Optimum Conditions for the Preparation of Red Blood Cell Suspensions for ABO Antibody Titration

Seon Joo Kang; Sung Ran Cho; Young Ae Lim

doi:10.3343/lmo.2015.5.1.15

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Kang, Cho, and Lim: Optimum Conditions for the Preparation of Red Blood Cell Suspensions for ABO Antibody Titration

Original Article

Transfusion Medicine

Laboratory Medicine Online 2015; 5(1): 15-19.

Published online: 1 January 2015

DOI: https://doi.org/10.3343/lmo.2015.5.1.15

Optimum Conditions for the Preparation of Red Blood Cell Suspensions for ABO Antibody Titration

Seon Joo Kang, M.D., Sung Ran Cho, M.D., Young Ae Lim, M.D.

Department of Laboratory Medicine, Ajou University School of Medicine, Suwon, Korea.

Corresponding author: Young Ae Lim. Department of Laboratory Medicine, Ajou University School of Medicine, 164 World cup-ro, Yeongtong-gu, Suwon 443-721, Korea. Tel: +82-31-219-5786, Fax: +82-31-219-5778, limyoung@ajou.ac.kr

Received 6 January 2014 Revised 11 March 2014 Accepted 10 April 2014

(open-access):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

There is significant inter-laboratory variation in the ABO antibody (Ab) titer levels of blood samples because a standardized method has not yet been developed. The aim of this study was to identify the best conditions for the preparation of the red blood cell (RBC) suspensions so as to aid the development of a standard ABO Ab titration method.

Methods

Serum samples from apparently healthy adults and RBCs from three different sources (residual EDTA blood from healthy adults, donor blood in citrate-phosphate-dextrose-adenine-1 [CPDA-1], and a commercially available RBC reagent) were used for Ab titrations. We measured the titers for each blood group under various conditions, including the time period of storage (days), the ratio of serum to RBC volume, and the RBC sources. The techniques for room temperature incubation and the indirect antiglobulin test were used for the tube and the gel card test.

Results

A storage period of 6 to 7 days significantly affected the Ab titers. Samples with 3% RBCs in a 1:1 serum to RBC volume ratio had significantly lower Ab titers than those with 2% RBCs in a 1:1 ratio or those with 3% RBCs in a 2:1 ratio. There were no significant differences in the Ab titers of RBCs from different sources.

Conclusions

To reduce inter-laboratory variations in ABO Ab titrations, using RBC suspension within five days of storage and applying ratio of serum to RBC volume to 2:1 with 3% RBC in the tube test will be helpful when using home-made RBC suspension.

Keywords: ABO blood group system, Titer, Red blood cell, Suspension

Figures and Tables

Table 1

Relationship between median values of the ABO antibody titer step and the storage day of RBC suspension in the tube test

Blood group	Antibody	N	Room temperature incubation														Indirect antiglobulin test
			1+ agglutination							w+ agglutination							1+ agglutination							w+ agglutination
			1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7
A	Anti-B	5	3	3	3	4	4	3	3	4	5	4	5	5	4	4	4	4	4	4	4	4	4	5	5	5	5	5	5	4
B	Anti-A	5	4	4	4	4	4	3	3	4	5	5	5	5	4	4	4	4	4	3	3	3	3	5	4	5	4	4	4	4
O	Anti-A	5	5	5	5	5	5	4	3	5	6	6	6	5	5	4	6	6	6	6	5	6	6	7	6	7	6	7	7	6
O	Anti-B	5	4	4	4	5	4	4	3	5	5	5	5	5	5	4	6	6	6	6	6	6	6	7	7	6	7	6	7	7
Total		20	4	4	4	4	4	4^*	3^*	5	5	5	5	5	5^*	4^*	6	6	6	6	5	6	6	7	6	6	6	6	7	6^†

^*P<0.01 vs. from the first day by the Friedman test; ^†P<0.05.

Table 2

Relationship between median values of the ABO antibody titer step and the storage day of RBC suspension in the gel test

Blood group	Antibody	N	Room temperature incubation														Indirect antiglobulin test
			1+ agglutination							w+ agglutination							1+ agglutination							w+ agglutination
			1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7
A	Anti-B	1	2	2	2	2	1	1	1	3	2	2	2	1	2	1	2	3	2	3	3	3	3	3	2	2	2	2	2	2
B	Anti-A	3	2	2	2	2	2	2	1	2	2	3	2	2	2	2	3	4	3	4	3	4	3	4	3	3	3	3	3	3
O	Anti-A	3	3	4	4	4	3	4	4	4	4	4	4	4	4	4	7	8	7	8	7	8	7	7	7	7	7	8	7	7
O	Anti-B	3	4	3	3	3	3	3	3	4	3	4	4	3	4	3	6	7	6	7	6	7	6	7	7	7	7	7	7	7
Total		10	3	3	3	3	3	3	3	4	3	4	4	3^*	4^*	3^*	6	7	6	7	6	7	6	7	6	7	6	7^*	6^*	7^*

^*P<0.01 vs. from the first day by the Friedman test.

Table 3

Relationship between median values of the ABO antibody titer step and ratio of serum to red blood cell volume in the tube test

Blood group	Antibody	N	Room temperature incubation						Indirect antiglobulin test
			1+ agglutination			w+ agglutination			1+ agglutination			w+ agglutination
			2%	3%	3%	2%	3%	3%	2%	3%	3%	2%	3%	3%
			1:1	2:1	1:1	1:1	2:1	1:1	1:1	2:1	1:1	1:1	2:1	1:1
A	Anti-B	10	4	4	3	5	5	4	4	4	4	5	5	5
B	Anti-A	10	4	5	4	5	6	5	4	4	4	5	5	5
O	Anti-A	10	5	5	4	5	6	5	6	6	5	7	7	6
O	Anti-B	10	5	4	4	6	5	5	6	6	5	7	7	6
Total		40	4^*	4^*	4	5^*	5^*	5	5^*	5^*	5	6^*	6^*	6

^*P<0.001 vs. 3% with 1:1 by the Friedman test and Wilcoxon signed rank test.

Abbreviation: S/R ratio, volume ratio for serum:RBC suspension.

Table 4

Relationship between median values of the ABO antibody titer step and three RBC sources in the tube test

Blood group	Antibody	N	Room temperature						Indirect antiglobulin test
			1+ agglutination			w+ agglutination			1+ agglutination			w+ agglutination
			EDTA	CPDA-1	CP	EDTA	CPDA-1	CP	EDTA	CPDA-1	CP	EDTA	CPDA-1	CP
A	Anti-B	15	4	4	4	5	5	4	4	4	4	5	5	5
B	Anti-A	15	4	4	4	5	5	5	4	4	4	5	5	4
O	Anti-A	15	4	4	4	5	5	5	6	6	6	7	6	7
O	Anti-B	15	4	4	5	5	5	5	6	6	6	7	7	6
Total		60	4	4	4	5	5	5	5	5	6	6	6	6

There were no significant differences between ABO antibody titers and three RBC sources (Kruskal-Wallis test).

Abbreviations: CDPA-1, citrate-phosphate-dextrose-adenine-1; CP, commercialized product.

Notes

This article is available from http://www.labmedonline.org

References

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4. Kumlien G, Wilpert J, Säfwenberg J, Tydén G. Comparing the tube and gel techniques for ABO antibody titration, as performed in three European centers. Transplantation. 2007; 84:12S. S17–S19.

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6. Roback JD, editor. Technical Manual. 17th ed. Bethesda: American Association of Blood Banks;2011. p. 873–907.

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