Evaluation of immunoglobulin M and immunoglobulin G titers of ABO blood group system in blood donors at a blood center of a tertiary care hospital

Abstract

BACKGROUND AND OBJECTIVES:

ABO antibodies have implications in platelet transfusions, solid organ, and bone marrow transplants. The study aimed at measuring the immunoglobulin G (IgG) and immunoglobulin M (IgM) ABO antibody titers in A, B, and O blood group blood donors and correlating the titers with age, gender, and blood group of donors.

SETTINGS AND DESIGN:

A prospective observational study was performed over 3 years at a tertiary care blood center.

MATERIALS AND METHODS:

Equal number of A, B, and O group blood donor samples chosen by random sampling were evaluated for titers by doubling dilution using tube method. IgM titers were determined at room temperature. IgG titers were tested at 37°C with anti-IgG antiglobulin reagent after inactivating IgM antibodies by dithiothreitol. The titers were correlated with age, gender, and blood group.

STATISTICAL ANALYSIS:

Mann–Whitney U-test, Kruskal–Wallis test, and Bonferroni correction were employed for various correlations using IBM SPSS v26 (Statistical Package for the Social Sciences) software. A two-tailed P < 0.05 was considered statistically significant.

RESULTS:

A total of 870 samples (290 of each of A, B, and O blood groups) were tested for IgG and IgM antibody titers. IgG anti-A and anti-B titers were significantly higher in blood group O as compared to blood groups B and A. Anti-B IgM titers in group A were significantly higher than anti-A IgM titer in B. The antibody titer showed decreasing trend with age. There was no correlation with gender.

CONCLUSIONS:

The study will help hospitals and blood centers in formulating policies regarding ABO antibody titer estimation in platelet donors and renal transplant recipients in the context of out-of ABO group platelet transfusions and ABO-incompatible renal transplants, respectively.

Introduction

Out of different blood group systems, ABO system is the only system that has naturally occurring (nonimmune) antibodies in the plasma directed to A and B blood group antigens.[1] These antibodies of ABO blood group system are implicated as a cause in hemolytic transfusion reactions, hemolytic disease of fetus and newborn, and chronic rejection of solid-organ transplants.[2] In ABO-incompatible marrow transplants, ABO antibodies can cause hemolysis, delaying the engraftment of red cells and megakaryocytes.[3] It is also important to understand the issues related to ABO incompatibility for the purpose of Platelet transfusions.[4,5,6]

The concentration of an antibody, as determined by finding the highest dilution at which it is still able to cause agglutination of the antigen, is called the titer of antibody. ABO antibody titration has been used to determine treatment strategies and evaluate patient outcomes in ABO-incompatible marrow transplants and solid-organ transplantation and to evaluate ABO-incompatible transfusion responses.[7,8]

A few authors from India have reported ABO titers only in O blood group populations.[4,9,10] Literature available from other countries has also discussed titers only in O group individuals[11,12,13,14] or only immunoglobulin M (IgM) antibody titer in all blood groups.[6] Furthermore, the focus of many of the studies was on comparison of techniques used for performing titers or on standardization of methods.[2,4,9,11,15,16] There are a few studies from different regions of India, where the researchers have performed ABO antibody titers in A, B, and O blood group populations.[17,18,19] However, only one of these studies is from Western region of India like ours.[18]

Hence, the authors performed the study with the overall goal of measuring the immunoglobulin G (IgG) and IgM ABO antibody titers in all three A, B, and O blood group healthy blood donors. The specific objectives were to correlate the IgM and IgG blood group titers with age, gender, and blood group of donors.

Materials and Methods

Study setting

A prospective, observational, cross-sectional study was performed at the blood center (department of transfusion medicine) of a tertiary care teaching hospital in Western India. The study was performed over a period of 3 years from January 2018 to December 2020. Institutional Ethics Committee approval was obtained (IEC (I)/OUT/2157/2017 dated 25/10/2017).

Selection of samples

Two days each week, preferably 3 days apart, were chosen for the study such that a minimum of 20 blood donors would have donated blood on the previous day. This was done to increase the possibility of getting more than one sample each of A, B, and O blood groups to enable random sample selection.

Sample size

The duration of the study was predecided to be 3 years. Considering the time and manpower required for the titer estimation, these tests were performed twice a week only. The samples were selected by random sampling method from the donors’ leftover samples of each of A, B, and O groups collected over the previous day. Each time, only one sample of each group was selected and tested, thus two samples of each group per week. Hence, the sample size for each blood group over 156 weeks in 3 years was 312.

Methodology

The results of ABO blood grouping and transfusion transmissible infections (TTI) screening tests performed on the blood donor samples of the previous day were reviewed. The Rh blood grouping results were not taken into consideration. The blood donor samples of AB blood group and/or any sample found reactive for any of TTI were excluded from the study. Both male and female blood donors (18–65 years of age) were included in the study. The titers were performed on ethylenediaminetetraacetic acid (EDTA) blood samples of the donors. A and B group EDTA samples with at least 1.5 ml plasma and O group samples with at least 2.5 ml plasma left in them were considered for sample selection (in O blood group, both anti-A and anti-B titers were tested; hence, more quantity of plasma was required, as compared to groups A and B).

The unique donor numbers were listed for each of A, B, and O groups fulfilling the above criteria. For each of these blood groups, one sample was selected by random chit method. The testing of plasma samples for ABO antibody titers was performed on the same day to ensure uniformity. All the samples included in the study were, thus, tested 24 h after collection with their preservation at 2°C–6°C overnight.

Antibody titration

Antibody titration was performed using doubling dilution method using tube technique as described in the Technical Manual, American Association of Blood Banks.[20]

Immunoglobulin M antibody titration

1. 200 μL of normal saline was added to tubes 2–10

2. 200 μL of plasma was added to tubes 1 and 2

3. After adequate mixing, 200 μL of diluted plasma was taken out from tube 2 and added to tube 3 and so on

4. To each of the tubes, 100 μL of 5% suspension of corresponding pooled red cells (freshly prepared in-house) was added

5. The contents were mixed properly and the tubes were incubated at room temperature for 15 min

6. They were centrifuged for 1000 rpm for 1 min. The tubes were examined for agglutination

7. The tube with the highest dilution showing 1+ agglutination was noted

8. The reciprocal of this highest dilution was considered the IgM antibody titer.

Immunoglobulin G antibody titration

Removal of immunoglobulin M antibodies using dithiothreitol

1. 1 mL of donor plasma was taken into two test tubes

2. To one of the tubes, 1 mL of 0.01 M dithiothreitol (DTT) was added (prepared by dissolving 0.154 g of DTT in 100 mL of phosphate-buffered saline [PBS], pH 7.3, stored at −18°C)

3. To the other tube, 1 ml of pH 7.3 PBS was added to act as a dilution control. Both the tubes were mixed and incubated at 37°C for 30 min.

Immunoglobulin G titration

1. For titration of IgG antibodies, two sets of ten tubes were taken one each for DTT-treated and dilution control plasma

2. 200 μL of normal saline was added to tubes 2–10

3. DTT-treated plasma and dilution control plasma 200 μL each were added to tubes 1 and 2 of the respective sets

4. After adequate mixing, 200 μL of diluted plasma was taken out from tube 2 and added to tube 3 and so on

5. To each of the tubes, 100 μL of 5% suspension of corresponding pooled red cells (freshly prepared in-house) was added

6. The tubes were centrifuged at 1000 rpm for 1 min and observed for agglutination or hemolysis

7. The tubes were incubated at 37°C for 60 min and then washed with normal saline 3 times every time centrifuging the tubes at 3000 rpm for 3 min and discarding the supernatant

8. Two drops of monospecific anti-IgG antiglobulin reagent (Erybank, Tulip Diagnostics (P), Ltd) was added to each tube

9. The tubes were centrifuged immediately at 1000 rpm for 1 min. The tubes were observed for agglutination

10. The tube with highest dilution showing 1+ agglutination was noted. The reciprocal of this highest dilution was considered the IgG antibody titer.

Note: Tubes were centrifuged at 1000 rpm for 1 min at room temperature and also after incubation at 37°C to ensure that there was no agglutination. Furthermore, the validity of negative antiglobulin test was confirmed by adding IgG-coated red cells.

Statistical analysis

Demographic data were summarized using descriptive statistics. Continuous data were presented as median (interquartile range [IQR]), while categorical data were summarized using frequency and percentage. The median titers of IgM and of IgG were compared across the three blood groups. Furthermore, the median titer was calculated and compared as per age groups and gender.

The Mann–Whitney U-test was utilized to compare blood group and gender with antibody titers, whereas the Kruskal–Wallis test was employed to compare age group with antibody titers. Pairwise testing was conducted using Bonferroni correction.

A two-tailed P < 0.05 was considered statistically significant. Statistical analysis was performed through appropriate statistical tests using IBM Corp. Released 2019. IBM SPSS Statistics for Windows, Version 26.0 (IBM Corp., Armonk, NY).

Results

The age and gender distribution in each blood group is shown in Table 1.

Table 1.

Age and gender distribution of blood donors in the study blood group wise

Age group (years)	Blood group A		Blood group B		Blood group O		Total
	Males	Females	Males	Females	Males	Females
18–29	94	9	109	13	90	10	325
30–41	118	12	106	8	119	12	375
42–53	41	5	42	3	47	3	141
54–65	11	0	7	2	7	2	29
Total	264	26	264	26	263	27	870
Total males		791		Total females		79

Table 2 shows the comparison of IgG and IgM titers between corresponding blood groups and their level of significance by Mann–Whitney U-test.

Table 2.

Comparison of titers between blood groups by Mann–Whitney U-test

Variable	Blood group	n	Median (IQR)	P
Anti A titer IgG	B	290	16 (8–16)	<0.001*
	O	290	64 (32–64)
Anti A titer IgM	B	290	64 (32–128)	0.624
	O	290	64 (64–128)
Anti B titer IgG	A	290	8 (4–16)	<0.001*
	O	290	32 (32–64)
Anti B titer IgM	A	290	128 (64–256)	<0.001*
	O	290	64 (32–128)
Anti B IgG	A	290	8 (4–16)	0.069
Anti A IgG	B	290	16 (8–16)
Anti B IgM	A	290	128 (64–256)	0.025*
Anti A IgM	B	290	64 (32–128)

*Significant. Ig=Immunoglobulin, IQR=Interquartile range

Table 3 shows no significant difference by Mann–Whitney U-test between the genders with regard to antibody titers in any of the blood groups.

Table 3.

Comparison of titers between genders by Mann–Whitney U-test

Blood group	Variable	Gender	n	Median (IQR)	P
B	Anti A titer IgG	Female	26	8 (4–16)	0.217
		Male	274	16 (8–16)
	Anti A titer IgM	Female	26	64 (32–128)	0.551
		Male	274	64 (32–128)
A	Anti B titer IgG	Female	26	8 (8–16)	0.462
		Male	274	8 (4–16)
	Anti B titer IgM	Female	26	128 (64–128)	0.769
		Male	274	128 (64–256)
O	Anti A titer IgG	Female	27	32 (32–128)	0.873
		Male	263	64 (32–64)
	Anti A titer IgM	Female	27	64 (32–128)	0.33
		Male	263	64 (64–128)
	Anti B titer IgG	Female	27	32 (32–64)	0.623
		Male	263	32 (32–64)
	Anti B Titer IgM	Female	27	64 (32–128)	0.232
		Male	263	64 (32–128)

IQR=Interquartile range, Ig=Immunoglobulin

Table 4 shows the median and IQR of antibody titers in the two extremes of age groups. The medians of both IgG and IgM titers were found to be highest in the age group of 18 to 29 years for all blood groups.

Table 4.

Titers in the extreme age groups of blood donors

Blood group	Variable	Age	n	Median (IQR)
B	Anti A titer IgG	18–29	122	16 (8–32)
		54–65	9	4 (4–16)
	Anti A titer IgM	18–29	122	128 (64–256)
		54–65	9	32 (32–64)
A	Anti B titer IgG	18–29	103	16 (8–32)
		54–65	11	4 (2–8)
	IgM	18–29	103	256 (128–256)
		54–65	11	16 (8–16)
O	Anti A titer IgG	18–29	100	128 (64–128)
		54–65	9	8 (8–16)
	Anti A titer IgM	18–29	100	128 (128–256)
		54–65	9	16 (16–16)
	Anti B titer IgG	18–29	100	64 (64–128)
		54–65	9	16 (16–32)
	Anti B titer IgM	18–29	100	128 (64–128)
		54–65	9	32 (16–64)

IQR=Interquartile range, Ig=Immunoglobulin

On comparing the titers between all age groups pair wise using Kruskal–Wallis test, it was found that median titers decreased with age. However, the difference was not statistically significant in O group after 29 years for anti-B (IgM and IgG) and after 42 years for anti-A (IgG and IgM). In blood group A, anti-B (IgM and IgG) decrease was not statistically significant after 42 years. In blood group B, anti-A IgM and anti-A IgG did not show statistically significant decrease after 42 and 29 years, respectively.

Discussion

Anti-A and anti-B titers were performed in healthy blood donors of A, B, and O blood groups with the aim of estimating difference of titers between blood groups, gender, and age groups. IgM antibody titers were tested at room temperature, while IgG titers were tested after removing IgM antibody using DTT.

The number of male donors in our study was 10 times higher as compared to female donors for all blood groups and age groups. Other studies have also shown that the number of female donors is much less in comparison to male donors in India.[21,22,23,24,25]

The highest number of blood donors (375/870) were in the age group of 30–42 years, similar to the study by Kumaran et al.[24] However, in another study by Uma et al., 61.3% were in the age group of 18–25 years, followed by 29% donors in the age group of 26–35 years.[23]

Anti-A IgG and IgM titers were compared between blood groups B and O, while anti-B IgG and IgM titers were compared between blood groups A and O. Both anti-A and anti-B IgG titers were significantly higher in blood group O as compared to blood groups B and A, respectively. Anti-B IgM titer was significantly higher in blood group A as compared to blood group O. However, there was no significant difference in the titer of anti-A IgM between blood groups B and O. Anti-B IgM titer in blood group A was significantly higher than anti-A IgM titer in blood group B. In a study by Park et al., high titers anti-A and anti-B were obtained using immediate spin technique in blood groups B and A, respectively, while high titers were obtained in O group donors using antiglobulin method. The median titers of both IgM and IgG antibodies were lower than those of corresponding antibodies in our study.[2] A study by Tendulkar et al. showed median titers of anti-A and anti-B IgM to be 128 in O group platelet donors. We found anti-A and anti-B IgM titers of 64 in O group blood donors.[4]

In a study by Josephson et al., both IgM and IgG anti-A titers were high in group O platelet donors.[13]

Bazigou et al. evaluated anti-A and anti-B IgM isoagglutinin titers in group O platelet donors and found anti-A titers to be significantly higher than anti B titers.[14] Kumar et al. found that O group donors had higher titers of both anti-A and anti-B than A and B groups had.[17]

In our study, the antibody titers were significantly highest in the age group 18–29 years. The antibody titers showed decreasing trend with age. There was no significant difference in any of the antibody titers between the genders. In a study by de França et al., both anti-A and anti-B IgM titers showed low titers in donors above 50 years of age. Young women showed high titers of anti-B IgM.[12] Sood et al. did not find a significant correlation of antibody titers with age or gender.[9]

Tendulkar et al. found significantly higher titers (P < 0.001) in female donors and also found an inverse relation between titer levels and age of plateletpheresis donors.[4]

Many studies have defined and used critical titers of antibodies. de Franca et al. performed ABO antibody titers in O blood group donors. They considered 64 as the critical titer.[12] Josephson et al. also defined critical titer for IgM and IgG antibodies to be 64 and 256, respectively.[13] On the other hand, a study by Karafin et al. reported that a critical titer of anti-A or anti-B is not the only factor that can predict the risk of hemolysis in patients receiving apheresis platelets with ABO-incompatible plasma.[26]

Taking 64 as the cutoff, 55.8% and 47.2% of group O donors had higher than critical titers for anti-A and anti–B, respectively, in a study by Bazigou et al.[14]

A study by Chatterjee et al. revealed a prevalence of dangerous blood group O to be 14.1%. High-titer A and B donors were found to be 3.52% and 10.5%, respectively. High titers of antibodies were seen to be associated with female gender, vegetarian diet, age under 30 years, and O blood group.[18]

Kumar et al. found that O group donors with anti-A and anti-B IgM titers above 64 were 39.42% and 36.85%, respectively. Those having IgG titers above 256 were 41.71% and 41.42%, respectively.[17] These were considered critical titers similar to the study by Kannan et al., who reported that high titers of anti-A IgM, anti-A IgG, anti-B IgM, and anti-B IgG were found in 36%, 9.6%, 32%, and 5.5% O group donors, respectively.[10] In a study by Ravikanth et al., an IgM titer of 1:64 was called a “high titer.” They found more than 1:64 titer of anti-A and anti-B IgM in 34.5% and 14.56% of B and A group donors, respectively. O group donors with more than 1:64 anti-A and anti-B IgM titers were 47.4% and 22.08%, respectively.[19] IgG titer was not done in this study.

However, in our study, we did not define any critical titer. The critical titer is particularly relevant to out-of-ABO group single donor platelet transfusions and ABO-incompatible renal transplants.

The risk of hemolysis can be lessened by not transfusing out-of-group platelets with ABO antibody titers above critical cutoff by universal screening of plateletpheresis donors for ABO antibody titers.[27] In ABO-incompatible renal transplant, “high titer” was considered to be 256 in the recipient.[28] In another study, the risk of antibody-mediated rejection of renal transplant was significantly higher among individuals with an elevated posttransplant ABO titer of ≥64. Therapeutic plasma exchange and immunosuppression are usually considered to reduce high antibody levels in a transplant recipient.[29]

However, determination of critical titer has been a challenging task.[13] Standardization of methods and systematic review of published reports can help in defining critical titers. Several studies in literature have thrown light on differences in titers using different methodologies. A study by Park et al. showed median titers of anti-B and anti-A in all blood groups to be higher by column agglutination technique (CAT) without DTT than by CAT with DTT, especially for group O individuals.[2] Kang et al. showed that IgG antibody titer by indirect antiglobulin test (IAT) using tube technique was higher than gel card IAT in blood groups A and B.[16] In a study by Sood et al., anti-A and anti-B IgM and IgG titers in O group donors were higher by tube technique as compared to CAT.[9] Some studies have attempted standardization of methods to circumvent interinstrument and interlaboratory variation in the titer.[8,15]

Furthermore, considering the influence of diet and ethnicity on ABO antibody titers, it will be judicious to establish critical ABO antibody titers for the population of each nation.[12,30]

Limitations of the study

Exact sample size for the study was not calculated using any statistical formula. Critical antibody titers were not used in our study. Only a fraction of our donor population was studied for titers.

Conclusions

IgG anti-A and anti-B titers were significantly higher in blood group O as compared to blood groups B and A. Anti-B IgM titers in group A were significantly higher than anti-A IgM titer in B. The antibody titer showed decreasing trend with age. The study will help hospitals and blood centers in formulating policies regarding the estimation of ABO antibody titer in platelet donors and renal transplant recipients in the setting of out-of ABO group platelet transfusions and ABO-incompatible renal transplants, respectively.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

We acknowledge the contribution of Ms. Mangal Auti, technician, Blood Centre, Seth GS Medical College and KEM Hospital, Mumbai.