"Blood grouping" conventionally means assignment of its ABO blood type.  The concept originated with the work of Landsteiner in 1901.  While investigating the properties of blood, he showed that serum separated from the blood of some individuals would cause clumping, or agglutination, of the RBCs isolated from some other individuals, but not RBCs from themselves.  Not all serum and not all RBCs would react.

He identified the response as being due to interaction of antibody in serum with antigens on red cell surfaces, and was able to describe four blood types and measure their frequency of occurrence in the population:

It took about 60 years for Landsteiner's work to be developed into a usable test for classifying blood type in stains.  The first partially successful attempt was introduced by Lattes in 1915.  The basic problem is that the RBCs are destroyed when a stain is formed, and so there is nothing to subject to an agglutination reaction.  Lattes realized that antibodies were less susceptible to degradation in stains and might be detectable.  He developed a method for extraction of antibody and identification with indicator A and B cells.  However, two problems remained.  The first is that the low levels of antibody extractable from stains made the test somewhat unreliable.  The second is that identification of AB blood depends on making a call from a negative observation (no agglutination with A and no agglutination with B indicator cells), which is not a scientifically acceptable thing to do.

Various attempts to type stains by identifying the antigen were tried for the next 45 years, and a sufficiently reliable method, absorption–elution, was eventually introduced in the early 1960s by Kind and by Outterridge. ^01, 02

Absorption-elution depends on detecting antibody that has been bound by A or B antigen on immobilized fragments of the lysed cell surface.  The antibody is then eluted and detected with A or B indicator cells.  Since group O blood has no antigen, there will be no bound antibody to elute and detect.  That means that almost half of the samples tested would produce no detectable result.  However, group O cells do in fact possess antigen on their surface.  It is a precursor of the A and B antigens and is designated as H substance.  Certain botanical extracts called lectins can act like antibodies, and the extract from Ulex europaeus reacts with H substance to agglutinate group O cells.  The extract is called H-lectin.  The chemistry of A, B and H is dealt with in the section on Semen. 

The procedure works because the IgM antibody molecules have an optimum reaction temperature of around 4°C. The first step, specific binding, is conducted at 4°C and then the temperature raised to 56°C which disrupts the non-covalent Ag-Ab interactions and elutes antibody from its bound state.  H-lectin behaves sufficiently similarly to permit identification of group O stains.  Note that since H is a precursor of A and B, and since absorption-elution is very sensitive, A and B stains will usually also display H activity.

Immobilization of stain can be achieved in various ways.  The two most widely used are fixing a single thread from the stain (or from a swab of the stain if it is not on fabric) onto a glass or plastic plate with glue (nail polish) or heat, or making an extract of the stain in 5% ammonia and heat fixing it to the plate.

Characterizing body fluid stains by absorption-elution typing for ABO group was one of the most significant advances in forensic biology.  However the technique has its problems.  The major difficulty is the presence of adventitious blood group substance.  That is, stains can sometimes produce false positive results, especially for group B.  Running appropriate negative controls is a vital step and results from any sample that shows a false positive has to be discarded.  Common evidential substrates such as denim fabric and soiled shoes often give false positives.

The end-point — agglutination of test cells by eluted antibody — is subject to some variability also.  Different observers may read weak results differently, and many samples will come up positive if left long enough.  Confirmation of test readings by a qualified second reader is a key quality control measure.

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