Global Journal of Transfusion Medicine

: 2020  |  Volume : 5  |  Issue : 2  |  Page : 187--191

Prevalence of high titered anti-A and anti-B antibodies among O blood group individuals and their associated factors

Sujitha Kannan, Rajendra Kulkarni, Abhishekh Basavarajegowda 
 Department of Transfusion Medicine, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, India

Correspondence Address:
Abhishekh Basavarajegowda
Department of Transfusion Medicine, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry


Background and Objectives: Severe hemolytic transfusion reactions due to minor incompatibility are usually associated with the so-called “High titred” antibodies in the plasma of donors especially the O group as it is generally regarded as universal donors. Critical titers of 1:64 or higher was considered high titered antibodies for IgM anti-A and anti-B. Critical titers of 1:256 or higher was considered high titered antibodies for IgG anti-A and anti-B. The objective of this study was to see the prevalence of such high titered antibodies among our donor population and to observe for an association if any with the age, gender, and diet type and probiotic intake of the donors. Methodology: This was a cross-sectional analytical study conducted over 20 months from September 2015 to May 2017. About 560 donors were chosen randomly after screening and counseling and titration of anti-A and anti-B antibodies was done by the double dilution tube agglutination technique. The information on age, gender, diet type, and probiotic intake was gathered from the donor history questionnaire and an association between the factors described and the distribution of critical titers of anti-A and anti-B was elicited. Results: In the present study, the prevalence of high titer anti-A and anti-B IgM antibodies was 36% and 32%, respectively. The prevalence of high titer anti-A and anti-B IgG antibodies was 9.6% and 5.5%, respectively. The median IgM antibody titers reduced with the increasing age of the donors starting 18 years. There was a statistically significant association between a vegetarian diet and high titers. Conclusion: There are a considerable proportion of O group donors with high titers of antibodies and hence a routine pretransfusion screening for such antibodies can prevent the development of hemolytic reactions by segregating units into “high titer” and “low titer” antibodies and issuing only low titer products for out of group transfusions.

How to cite this article:
Kannan S, Kulkarni R, Basavarajegowda A. Prevalence of high titered anti-A and anti-B antibodies among O blood group individuals and their associated factors.Glob J Transfus Med 2020;5:187-191

How to cite this URL:
Kannan S, Kulkarni R, Basavarajegowda A. Prevalence of high titered anti-A and anti-B antibodies among O blood group individuals and their associated factors. Glob J Transfus Med [serial online] 2020 [cited 2021 Jun 25 ];5:187-191
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Despite the widespread adoption of component preparation and the use of group specific packed red cells, hemolytic transfusions although greatly reduced, continue to be reported. Component preparation and usage dilute the possible passive transfer of antibodies due to the minimal amount of plasma present in the packed cells and platelets. However, rarely minor incompatibility, i.e., incompatibility between the donor's serum and recipient's cells result in massive, near fatal, and fatal hemolytic reactions in susceptible patients.[1],[2],[3],[4],[5],[6] Issue of ABO identical platelets is not possible across most of the centers due to logistics and inventory management issues. Switching over to ABO compatible groups is especially common with platelets though less common among packed red blood cells. More and more reports of the passive transfer of antibody-induced hemolytic reactions are becoming evident, especially with the increasing use of apheresis derived platelets which carried a high volume of plasma.[1],[2],[3],[4] In many of the severe hemolytic reactions reported, the anti-A and anti-B titers of the incriminated unit were retrospectively found to be extraordinarily high, which if it had been identified in the pretransfusion testing stage, would have prevented the hemolytic reaction in the first place.[7]

Aims and objectives

This study aimed to identify the prevalence of these high titer anti-A and anti-B antibodies in group O donors and identify their hemolyzing capacity. We also determined the presence of IgG-specific anti-A and anti-B antibodies if any, which are unique to O group individuals. We attempted to look for specific factors if any, which contribute to the high titers.

Critical titers of 1:64 or higher was considered high titered antibodies for IgM anti-A and anti-B.[7] Critical titers of 1:256 or higher was considered high titered antibodies for IgG anti-A and anti-B.[8]


Study setting, sample size, and participants

This was a cross-sectional analytical study conducted over 20 months from September 2015 to May 2017 in a tertiary level care teaching hospital blood center in southern India.

The sample size was estimated using the statistical formula for estimating a proportion. With an expected prevalence of high titer anti-A and anti-B antibodies as 63% at 4% absolute precision and 95% confidence interval the required sample size was estimated at 560. Systematic random sampling was followed wherein the sample from every 5th O group blood unit was included in the study. Blood donors who were grouped as O and met the departmental criteria for blood donation were identified by screening and counseling. Once they were willing to be a part of the study, every fifth donor was included purposively till the required sample size was achieved. Repeat donors, the units which tested positive for transfusion transmittable infections and units with insufficient quantity were excluded from the study. The information on demographic profile was gathered from the donor history questionnaire. An additional note was added to record the type of diet and probiotic intake.

Antibody titration

Five milliliters (ml) of clotted sample collected post donation was centrifuged at 3000 rpm for 3 min and serum was separated. ABO forward and reverse grouping were carried out by conventional tube technique methods.

Titration of anti-A and anti-B antibodies was done by the double dilution technique in tubes. This method was used for the identification of both IgM and IgG titers. IgM titers were assessed at room temperatures, whereas IgG titers were assessed at 37°C with Anti-Human Globulin technique. The tubes were serially read and the last tube showing 1+ grade agglutination macroscopically was considered the titer of that antibody.[9]

Hemolysin test

Freshly prepared 5% suspensions of pooled A-cells and B-cells were added to two tubes (for anti-A and anti-B antibodies) and serum from the donor was added along with fresh AB serum as source of complement. The tubes were then incubated at 37°C for 2 h. At the end of 2 h, hemolysis in the supernatant indicated a positive hemolysin test. More than 50% hemolysis (grade 2+ and 3+) was taken as positive test.[10] Samples that were hemolysin test positive were taken for downstream testing for IgG titers after dithiothreitol (DTT) treatment.[11]

Statistical analysis

All the data were collected in a predesigned pro forma and entered into Microsoft Excel sheets and analyzed using IBM SPSS Statistics for Windows, Version 21.0., 2013 (Armonk, NY, USA, IBM Corp.). Ordinal variables like titers were expressed as median and as proportions with respect to the demographic groups. Comparison of the distribution of titers between the diet groups was done by Chi-square test. A P < 0.05 was considered as statistically significant.


The study was approved by Institutional Ethics committee vide letter no JIP/IEC/SC/2015/19/799 dated 26.08.2015.


During the study, 11,250 O group donors donated blood in our department. A total of 560 samples, were included in the study as per our study protocol and inclusion criteria. Three percentage of these samples were Rh “D” negative. The distribution in various titer levels for antibodies (anti-A, anti-B) and their subtypes (IgM, IgG) in the study samples in percentages are shown in [Figure 1]. In the present study, the prevalence of high titer anti-A and anti-B IgM antibodies was 36% and 32%, respectively. The corresponding prevalence of high titer IgG antibodies was 9.6% and 5.5%, respectively. The distribution of general prevalence of various titers in the study population is shown in [Figure 1].{Figure 1}

Association between donor age and gender distribution and high titer anti-A and anti-B antibodies

The eligible age group for blood donation is between 18 and 65 years. The blood donor pool that was part of the study had a relatively young average age. The mean age of the O group donor among the 560 samples analyzed was 33 ± 2.33. Majority of the donations (95.2%) were from males. The total number of females in the sample is 27 (4.8%). The age and gender distribution with their median titers of anti-A and anti-B is shown in [Table 1].{Table 1}

Dietary preferences and distribution of anti-A and anti-B titers

Majority of the donors (81.1%) donors answered that they consumed a mixed diet. The median anti-A and anti B titer among the mixed diet donors were 1:64 and 1:32, respectively. The median IgG anti-A and anti B titers among the mixed diet donors were 1:16 and 1:8, respectively. The distribution of titers among the diet patterns is shown in [Table 2].{Table 2}

[Table 3] compares the association between the diet type and high titers. There was an association between high titered antibodies and the mixed diet type and it was statistically significant by Chi-square test (X2 [1, N = 560] = 5.56, P < 0.05).{Table 3}

Hemolysin test

Of the 560 samples which were tested for hemolysin test, 222 (39.6%) of the samples were positive. On down streaming with DTT treatment 163 samples, i.e., 73.2% of them showed the presence of IgG antibodies. Out of these samples, 54 (33%) showed complete lysis whereas the other 109 samples showed partial lysis. [Table 4] and [Table 5] show the cross tabs for the high titered antibodies and the hemolysin test positivity for anti-A anti-B antibodies, respectively. There was no statistically significant association between the two as shown in tables.{Table 4}{Table 5}

Probiotic intake and high titer anti-A and anti-B antibodies

Of the 11,250, probiotic history was elicited from 80 donors. A positive prophylactic intake without any symptoms was elicited from 20 of the 560 donors in whom titers were performed. The median critical titer of anti-A and anti-B in the probiotic positive donors was 1:32 and 1:8, respectively. A history of probiotic intake was not associated with a rise in critical titer of IgM and IgG antibodies in the donors studied. This is pictorially depicted in [Figure 2].{Figure 2}


The prevalence of high titer anti-A and anti B IgM antibodies was 36% and 32% respectively in our study which is comparable to other studies Kulkarni et al. (32.3%) Olawumi and Olatunji (23%).[12],[13] This study was done by the tube technique with the use of the double dilution method. The reportage of titers depends on the method of dilution used. Various studies that used gel cards for titers or used another method of dilution have presented their data in increasing dilutions of 100, starting from 1:100, 1:200 and so on or as doubling dilution such as 1:2, 1:4, 1:8, 1:16, and so on.[12],[13],[14] In some studies, where universally all samples were done with a single dilution like the study by Quillen et al., a single cut off was used and the resulted or reported as positive or negative for high titers.[14] In the study, they reported 50% high titers when they considered 150 as the titer cut off, 33% when they considered 200 and 25% when they considered 250 as the cut off. This is semi-quantitative as the exact level of high titer is not known.

A recently published study by Bastos et al., showed an association of anti-A and anti-B with hemolysin test at a titer of above 64. However, even among anti-A and anti-B IgM titers ≤64, there were 21.9% and 12.8% donor sera graded as total hemolysis in their study.[15] Establishing the titer of anti-A and anti-B antibodies of group O donors, quantifying and documenting it pretransfusion, may predict the risk of hemolysis and hence may help us in avoiding using such O group donors as “Universal Donors.”

Association of age and gender with high titer anti-A and anti-B antibodies

The average age of the donor in the present study was 33 ± 2.3. A relatively young donor population donates blood at our blood bank. In the study, it has been shown that the median anti-A and anti-B titers were highest in the 4th decade 1:64 and 1:32, respectively, compared to the least titers of 1:8 in the donors aged >50. Our findings are alike the study from Brazil wherein it showed titers falling in men aged more than 40 years and is more accentuated beyond the age of 50 years.[7] Studies gathering prospective information of age and gender and its association with high titer anti-A and anti-B antibodies have shown a consistent association with female gender and high titer antibodies. Antibody titers also peak in middle age with titers falling to below median values in donor above 50.

In the study by De França et al., they have shown a statistically significant association between donor gender (P = 0.008), i.e., female gender and critical titers.[7] This is similar to the present study which shows that females are more frequently associated with high titers. Titers were also high in women in the age group of 30–39 years as shown in our study especially the IgG anti-A titers. This is thought to be due to pregnancy and its related immunizations.[7] They were also high in women more than 50 years, but there were no donors in that age group in females in our study. In this study, an association between gender and titers could not be proved to be statistically significant because of skewed data distribution between male and female donors. Of the 560 samples collected from O group donors, only 27 were female. This is consistent with the donor distribution demographic of our blood bank in which <5% of donations are contributed by female donors.

In the study by Olawumi and Olatunji hemolytic anti-B was twice common than hemolytic anti A whereas titer of hemolytic anti-A was higher than hemolytic anti-B. There was no relationship between sex and age and the prevalence of hemolysins, but they noticed lysis at titers as low as 8. However, their hemolysis was determined by spectrophotometric methods and not visual as in our study.[12]

Association between dietary preferences and probiotic intake and high titers of anti-A and anti B

As with many of the triggering factors such as vaccination and probiotic intake the association between the inciting factor and the development of high titer antibodies is not absolute. The subject may or may not respond to the trigger. Hence, prospective collection of information about such triggers in apparently healthy donors has a very low positive predictive value. In the literature on these trigger factors that have previously reviewed, hardly any study that assessed the prevalence of high titer antibodies was able to prospectively collect this information.[16],[17] In most cases, the index case is a severe or fatal hemolytic reaction which on further investigation was found to be have happened because of passive transfer of high titer anti-A or anti-B antibodies.

The incriminated donor was then be retrospectively traced, either called back for a repeat sampling or retested from the stored sample and found to have such high titered antibodies. These donors are then retrospectively questioned on these so called trigger factors and history of probiotic intake or recent pregnancy, etc., elicited as evidenced in the studies by Boothe et al., and Daniel-Johnson et al., respectively.[16],[17] This approach while providing useful information on the pathogenesis of development of such antibodies, helps devise screening mechanisms. This is however riddled with biases, especially “recall bias.” Furthermore, like in the present study, a good majority of donors who were taking probiotics were deferred due to the primary diagnosis like diarrhea for which they were taking it in the first place, thus falling short to provide robust evidence to prove its association.

Probiotics are dietary supplements which contain bacteria which are alive and are known to be beneficial by deterring bowel colonization, adhesion, and invasion by pathogenic organisms and modulate host immune response. Prophylaxis for gastroenteritis with increased stool frequency is the most common prescription.[18] These bacteria have A or B like substances thereby stimulating antibodies and hence reaching higher titers. Donors who are on probiotics were deferred from donating due to the infectious risks (from the loose stools) involved and hence we had a very small population which could be included in the study.

There were many caveats to this testing. The titration procedure was difficult to standardize resulting in poor reproducibility of results. Significant donor behavioral factors such as probiotic use, and subclinical infections influence these titers. Geographic variation in titers of these antibodies has been described.[12] The anti-A and anti B titers and their relationship with the risk of hemolysis were not linear. A consensus on a universally dangerous lower limit above which the risk of hemolysis begins, could not be arrived at. Donor samples with so called low titers of anti-A and anti-B antibodies could still result in hemolytic transfusion reactions in certain cases. Despite several limitations to this technique, titration studies of anti-A and anti B antibodies are the most cost-effective, feasible, and useful test available for predicting hemolysis. An institute wide audit of the extent of hemolytic transfusion reactions attributable to the passive transfer of high titer anti-A anti-B antibodies will help in establishing the protocol for universal prescreening in O group donors. Due to the wide variation in geographical and environmental impact on the titer of these antibodies, local guidelines based on individual experience will benefit in patient safety rather than universal/nationalized guidelines.[19]

The strength of the study was that a fairly good number of samples were included given the method used for titration was manual which is very cumbersome. Hemolysin test was also performed which is more meaningful clinically than mere titers.

The limitation of the study was that the number female donors in our population was too less to derive a meaningful conclusion with regard to gender and antibody titers. The same was also an issue with probiotic whose intake was in <5% of study population. Although the study gives us a fair idea of prevalence of the high titer antibodies, much larger sample size is required to show its association with specific characters.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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