A Questionnaire Survey of HLA Crossmatch Tests in Korea (2015)

Shinae Yu; Eun-Suk Kang; Myoung Hee Park

doi:10.3343/lmo.2017.7.3.147

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Yu, Kang, and Park: A Questionnaire Survey of HLA Crossmatch Tests in Korea (2015)

Original Article

Lab Med Online 2017;7(3):147-156.

Published online: 17 July 2017

DOI: https://doi.org/10.3343/lmo.2017.7.3.147

A Questionnaire Survey of HLA Crossmatch Tests in Korea (2015)

Shinae Yu, M.D.^1,^∗, Eun-Suk Kang, M.D.^1,, Myoung Hee Park, M.D.^2,

¹Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

²Korea Organ Donation Agency Laboratory, Seoul, Korea

^∗ Currently affiliated with the Department of Laboratory Medicine, Haeundae Paik Hospital, Inje University College of Medicine, Busan, Korea.

Corresponding author: Eun-Suk Kang Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro Gangnam-gu, Seoul 06351, Korea Tel: +82-2-3410-2703, Fax: +82-2-3410-2719, E-mail: eskang@skku.edu

Co-corresponding author: Myoung Hee Park Korea Organ Donation Agency Laboratory, 6F, 12 Nonhyeon-ro 132-gil, Gangnam-gu, Seoul 06052, Korea Tel: +82-2-548-5639, Fax: +82-2-548-5631, E-mail: parkmhee@snu.ac.kr

Received 3 January 201717 April 2017 Accepted 19 April 2017

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.

Abstract

Background

We carried out a questionnaire survey for laboratories performing human leukocyte antigen-crossmatch (HLA-XM) to provide a basis for laboratory standardization of HLA-XM tests in Korea.

Methods

The questionnaires were distributed to 51 HLA laboratories participating in the HLA-XM part of the HLA proficiency survey program organized by the Korean Society for Laboratory Medicine and replies from 50 laboratories were analyzed. The questionnaires included following items: 1) HLA-XM methods performed and annual number of tests, 2) types of the specimen and lymphocyte separation methods, 3) test procedures and reagents for complement-dependent cytotoxicity crossmatch (CDC-XM) and flow cytometry crossmatch (FCXM).

Results

The number of laboratories performing anti-human globulin (AHG) CDC-XM (47/49, 96%) and FCXM (30/50, 60%) was considerably increased compared to the 2005 survey (AHG CDC-XM, 35/43, 81%; FCXM, 7/44, 16%). As for the annual number of XM tests, more than 50% of the laboratories were low volume laboratories performing ≤50 tests, and only 10% of the laboratories were performing >500 tests. For cell isolation methods, negative selection was used by 43% (21/49) of laboratories performing CDC-XM. Number of cells reacted per 1 μL of serum varied among different laboratories in both CDC-XM (1,000–8,000) and FCXM tests (1,300-20,000). For the interpretation of FCXM, log fluorescence ratio (26/30, 87%) was more commonly used than channel shift values (5/30, 17%).

Conclusions

Considerable variation is noted in both CDC-XM and FCXM methods performed by different laboratories. A continuous effort for laboratory standardization is needed to reduce inter-laboratory variation in the HLA-XM test results.

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Keywords: Questionnaire survey, HLA crossmatch, Complement-dependent cytotoxicity, Flow cytometry, Standardization

REFERENCES

1. Tait BD, Susal C, Gebel HM, Nickerson PW, Zachary AA, Claas FH, et al. Consensus guidelines on the testing and clinical management issues associated with HLA and non-HLA antibodies in transplantation. Transplantation. 2013; 95:19–47.

2. Hopkins KA. The basic lymphocyte microcytotoxicity tests: standard and AHG enhancement. Hahn AB, Land GA, Strothman RM, editors. ASHI laboratory manual. 4th ed.American Society for Histocompatibility and Immunogenetics;2000. I.C.1.1-I.C.1.7.

3. Bray RA, Lebeck LK, Gebel HM. The fow cytometric crossmatch. Dual-color analysis of T cell and B cell reactivities. Transplantation. 1989; 48:834–40.

4. Scornik JC, Bray RA, Pollack MS, Cook DJ, Marrari M, Duquesnoy R, et al. Multicenter evaluation of the fow cytometry T-cell crossmatch: results from the American Society of Histocompatibility and Immunogenetics-College of American Pathologists profciency testing program. Transplantation. 1997; 63:1440–5.

5. Gebel HM, Bray RA. Sensitization and sensitivity: defning the unsensitized patient. Transplantation. 2000; 69:1370–4.

6. Bray RA, Pollack MS, Gebel HM. The HLA System. Fung MK, Grossman BJ, editors. eds.Technical manual. 18th ed.Bethesda: American Association of Blood Banks;2014. p. 475–97.

7. Park MH, Whang DH, Kim BC. A two-year study on the HLA typing profciency survey in Korea, 1996-1998. Korean J Clin Pathol. 1999; 19:714–22.

8. Park MH, Kim BC, Han BY. Results of the HLA typing profciency survey in Korea, 2000-2002. Korean J Lab Med. 2005; 25:329–39.

9. Park MH. A questionnaire survey of HLA laboratories in Korea (1993). J Korean Soc Transplant. 1993; 7:245–8.

10. Park MH, Yang YS. A questionnaire survey of HLA laboratories in Korea (1995). Korean J Lab Med. 1996; 16:987–1000.

11. Park MH, Whang DH. A questionnaire survey of HLA laboratories in Korea (1997). Korean J Lab Med. 1998; 18:650–9.

12. Lim JH, Hwang SH, Oh HB. A questionnaire survey of HLA laboratories in Korea (2005). Korean J Lab Med. 2005; 25:425–33.

13. Tinckam K. Histocompatibility methods. Transplant Rev (Orlando). 2009; 23:80–93.

14. Ayna TK, Soyoz M, Kurtulmus Y, Dogan SM, Ozyilmaz B, Tugmen C, et al. Comparison of complement-dependent cytotoxic and fow-cy-tometry crossmatch results before cadaveric kidney transplantation. Transplant Proc. 2013; 45:878–80.

15. Tian J, Li D, Alberghini TV, Rewinski M, Guo N, Bow LM. Pre-transplant low level HLA antibody shows a composite poor outcome in longterm outcome of renal transplant recipients. Ren Fail. 2015; 37:198–202.

16. Mongkolsuk T, Ingsathit A, Worawichawong S, Jirasiritham S, Kitpoka P, Thammanichanond D. Shared molecular eplet stimulates acute antibody-mediated rejection in a kidney transplant recipient with low-level donor-specifc antibodies: a case report. Transplant Proc. 2014; 46:644–7.

17. Wu P, Jin J, Everly MJ, Lin C, Terasaki PI, Chen J. Impact of alloantibody strength in crossmatch negative DSA positive kidney transplantation. Clin Biochem. 2013; 46:1389–93.

18. Bachler K, Amico P, Honger G, Bielmann D, Hopfer H, Mihatsch MJ, et al. Effcacy of induction therapy with ATG and intravenous immunoglobulins in patients with low-level donor-specifc HLA-antibodies. Am J Transplant. 2010; 10:1254–62.

19. Kute VB, Vanikar AV, Trivedi HL, Shah PR, Goplani KR, Patel HV, et al. Desensitization protocol for highly sensitized renal transplant patients: a single-center experience. Saudi J Kidney Dis Transpl. 2011; 22:662–9.

20. Jeong JC, Jambaldorj E, Kwon HY, Kim MG, Im HJ, Jeon HJ, et al. Desensitization using bortezomib and high-dose immunoglobulin increases rate of deceased donor kidney transplantation. Medicine (Baltimore). 2016; 95:e2635.

21. Saiz PA, Blanck CE. Lymphocyte crossmatch: extended incubation and antiglobulin augmented. Hahn AB, Land GA, Strothman RM, editors. ASHI laboratory manual. 4th ed.American Society for Histocompatibility and Immunogenetics;2000. I.C.9.1-I.C.9.5.

22. Lou C, Garovoy MR. Current crossmatch techniques. Moulds JM, Fawcett KJ, Garner RJ, editors. Scientifc and technical aspect of the major histocompatibility complex. Arlington: American Association of Blood Banks;1989. p. 187–205.

23. Hamrick CW, Lebeck LK. Flow cytometric T and B cell crossmatching. Hahn AB, Land GA, Strothman RM, editors. ASHI laboratory manual. 4th ed.American Society for Histocompatibility and Immunogenetics;2000. VI.B.4.1-VI.B.4.5.

24. Book BK, Agarwal A, Milgrom AB, Bearden CM, Sidner RA, Higgins NG, et al. New crossmatch technique eliminates interference by humanized and chimeric monoclonal antibodies. Transplant Proc. 2005; 37:640–2.

25. Lobo PI, Spencer CE, Isaacs RB, McCullough C. Hyperacute renal allograft rejection from anti-HLA class 1 antibody to B cells–antibody detection by two color FCXM was possible only after using pronase-digested donor lymphocytes. Transpl Int. 1997; 10:69–73.

26. Vaidya S, Cooper TY, Stewart D, Gugliuzza K, Daller J, Bray RA. Pronase improves detection of HLA antibodies in fow crossmatches. Transplant Proc. 2001; 33:473–4.

27. Vaidya S, Cooper TY, Avandsalehi J, Barnes T, Brooks K, Hymel P, et al. Improved fow cytometric detection of HLA alloantibodies using pronase: potential implications in renal transplantation. Transplantation. 2001; 71:422–8.

28. Lee YS, Won DI. Analysis of positive fow cytometric crossmatch in organ transplantation. Lab Med Online. 2011; 1:43–50.

29. Ta M, Scornik JC. Improved fow cytometric detection of donor-specifc HLA class II antibodies by heat inactivation. Transplantation. 2002; 73:1611–4.

30. Al-Muzairai IA, Mansour M, Almajed L, Alkanderi N, Alshatti N, Sam-han M. Heat inactivation can differentiate between IgG and IgM antibodies in the pretransplant cross match. Transplant Proc. 2008; 40:2198–9.

31. Hetrick SJ, Schillinger KP, Zachary AA, Jackson AM. Impact of pronase on fow cytometric crossmatch outcome. Hum Immunol. 2011; 72:330–6.

32. Park H, Lim YM, Han BY, Hyun J, Song EY, Park MH. Frequent false-positive reactions in pronase-treated T-cell fow cytometric crossmatch tests. Transplant Proc. 2012; 44:87–90.

33. Duquesnoy RJ, Marrari M. Multilaboratory evaluation of serum analysis for HLA antibody and crossmatch reactivity by lymphocytotoxicity methods. Arch Pathol Lab Med. 2003; 127:149–56.

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Table 1.

HLA crossmatch methods used by the participant laboratories (N=50)

T-CDC^∗	B-CDC^∗	T-FC^†	B-FC^†	No. (%)
+	–	–	–	8 (16)
+	+	–	–	12 (24)
+	+	+	–	6 (12)
+	+	+	+	17 (34)
+	–	+	–	1 (2)
+	–	+	+	5 (10)
–	–	+	+	1 (2)

^∗ The number of laboratories performing CDC method among 50 participants: CDC 49 (98%); T-CDC and B-CDC, 35 (70%); T-CDC only, 14 (28%);

^† The number of laboratories performing FC method among 50 participants: FC, 30 (60%): T-FC and B-FC, 23 (46%); T-FC only, 7 (14%). Abbreviations: CDC, complement-dependent cytotoxicity; FC, flow cytometry.

Table 2.

Phases of CDC crossmatches used by the participant laboratories (N=50)

Crossmatch	NIH	Amos wash	Long incubation	AHG	No. (%)
T-CDC^∗	+	–	–	+	39 (68)
	+	–	+	+	4 (8)
	+	–	+	–	2 (4)
	–	–	+	+	2 (4)
	–	+	+	+	1 (2)
	–	–	–	+	1 (2)
	–	–	–	–	1 (2)
B-CDC^†	+	–	–	–	32 (64)
	+	+	–	–	1 (2)
	+	–	–	+	2 (4)
	–	–	–	–	15 (30)

^∗ T-CDC, 49/50 (98%): NIH and AHG, 43 (86%);

^† B-CDC, 35/50 (70%): NIH only, 32 (64%). Abbreviations: CDC, complement-dependent cytotoxicity; NIH, National Institutes of Health; AHG, anti-human globulin.

Table 3.

Annual number of HLA crossmatch tests performed by the participant laboratories in 2013 and 2014

No. of tests	2013, No. (%)				2014, No. (%)
No. of tests	T-CDC (N=46)	B-CDC (N=34)	T-FC (N=27)	B-FC (N=18)	T-CDC (N=48)	B-CDC (N=35)	T-FC (N=30)	B-FC (N=23)
≤10	7 (15)	7 (21)	5 (19)	2 (11)	9 (19)	11 (31)	8 (27)	7 (30)
11-50	19 (41)	18 (53)	14 (52)	12 (67)	16 (33)	12 (34)	10 (33)	10 (43)
51-100	3 (7)	1 (3)	1 (4)	0 (0)	6 (13)	5 (14)	4 (13)	1 (4)
101-300	10 (22)	3 (9)	2 (7)	1 (6)	10 (21)	3 (9)	2 (7)	2 (9)
301-500	3 (7)	3 (9)	3 (11)	1 (6)	2 (4)	3 (9)	3 (10)	1 (4)
>500	4 (9)	2 (6)	2 (7)	2 (11)	5 (10)	1 (3)	3 (10)	2 (9)
Median (range)	39 (2-1,903)	26 (1-599)	26 (2-1,607)	33 (2-769)	42 (2-3,089)	25 (0-513)	33 (0-3,065)	29 (0-734)

Abbreviations: CDC, complement-dependent cytotoxicity; FC, flow cytometry.

Table 4.

Types of the cells and cell separation methods used for HLA crossmatch by the participant laboratories (N=50)

Crossmatch methods (N)	Types of the cells (N, %)	Nylon wool No. (%)	Negative selection No. (%)	Positive selection No. (%)	Nylon wool or negative selection No. (%)
T-CDC (49)	MNC^∗ (8, 16)
	T cell (34, 69)	15 (31)	11 (22)	5 (10)	3 (6)
	Total lymphocyte (7, 14)	0 (0)	7 (14)	0 (0)	0 (0)
B-CDC (35)	MNC^∗ (1, 3)
	B cell (34, 97)	14 (49)	12 (34)	5 (14)	3 (9)
T-FC (30)	MNC^∗ (20, 67)
	T cell (3, 10)	0 (0)	2 (7)	0 (0)	1 (3)
	Total lymphocyte (7, 23)	0 (0)	7 (23)	0 (0)	0 (0)
B-FC (23)	MNC^∗ (15, 65)
	B cell (2, 9)	0 (0)	1 (4)	0 (0)	1 (4)
	Total lymphocyte (6, 26)	0 (0)	6 (26)	0 (0)	0 (0)

^∗ Separated by ficoll density gradient.

Abbreviations: CDC, complement-dependent cytotoxicity; FC, flow cytometry; MNC, mononuclear cell.

Table 5.

Number of cells and volume of sera used for CDC crossmatc by the participant laboratories (N=49)

No. of cells per well	Volume of sera per well (uL)	No. (%)
2,000-3,000	1	35 (71)
2,000-3,000	1.4	1 (2)
2,000-3,000	2	3 (6)
2,000-3,000	1 or 2^∗	1 (2)
2,000-5,000	1	1 (2)
2,000-5,000	2	1 (2)
3,000-4,000	1	2 (4)
3,000-5,000	1	2 (4)
5,000	2	1 (2)
7,000	2	1 (2)
8,000	1	1 (2)

^∗ 1 μL used for NIH and 2 μL used for AHG.

Abbreviation: CDC, complement-dependent cytotoxicity.

Table 6.

Type of media and percentage of FCS used for serum dilution of CDC crossmatch by the participant laboratories (N=49)

Media	Percentage of FCS used, No.(%)							Total No. (%)
Media	0%	2%	3%	5%	10%	15%	20%	Total No. (%)
RPMI 1640	6 (12)	3 (6)	1 (2)	16 (33)	8 (16)	2 (4)	1 (2)	37 (76)
IMDM				3 (6)			6 (12)	9 (18)
McCoy				2 (4)				2 (4)
PBS		1 (2)						1 (2)

Abbreviations: FCS, fetal calf serum; CDC, complement-dependent cytotoxicity; RPMI, Roswell Park Memorial Institute; IMDM, Iscove's modified Dulbecco's media; PBS, phosphate buffered saline.

Table 7.

Incubation conditions of CDC crossmatch used by the participant laboratories (N=49)

CDC methods (N)	Cell and serum incubation		Complement incubation		No. (%)
CDC methods (N)	Temperature	Time, minutes	Temperature	Time, minutes	No. (%)
T-CDC NIH (45)	RT	30	RT	60	29 (64)
	37°C	30	RT	60	12 (27)
	RT	60	RT	60	2 (4)
	RT	60	RT	90	1 (2)
	4°C and 37°C	30	RT	60	1 (2)
T-CDC Amos-wash (1)	RT	30	RT	60	1 (100)
T-CDC long incuba-	RT	60	RT	120	9 (90)
tion (10)	RT	30	RT	120	1 (10)
T-CDC AHG (47)	RT	60	RT	120	29 (62)
	RT	30	RT	60	6 (13)
	RT	60	RT	90	4 (9)
	RT	30	RT	120	2 (4)
	37°C	60	RT	120	2 (4)
	RT	45	RT	60	1 (2)
	RT	60	RT	60	1 (2)
	RT	90	RT	90	1 (2)
	37°C	30	RT	120	1 (2)
B-CDC NIH (35)	37°C	60	RT	120	22 (63)
	37°C	60	RT	60	4 (11)
	37°C	30	RT	60	2 (6)
	RT	60	RT	120	2 (6)
	4°C and 37°C	60	RT	120	2 (6)
	RT	30	RT	60	1 (3)
	4°C	60	RT	120	1 (3)
	4°C	60	4°C	120	1 (3)
B-CDC Amos-wash (1)	) 37°C	60	RT	120	1 (100)
B-CDC AHG (2)	RT	60	RT	120	2 (100)

Abbreviations: CDC, complement-dependent cytotoxicity; NIH, National Institutes of Health; AHG, anti-human globulin; RT, room temperature.

Table 8.

Number of cells and volume of sera used for flow cytometry crossmatch by the participant laboratories (N=30)

No. of cells per tube	Volume of sera per tube (μL)	No. of cells/1 μL serum	No. (%)
50,000	5	10,000	1 (3)
100,000	20	5,000	2 (7)
100,000-150,000	50	2,000-3,000	3 (10)
130,000	100	1,300	1 (3)
200,000	50	4,000	3 (10)
200,000-300,000	20	10,000-15,000	4 (13)
200,000-300,000	25	8,000-12,000	3 (10)
200,000-300,000	30	6,667-10,000	1 (3)
200,000-300,000	50	4,000-6,000	3 (10)
200,000-300,000	70	2,857-4,286	1 (3)
200,000-300,000	100	2,000-3,000	1 (3)
300,000	25	12,000	1 (3)
300,000-500,000	50	6,000-10,000	1 (3)
500,000	25	20,000	1 (3)
500,000	50	10,000	3 (10)
500,000	100	5,000	1 (3)

Table 9.

Incubation conditions of flow cytometry crossmatch used b the participant laboratories (N=30)

Cell and serum incubation		Conjugate incubation		No. (%)
Temperature	Time, minutes	Temperature	Time, minutes	No. (%)
RT	15	RT	20	1 (3)
RT	20	RT	20	4 (13)
RT	20	4°C	30	2 (7)
RT	30	RT	15	1 (3)
RT	30	RT	20	5 (17)
RT	30	RT	30	3 (10)
RT	30	4°C	20	2 (7)
RT	30	4°C	30	5 (17)
RT	60	RT	45	1 (3)
RT	30^∗/60^†	RT	30^∗/60^†	1 (3)
37°C	20	RT	20	2 (7)
37°C	20	RT	30	1 (3)
37°C	30	4°C	30	2 (7)

^∗ T cell incubation time;

^† B cell incubation time. Abbreviation: RT, room temperature.

Table 10.

Analysis of flow cytometry crossmatch results by the participant laboratories (N=30)

FC methods (N)	MFI ratio (log)	MCS (channel)	Channel ratio (channel)	% Dead cel (7-AAD)	l No. (%)
T-FC (30)	+	–	–	–	23 (77)
	+	+	–	–	2 (7)
	–	–	+	–	2 (7)
	–	+	–	–	1 (3)
	–	+	+	–	1 (3)
	+	+	+	–	1 (3)
B-FC (23)	+	–	–	–	17 (74)
	+	+	–	–	2 (9)
	–	–	–	+	2 (9)
	+	+	+	–	1 (4)
	–	–	+	–	1 (4)

Abbreviations: FC, flow cytometry; MFI, median fluorescence intensity; MCS, median channel shift; 7-AAD, 7-aminoactinomycin D.

Table 11.

Cut-off values of log MFI ratio for positive flow cytometry crossmatch by the participant laboratories (N=26)

FC methods (N)	Cut-off	No. (%)
T-FC (26)	1.1	1 (4)
	1.2	4 (15)
	1.3	2 (8)
	1.44	1 (4)
	1.5	2 (8)
	1.6	1 (4)
	1.8	1 (4)
	2	13 (50)
	1.5-2	1 (4)
B-FC (20)	1.1	1 (5)
	1.4	2 (10)
	1.5	1 (5)
	1.7	1 (5)
	1.8	1 (5)
	2	10 (50)
	2.35	1 (5)
	2.7	1 (5)
	3	1 (5)
	1.5-2	1 (5)

Abbreviations: MFI, median fluorescence intensity; FC, flow cytometry.

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