Blood transfusion. Blood groups, Rh – factor. Methods of determination, typical mistakes.


Hemotransfusion therapy includes transfusion of a whole blood, blood components and products. Blood transfusion should be considered as serious operation - transplantation of a living tissue, which fulfills its biological functions in a recipients organism.


Main historical events

1628 — English physician William Harvey described completely and in details blood circulatory system and properties of blood being pumped to the body by the heart;


1665 — First official registered blood transfusions: British physician Richard Lower showed the possibility for blood to be transfused from animal to animal and from animal to man intravenously;


1667 — Jean-Baptiste Denys in France and Richard Lower in UK noted separately about successful blood transfusion from a ship to a man. But next 10 years blood transfusions from animals to men were denied due to very high mortality;


1818 — James Blundell, British obstetrician, carried out first successful transfusion of human blood to the women with postpartum hemorrhage;


1900 — Karl Landsteiner, Austrian physician, discovered first three groups of blood – A, B and C. Later group C was recalled as O. For his discoveries he received the Nobel Prize in 1930;


1914 — Long-term anticoagulants were invented which allowed to preserve donor blood.

Blood consists of two main compounds – blood plasma and suspended blood cells. RBCs, WBCs, blood platelets and plasma are four blood components. In adults blood cells are about 40-50 %, plasma — 50-60 %. This ratio is called hematocrit (Ht) or packed cell volume (PCV) or erythrocyte volume fraction (EVF).

All blood antigens are divided on cellular and plasma antigens. The main role in transfusiology plays cellular antigens.

Cellular antigens:

RBC antigens;

WBC antigens;

Platelet antigens

Human erythrocytes contain antigens of several systems simultaneously. Identical combinations of blood groups of two persons are rare. It is possible only at uniovular twins, whose blood group antigens are identical.


The ABO system

Today is the main antigen system determining compatibility or incompatibility of transfused blood.

А and В Agglutinogens (antigens) are located on the surface of erythrocytes.

α and β Agglutinins (antibodies) are located in blood plasma.

α agglutinin is antibody for А agglutinogen;

β agglutinin is antibody for В agglutinogen.

Blood of a man can not contain the same-called agglutinogens

and agglutinins.


ABO antigens are expressed on 3rd month of gestation at high density on almost all human cells. And do not change during all life.


ABO antibodies do not arise spontaneously but are normally induced during the first year of life by exposure to ABO-like substances common to flora colonizing the normal gut. Infants then develop antibodies to the A antigen or В antigen, or both, which are absent in their own cells.



Classic blood groups

Group 0(I) There are no agglutinogens in erythrocytes, blood plasma contains α and β agglutinins;

Group A(II) There is A agglutinogen in erythrocytes, blood plasma contains β agglutinins;

Group B(III) There is B agglutinogen in erythrocytes, blood plasma contain α agglutinins;

Group AB(IV) There are А and В agglutinogens in erythrocytes, there are no agglutinins in blood plasma;


Subtypes of agglutinogen А.

There are 2 main subtypes: А1 (≈ 88%) А2 (≈ 12%);

According to it group А(II) has 2 subgroups: А1(II) and А2(II); group АВ(IV) – A1B(IV) and A2B(IV)

А1 subtype has high ability to agglutination;

А2 subtype has low ability to agglutination;

Blood plasma of А2(II) and A2B subgroups in 20% cases contains α1 extraagglutinin, which agglutinates only with erythrocytes А1;

There are subtypes of erythrocytes with much lower ability to agglutination due to other subtypes of agglutinogens: А3, А4, АZ



Bombay blood phenomenon.

All types of erythrocytes contain H antigen, which is precursor of antigens A and B.

Individuals with the rare Bombay phenotype do not express H antigen, the antigen which is present even in blood group O. As a result, they cannot make A antigen or B antigen. For this reason people who have Bombay phenotype can donate blood to any member of the ABO blood group system, but they cannot receive any member of the ABO blood group system's blood (which always contains one or more of A and B and H antigens), but only from other people who have Bombay phenotype.



Rh Antigen System.

· The are 5 most important antigens: D, C, c, E, e;

· An individual either has, or does not have, the D antigen on the surface of their red blood cells;

· The status is usually indicated by Rh positive (Rh+ does have the D antigen) or Rh negative (Rh- does not have the D antigen) suffix;

· Immunogenicity of other antigens is much lower and decreases as follows: с > Е > С > е


·Blood groups identification.

By standard serum;

By standard serum and standard erythrocytes (crossing method);

By monoclonal antibodies (anti-A, anti-B)


Blood groups identification by standard serum.

Equipment:

Standard serum of 2 different series

0(I) – white;

A(II) – blue;

B(III) – red;

AB(IV) – yellow


This colors are used to mark all labels on blood components (red blood cells, plasma etc.) Dry white porcelain, enameled or plastic plates, marked on areas 0(I), A(II), B(III), AB(IV); Isotonic sodium chloride solution;Needles, droppers, sterile swabs etc.


Method:

Divide the plate into 4 parts with a colour pencil and label the parts clockwise - I (0), II (A), III (B)

Place the serum (each drop is about 0,1ml) of two different series of groups I (0), II (A), III (B) on the corresponding areas using their individual pipettes.

Place the blood drops (about 0,01ml) near the serum and mixed them. Blood drop should be in 5-10 times smaller than drop of standard serum!!!

Shake the plate mixing the serum and blood. Agglutination starts in first 10-30 sec., but the observation should be made for 5 min!!!

Add a few drops of normal saline, and shake the plates again to mix the drops.

Assess the results

Result.

Positive agglutination:

Small red grains appear in the mixture, which are consisted of agglutinated erythrocytes. Small grains merge in larger grains or sometimes in flakes.


Negative agglutination:

The drop remains uniformly colored, there is no observed any grains (agglutinates).

Results in drops of different series should be the same!!!

Observation is done for 5min!!!


Blood groups identification by crossing method

Includes 2 stages:

Blood groups identification by standard serum;

Blood groups identification by standard washed red blood cells

Blood is taken from a vein into a tube. Blood plasma is isolated by centrifugation.

Place 3 large drops (0,1ml) of patient's plasma on the labeled plate. Then add 3 small drops (0,01ml) of standard erythrocytes;

Mix the drops, shake the plate slightly, observe during 5min;

Add the sodium chloride into agglutinated drops;

Assess the results


·Identification with monoclonal antibodies

·This reagent is a diluted ascetic fluid of mice carriers of hybridomas that are producing of IgM against antigens A or B. As distinct from the standard ABO-serum, this reagents provides a quicker and more pronounced reaction of agglutination.

·Technique:

· Place large drops (0,1ml) of anti—A and anti-B antibodies on a labeled plate or a flat plastic surface ;

· Place small drops (0.01ml) of testing blood and mix them with the reagent;

· Shake the plate slightly and observe for about 2,5 minutes (the reaction normally occurs with­in 3—5 seconds to form small red aggregates fol­lowed by flakes)


In contrast to the standard ABO-serum, the monoclonal antibodies provides a quicker and more pronounced reaction of agglutination. Using of monoclonal antibodies eliminates the risk of transmission of hepatitis B or C viruses or HIDV.


Possible mistakes of blood groups identifications

Low quality of reagents;

Technical mistakes:

· Low lighting;

· Temperature above 25°С sharply slows down the reaction;

· Cold panagglutination (temperature below 15°С);

· Early assessment of the results (especially when А2 antigen is present);

· Not adding of saline (pseudoagglutination is possible)

·Features of testing blood

· Panagglutination;

· Autoagglutination;

· Decreased ability of agglutinogens to agglutination

·In all cases of doubtful or careless results reidentification should be made with standard serum of 2 series or with crossing method.


Rh-factor identification.

Routinely detection of D antigen is enough for Rh-identification;

E and C antigens should be assessed only in donor blood. Only donors without D, C and E antigens in their blood are considered as rhesus-negative.


Methods of Rh identificationApplicable in clinical practice

Express method with universal anti-Rh reagent in a test tube;

Express method with anti-Rh serum of group IV (AB) diluted in 20—30% albumin;

Reaction with anti-D monoclonal antibodies

Laboratory methods

Agglutination in saline solution;

Agglutination with gelatin;


Indirect antiglobulin test (Coombs` test);

Express method with universal anti-Rh reagent in a test tube requires special reagent — anti-Rh serum of group IV (AB) diluted by 33% “Dextran 70” (“Polyglucin”) used as the

conglutin, i.e. that substance that allows agglutination of red blood cells at room

temperature.

Procedure

• Put a drop of the universal anti-Rh reagent into a test tube;

• Add 1 drop of RBCs, which Rh-factor should be identified;

• mix these with turning of a test tube to let a mixture be smeared on the walls of a test tube from inside during 3 minutes;

• add 2-3 ml of normal saline to the mixture turning a test tube;

• Observe the results.

Results

1. Agglutination of the red blood cells present with the universal anti-Rh reagent indicates Rh-positive blood.

2. The absence of agglutination with the universal anti-Rh reagent indicates Rh-negative blood.

An express method of Rh identification on a slide requires special reagents — anti-Rh serum of group IV (AB) diluted in 20—30% albumin used as the conglutin, i.e. that substance that allows aggregation of red blood cells under room temperature.


Procedure

• Put a drop of the anti-Rh serum of group IV (AB) and nearly a drop of Rh-negative serum of group IV (AB) free of antibodies on a ground slide or a Petri dish;

• add to each drop of serum 2—3 times less than the patient's blood in the amounts half even less as much as those of the serum;

• mix these with a glass rod or by shaking for 3-4 minutes;

• add one drop of normal saline to each mixture;

• Observe the results in 5 minutes.


Results

1. Agglutination of the red blood cells present with the anti Rh serum and absent with the control serum implies Rh-positive blood.

2. The absence of agglutination with the serum is indicative of the patient's blood being Rh-negative.

3. In case of agglutination with both sera the reaction has to be regarded as unclear.

In emergency, transfusion of only Rh-negative blood is possible, and if it is not available and the patient's condition requires blood transfusion, Rh-positive blood may be given following crossmatching the blood for Rh compatibility.



Rh identification with monoclonal reagent “Anti-D-super”.

The monoclonal reagent “Anti-D-super” is a diluted ascitic fluid of mice, carriers of hybridoma, which produces Ig I (antibodies) against D-antigen.

Procedure

• Place big drops of “Anti –D-super” reagent on a labelled plate or a flat plastic surface.

• Put the drop of blood in question (which should be one-tenth as big in size nearby and mix).

• Shake the plate slightly and observe for about 3 minutes (the reaction normally occurs within 30-60 seconds to form small red aggregates followed by flakes). If the reaction with anti –D-super reagent is positive, i.e. the blood is supposed to be Rh-positive blood. Otherwise it is Rh-negative.


Contraindications to blood transfusion.

decompensated cardiovascular failure IIIB-IV degree (heart defects, myocarditis, myocardiosclerosis; septic endocarditis);

disturbance of cerebral blood circulation; pulmonary edema;

thromboembolism, hemorragic vasculitis; acute glomerulonephritis; acute hepatic, hepato-renal or reno-hepatic failure; general amyloidosis;

active disseminated pulmonary tuberculosis;allergic condition (i.e. bronchial asthma).


Main actions of hemotransfusiologist before blood transfusion .

a.to define the indications to blood transfusion;

b. to define contraindications to blood transfusion;

c. to choose product for transfusion, method and type of transfusion;

d. to determine blood group and Rh- factor of the patient and to compare the results with a record in a case history;

e. to perform visual evaluation of blood or blood component / product, correct labeling of a blood unit;

f. to determine donors blood group and Rh- factor, taken from blood unit, and to compare result with a record on a label;

g. to perform individual АВО and Rh- compatibility crossmatches;

h. to perform biological compatibility crossmatch.

Main actions of hemotransfusiologist during blood