyping results. The range of the HBsAg concentration of the panel members was 0.05~1.28 IU/mL. Two HBsAg negative units were also included as negative controls. CONCLUSION: As a result of this study, a low titer performance panel [KFDA standard (08/028); HBsAg low titer performance panel (BTRL HBV/LP)] for validation of HBsAg immunoassays has been established as a Korean national standard. Use of this panel will improve performance assessment of HBsAg immunoassays. Because the performance of immunoassays cannot be assessed properly with a limited number of panels, continuous efforts are needed to develop a range of performance panels.

A Proposal for Developing a National Quality Assurance Program for Donor Bloodassays / 대한수혈학회지

BACKGROUND: Viral screening assays performed for blood donors are required to have high sensitivity because false negative results can lead to transfusion-transmitted infections. To minimize the number of false negative cases, a systematic quality assurance program is required to verify donor screening tests. METHODS: The current status of quality assurance (QA) for blood donor screening tests in Korea and other countries was reviewed. A quality assurance program using the national standards of the Korea Food and Drug Administration (KFDA) was done as a pilot study to evaluate both the need for such a program and the feasibility of such a program. RESULTS: Singapore had a national quality assurance programs for the anti-HIVdonor screening tests. In the United Kingdom, all laboratories use the NIBSC working standards as QA materials for the donors screening. Ninety-five % (84/80) of blood centers replied that they would participate in a national quality assessment program and 92% (84/77) of the blood centers also felt that an independent organization should be designated to operate the program. Quality control materials with a weak reactivity should be included in a quality assessment program for donor screening. CONCLUSION: We propose 2 models for a National Quality Assurance Program (NQAP). In the first model, an independent national reference laboratory (NRL) needs to be established that operates the national quality assurance program. The second model involves the integration of the national quality assurance program for donor screening into the External Quality Assurance Survey run by the Korean Association of Clinical Assurance for Clinical Laboratory (KAQACL) using the national standards.

A Case of Splenic Marginal Zone Lymphoma / 대한진단검사의학회지

Splenic marginal zone lymphoma (SMZL) is a rare B-cell neoplasm characterized by massive splenomegaly, moderate lymphocytosis, bone marrow intrasinusoidal involvement of lymphocytes and a relatively indolent course. We report a case of SMZL diagnosed by bone marrow studies using immunophenotyping and immunohistochemical stain, and confirmed by splenectomy. The patient was a 61-year old male, who showed mild lymphocytosis in peripheral blood and bone marrow aspirates. Immunophenotyping of bone marrow aspirates showed lymphocytes positive for CD19, CD20, CD22 (dim), CD23 (dim) and negativie for CD5 and CD10. The immunohistochemistry of bone marrow and spleen also showed lymphocytes positive for CD20 and negative at for cyclin D1. Now he is being treated for chronic obstructive pulmonary disease and will receive chemotherapy.

Confirmation of HCV Positivity for Indeterminate Donors in Anti-HCV Antibody Immunoblot Assay among Blood Donors / 대한수혈학회지

BACKGROUND: In Korean Red Cross, recombinant immunoblot assay (RIBA) has been used for the confirmatory test of HCV positive units since 1995. To certify the HCV infection in blood donors who showed the 'indeterminate result on the RIBA test, this study was performed. METHODS: Three enzyme immunoassay (EIA) kits (LG HCD 3.0, DONG-A HCV 3.0, and ORTHO HCV 3.0)and RNA detection method were employed to evaluate infection state of 135 samples of the 'indeterminate in the RIBA test. RESULTS: The 52.6% of the samples showed the same test results with three EIA kits. Fifteen samples (11.1%) were HCV RNA positive with RT-PCR-hybridization technique. Among 15 samples of HCV RNA positive, 13 (86.7%), 13 (86.7%), and 14(93.3%) of samples were positive in LG HCD 3.0, Ortho HCV 3.0 and Dong-A HCV 3.0 EIA, respectively. In the analysis of RIBA band reaction, HCV RNA positivity were correlated with core14, core518, and 897 antigen. However, among 64 samples which react with core antigen only, five samples (7.8%) were HCV RNA positive. CONCLUSION: Based on the results of the present study, it is recommend that the HCV RNA test be used as a method of confirmatory test in order to notify exact HCV positivity status to blood donor who showed indeterminate RIBA result.

Experience of Anti-HCV antibody immunoblot test in Korean Blood Donors / 대한수혈학회지

BACKGROUND: All donated bloods collected by the Korean Red Cross Blood Centers are tested for anti-HCV (Hepatitis C Virus) antibody by enzyme immunoassay (EIA) kits made in Korea. EIA test has sustaining problem of false positivity in spite of great progress in manufacturing kits. So, many healthy donors have been reported as being infected with HCV and excluded from next donation. METHODS: Among blood samples of 2,040,151 donors which were tested by two kinds of EIA kits (DONG-A HCV 3.0 and LG HCD 3.0) from 16 blood centers during 12 months, repeatably reactive samples, total 6,851 samples, were supplementally tested by LG HCD CONFIRM immunoblot test. RESULTS: Positive, indeterminate and negative rate in immunoblot tests were 39%, 9%, and 12% respectively among 6,851 repeatably reactive samples. Estimated true positive rate of anti-HCV antibody in Korean blood donors was 0.13%, showing geographical difference between 0.03% and 0.46%. Of EIA repeatably reactive samples, 28% showed greater than 5 signal to cutoff (S/C) ratio and most of them (94%) was revealed to be positive. CONCLUSION: True positive rate of EIA test results is so low that it would be necessary to increase the confidence of such results by immunoblot tests.

Genotyping of ABO and D Antigens / 대한수혈학회지

BACKGROUND: Genes for ABO and Rh phenotypes were recently identified. Although ABO genotyping don't find wide application in hospital transfusion services, it can play an important role in paternity and forensic investigation. In case of Rh system, however, DNA typing may find several practical applications such as prenatal determination of fetal Rho(D) genotype. METHODS: 64 blood samples for ABO genotyping were collected from blood donors (34 A, 30 B) and 18 samples for D genotyping (10 D+, 8 D-). To distinguish A, B and O alleles, we analyzed nucleotide positions 261 and 803 using polymerase chain reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP). PCR products containing nucleotide position 261 were restricted with KpnI and BstEII. Rh genotyping was done by two sets of primers, one set for both RhD and RhCcEe gene amplification, and the other set for RhD only. RESULTS: The frequencies of ABO genotypes found in Korean blood donors were as follows: in the phenotype A group, AO=79% and AA= 21%; and in the phenotype B group, BO=93% and BB=7%. Of 18 blood samples for D genotytping, 10 were typed as RhD positive and 8 as RhD negative, showing full agreement with serological typing. CONCLUSION: ABO and D genotyping can be used when RBCs suitable for serological phenotyping are not available. Futhermore, these will be useful as a supplemental test to solve the problem of blood group typing caused by weak ABO and Rh phenotype.

Irregular Antibody Positivity in Women Blood Donors with History of Parturition / 대한수혈학회지

BACKGROUND: Antibody screening for donated blood is not yet being performed in Korea. Positive rate of irregular antibodies in Korean patients or blood donors has been thought to be much lower than that of foreign contries. We studied to know the actual frequency of irregular antibodies in blood donors with history of parturition using gel card, which was recently introduced in the field of blood banking and considered to be easy to standardize and sensitive to detect irregular antibodies. METHODS: 706 samples were collected from four blood centers in Seoul for 4 months. Antibody screening and identification were done by two kinds of Gel Card (DiaMed-ID corp, DiaMed, Murten, Switzerland) such as Nacl/Enzyme and LISS/Coombs' card. Adsorption- elution test was done in samples of which we could know the antibody specificity. RESULTS: Irregular antibodies were identified in 24 cases among 706 samples, therefore the overall frequency was 3.4% (95% CI: 3.4% +/- 1.3%). Only 4 cases, however, showed positive reaction in both enzyme and Coombs' phase, therefore frequency of clinically significant antibodies was 0.57% (95% CI: 0.57% +/- 0.55%). The identified irregular antibodies were anti-Lea (8 cases), Anti-Rh (3 cases) and Anti-P1 (1 case). Adsorption-elution test showed positive reaction only in 3 cases with anti-Rh antibodies. CONCLUSION: Considering that blood donors with history of parturition comprize just little proportion of total donors in Korea and frequency of irregular antibody is relatively lower than that of foreign countries in same group (0.57% vs 3.8%), it can be concluded that antibody screening be not urgent problem in Korean blood donation program.

Detection Rate of Hepatitis B Virus DNA among Blood Donors showing HBsAg Positivity or Negativity and relatedness with other hepatitis B viral markers / 대한수혈학회지

BACKGROUND: Serologic assay for the detection of hepatitis B virus (HBV) surface antigen (HBsAg) have been used routinely in the screening of blood donors in Korea since 1973. However some investigators have reported the presence of HBV DNA in HBsAg negative blood. So this study is designed to determine the detection rate of HBV DNA according to various patterns of HBV markers in Korean blood donors. METHODS: The presence of HBV DNA in plasma from 469 donors was determined by polymerase chain reaction using commercial kit (Bioneer HBV Detection Kit, Bioneer Corp., Chungbuk, Korea). 289 donors showing all negative results by donor screening tests and 120 donors showing positive results only in HBsAg test and 60 donors showing abnormal result only in alanine aminotransferase (ALT) test (> or =65 IU/L) were included in this study. Other markers for HBV infection such as anti-HBsAb, anti-HBcAb, HBeAg were also tested. RESULTS: 65 (54%) of 120 donors with positive for HBsAg and 5 (1.7%) of 289 donors without abnormal results in screening tests and 3 (5.0%) of 60 donors with elevated ALT were found to have HBV DNA in their plasma. Among 54 cases showing HBsAg-positive/HBeAg-positive, 52 cases (96%) were found to have HBV DNA. HBV markers in 5 cases showing HBsAg-negative/HBV DNA-positive were as follows: 2 (1.3%) among 159 cases showing anti-HBs-positive/anti-HBc-negative and 1 (20%) among 5 cases showing anti-HBs-negative/anti-HBc-positive and 1 (1.8%) among 55 cases showing anti-HBs-positive/anti-HBc-positive and 1 (1.5%) among 65 cases showing no viral markers. 3 cases with HBV DNA among elevated ALT groups were positive only in anti-HBs test. CONCLUSION: This study indicates that serological markers for HBV infection are insufficient to guarantee the safety of donated blood. To improve the safety, it may be suggested that (1) donors with history of viral hepatitis or with history of HBsAg positivity shoud be indefinitely deferred from donation, (2) blood collected from donors who have showed HBsAg positive result at previous donation shoud be discarded, (3) HBsAg-negative /anti-HBs-negative /anti-HBc-positive blood should be discarded, (4) ALT screening should be continuously done because it could screen out HBsAg-negative /HBV DNA-positive blood irrespective of anti-HBc result, (5) HBV detction kit that can also detect HBV mutant shoud be developed, (6) governmental support for HBV vaccination program shoud be done especially for recruits.