Blood groups and the process of cross matching

This chapter is relevant to Section Q1(ii) of the 2023 CICM Primary Syllabus, which expects the exam candidates to "explain the major blood groups and principles of cross matching". This contrasts with the old 2017 syllabus entry, which asked for the "process" of crossmatching. It has appeared in Question 2 from the second paper of 2010 and Question 9(p.2) from the first paper of 2008, as well as in some Part Two exam papers, where an abbreviated revision page reminds people how this works in case they forgot. In a refreshing change for these syllabus items, this one has discrete boundaries for what is expected, and you can see the edges of it when you study it.

In summary:

  • Blood group is defined by the antigens found on the surface of red cells
    • Numerous antigen molecules exist, of which A, B and the Rhesus antigens are the most common
    • A and B are carbohydrate (oligosaccharide) molecules on the glycocalyx of red cells
    • The presence of the antigen identifies the blood group, i.e. the patient with the AB blood group has A and B antigens on the surface of their red cells
    • Antibodies (IgM) to the other antigens usually develop in the first months of life
    • O, A, B and AB are phenotypes. 
    • An "O" phenotype indicates that the red cells have neither A nor B antigens.
      Group  Antigens  Antibodies Can receive blood from:  Can donate blood to:
      A A Anti-B A and O A and AB
      B B Anti-A B and O B and AB
      AB A and B None Everybody AB
      O None

      Anti-A and Anti-B

      Only O Everybody
  • The presence of blood groups in the general population:
    • O group: 49% of the population
    • A group: 38% of the population
    • B group: 10% of the population
    • AB group: 3% of the population
    • 85% of the population is Rh-positive
  • Laboratory determination of ABO grouping:
    • Forward grouping: patient RBCS mixed with known anti-A and anti-B antibodies
    • Reverse grouping:  patient's plasma is mixed with known Type A or Type B red cells
    • Both must agree to formally identify the blood group
  • Compatibility testing:
    • Determines compatibility for tranfusion by directly testing donor and recipient samples
    • Saline agglutination: looks for anti-ABO IgM in the blood of the recipient.
      • Immediate-spin crossmatch: patient serum and donor red cells are centrifguged for 10 minutes and checked for agglutination
    • Indirect antiglobulin test: looks for clinically significant IgG antibodies.
      • Patient's plasma is  incubated together with the donor red cells

For something as ubiquitous and straightforward as this, it was surprisingly difficult to find a good single article to explain all the important parts. Fortunately Pourazar et al (2007) is close enough, and has the benefit of being free. Though it is not a peer-reviewed resource, transfusion.com.au is a pretty good resource for this topic, and contains extensive (but readable) documentation for health professionals, with a more fastidious referencing style then what you might get from many textbooks.

Erythrocyte antigens

The need to be able to safely perform transfusion is the main reason we even have blood groups as a concept. The basis of the blood antigen grouping system is that some people have red cells with antigens which will trigger an impressive immune reaction if they are infused into other people, because those other people have antibodies that recognise them. If it were not for this inconvenient problem, we would not look twice at most of these antigen molecules, as their physiological role can be described as "background extras". For example, A and B antigens are oligosaccharides thought to be a part of the glycocalyx, in the sense that we find them sitting around in the glycocalyx of the red cell, and the Rhesus antigen is a transmembrane transport protein.  For even more example, here's a breakdown of about thirty blood group antigens from Daniels (2006), featuring their biochemistry and possible function.

blood group antigenss and their supposed functions from Daniels (2006)

As you can see, they are numerous, all totally different, and none of them have anything in common, except that they tend to resemble some naturally occurring chemical for which you do not develop self-tolerance. That forms the basis of their antigenicity. For example, for the AB group, at some stage in your life (usually in the first 3-6 months after birth) you will encounter a whole host of different oligosaccharides and develop antibodies to the ones which do not resemble your own native oligosaccharides. 

Consider an example. Let's say your own red cells may be sprouting Oligosaccharide A. Then, in the process of exploring a Duplo block with your mouth you encounter a species of kitchen floor yeast (Horrendosporidium sp.) with a cell envelope looking exactly like Oligosaccharide A. You will not form an antibody to this yeast envelope component, as self tolerance must be maintained. Then, on the other side of the kitchen, you may encounter a species of Horrendomyces sp. which has a cell envelope closely resembling Oligosaccharide B. Through your exposure to this antigen, you will develop antibodies (IgM) which will forever recognise and destroy Horrendomyces on sight. Unfortunately, they will also react to any blood cells with the B antigen on their surface. 

Why, one might ask, do we tolerate this seemingly useless and inefficient diversity, as a species?  Why indeed. The physiological purpose of anything is of course a purely speculative philosophical exercise, and so with this. But intelligent people have had a poke at this question. For example, in the middle of the massive biochemistry textbook by Berg (2007), the author speculates that a diverse range of self-tolerance is a necessary strategy for a species to remain robust in the face of pandemic infections. If all individuals in a population had a uniform level of self-tolerance for Horrendomyces and their immune systems ignored it completely, an epidemic of horrendomycosis could conceivably come along and wipe out the entire species. This sort of diversity comes from vigorous genetic mixing; human and primate societies which are characterised by their isolation (mountain gorillas, Siberians, the Swiss) tend to have antigenic monoculture (eg. 88% of all chimpanzees are blood group A).

ABO antigen phenotype groups

To recover from the digressions above, the following facts can be firmly stated about ABO blood group phenotypes:

Blood group phenotype Antigens Antibodies Can receive blood from:
A A Anti-B A and O
B B Anti-A B and O
AB A and B None Everybody
O None

Anti-A and Anti-B

Only O

Additionally, there is the Rhesus antigen, named in error after the rhesus macaque. This system remains relevant because of the possible haemolytic disease of the newborn which could occur if the Rh-negative mother becomes exposed to foetal Rh-positive blood.

How common are these phenotypes in the human population?

  • O group: 49% of the population
  • A group: 38% of the population
  • B group: 10% of the population
  • AB group: 3% of the population
  • Weird antigens is how one might colloquially refer to the 400+ other antigenic molecules which can be expressed on the surface of red cells, and these are usually present in less than 1% of the population.
  • Rh antigen is positive in 85% of Caucasians, and can be up to 100% in Asian populations

In case you were about to completely ignore these irrelevant-seeming statistical factoids, be warned: the "prevalence of blood groups in the general population" was considered an essential part of the answer to Question 9(p.2) from the first paper of 2008. Fortunately for the exam candidate, these numbers must all be completely random, as the prevalence of red cell antigens in the community varies considerably with the ethnicity of the population and the geographic region, which means that theoretically one could pull out any plausible-sounding numbers and still be accurate. The ones quoted above come from the Australian Red Cross.

To summarise:

  • Everyone can receive O blood (the cells have no agglutinins on them).
  • O people can have O blood only (they have antibodies to both A and B agglutinins)
  • A people can get A or O blood only (they have anti-B agglutinins)
  • B people can get B or O nlood only (they have anti-A agglutinins)
  • AB people can receive any blood: they have no anti-aggutinin antibodies, but their AB cells are covered in agglutinins.
  • Nobody other than other AB people can receive AB blood (as it is covered in A and B agglutinins)

The laboratory tests for red cell antigens

Otherwise known as "typing" or "grouping", this is an essential pre-transfusion test. 

  • Forward grouping is where you take the patient's red cells and mix them with anti-A and anti-B antibodies. If the blood agglutinates, it reveals the presence of the antigen in question, i.e. logically if the anti-A antibodies make it clag, the A antigens are present.
  • Reverse grouping  is where you take the patient's plasma and mix it with known Type A or Type B red cells to see if the cells agglutinate; this looks for anti-A and anti-B antibodies in the patient's plasma. 
  • Both forward and reverse grouping must agree in order for the result to be formally accepted as valid; an ambiguous result suggests that the patient has some sort of weird subgroup, or has recently received a bunch of incorrectly matched cells, or has experienced foetomaternal haemorrhage, etc.
  • RhD typing is carried out routinely on all blood, and involves testing the patient's blood for the presence of Rhesus D antigen using known anti-D sera. 
  • Antibody screening is a much more involved process which tests for the presence of clinically significant antibodies in the patient's plasma.  The patient’s plasma is tested by an indirect antiglobulin test against a selection of red cells each of which has a known antigen profile. 

Crossmatching

Pretranfusion compatibility testing ("group and hold" or "crossmatch") tests the patient's blood with a donated sample to make sure they are compatible, i.e. no agglutination takes place. This typically detects ABO incompatibilities, but could also pick up weird antibody groups which you might not have expected. The benefit of this process is that it labels a donated sample of blood as "compatible", meaning it is safe to transfuse that bag of donated cells into the recipient. The Australian Red Cross has a page that describes the process of compatibility testing with references. 

  • Saline agglutination: This is a test to detect anti-ABO IgM in the blood of the recipient. It is usually done by immediate-spin crossmatch. A couple of drops of a patient's serum are added to a drop of the donor red cells and then centrifuged with some saline. It is possible to read the test immediately after ten minutes of centrifuge time, i.e this is a relatively rapid test. 
  • Indirect antiglobulin)  is a test which is usually not necessary, and is only performed where the patient is expected to have clinically significant IgG antibodies. The test requires that the patient's plasma be incubated together with the recipients' red cells, which typically takes longer.

A much more detailed account of what is (has to be) an incredibly precise piece of laboratory work is described in the ANZSBT Guideline from 2016, which will surely be out of date by the time that you are reading this. It, in turn, is heavily based on the British version (2014).

References

Pourazar, Abbasali. "Red cell antigens: Structure and function." Asian journal of transfusion science 1.1 (2007): 24.

Daniels, G. "Structure and function of red cell surface antigens." ISBT Science Series 1.1 (2006): 3-8.

Pawlak, Z., and M. Lopez. "Development of antigens ABH and Ii in children between 0 to 16 years old." Revue francaise de transfusion et immuno-hematologie 22.3 (1979): 253-263.

Berg, J.M., Tymoczko, J.L. & Stryer, L. (2007). Biochemistry, 6th Ed. (p. 315). New York, NY: W.H. Freeman

ANZSBT: GUIDELINES FOR THE ADMINISTRATION OF BLOOD PRODUCTS (2018)

British Committee for Standards in Haematology, et al. "Guidelines for pre‐transfusion compatibility procedures in blood transfusion laboratories." Transfusion Medicine 23.1 (2013): 3-35.