Construction and Application of Internal Control for Laboratory-Developed HTLV PCR / 대한수혈학회지

BACKGROUND: For donor samples showing reactive results in a human T-cell lymphotropic virus (HTLV) antibody test along with indeterminate results in Western blot assay, HTLV nucleic acid amplification test using laboratory-developed polymerase chain reaction (PCR) was performed. It is necessary to construct an adequate internal control (IC) to evaluate the accuracy of the results since we did not use an IC in the laboratory-developed PCR. METHODS: As a competitive IC, plasmid DNA containing the primer recognition sequence for amplification of the HTLV pX region was constructed. We determined the adequate concentration of the IC, which was added to the samples to evaluate the accuracy of the test results. RESULTS: When the plasmid DNA was added to the HTLV-positive samples, the amplified product of IC (400 bp) was detected with the HTLV gene (230 bp). The adequate concentration of plasmid DNA added as an IC was 1 pg. CONCLUSION: The construction of plasmid DNA as a competitive IC is an efficient method to evaluate accuracy of the test results. However, the production process for the competitive IC must be further developed. Therefore, it is necessary to compare with the performance of a non-competitive IC.

Reduction Plan of ‘Undeterminable’ in the Unexpected Antibody Screening by the Microcolumn Agglutination Automated Instrument / 대한수혈학회지

BACKGROUND: Among the results of an unexpected antibody screening test using IH-1000, ‘undeterminable’ results can be obtained. Repeated tests not only use reagents and consumables but also cause a turnaround time delay. Therefore, it is important to reduce the ‘undeterminable’ results and to determine the effects. METHODS: From January to early June, 2016, 2,872 cases/259,455 tests (1.11%) of ‘undeterminable’ were detected in the screening test. The factors considered to affect the ‘undeterminable’ were classified into four categories: ① reagent, ② consumables, ③ inspection environment & specimen, and ④ enhancing the equipment management. For data comparison, a chi-square test was conducted (95% confidence interval, 0.05 significant level). RESULTS: The incidence of ‘undeterminable’ cases decreased from 1.11% before management to 0.66% (P < 0.001) after Pool Cells management. The consumption of ‘LISS/Coombs Card’ decreased from 1.07% before management to 0.51% (P < 0.001) after management. By maintaining a clean inspection environment and strengthening sample management, the rate decreased from 1.11% before management to 0.66% (P < 0.001) after management. On the other hand, there was no difference in the incidence of ‘undeterminable’ between before and after IH-1000 management reinforcement. CONCLUSION: Among the factors predicted to affect the decrease in the incidence of ‘undeterminable’, the management of Pool Cells and keeping the inspection environment clean as well as improving sample management contributed the most to the reduced ‘undeterminable’. Improvements in the management of consumables, and removing dust from the inside of the equipment, had a positive impact. A continuous quality improvement theme has been adopted and it is helpful for managing and improving the predicted factors.

Proposal of Evaluation Method for Leukoreduction Blood Filter and Evaluation of Domestic Filter / 대한수혈학회지

BACKGROUND: A leukoreduction filter was recently developed in Korea to reduce various kinds of adverse transfusion reactions. The objective of this study was to propose a domestic evaluation system for leukoreduction filters and to apply this evaluation system to assess the newly developed leukoreduction filter. METHODS: We prepared packed red blood cells from 60 units of whole blood (400 mL) collected from 60 normal individuals and evaluated the efficacy of the newly developed filter (FINECELL, KOLON INDUSTRIES, Gumi, Korea) and a control filter (RCM1, Haemonetics, MA, USA). To verify the evaluation system, we assessed the filtration time, residual leukocyte count, RBC recovery, RBC hemolysis, hemoglobin concentration, and hematocrit using a control filter RCM1 and compared the results with those of an evaluation performed by the American Red Cross (ARC) in 2013. We then evaluated the efficacy of the test filter FINECELL using the methods established in this study and compared the results with those of the control filter RCM1. RESULTS: The results of the current study were similar to those of the ARC with the control filters. The test filters developed in Korea were not inferior to commonly used control filters regarding residual leukocyte count, RBC recovery, and RBC hemolysis at 35 days after filtration. All of the results in the evaluation satisfied the international standards. CONCLUSION: These results of this study showed that the efficacy of the newly developed domestic leukoreduction filter were satisfactory and will contribute to improvement of quality of blood components in Korea.

Estimation of the Outbreaks of Transfusion-Transmissible Emerging Infectious Diseases in Korean Blood Donors by Public Data / 대한수혈학회지

BACKGROUND: Transfusion transmissible emerging infectious diseases (EIDs) is a potential risk to the safety of blood transfusions due to the lack of donor screening assays. To prevent the spread of EIDs through blood transfusions, we attempted to predict the possibility of blood donations from people with EIDs using a public database. METHODS: We used the Disease Web Statistics System of the Korean Centers for Disease Control and Prevention and Korean Statistical Information Service. We estimated the possibility of blood donations from people with EIDs using the public database combined with the database made available by the Blood Information Management System of the Korean Red Cross. RESULTS: Among the transfusion transmissible EIDs, Babesiosis, Leishmaniasis, West Nile fever, Chikungunya, and Dengue fever were reported in Korea. All of them were cases imported from abroad. Although the number of reported cases of Babesiosis, Leishmaniasis, West Nile fever, and Chikungunya were less than 10 per year until 2016, the reported cases of Dengue fever gradually increased from 2001, and there were 318 cases of Dengue fever in 2016. CONCLUSION: The possibility of blood donation from people with transfusion-transmissible EIDs was low because all reported transfusion-transmissible EIDs in Korea were from foreigners and blood donation from Koreans who returned from abroad was restricted for a period of a month. Nonetheless, preventive strategy for donation from people is necessary given the recent increase in Dengue fever.

Evaluation of Quality of Washed Platelets Stored in Platelet Additive Solutions / 대한수혈학회지

BACKGROUND: Because of a lack of substances for platelet (PLT) metabolism and preservation, normal saline (NS) washed PLTs can only be stored for short lengths of time. However, the use of platelet additive solutions (PAS) could help solve this problem. In this study, the in vitro quality of NS washed platelets (wPLTs) stored in two types of PAS were compared with those of wPLTs stored in NS. METHODS: Five units of NS washed apheresis platelets were pooled aseptically and separated into five aliquots for storage in NS only as well as T-PAS+ (Terumo BCT, Lakewood, CO, USA) and CompoSol PS (Fenwal, Lake Zurich, IL, USA) with or without 15 mM glucose. The parameters of wPLTs quality were assessed up to 48 hrs after washing and the whole experiment was repeated 10 times independently. RESULTS: wPLTs in two kinds of PAS had better quality than wPLTs in NS, and wPLTs in T-PAS+ showed better quality than those in CompoSol PS. PAS-stored wPLTs with added glucose maintained stable CD62P and Annexin V expression during storage, but exhibited increased lactate accumulation. Evaluation of in vitro quality revealed that all wPLTs had a rating of 4 immediately after washing. However, only T-PAS+-stored wPLTs with glucose maintained a rating of 4 up to 48 hrs of post-washing. CONCLUSION: Using PAS storage for wPLTs may be beneficial compared to NS. The results presented herein suggest that T-PAS+ containing glucose has the potential to extend storage time by up to 48-hours.

Consideration of Discontinuance of Malaria Antibody Test for Blood Donor Screening / 대한수혈학회지

BACKGROUND: Since 2001, the Korean Red Cross has performed malaria antibody test for blood donors in malaria-risk areas to prevent transfusion-transmitted malaria. However, due to insufficient sensitivity and specificity the malaria antibody assay is not considered an efficient screening method. Therefore, we have considered discontinuing malaria antibody testing for blood donors. METHODS: We analyzed the results of malaria antibody test from 2001 to 2014 utilizing data from the Blood Information Management System of the Korean Red Cross. RESULTS: Among 16,650,812 donations tested from 2001 to 2014, 50,143 donations (0.30%) showed positive results. However, there was no truly infected case at the time of donation. The positive rate among blood donations was between 34 and 39 per 10,000 in 2001, but between 9 and 10 per 10,000 in 2014. There was no interregional disparity in the positive rate of blood donations. CONCLUSION: Korea is in a malaria elimination phase and malaria antibody testing in limited areas is not effective, therefore we propose discontinuing the malaria antibody test.

Analysis of the Reasons for Donor Deferral after Introduction of the Electronic Donor Health Questionnaire Program / 대한수혈학회지

BACKGROUND: Electronic Donor Health Questionnaire (e-DHQ) could prevent omissions in filling in the questionnaire. Compared with the paper Donor Health Questionnaire (p-DHQ), it can expect honest answers from donors to the questions relating to high risk behaviors. The aim of this study is to evaluate the effect of e-DHQ on the answers from donors by analyzing variations of the reasons for deferral after the introduction of e-DHQ to the Korean Red Cross (KRC). METHODS: The reasons for deferral determined by p-DHQ in 2008~2011 and by e-DHQ in 2011~2014, which have been accumulated in the database of the Blood Information Management System in KRC, were analyzed and compared. RESULTS: The results showed that the deferral rates for the general health status and medication taken were 0.47% (P=0.0100) and 0.16% (P=0.0103) higher in e-DHQ than in p-DHQ. In particular, for questions including hunger, lack of sleep, fatigue, endoscopy, dental treatment, surgery, tattoo, and acupuncture, the proportion of answers was higher in e-DHQ than in p-DHQ. CONCLUSION: The deferral rates for general health status and medication taken increased after the implementation of e-DHQ. The rate of deferral by some details of general health status, medical treatment for recent one month, and history for recent one year also increased because e-DHQ induced donors to give straightforward answers. e-DHQ is expected to contribute to the strengthening of health protection of blood donors and recipients. However the questions relating to high risk behaviors should be reformed so that honest answers can be induced from donors.

Emerging Infectious Disease and Safety of Blood Components / 대한수혈학회지

Recently, the emergency of Middle East Respiratory Syndrome in Korea raised public concern regarding emerging infectious disease (EID) and affected the strategy for blood safety. Although some cases of EID such as West Nile virus in the United States and hepatitis E virus in Japan emerged in a restricted area, the emergence can rapidly affect the situation in other countries in a globalized society with advancements in transportation and international exchange. Because the risk of transfusion-mediated infection may occur by the agent of EID which can be transmitted through blood, the strategy for safety must be considered for agents that were not tested in the stage of blood donor screening. We reviewed the characteristics of transfusion-transmissible EIDs raising concerns worldwide and the strategies which had been adopted.

Determining the Cause of Discrepant Results in HCV NAT Comparative Tests / 대한수혈학회지

BACKGROUND: The Korean Red Cross blood laboratory centers have been performing comparative tests for NAT reactive specimens since February 2005. However, five discrepant specimens were found in HCV-diluted specimens between 2007 and 2008 and the reasons for this has been investigated. METHODS: For the five discrepant specimens, the HCV RNA concentration was measured in 5 tubes for each speciment. Subsequently, in order to compare the sensitivity of the low titer specimens measured by RT-PCR and TMA, comparative tests of diluted samples were examined six times per sample. Finally, the genotype was tested in order to determine the characteristics of the discrepant samples. RESULTS: Result of the quantitative tests for HCV RNA demonstrated that aliquots of the plasma bag were homogenous in term of viral load quantitation. As a result of the comparative test, all samples were found to contain over 1.0x10(1) IU/mL as detected by the two analytical systems. In contrast, those less than 1.0x10(1) IU/mL were not entirely detected by the two systems. CONCLUSION: It was impossible to completely detect using the two NAT system and the detection rates for both systems were equivalent for the samples examined. In particular, with respect to HCV, it may be undetectable on the NAT test because viral load decreases rapidly before and after sero-conversion. This result indicates that anti-HCV and NAT should be performed together as an HCV screening test prior to blood donation.

Characteristics and Follow-up Results of the Window Period Blood Donors for Hepatitis C Virus / 대한수혈학회지

BACKGROUND: To improve the safety of the blood supply in Korea, nucleic acid amplification testing (NAT) for HCV and HIV-1 as a blood screening test has been used by the Korean Red Cross since February 1st, 2005. Until October 31st 2006, a total of 4,037,618 donor samples had been tested and 436 cases showed reactivity for HCV RNA. Only 10 of these cases showed serologically negative antibody results. METHODS: We investigated the characteristics and the follow-up results of the ten donors who showed HCV RNA reactivity and serologically negative results. RESULTS: All the ten donors were identified to be "window period" blood donors with confirmation of their seroconversion by follow-up testing. The average RNA titer of the donors was 9.21x106 IU/mL. Five donors showed high levels of ALT. One donor showed the reversed pattern on the test results for antibody and RNA. CONCLUSION: By detecting five cases of HCV "window period" infection, NAT has increased the safety of the Korean blood supply. But it is considered that NAT can not be a substitute serological test and an adequate follow-up strategy is required for verification of the accurate seroconversion time.

The Analysis of the Effect of Mixing of Anticoagulant on Nucleic Acid Amplification Tests for Donated Blood / 임상검사와정도관리

BACKGROUND: In the process of implementing the nucleic acid amplification tests (NAT) for the blood screening, it was needed to change plain tube to EDTA tube for the sampling. Because the sample is taken from the CPDA-1 anticoagulated whole blood, the EDTA of tube could be mixed with the CPDA-1. So, we studied the effect of the mixing of two anticoagulants on the NAT. METHODS: Using HIV-1 and HCV RNA standards, we made the qualitative and quantitative test panels for the EDTA anticoagulant and the EDTA/CPDA-1 anticoagulant containing blood. The reverse transcription-polymerase chain reaction of Roche and transcription-mediated amplification of Chiron were used for the RNA qualitative and quantitative test. RESULTS: On the qualitative HIV-1 and HCV RNA tests for the EDTA, CPDA-1 alone and the CPDA-1/EDTA mixture, false negative and false positive reactions were not observed. On quantitative test, viral loads were not different statistically. CONCLUSIONS: Since there were no statistically significant differences between CPDA-1 alone and EDTA/CPDA-1 mixture in both qualitative and quantitative tests for HIV-1 and HCV RNA, it was concluded that mixing of anticoagulants, EDTA and CPDA-1, would not cause an significant effect on the NAT for the donated blood.

A Study on the Prevalence of HBsAg and Anti-HCV in Patients with Hepatocellular Carcinoma: Comparative Study with Healthy Blood Donors / 대한임상병리학회지

BACKGROUND: The aim of this study was to evaluate the positive rates of hepatitis B surface antigen (HBsAg) and antibody to hepatitis C virus (anti-HCV) in patients with hepatocellular carcinoma (HCC), and to estimate the risk of developing HCC in association with HBsAg or anti- HCV positivity. METHODS: HBsAg and anti-HCV (anti-c22-3 and anti-c200) were tested by enzyme immunoassay in 892 patients with HCC from 1991 to 1994. Data regarding the prevalence of these hepatitis markers in 4,269 healthy blood donors were obtained from the Central Blood Center of the Korea Red Cross and used for case-control study. RESULTS: The positive rate of HBsAg was 72.3% (645/892) in patients with HCC and 2.7% (117/ 4,269) in blood donors, while that of anti-HCV was 7.6% (68/892) in patients with HCC and 0.3% (11/4,269) in blood donors. Six hundreds and thirty-six among 892 patients with HCC (71.3%) were only positive for HBsAg, 59 (6.6%) were only anti-HCV positive, and 9 (1.0%) were positive for both HBsAg and anti-HCV. The odds ratio (with 95% confidence interval: CI) comparing patients with HCC to healthy blood donors were 17.8 (CI: 4.7-61.5) for HBsAg positive, 11.9 (CI: 2.8-52.2) for anti-HCV positive, and 208.9 (CI: 18.6-2,345.7) for both HBsAg and anti-HCV positive. The risk estimates for both HBsAg and anti-HCV positve were not different from those for HBsAg positive or anti-HCV positive because the confidence interval of patients with both HBsAg and anti-HCV positive overlapped that of patients with HBsAg positive, or that of patients with anti-HCV positive. CONCLUSIONS: Our results suggested that HBsAg was more important risk factor of HCC than anti-HCV and there was no interaction between HBsAg and anti-HCV in the development of HCC.

Deletion of Rb1 gene in late osteosarcoma from survivor of unilateral retinoblastoma: a case report

It has been well known that the survivors of retinoblastoma are prone to have osteosarcoma. But the secondary tumor usually occurs in bilateral, hereditary type of retinoblastoma. We report one case of osteosarcoma in a survivor of unilateral, sporadic retinoblastoma. A fourteen year old male presented with a painfully swollen distal forearm of 2 month duration. He had enucleated his left eye 10 years ago due to retinoblastoma with no other adjuvant therapy. We managed him with our conventional protocol and identified deletion of Rb gene from his pathological specimen by using the PCR-RFLP method. This result is unusual for unilateral nonhereditable retinoblastoma and may suggest gene level change even in sporadic cases. And Rb gene study may be helpful for unilateral, sporadic retinoblastoma patient in detecting the possibility of late osteosarcoma.