Determination of blood group HAP Lab Manual PDF MCQ Video Synopsis
To Determine the Blood Group of one’s own blood By ABO System & Rhesus System.
- Glass slide
- Glass marking pencil
- Sterile cotton
- Pricking needle / lancet
- Test tube
- Convex lens
- anti A serum, anti B Serum, anti D serum.
- Normal saline
- 70% alcohol
Theory / Principal :
It was in 1901, that Austrian-American immunologist and pathologist Karl Landsteiner discovered human blood groups. Karl Landsteiner’s work helps to determine blood groups and thus opens a way for blood transfusions that can be carried out safely. He was awarded the Nobel Prize in Physiology or Medicine in 1930 for this discovery.
Death of the patient was the result in most cases before 1900 when blood transfusion was attempted. Blood transfusion was made much safer by the discovery of blood groups, as the blood of the same ABO group could be chosen for each patient. However, there were still many cases of unexplained blood transfusion reactions. Biologists still went in search of these unexplained questions.
In 1902, the fourth main type, AB, was founded by Decastrello and Sturli. It was the observations of Levine and Stetson in 1939, and Landsteiner and Weiner in 1940 that laid the foundations of our knowledge about the remaining major blood group, the Rhesus system.
The Components of Blood
The circulatory system distributes about 4-6 litres of blood to the adult human body. The blood mainly has 2 portions:
Plasma and Blood Cells. Plasma is mainly composed of water but contains different types of proteins and other chemicals such as enzymes, glucose, fat particles, salts, hormones, antibodies etc. It constitutes about 60% of the blood. Blood cells can be observed under a microscope on staining. The formation of blood cells occurs in the bone marrow by the ‘Hematopoietic stem cells. They can be divided into 3 basic cell types: RBC, WBC, Platelets.
Erythrocytes- Red Blood Cells (RBC):
As the name suggests, these red coloured cells give blood its red colour. (The word erythrocyte is from erythro in Greek meaning red and Latin-cytos meaning cell.)
1 ml of blood contains approximately 5 million RBCs. The proportion of blood occupied by red blood cells is referred to as the hematocrit and is normally about 45%. Mature RBCs are biconcave in shape, lack a Nucleus and many other organelles. They circulate in the system for about 120 days, carrying out their job, i.e., to supply oxygen.
Individuals have different types of antigens on the surfaces of their red blood cells. These antigens, which are inherited, determine the individual’s blood group. In addition, individuals make antibodies to these antigens, but not to their own type of antigen, since if they did the antigens and antibodies would react causing a transfusion reaction. The main signs are clumping of red blood cells, haemolysis, and shock and kidney failure.
These antibodies circulate in the bloodstream and the ability to make them, like the antigens, is genetically determined and not associated with acquired immunity. If individuals are transfused with blood of the same group, i.e. possessing the same antigens on the surface of the cells, their immune system will not recognise them as foreign and will not reject them.
However, if they are given blood from an individual of a different blood type, i.e. with a different type of antigen on the red cells, their immune system will attack upon them and destroy the transfused cells. This is the basis of the transfusion reaction; the two blood types, the donor and the recipient, are incompatible.
There are many different collections of red cell surface antigens, but the most important are the ABO and the Rhesus systems.
About 55% of the population has either A-type antigens (blood group A), B-type antigens (blood group B) or both (blood group AB) on their red cell surface. The remaining 45% have neither A nor B type antigens (blood group O). The corresponding antibodies are called anti-A and anti- B. Blood group A individuals cannot make anti-A (and therefore do not have these antibodies in their plasma), since otherwise, a reaction to their own cells would occur: they do, however, make anti-B. Blood group B individuals, for the same reasons, make only anti-A. Blood group AB makes neither, and blood group O makes both anti-A and anti-B
Because blood group AB people make neither anti-A nor anti-B antibodies, they are known as universal recipients: transfusion of either type A or type B blood into these individuals is safe since there are no antibodies to react with them. Conversely, group O people have neither A nor B antigens on their red cell membranes, and their blood may be safely transfused into A, B, AB or O types; group O is known as the universal donor.
The Rhesus system:
This system was first discovered in Rhesus monkeys, hence called the Rh system. In this system, there are six antigens but there are no naturally occurring antibodies. The antigens are C, D, E. c. d and e. Out of these D is most significant immunologically.
The Rh system has two blood groups:
i. Rh positive
D antigen is present
D antigen is absent
In the Indian population, 95-98% are Rh-positive and 2-5 % are Rh-negative.
The red blood cell membranes have an important antigen, the Rhesus (Rh) antigen, or Rhesus factor. About 85% of people have this antigen; they are Rhesus positive (Rh+) and do not, therefore, make anti-Rhesus antibodies.
The remaining 15% have no Rhesus antigen (they are Rhesus negative individuals capable of making anti-Rhesus antibodies, but are stimulated to do so only in certain circumstances. The result of an incompatible blood transfusion.
A person with Rh-blood does not have Rh antibodies naturally in the blood plasma. But a person with Rh- blood can develop Rh antibodies in the blood plasma if he or she receives blood from a person with Rh+ blood, whose Rh antigens can trigger the production of Rh antibodies (as the immune system is triggered by the presence of an unknown antigen in the system). A person with Rh+ blood can receive blood from a person with Rh-blood without any problems.
The principle behind blood tests: Blood clumping or Agglutination observation.
Compatibility between the blood groups of donor and recipient determines the success of a blood transfusion.
The ABO and Rh blood groups are looked at while conducting the test. In a diagnostic lab, Monoclonal antibodies/substitute antibodies (laboratory-made proteins that mimic the immune system’s ability to fight off harmful pathogens) are available for A, B and Rh antigens. Monoclonal antibody against Antigen A (also called Anti-A), comes in small bottles with droppers; the monoclonal suspension being BLUE in colour. Anti-B comes in YELLOW colour. Anti-D (monoclonal antibody against Rh) is colourless. The monoclonal antibody bottles should be stored in a refrigerator. It is recommended to tilt the bottle a couple of times before use in order to resuspend the antibodies that have settled at the bottom of the bottle.
All the colour codes are universal standards. When the monoclonal antibodies are added one by one to wells that contain the test sample (blood from patient), if the RBCs in that particular sample carry the corresponding Antigen, clumps can be observed in the corresponding wells. A drop of blood is left without adding any of the antibodies; it is used as a control in the experiment. The monoclonal antibody bottles should be stored in a refrigerator. It is recommended to tilt the bottle a couple of times before use in order to resuspend the antibodies that have settled at the bottom of the bottle.
Clinically, the important blood groups are:
1. ABO system
2. Rh system
3. The M and N system is important from a medico-legal point of view.
The ABO blood group system is important for all blood group systems. In this system, there are two antigens, antigen A and antigen B. Based on the presence or absence of these antigens, blood groups are classified into four classes:
i. Blood group A
Antigen A is present
ii. Blood group B
Antigen B is present
iii. Blood group AB
Both A and B antigens are present
Neither A nor B antigen is present
iv. Blood group O
A blood type (blood group) is a classification of blood-based on the presence and absence of antibodies and also based on the presence or absence of inherited antigenic substances on the surface of RBCs.
Time needed: 30 minutes
Determination of blood group
- Glass slide preparation
Divide the glass slide into three parts and mark them as anti-A, anti-B and anti-D.
- Finger pricking
Get a deep finger prick under aseptic conditions to get free-flowing blood
Make a perfect one drop
- Take sample
Put it in 3 different places on a slide. Or one can go for an alternative method By taking 2 ml of normal saline in a watch glass add a drop of blood after pricking the finger in all aseptic conditions in saline. Add one drop of saline suspension of cells with the help of dropper into every pit of slide marked as anti- A, anti- B, anti- D.
- Add Antiserum
Then add Antiserum A, Antiserum B and Antiserum D to the 3 drops simultaneously
- Take control sample
Also one can go for control Add two drops of this saline suspension of cells where you have marked S on tile or on a glass slide. It will act as a control to compare with agglutinated cells.
Then mix the two with the help of the separate toothpicks.
- Agglutination reaction
Wait for 8-10 minutes and observe the slide for agglutination reaction i.e. clumping and haemolysis of red cells
- Observe result
Then see the results. Agglutination can be compared with a saline mixture of cells which is taken as control.
- Observe under microscope
If clumping is not visible with naked eyes, observe the slides under a low power microscope.
1) If clumping of RBC in Antiserum A it is blood group A.
2) If clumping of RBC in Antiserum B it is blood group B.
3) If clumping of RBC in Antiserum A & Antiserum B it is blood group AB.
4) If there is no clumping of RBC in Antiserum A & Antiserum B it is blood group O.
5) If clumping of RBC in Antiserum D it is Rh-Positive.
6) If no clumping of RBC in Antiserum D it is Rh Negative
Determination of blood group is of great clinical importance especially in
- blood transfusion.: In blood transfusion cases, the blood group of recipient and donor must be matched; otherwise, agglutination or clumping of RBC occurs and results in fetal reactions.
- In Hemolytic disease of newborn ( HDN), / erythroblastosis fetalis,
- In Paternity diseases of newborn and also in medico-legal practice for paternity disputes.
1. Tile or slide should be clean, dry and grease-free.
2. Mark the slides as A, B and D.marking them is very important for accurate results.
3. Use applicator sticks separately for each antiserum.
4. There should be no intermixing of the mixture to avoid contamination.
5. Finger should be pricked deep enough.
6. Ensure free flow of blood.
7. The antiserum should be stored immediately in the refrigerator after use.
8. Check the conditions and expiry date of antiserum before use.
9. The skin should be dry if the puncture is 3-4 mm deep to give free-flowing blood the blood flow.
Different blood group systems are ABO, Rh MN, S, P, E, Kell, Duffy, Kidd etc.
The ABO group system has A, B, AB and O blood groups. The ABO blood group is based on two glycolipid antigens (agglutinogens) called A and B.
On the basis of the presence of these antigens blood group systems are classified.
Human RBCs contain on their surface (cell membrane) a series of glycoproteins and glycolipids which constitute blood group antigens. The development of these antigens is genetically controlled. They appear early in fetal life and remain till death.
People whose RBCs display only antigen A have type A blood group. Those who have only antigen A are type A. Individuals with both A and B antigens are type AB, whereas those who have neither antigen A nor B are type O.
The MN system is usually required for paternity tests. It is important in medico-legal cases of paternity disputes. MN groups are also useful in genetic and anthropological studies.
Agglutination is the clumping of red cells due to an antigen-antibody reaction that is visible to the naked eyes. Mixing of incompatible blood causes agglutination.
Karl Landsteiner, Austrian American immunologist and pathologist who received the 1930 Nobel Prize for Physiology or Medicine for his discovery of the major blood groups and the development of the ABO system of blood.
Landsteiner’s law states that, for whichever ABO antigen is not present on the red cells, the corresponding antibody is found in the plasma or vice versa. This law is applicable to the ABO group system and the only first half of the law is applicable to the Rh group system.
Universal donor – A person who can donate blood safely anyone is a ‘universal donor”. Persons with blood group ‘O’ negative are considered as universal donors, as their red cells contain no antigens.
Universal Recipient Person who can receive blood safely from anyone is ‘universal recipient. Persons with blood group ‘AB’ positive are considered to be universal recipients as their plasma contains no antibodies.
1) To study human genetics.
2) Problems of identity and parentage.
3) Study of anthropogeny.
4) Association and proneness to disease.
6) In Forensic medicine e.g. group A people suffer more from gastric carcinoma
7) Rh group is important for marriage counselling.
8) To ensure compatible blood transfusion.
Blood transfusion is defined as a collection of whole blood from the donor and its infusion into the venous blood of the recipient.
Transfusion can be of whole blood or it can be as packed red cells, white cells, platelets or fresh frozen plasma. Even if carefully supervised the blood transfusion carries a definite risk. Thus transfusion should be given only if another simpler and safer therapy has proved ineffective.
i. Chronic anaemia
iii. Deficiency of clotting factors
ii. Severe haemorrhage
iv. Bone marrow suppression
Common indications for blood transfusion are –
Pre and postoperative.
Acute blood loss
Bone marrow failure
Purpura Clotting factor deficiencies.
Complications of blood transfusion are
Febrile reactions Fever, rigor. Allergic reactions itching, urticaria, erythema, anaphylactic reaction
Circulatory overload – heart failure, pulmonary edema
Haemolytic reactions – jaundice, haemoglobinuria
Reactions due to infected blood fever, rigors, peripheral circulatory failure Transmission of diseases hepatitis, malaria, AIDS, syphilis.
Transfusion hemosiderosis (excessive accumulation of iron deposits called hemosiderin in the tissues.)
Post transfusion purpura ( is a delayed adverse reaction to a blood transfusion or platelet transfusion that occurs when the body has produced alloantibodies (an antibody formed in response to pregnancy, transfusion, or transplantation targeted against a blood group antigen that is not present on the person’s red blood cells.) to the allogeneic transfused platelets’ antigens. These alloantibodies destroy the patient’s platelets leading to thrombocytopenia, a rapid decline in platelet count.)
Thrombophlebitis ( inflammation of the wall of a vein with associated thrombosis)
Air embolism due to faulty technique of transfusion.
Citrate toxicity (Tetany) due to the rapid rate of transfusion.
Blood is collected from the donor and the recipient. Plasma and red cells are separated in each. Then the donor’s cells are mixed with the recipient’s plasma. (major cross). The recipient’s cells are matched with the donor’s plasma. (minor cross). This is called cross matching and agglutination is observed. If agglutination does not occur, the recipient does not have antibodies that will attack the donor RBCs.
Alternatively, the recipient’s serum can be screened against a test panel of RBCs having antigens.
When incompatible blood is transfused in a recipient, the donor’s cells get agglutinated because against their agglutinogen, there is a high agglutinin concentration in the recipient’s plasma. Because of the high concentration of agglutinins, the reaction can easily take place (between donor’s cells and recipient’s plasma) and therefore matching of donor’s cells with the recipient’s plasma is called major cross-matching.
The reaction of donor’s plasma and recipient’s cells is not very important and usually does not occur on giving mismatched transfusion therefore it is called minor cross-matching. Normally the amount of donor’s blood given is about 500 ml. Half of it is only plasma (about 250 ml). This donor’s plasma when enters the recipient’s circulation, gets diluted with the recipient’s blood. Therefore the concentration of donor’s agglutinins in the recipient’s blood is very low (not sufficient to cause agglutination of recipient’s cells).
When incompatible blood is transfused it is called mismatched transfusion.
Causes of mismatched blood transfusion are:
1) Most important and commonest cause is an error in blood grouping or cross-matching (blood
2) Error in the identification of blood due to incorrect labelling.
Due to mismatched blood transfusion mainly donor’s cells agglutinate in the recipient’s circulation. Agglutinated red cells get haemolysed. The immediate severe haemolytic reaction occurs.
Impacts of mismatched blood transfusion: –
1) Phase of haemolytic shock – Fever, chills, nausea, vomiting, breathlessness, hypotension, peripheral circulatory failure. The severity and onset of symptoms depend on the rate of intravascular red cell destruction.
2) Post Shock phase – Because of red cell destruction there is the presence of free haemoglobin in the blood (hemoglobinemia). This in turn causes the greater formation of bilirubin leading to jaundice and haemoglobinuria. Since free haemoglobin binds with
3) Oliguric phase – In many (but not all) the patients with the haemolytic reaction the kidneys.
are damaged due to the development of acute tubular necrosis and renal failure. This occurs
mainly due to
The Rh blood group is so named because the antigen was discovered in the blood of the Rhesus monkey.
People whose, RBCs have Rh antigen are designated Rh* (Rh-positive); those who lack Rh antigen are designated Rh (Rh-negative).
Rh blood group is determined by the agglutination test. In this test, blood is mixed with antisera D which contains anti-D antibodies. If RBCs contain Rh antigen then agglutination is observed and the person is known as Rh. If agglutination is absent, the person is known as Rh.
Rh system has six agglutinogens known as C, D, E, c, d and e. Out of these D antigen is of clinical importance. The red cells which are agglutinated are Rh-positive, whereas those not agglutinated are called Rh-negative.
In Rh group system natural antibodies against Rh are not present i.e. an Rh -ve person does not have agglutinins against Rh antigen. Therefore by any chance Rh-negative person receives Rh +ve blood for the first time, Rh antigen causes sensitization and a large number of agglutinins are formed against Rh antigen. If a person is given Rh-positive blood next time, he gets a severe reaction. Similarly in Rh-negative women bearing Rh +ve fetus for the first time sensitization can occur.
At the time of delivery, fetal RBCs enter the maternal circulation, the mother is sensitized and agglutinins against Rh antigen are formed. If next time also woman bears Rh +ve fetus, then the fetus is likely to suffer because agglutinins pass from mother to fetus through placenta causing agglutination of fetal RBCs leading to a disease known as erythroblastosis fetalis. It is treated by giving exchange transfusion.
Hemolytic disease of the newborn is also called as erythroblastosis fetalis. This is an example of Rh incompatibility which occurs during pregnancy.
When a woman is pregnant, there is no direct contact occurs between maternal and fetal blood. It occurs when the Rh mother carries the Rh fetus. In this case, a small amount of Rh blood leaks from the fetus through the placenta into the bloodstream of Rh mother and evoke anti-Rh antibodies. The fetal blood leakage into the maternal circulation occurs at delivery, the firstborn baby usually is not affected. If the mother becomes pregnant again, her anti-Rh antibodies can cross the placenta and pass into a fetus.
If the fetus is Rh, there is no problem (Rh blood does not have the Rh antigen). But if the fetus is Rh antigen-antibody reaction causes hemolysis of fetal RBCs.
Hemolytic disease of the newborn can be prevented by an injection of anti-Rh antibodies called anti-Rh gamma globulin. These antibodies bind to and inactivate the fetal Rh antigens if they are present. Therefore, the mother’s immune system does not respond to the foreign antigens by producing antibodies.
All Rh women should receive anti-Rh gamma globulin soon after every delivery, miscarriage, or abortion.
Erythroblastosis fetalis can be prevented by desensitizing the pregnant Rh-negative mother (having Rh +ve fetus) by injecting anti-Rh antibodies.
Normally, plasma does not contain anti-Rh antibodies. If an Rh person receives Rh blood transfusion the immune system starts to make anti-Rh antibodies that will remain in the blood. If a second transfusion of Rh blood is given later, the previously formed anti-Rh antibodies will cause hemolysis of RBCs in the donated blood which leads to a severe reaction
Secretor status refers to the presence or absence of water-soluble ABO blood group antigens in a person’s bodily fluids, such as saliva, tears, breast milk, urine, and semen. (except the CSF) People who secrete these antigens in their bodily fluids are referred to as secretors, while people who do not are termed non-secretors.
Blood is collected with complete aseptic precautions. It is stored in a sterile container. To every 480 ml of blood 120 ml of ACD mixture is added. Blood is stored at 4°C. Blood can be used up to 21 days if stored by taking the above precautions. If blood is stored for a long time there can occur lysis of RBCs, WBCs. Various platelet coagulation factors (factor V, VIII) are destroyed.
Acid Citrate Dextrose (ACD) Solution A, also known as Anticoagulant Citrate Dextrose Solution is used as an anti-coagulant for whole blood and erythrocyte survival, routinely used for blood storage. The ideal red blood cells shelf life is 21 days when stored in ACD solution.
ACD mixture contains
i) Acid citric (monohydrous) – 0.48 g
ii) Trisodium citrate – 1.32 g
iii) Dextrose – 1.47 g
iv) Distilled water to make 100 ml
RBCs in the blood tend to swell and haemolyse unless the Na K pump remains active for maintenance of their size. This pump activity requires energy. When blood is taken out. this energy is provided by glucose added to the ACD mixture. If glucose is not added rate of haemolysis of RBCs would be very high
Following precautions are taken for selecting the donor –
i) Donors should be healthy.
ii) Donors should not suffer from diseases like syphilis, malaria, AIDS etc, which are spread
iii) Donor’s haemoglobin and PCV should be within normal range. This is grossly determined
by measuring the specific gravity of fresh capillary blood.
Following precautions should be taken during blood transfusion
i) Transfusion should be done only if it is absolutely indicated.
ii) Donor’s and recipient’s blood groups should be checked. Cross-matching should be done
to exclude mismatching of blood groups other than ABO.
iii) Rh blood group of the donor and recipient should be determined. Rh +ve blood should
never be transfused to Rh -ve person.
iv) Before starting the transfusion, the label on the blood bottle should be checked for the name and the blood group. Warm the blood up to room temperature.
v) Transfusion should be given at a slow rate (not more than 20 drops per minute). If
it is given fast, citrate present in the stored blood may cause chelation of calcium in the recipient’s blood leading to tetany.
vi) Proper aseptic precautions must be taken during transfusion.
1) Packed red cells –
2) Platelet concentrate
3) Plasma, human albumin –
4) Cryoprecipitate, fibrinogen
5) Fresh frozen plasma
Human Anatomy and Physiology Practical Syllabus
- Study of a compound microscope.
- Microscopic study of epithelial and connective tissue
- Microscopic study of muscular and nervous tissue
- Identification of axial bones
- Identification of appendicular bones
- Introduction to hemocytometry.
- Enumeration of white blood cell (WBC) count
- Enumeration of total red blood corpuscles (RBC) count
- Determination of the bleeding time
- Determination of clotting time
- Estimation of haemoglobin content
- Determination of blood group.
- Determination of erythrocyte sedimentation rate (ESR).
- Determination of heart rate and pulse rate.
- Recording of blood pressure.
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