Practical laboratory Physiology I

BLOOD GROUP SYSTEMS

Human erythrocytes have a large number of antigens (Ag) on their surface. The Ag are grouped
in 30 systems who defy blood groups, the most important being ABO and Rh (Table I). The clinical
importance is related with alloantibody (antibody against some antigens the patient doesn`t have)
capacity to cause transfused erythrocytes destruction or to pass through placenta and starts
hemolisis in fetal circulation.

Table I. Main antigenic systems in blood groups.

SYSTEM FENOTYPE FREQUENCY
CAUCASIANS (%)
ABO 0 44
A 42
B 11
AB 4
Rh Dce 2
DCcEe 13
Dce 15
Dce 19
Dcce 35
DcE 12
DCE 2

1. ERITROCYTIC ANTIGENS

Erytrocytic antigens (aglutinogens): are found only on erythrocytes (Rh antigens), on

erythrocytes and tissues (ABO antigens) or in other blood cells, and there are carbohydrates or
peptides with genetic determination.

2. ANTIERITROCYTIC ANTIBODY

Antibody against erytrocytic antigens (aglutinines): are natural occurring antibodies and post
immune antibody.

2.1. NATURAL ANTIBODY

Most of them are IgM type. It is believed that they are produced by exposing to substances
from environment or diet that has similar structure with erytrocytic antigens. Antibodies anti A, B
and A+B are active at 37oC. Most of the other antibodies aren`t active at body temperature, and
they are without clinical importance.

2.2. POSTIMMUNE ANTIBODY

Most of them are IgG and there are formed by exposing to foreign erythrocytes, by transfusion
or pregnancy. D antigen has intense immunogenity, but also antigens K and c determine immune

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reactions. The capacity to perform an immune reaction variant, for example the patients with
autoimmune diseases have a bigger predisposition to develop antierytrocytic antibody (32% of
hemolytic anemia), the probability to develop antibody is very small in hipogamaglobulinemia
(chronic lymphatic leukemia) and in the first months.
IgM are more efficient than IgG in complement activation (one molecule of IgM can activate
the complement, to activate the complement by IgG requires 2 molecules linked in close sites),
producing intense hemolisis. IgG are represented by 4 fractions with different capacity to activate
the complement and they can pass through placenta, with fetal erythrocytes hemolisis.

3. ABO SYSTEM

ABO system was discovered by Landsteiner in 1901, who observed that the erythrocytes of one
person mixed with other persons plasma can agglutinate.
Hemagglutination represents the gathering of erythrocytes in visible piles through antigen-
antibody binding.
Hemolisis represent the destruction of erythrocytes and can occur extra vascular as a
consequence of antigen-antibody binding or intravascular through antigen-antibody binding and
complement activation.
Is a system formed of two antigens (agglutinogens) A and B expressed on red cell surface and
two antibodies (agglutinins) α and β, in plasma. In routine clinical practice, individuals are grouped
as O, A, B and AB. Several subgroups of A and B are known, but they are rarely of clinical
significance. Group A individuals may rarely acquire a B antigen, associated with gastrointestinal
carcinoma, by releasing an enzyme that converts the membrane structure. Individuals with
acquired B being transfused with AB red cells develop severe hemolytic reactions, following the
production of hyper immune antiB antibodies.

Fig. 1. Characteristics of blood groups.

ABO blood groups are defined by the presence of antigens on erythrocytes surface and antibody
in plasma (Fig. 1)

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ABO system is the most important blood group system due to the presence of complement
fixing antibody at 37oC which can cause intravascular hemolisis of transfused red blood cells. ABO
antigens are associated with the predisposition to gastric neoplasm (group A), or ulcer (group 0).

4. RHESUS SYSTEM (Rh)

Rh system is a complex system with 45 antigens. From those RhD antigens have high
imunogenity and are present in 85% of Caucasians.
Anti Rh antibodies are not normally found in plasma, they appear in two situations:
- Following D-mismatched transfusion, there is a 90% chance that a D negative recipient will
form anti-D. A second exposure to D positive red cells will produce a prompt anamnestic
immune response, with hemolysis.
- A D-negative woman carrying an ABO compatible D-positive fetus has a 1 in 6 chance of
forming anti-D antibodies. These antibodies can cross the placenta giving rise to the hemolytic
disease of new born. We can prevent this disease by stopping the formation of Anti-RhD
antibodies by RhD negative mothers with an injectable medication called Rho (D) immune
globulin.
Apart from D, four antigens C, c, E, e are associated with antibodies that account for almost all
Rh related transfusion problems, but their immunogenity is low.

5. METHODS FOR BLOOD GROUP TYPING IN ABO AND Rh SYSTEMS

5.1. BETH-VINCENT-TZANK METHOD

PRINCIPLE
Mix on a slide the serum test (contains known group antibodies) with patient’s red cells.
Haemaglutination appears in the presence of corresponding antigens and antibody-antigen
complex development.

MATHERIALS
 antiAB serum test (contains α and  antibodies)
 antiB serum test (contains  antibodies)
 antiA serum test (contains α antibodies)
 glass or white plastic slides, sterile niddle, cotton pads, alcohol.
 syringe, test tube and glass wand for venopuncture.

PROCEDURE
We can use capillary blood, from finger or venous blood without anticlothing agent.
On a clean and dry slide place a drop of anti-AB serum, a drop of anti-B serum and a drop of
anti-A serum.
Disinfect the patients finger with alcohol, puncture with a sterile needle, collect the blood with
the corner of a slide (with the first corner mix blood with anti AB serum, with the second corner
mix blood with antiB serum, with the third corner mix blood with antiA serum and with the last
corner mix blood with antiRh serum).

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The blood/serum test ratio should be 1/10-1/20, to avoid fibrinogen pseudo agglutination.
The slide is shacked to mix test serum with blood and the result is read in max. 3 minutes
against a white surface.
The presence or absence of agglutination in each weal is noted.

RESULTS

anti AB serum anti B anti A

Gr O
(No Ag)

Gr A
(Ag A)

Gr B
(Ag B)

Gr AB
(Ag A, B)
Beth Vincent Tzanck method

5.2. SIMONIN METHOD

The method is based on the same principle. The patient’s serum (contains unknown antibodies)
is mixed with test red cells (group O, A and B red cells).

5.3. BLOOD GROUP TYPING IN Rh SYSTEM

PRINCIPLE
Monoclonal antibodies (against D antigen) or polyclonal antibodies (against D and other
antigens) are mixed with patient’s blood.

MATHERIALS
 antiRh (D) serum antibodies;
 Rh (D)+ red cells (control);
 Rh (D)- red cells (control);
 glass or white plastic slides;
 test tube and glass wand for venopuncture;
 incubator.

PROCEDURE
Blood is obtained by finger puncture or venopuncture without aticlothing agent, when after
clothing; the clot is dispersed and is obtained the red blood cells in serum.
On the slide drop patient’s red cells, Rh (D)+ red cells, Rh (D)- red cells.

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The test serum/red cells report should be 1/10-1/20.
The slide is placed in a box on wet absorbent paper and placed into incubator (37oC). The result
is read after 6 min.

RESULTS
 if agglutination appears, D antigen is present  Rh (D)+ blood group;
 if agglutination does not appears, D antigen is absent  Rh (D)- blood group.
Results are related to control wails.
ERROR FACTORS IN BLOOD GROUP TYPING
 expired serum, infected, the presence of unspecific agglutinin at cold (inactive antibody at 37oC
but active at room temperature);
 hemolised red blood cells, with weak antigens A2 (does not agglutinate), infected
(agglutination in all samples);
 infected blood, anexines in serum (membrane proteins which can determine hemolisis or lack
of agglutination), unspecific agglutinins, weak antigens;
 inappropriate temperature and incubation (false negative Rh)

6. CLINICAL REVELANCE IN BLOOD GROUP TYPING IN ABO AND Rh SYSTEMS

6.1. BLOOD TRANSFUSION

Donated blood can be administered as whole blood, or for a better efficiency, as fractionated
blood products..
The main blood products are:
 erythrocytes concentrate;
 trombocytes concentrate;
 cryoprecipitate (concentrate with clot factors);
 fresh frozen plasma (contains labile clotting factors V and VII). Clot factors used in hemophilia
can be sintetised also by genetic recombination, so transfusion complications are avoid.

BLOOD GROUP AB
Individuals have both A and B antigens on the surface of their RBCs and their blood serum do
not contain any antibodies against either A or B antigen. Therefore, an individual with type AB
blood can receive blood from any group (with AB being preferable), but can donate blood only to
another type AB individual.

BLOOD GROUP A
An individual have the A antigen on the surface of their RBCs, and blood serum containing IgM
antibodies against the B antigen. Therefore, a group A individual can receive blood only from
individuals of groups A or O (with A being preferable), and can donate blood to individuals with
type A or AB.

BLOOD GROUP B
Individuals have the B antigen on the surface of their RBCs, and blood serum containing IgM
antibodies against the A antigen. Therefore, a group B individual can receive blood only from

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individuals of groups B or O (with B being preferable), and can donate blood to individuals with
type B or AB.

BLOOD GROUP O
Individuals do not have either A or B antigens on the surface of their RBCs, but their blood
serum contain IgM anti-A antibodies and anti-B antibodies against the A and B blood group
antigens. Therefore, a group O individual can receive blood only from a group O individual, but can
donate blood to individuals of any ABO blood group.

Rh NEGATIVE PATIENTS
Can receive blood Rh negative compatible ABO, and patients Rh positive can receive blood Rh
positive or negative (is preferred positive, Rh negative is present in only 15% of population).
Transfusion will be done only after direct compatibility test, in the absence of unspecific
antibody (negative cross-mach, the absence of agglutination of red blood cells of donor with
patients’ plasma). If a patient requires emergency blood and there is no time for tests will be
administrate blood group O Rh negative.

6.2. POSTTRANSFUSION COMPLICATIONS

 acute hemolisis can occur after transfusion with incompatible ABO red blood cells or plasma
with increased antibody concentration and it occurs intravascular, secondary to complement
activation with IgM antibody. Clinically the patient presents with fever, frison, hypotension
tachycardia, dispneea, hemoglobinuria and the evolution can be severe by disseminated
intravascular clotting and renal failure.
 Delayed hemolisis, after 5-7 days is due to an immune response secondary to reexposure to an
erytrocytic antigen, after a first exposure in pregnancy or transfusion. It is extra vascular,
without complement activation, with antibody IgG anti Rh, Kidd, Duffy or Kell. Clinically the
patient presents fever and decreased hemoglobin, rarely with severe evolution.
 Transfused blood can be hemolised by bacteria contamination, temperature variations cooling
or heating, drug administration on the same vein, enzyme deficiency.

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 Fever occurs at patients previously sensitized (through pregnancy or transfusions) at leucocytic
or trombocytic antigens.
 Acute pulmonary edema (TRALI syndrome) through elimination of acute phase reactives from
transfused leucocytes and increased capillary permeability.
 Anaphylactic reaction and urticaria through mastocytic degranulation.
 Bacterial infection (Yersinia enterocolitica, Pseudomonas, Staphylococus aureus), viral (viruses
hepatitis, HIV), parasitic.
 thrombocytopenic purpur, through unspecific antibody.
In order to avoid mistakes and diseases was implemented the following protocol, mandatory
before any blood transfusion:
- verify every blood of groups ABO, Rh and Kell trough 2 methods in 2 different laboratory;
- determine patient`s blood group ABO and Rh;
- bacteriological and virusological verification of transfused blood;
- perform direct compatibility reaction with Jeanbreau method by putting in contact donor
erythrocytes with receiver serum.

6.3. IN NEW-BORN HAEMOLYTIC DISEASE (see Rh system).

6.4. IN FORENSIC MEDICINE to exclude paternity when DNA testing is not available.

6.5. IN ORGAN AND BONE MARROW TRANSPLANT HLA compatible. Because ABO antigens are
present on most epithelial surface and endothelia, the transplant incompatible solid organs
increases the potential for hyperacute graft rejection. Major ABO incompatible stem cell
transplants will provoke hemolysis, unless the donation is depleted of red cells.

IMPORTANT!!!
The ambulance brings victims from an accident that requires blood transfusions. You must
determine patient`s blood group and transfuse compatible blood.
Attention! If you transfuse incompatible blood the patient`s life will be in danger......

MULTIPLE CHOICE QUESTIONS (ONE CORRECT ANSWER)

1. A blood group red cells express on the cell surface:

A. A antigen
B. B antigen
C. No ABO antigens
D.  antibodies

2. B blood group red cells express on the cell surface:

A. A antigen
B. B antigen
C. A and B antigens
D. No ABO antigens

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3. O blood group red cells express on the cell surface:
A. A antigen
B. B antigen
C. No ABO antigens
D. Both A and B antigens

4. A blood group plasma contains :

A.  antibodies
B.  antibodies
C.  and  antibodies
D. No ABO antigens

5. B blood group plasma contains:

A.  antibodies
B.  antibodies
C.  and  antibodies
D. A antigen

6. AB blood group plasma contains:

A.  antibodies
B.  antibodies
C.  and  antibodies
D. No ABO system antibodies

7. A Rh (D)+ blood group persons have:

A. D antigen on the red cell surface
B. A antigen on the red cell surface
C. Anti Rh(D) antibodies in plasma
D.  antibodies in plasma

8. The following sentences are correct:

A. D antigen is higly immunogenic
B. Anti Rh antibodies concentration follow-up is mandatory in pregnancy
C. If Rh- blood is transfused, antiRh antibodies can lead to haemolysis
D. AntiRh antibodies are natural and present at birth

9. One of the following sentences regarding blood groups is not correct:

A. Two methods typing is mandatory
B. Simonin method is based on patient’s serum and test red cell agglutination
C. Rh typing requires 37oC incubation
D. Direct compatibility test is not mandatory

10. Immune agglutinins:

A. Are mainly IgG
B. Can appear in pregnancy

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C. Appear after Rh- blood transfusion
D. Appear at environmental exposure

Answers: 1-A, 2-B, 3-C, 4-B, 5-A, 6-D, 7-A, 8-A, 9-D, 10-B