The ABO Blood Group System: Genetics and Serology

The ABO locus sits on chromosome 9q34.2 and contains three common alleles: A, B, and O. The A and B alleles encode glycosyltransferases that differ from each other at only four amino acid positions, yet those differences determine which sugar is added to H antigen and therefore which antigen is produced. The O allele contains a single nucleotide deletion that causes a frameshift and a premature stop codon, resulting in a truncated, non-functional enzyme. A person who inherits two O alleles (genotype OO) produces no ABO transferase activity; their red cells and secretions carry H antigen without further modification, and they are phenotypically group O.

Because A and B are both expressed when both alleles are present, AB individuals produce both antigens on their red cells. Both A and B are dominant over O at the protein level. There are also subgroups within A and B: A1 and A2 are the most common A subgroups, with A1 cells carrying more A antigen sites per cell than A2 cells. The distinction matters in transfusion medicine and occasionally in forensic interpretation, because anti-A1 lectin (from Dolichos biflorus seeds) can be used to distinguish A1 from A2 cells.

The serum antibodies in adults are produced naturally, without prior exposure to red cells of the relevant type, and are predominantly IgM class. They develop in the first months of life, likely stimulated by cross-reactive carbohydrate antigens on gut bacteria. Anti-A and anti-B are clinically important because they cause rapid intravascular haemolysis if ABO-incompatible blood is transfused, and forensically important because they are the reagents used in reverse grouping.

H antigen is a carbohydrate structure built on glycoproteins and glycolipids on the red cell surface. Its synthesis is directed by the FUT1 gene (also called H gene), which encodes an alpha-2-L-fucosyltransferase that adds fucose to the terminal galactose of a precursor chain. The ABO transferases then act on H antigen: without H antigen, neither A nor B antigens can be assembled.

A separate gene, FUT2 (Se gene), controls H antigen secretion in exocrine glands. Individuals who are homozygous for non-functional FUT2 alleles are non-secretors: their red cells carry normal ABO antigens, but their saliva, semen, vaginal secretions, and other body fluids contain no detectable ABO antigens. Secretors, who carry at least one functional FUT2 allele, produce H antigen in these fluids, on which ABO transferases can add A or B specificities. Approximately 80 percent of Europeans and most other populations studied are secretors, though the proportion varies by ethnic group.

H antigen itself varies in quantity across ABO groups: group O cells carry the most H antigen because no transferase acts on it; group A1 cells carry the least because most H antigen has been converted to A antigen. This relative H content is exploited using lectins such as Ulex europaeus agglutinin (anti-H), which agglutinates O cells strongly and A1 cells weakly. The anti-H lectin test is used to confirm suspected group O samples and to detect the Bombay phenotype.

ABO grouping in a clinical or forensic reference laboratory uses two complementary tests performed simultaneously. Forward grouping tests the patient's or donor's red cells against two reagent antibodies: monoclonal or polyclonal anti-A and anti-B. Where the reagent antibody reacts with the cell antigen, agglutination occurs. Reverse grouping tests the patient's serum against two reagent cell suspensions: A1 cells (from group A1 donors) and B cells (from group B donors). The expected result is that a group A patient's serum will agglutinate B cells (because it contains anti-B) but not A1 cells.

Discrepancies between forward and reverse grouping results must be resolved before a blood group is reported. Common causes include: weak or absent antibodies in neonates (who have not yet developed their own anti-A and anti-B) or elderly or immunosuppressed adults; ABO subgroups such as A2 with anti-A1 in the serum; rouleaux formation (a false agglutination pattern); cold-reactive alloantibodies that interfere with reverse grouping; and acquired changes to ABO antigens in disease states. In forensic serology, where serum is often unavailable, only forward grouping is possible; the interpretation is stated accordingly.

Several methods have been used to determine ABO type from biological crime scene stains. The method of choice depends on the specimen type, age, and condition of the stain.

Absorption-inhibition is the classic method for secretion stains. A known quantity of anti-A or anti-B antibody is incubated with an extract of the stain. If the stain contains A or B antigen, it will absorb (bind and neutralise) the corresponding antibody. The residual antibody activity is then tested against known reagent red cells. Reduced agglutination in the indicator step indicates that the stain absorbed the antibody, confirming the corresponding antigen was present. The method requires careful controls and is semi-quantitative at best.

Absorption-elution is used for bloodstains where red cell remnants may be present. Anti-A or anti-B antibody is absorbed onto the stain, then eluted by heating. The eluate is tested against known reagent cells. Agglutination in the eluate test indicates the antibody was bound and released, confirming the corresponding antigen. Absorption-elution is more sensitive than absorption-inhibition for small or degraded bloodstains.

Mixed agglutination is a variant in which antibody is absorbed onto the stain cells and indicator cells of known type are then added. The indicator cells become trapped in agglutination complexes with the stain cells if the antibody linked them. This method can detect ABO antigens on non-erythroid cells such as epithelial cells in saliva stains.

Modern forensic laboratories have largely replaced these methods with DNA-based ABO genotyping, which determines the ABO alleles directly from the biological evidence without requiring intact antigens. PCR amplification of the ABO gene region, followed by restriction enzyme analysis or sequencing, can identify A, B, and O alleles and distinguish common A subgroups. DNA genotyping is more sensitive, more reproducible, and more amenable to mixtures, but it does not replace antigen-based methods in all settings. Genotyping gives the underlying allele, which predicts the phenotype; it does not directly detect the antigen and cannot determine secretor status unless FUT2 is also genotyped.

When the biological evidence is a secretion stain (saliva, semen, vaginal fluid, breast milk), the first step is to determine whether the contributor is a secretor. If the individual is a non-secretor, ABO typing of the stain will yield a negative result regardless of their blood group, because non-secretors produce no ABO-active substances in their body fluids. A negative ABO result from a secretion stain is therefore not informative about blood group; it tells the analyst only that the contributor may be a non-secretor.

Secretor status is determined by testing the body fluid stain for H antigen using anti-H lectin (Ulex europaeus agglutinin). Group O secretors will show strong H activity; group A or B secretors will show moderate H activity (because some H is converted to A or B). Non-secretors of all ABO groups will show no H activity in their secretions. This test must be performed before ABO typing is attempted and must be included in the report alongside the ABO result.

The forensic significance of secretor typing has shifted since DNA profiling became routine. In a rape investigation, the semen stain was historically ABO-typed and secretor-tested to narrow the suspect pool. Today, DNA profiling provides far greater discrimination power. However, where the DNA is degraded or insufficient for full STR profiling, ABO and secretor typing can still provide probative information. In resource-limited settings across parts of South Asia, sub-Saharan Africa, and Latin America, serological blood group typing remains in routine forensic use.

ABO blood group evidence is primarily exclusionary in forensic casework. If the ABO group of a crime scene stain differs from that of the suspect, the suspect is excluded as the donor of that stain, subject to the caveat that ABO typing is correct and the stain is from a single source. This exclusionary power is the strongest argument for retaining ABO typing in jurisdictions where DNA evidence is not available for every specimen. An exclusion requires no probabilistic interpretation; it is categorical.

Where the ABO group of the stain matches the suspect, the finding cannot individualise. Because ABO groups are shared by large proportions of the population, a match is consistent with the suspect being the donor but does not prove it. The strength of a concordant ABO result depends on the rarity of the group in the relevant population. A match on group AB (approximately 3 to 5 percent in most European populations) is stronger evidence than a match on group O (approximately 44 percent). In most jurisdictions today, concordant ABO alone is not presented as a significant incriminating finding; it is used as preliminary screening or corroboration.

The legal weight given to ABO evidence varies across jurisdictions. Under the Bharatiya Sakshya Adhiniyam 2023 in India (which replaced the Indian Evidence Act 1872), serological evidence is admissible as expert opinion; courts have used ABO typing in paternity disputes and in cases where DNA was not available. In the United States, ABO typing evidence has been admitted under Federal Rule of Evidence 702, subject to Daubert reliability standards, and has featured in both inclusion and exclusion contexts. UK courts under the Police and Criminal Evidence Act 1984 framework and the Criminal Procedure Rules require disclosure of both the method and its limitations, including population frequencies. EU member states vary in their rules on scientific evidence, but the trend across the European Investigation Order framework is toward disclosure of uncertainty and population statistics.

Mixed stains present a particular problem for ABO typing. If a sample contains blood or secretions from two donors of different ABO groups, the typing result may show both antigens, which can be misinterpreted as a single group AB donor. Forensic serologists must consider mixed stain possibilities and, where they are suspected, refer the question to DNA profiling rather than relying on ABO alone. This limitation was recognised in historical cases where incorrect ABO interpretations contributed to miscarriages of justice, including cases reviewed by appellate courts in the UK and Australia in the 1990s and 2000s.