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Original Article
BCR::ABL1 정량 검사의 국내 현황 조사(2022)
Current Status of BCR::ABL1 Quantitative PCR Analysis in Korea (2022)
인제대학교 부산백병원 진단검사의학과1, 양산부산대학교병원 진단검사의학과2, 삼성서울병원 진단검사의학과3, 연세대학교 세브란스병원 진단검사의학과4, 서울아산병원 진단검사의학과5
Department of Laboratory Medicine1, Inje University Busan Paik Hospital, Busan; Department of Laboratory Medicine2, Pusan National University Yangsan Hospital, Yangsan; Department of Laboratory Medicine and Genetics3, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul; Department of Laboratory Medicine4, Yonsei University College of Medicine, Seoul; Department of Laboratory Medicine5, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
Correspondence to:This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Lab Med Online 2023; 13(4): 356-363
Published October 1, 2023 https://doi.org/10.47429/lmo.2023.13.4.356
Copyright © The Korean Society for Laboratory Medicine.
방법: 대학병원, 3차 의료기관 및 전문 수탁검사기관 분자유전검사 책임전문의에게 전자 메일을 통한 설문을 시행하였다. 설문 문항은 BCR::ABL1 정량 PCR 검사방법(8문항), 결과보고(6문항), 최소검출한계 및 최소정량한계 설정(7문항) 및 내·외부 정도관리 방법(2문항)에 대한 질문을 포함하였다.
결과: 15개 기관의 회신을 받았다. 설문조사 결과 모든 기관에서 상품화된 키트를 이용한 실시간 PCR법으로 시행하고 있었다. 모든 기관에서 ABL1 유전자를 참고유전자로 이용하고 깊은 분자유전학적 반응 판정에 요구되는 10,000 이상의 ABL1 유전자 복제수 범위를 가지고 있었다. 대부분의 기관이 깊은 분자유전학적 반응 판정에 적합한 검사실 자체 검출 한계를 설정하고 있었다. 검사기관별 결과보고방식 및 내부 및 외부정도관리 방법이 다양하였다.
결론: 설문조사를 통해 국내 검사기관들이 표준화된 검사방법을 시행하고 있는 것을 확인하였으나 결과의 정확성 및 민감도를 높일 수 있는 검출한계 검증 및 적절한 내부 및 외부정도관리 시행이 필요하겠다. 검사기관 간 검사결과보고의 일치도를 높이기 위한 표준화 및 가이드라인 정립이 필요하다.
Methods: The survey was provided by e-mail to the laboratory experts in molecular genetic testing at university hospitals, tertiary medical institutions, and specialized testing institutions. Questionnaires covered each institution’s BCR::ABL1 RQ-PCR test (eight questions), reporting format (six questions), verification of the lower limit of detection and quantitation (seven questions), and internal and external quality control (two questions).
Results: Responses were received from 15 institutions. Every institution utilized RQ-PCR kits that were commercially available. All of them used the ABL1 gene as a reference gene and had a copy number range of at least 10,000, which is necessary for determining a deep molecular response (DMR). The majority of laboratories established their own laboratory detection limits suitable for determining a DMR. The reporting of results and internal and external quality control procedures differed by institution.
Conclusions: The survey confirmed that testing institutions are implementing standardized RQ-PCR methods, but it is necessary to perform detection limit verification and internal and external quality control to improve the precision and sensitivity of the results. Standardization and guidelines are required to enhance the consistency of testing result reports across institutions.
Keywords
본 연구에서는 국내
설문대상자는 해당 연도 기준 대한진단검사의학회 회원 중 대학병원, 3차 의료기관 및 전문 수탁검사기관 분자유전검사 책임 전문의로 2021년 7월 5일에서 8월 30일까지 65기관에 이메일을 발송하여 온라인 설문조사를 실시하였다.
설문 항목은 기관의
설문조사에 응한 15기관의 답변을 분석하였다. 설문에 회신한 기관은 대학병원 및 3차 의료기관 11기관, 전문 수탁검사기관 4기관이었으며, 자세한 설문 내용은 Table 1에 정리하였다.
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Table 1 Response to the questionnaire about the
BCR::ABL1 quantitative PCR in KoreaQuestionnaires & Answer options Response, % (institution) 1 Select the institution-recommended BCR::ABL1 quantitative PCR sample storage period (the time between drawing blood and receiving the sample).1) Less than 4 h 53.3 (8/15) 2) Less than 24 h 33.3 (5/15) 3) Less than 48 h 13.3 (2/15) 4) Others 2 What methods are used for RNA extraction? 2-1 Please describe the product name of RNA extraction kit 1) QIAamp RNA Blood Mini Kit 73.3 (11/15) 2) RNAqueous™ Total RNA isolation 13.3 (2/15) 3) High Pure RNA isolation 6.7 (1/15) 4) Others (manual extraction using trizol) 6.7 (1/15) 2-2 If an automated RNA extraction system is used, describe the system name 1) QIAcube 20.0 (3/15) 3 How can the quality of isolated nucleic acids be evaluated? 1) Spectrophotometry (e.g., NanoDrop) 93.3 (14/15) 2) Microfluidic analysis (e.g., Bioanalyzer) 0.0 (0/15) 3) Capillary gel electrophoresis 0.0 (0/15) 4) Fluorescent dye detection (e.g., RiboGreen) 0.0 (0/15) 5) Others (4200 TapeStation) 6.7 (1/15) 4 How do you determine the concentration of BCR::ABL1 ?1) Real-time PCR 100.0 (15/15) 2) Digital droplet PCR 0.0 (0/15) 3) Others 0.0 (0/15) 5 What reference gene is used for real-time PCR? 1) ABL1 gene 100.0 (15/15) 2) GUSB gene 0.0 (0/15) 3) BCR gene 0.0 (0/15) 4) Others 0.0 (0/15) 6 How many average copies of the reference gene are detected by quantitative BCR::ABL1 PCR?1) 100,000 or more 40.0 (6/15) 2) 50,000–100,000 20.0 (3/15) 3) 32,000–50,000 20.0 (3/15) 4) 10,000–32,000 0.0 (0/15) 5) Less than 10,000 0.0 (0/15) 6) Others (10,000–50,000/10,000–100,000/32,000–100,000) 20.0 (3/15) 7 Please provide the name of the product used for BCR::ABL1 quantitative PCR.1) Ipsogen BCR::ABL1 Mbcr Is-MMR kit100.0 (15/15) 8 To report quantitative results, how many tests are performed and reported for each sample? 1) One test 86.7 (13/15) 2) Two tests 13.3 (2/15) 3) Three tests 0.0 (0/15) 4) Others 0.0 (0/15) 9 Do you describe the copy number of the reference gene in the BCR::ABL1 quantitative PCR report?1) Yes 60.0 (9/15) 2) No 40.0 (6/15) 10 Do you include the copy number of the BCR::ABL1 fusion transcript in the quantitative PCR report?1) Yes 53.3 (8/15) 2) No 46.7 (7/15) 11 Do you describe the normalized copy number in the BCR::ABL1 quantitative PCR report (copy number of theBCR::ABL1 fusion transcript or copy number of the reference gene)?1) Yes 66.7 (10/15) 2) No 33.3 (5/15) 12 Do you include the international scale (IS) in the quantitative PCR report for BCR::ABL1 ?1) Yes 100.0 (15/15) 2) No 0.0 (0/15) 13 What format is used to describe the IS in the BCR::ABL1 quantitative PCR report?1) Report only %IS 73.3 (11/15) 2) Report %IS and molecular response 26.7 (4/15) 3) Report only molecular response 0.0 (0/15) 4) Others 0.0 (0/15) 14 How do you determine the IS? 1) Calculate the results of each test using the IS calibrator (e.g., Normalized copy number IS-calibrator value / IS-normalized copy number of the IS calibrator) 100.0 (15/15) 2) Calculate using a fixed conversion factor (e.g., number of normalized copies multiplied by conversion factor) 0.0 (0/15) 3) Others 0.0 (0/15) 15 Are the lower limits of detection (LOD) and quantification (LOQ) established for BCR::ABL1 quantitative PCR?1) Both LOD and LOQ are set 33.3 (5/15) 2) Only LOD is set 40.0 (6/15) 3) Only LOQ is set 13.3 (2/15) 4) Neither set 13.3 (2/15) 16 The LOD and LOQ of BCR::ABL1 quantitative PCR were established for which of the following values?1) NCN 53.8 (7/13) 2) %IS 46.2 (6/13) 3) Others 0.0 (0/13) 17 Please describe the LOD for BCR::ABL1 quantitative PCR established by your institution.1) 0.0069 45.5 (5/11) 2) 0.0022 18.2 (2/11) 3) 0.0001 18.2 (2/11) 4) 0.01 9.1 (1/11) 5) Three copies 9.1 (1/11) 18 Please describe the LOQ for BCR::ABL1 quantitative PCR established by your institution.1) 0.0069 42.9 (3/7) 2) 0.0032 14.3 (1/7) 3) 0.0017 14.3 (1/7) 4) 0.01 14.3 (1/7) 19 If the BCR::ABL1 quantitative PCR result is below the LOD or a value is detected, do you perform a retest?1) No, we do not test again 83.3 (10/12) 2) Yes, the retest is performed once 8.3 (1/12) 3) Yes, the retest is performed twice 0.0 (0/12) 4) Others (the necessity of a retest is determined by the first or second visit of the patient, previous test results, and related test results) 8.3 (1/12) 20 How do you report the BCR::ABL1 quantitative PCR result if they are below the LOD but a value is detected? (If the LOD is not specified and only the LOQ is specified, the value is considered to be smaller than that of the LOQ)1) Report the value as is with a comment indicating that it is less than the LOD or LOQ. 38.5 (5/13) 2) Report a positive result below the LOD or LOQ 23.1 (3/13) 3) Indicate that the result is negative 23.1 (3/13) 4) Provide the value as is 7.7 (1/13) 5) Others (report the value is formed as %IS with a comment indicating that it is less than the analytical measurement range) 7.7 (1/13) 21 How do you report the BCR::ABL1 quantitative PCR result if they are below the LOQ and above the LOD?1) Report the positive result below the LOQ 50.0 (2/4) 2) Report the value as is with a comment indicating that it is less than the LOD 25.0 (1/4) 3) Provide the value as is 25.0 (1/4) 22 Are you performing internal quality control procedures for BCR::ABL1 quantitative PCR ? If yes, please describe in detail how to implement1) Yes (e.g., use positive and negative samples for each test) 100.0 (15/15) Use positive, weakly positive, and negative samples for each test 40.0 (6/15) Use positive and negative samples for each test 26.7 (4/15) Use positive samples of two levels for each test 6.7 (1/15) Use a positive sample for each test 6.7 (1/15) Use a laboratory-created low-positive sample (0.1–0.001%IS) for each test 6.7 (1/15) Use the supplied control for each test 6.7 (1/15) Use the IS calibrator for each test 6.7 (1/15) 2) No 0.0 (0/15) 23 Are you performing external quality control procedures for BCR::ABL1 quantitative PCR?1) Participate in the external quality control program (please describe all participating programs) 73.3 (11/15) The College of American Pathologists survey 72.7 (8/11) Molecular program of the Korean Association of External Quality Assessment Service 45.5 (5/11) Korean institute of genetic testing evaluation 27.3 (3/11) 2) Comparison with other laboratories 27.3 (3/15) 3) Others (interobserver comparison) 6.7 (1/15)
1. BCR::ABL1 정량 PCR 검사방법
2. BCR::ABL1 정량 PCR 결과보고
결과 보고서에 ABL1 참고유전자의 유전자 복제수(copy number)를 기술하는 기관은 60% (9/15)였다.
3. BCR::ABL1 정량 PCR 검사의 최소검출한계 및 최소정량한계 설정
검사실 자체 최소검출한계(lower limit of detection, LOD) 및 최소정량한계(lower limit of quantification, LOQ) 설정에 대한 설문조사에서 LOD만 설정된 기관 40% (6/15), LOD 및 LOQ가 모두 설정되어 있는 기관 33.3% (5/15), LOQ만 설정된 기관 13.3% (2/15)순이었으며 LOQ 및 LOD가 모두 설정되지 않은 기관도 2기관 있었다. LOD 및 LOQ 값을 NCN으로 설정한 기관 53.8% (7/13), %IS로 설정한 기관 46.2% (6/13)이었으며 기관별 LOD는 0.0069인 기관이 45.5% (5/11)로 가장 많았으나 0.0022, 0.0001, 0.01, 3 copies 등 다양하였다. 기관별 LOQ는 LOD와 동일한 0.0069인 기관이 42.9% (3/7)이었으며 이외에 0.0032, 0.0017, 0.01로 답하였다. 정량 PCR 결과가 LOD 미만이나 값이 나올 경우 재검하지 않는 기관이 83.3% (10/12)로 다수를 차지하였으며 1기관은 1회 재검, 1기관은 환자의 초진 혹은 재진 여부, 이전 검사 결과 및 연관 검사 결과 리뷰 등을 통해 재검 여부를 결정한다고 답하였다. 정량 PCR 결과가 LOD 혹은 LOQ 미만이나 값이 나올 경우 값을 그대로 보고하고 LOD 혹은 LOQ 미만이라는 코멘트를 추가하는 기관이 38.5% (5/13)로 가장 많았으며, 양성이나 LOD 혹은 LOQ 미만으로 보고하는 기관 23.1% (3/13), 음성으로 보고하는 기관 23.1% (3/13)이었으며 값을 그대로 보고하거나 %IS로 보고하고 분석범위 미만으로 보고하는 기관도 각각 1기관이 있었다. 정량 PCR 결과가 LOQ 미만, LOD 이상의 값일 경우 보고방법에 대한 설문에 4기관만 답하였는데 양성이나 LOQ 미만으로 보고하는 기관이 50% (2/4), 값을 그대로 보고하고 LOQ 미만이라는 코멘트를 추가하거나 값을 그대로 보고하는 기관이 각각 1기관 있었다.
4. BCR::ABL1 정량 PCR 검사의 내/외부 정도관리
설문에 답한 모든 기관이 내부정도관리를 시행하고 있었으며 주관식으로 설문한 정도관리방법은 다양하였다. 매 검사 시 양성, 약양성 및 음성 검체를 이용하여 내부정도관리를 시행하는 기관이 40% (6/15)로 가장 많았으며 양성 및 음성 검체를 이용하는 기관 26.7% (4/15)였다. 1개 혹은 2개의 양성 물질을 이용하거나 검사실에서 제작한 약양성 물질(IS 0.1–.0.001%), 키트 내 대조군 혹은 IS 보정물질을 이용하여 내부정도관리를 시행하는 기관도 1기관씩 있었다.
모든 기관이 외부정도관리를 시행하고 있다고 답하였으며 외부정도관리 프로그램에 참여하는 기관이 73.3% (11/15)였다.
정량 분석을 위한
Digital PCR 검사가 실시간 정량 PCR보다 낮은 값을 좀 더 정확하게 검출하여 MR5 이상의 MR 판정에 더 유용하게 사용할 수 있다[15, 16]. 그러나 설문조사 시행 시 digital PCR 방법을 사용하고 있다고 답한 기관은 없었다.
%IS는 참고 유전자 및 분석 장비에 따라 값의 차이를 보여 보정 및 CF가 필요하며 검사기관에서는 측정된
본 설문조사 시행 시 질문의 응답률을 높이기 위해 예시를 제시한 것이 도움이 되었으나 일부 주관식 답변을 요구하는 설문의 경우 객관식 문항에 비해 응답률이 떨어지는 한계가 있었다. 또한 각 기관에서 사용중인 정량 PCR 검사 장비 및 spectrophotometer 장비의 종류, cDNA 합성에 필요한 RNA 농도의 최솟값 등에 대한 검사실 간 일치도를 확인할 수 있는 설문을 일부 시행하지 못한 한계점이 있다.
이번 설문조사를 통해 국내 검사기관의
본 연구는 대한진단유전학회 분자혈액분과 지원으로 이루어졌습니다.
저자들은 본 연구와 관련하여 어떠한 이해관계도 없음을 밝힙니다.
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