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Blood Group Test Procedure and Results

Blood group test is a way to classify blood based on the presence or absence of certain antigens and antibodies. The most common blood grouping system is the ABO system, which has four main blood types: A, B, AB, and O. Additionally, there is the Rh (Rhesus) system, which classifies blood based on the presence or absence of the RhD antigen. Understanding blood group test is important in the field of medicine, as it plays a crucial role in blood transfusions and transplantation. In this blog, we will explore the different blood groups, how they are determined, types and their clinical significance.

What is Blood Grouping?

Blood grouping is a method of classifying blood based on the presence or absence of certain antigens and antibodies. The discovery of blood grouping was made by Karl Landsteiner in 1901, an Austrian-American immunologist and pathologist, who received the Nobel Prize in Physiology or Medicine for this discovery. His work laid the foundation for safe blood transfusions.

There are two types of blood grouping, forward and reverse. In forward grouping, a known source of antibodies is used to detect antigens on red blood cells. In reverse grouping, reagent cells with known ABO antigens are used to test a patient’s serum for ABO group antibodies.

The ABO and Rh systems are the most clinically significant and important blood group systems. The ABO system classifies blood into four groups based on the presence or absence of inherited antigenic substances on red blood cells. These groups are A, B, AB, and O. Each group has a specific combination of antigens and antibodies.

The Rh system, also known as the Rhesus system, is the second most important blood group system in humans. The most significant antigen in this system is the RhD antigen. People can be classified as Rh+ or Rh- based on the presence or absence of this antigen on their red blood cells. Rh- individuals do not have Rh antibodies, but they can develop them if they receive blood from an Rh+ individual. Rh+ individuals can receive blood from Rh- individuals without any issues. The Rh system is controlled by two allelic genes, RhD and RhCE.

Objective of Blood Grouping

The purpose of blood group test is to classify blood according to the presence or absence of specific antigens and antibodies, as well as to evaluate the compatibility of blood between persons. This is significant in the medical world, especially in the areas of blood transfusions and transplantation.

Principle of Blood Grouping

The ABO and Rh blood grouping system uses agglutination reactions to determine an individual’s blood type. This is done by exposing red blood cells carrying specific antigens to corresponding antibodies, which causes visible clumping or agglutination.

The ABO antigens, for example, are O-linked glycoproteins that are located on the surface of red blood cells. The type of sugar residue present determines whether the antigen is A or B. Individuals with blood group A have A antigens on their red blood cells and anti-B antibodies in their serum. Similarly, blood group B individuals have B antigens on their red blood cells and anti-A antibodies in their serum. 

Blood group AB individuals have both A and B antigens on their red blood cells and no anti-A or anti-B antibodies in their serum. On the other hand, individuals with blood group O have neither A nor B antigens on their red blood cells, but possess both anti-A and anti-B antibodies in their serum. The Rh antigens, on the other hand, are transmembrane proteins that are found on the surface of red blood cells and interact with corresponding antibodies.

ABO and Rh blood grouping system diagram

Figure 1: ABO and Rh blood grouping system

Materials for Blood Grouping

Anti A Sera

Anti B Sera

Anti RhD Sera

Cavity slide

Disposable Mixing Stick

Blood Lancet

70% Alcohol/ Spirit

Cotton Swab

ABO Blood Grouping Procedure

The Blood Grouping test can be performed using either the Tile or Slide method or the Tube method.

a) Slide method

  1. Label a slide or tile and place 1 drop each of anti A (blue) and anti B (yellow) reagents, and D reagent separately. 
  2. Add 1 drop of blood to each of the typing antiserum drops. Mix the cells and reagents with a clean stick, spreading the mixture evenly on the slide or tile in a 10-15mm diameter area. 
  3. Tilt the slide or mix with a stick and observe for agglutination with the naked eye for 5 minutes at room temperature. If agglutination is minimal, confirm with a microscope.

a) Tube method

  1. Label five test tubes as 1, 2, 3, 4, and 5. Add one drop of anti-serum A in tube 1, anti-serum B in tube 2, and anti-serum AB in tube 3. 
  2. Add one drop of the patient’s serum in tubes 4 and 5. Add one drop of known RBC in A1 and B in tube 4 and 5.
  3. Add one drop of a 5% RBC suspension of the patient in tubes 1, 2, and 3. Incubate the tubes at room temperature for 60 to 90 minutes or centrifuge for 15 seconds. 
  4. Observe microscopically for agglutination or lysis using optical assistance, referring to table 2 for guidance.

Result of blood Group Test

a) Slide method

ABO Blood grouping slide or tile Method Procedure and result

Figure 2: ABO Blood grouping slide or tile result

S. No.Anti AAnti BAnti RhDBlood Group
Slide 1AgglutinationNo agglutinationAgglutinationA +ve
Slide 2No agglutinationAgglutinationAgglutinationB +ve
Slide 3AgglutinationAgglutinationAgglutinationAB +ve
Slide 4No agglutinationNo agglutinationAgglutinationO +ve

a) Tube method

ABO Blood grouping Tube Method Procedure and result

Figure 3: ABO Blood grouping tube result.

Tube 1Tube 2Tube 3Tube 4Tube 5Blood group
anti-Aanti-Banti-ABA1- RBCB-RBC 
NegativeNegativeNegativePositivePositiveO
PositiveNegativePositiveNegativePositiveA
NegativePositivePositivePositiveNegativeB
PositivePositivePositiveNegativeNegativeAB

Rh Blood Group Test Procedure

Both the slide method and the tube method can be used to figure out the Rh blood group.

a) Slide method

  1. Obtain two slides, one labeled as “patient” and the other as “control.” On the patient slide, add one drop of Anti-Rh D. On the control slide, add one drop of albumin. 
  2. Next, add one drop of a 40-50% red blood cell suspension to both slides. 
  3. Mix the contents of each slide thoroughly. Observe for any agglutination on both slides.

a) Tube method

  1. Obtain two tubes, one labeled as “patient” and the other as “control.” In the patient tube, add one drop of Anti-Rh D. In the control tube, add one drop of albumin. 
  2. Next, add one drop of a 2-5% suspension of the patient’s red blood cells in both tubes. Mix the contents of each tube thoroughly, then centrifuge them. 
  3. Gently resuspend the samples tubes and observe for any agglutination.

Rh Blood Group Test Result

a) Slide method

A positive result occurs when the patient sample displays agglutination and the control sample remains in suspension. A negative result occurs when both the patient and control samples remain in suspension.

a) Tube method

An Rh-D positive result is indicated by agglutination in the patient’s tube and smooth suspension in the control tube. An Rh-D negative result is indicated by smooth suspension in both tubes.

Interpretation of Blood Grouping

An individual’s blood group can be determined by the presence or absence of agglutination when their blood is mixed with specific reagents.

  1. If agglutination is observed when blood is mixed with Anti A reagent, the individual is blood group A.
  2. If agglutination is observed when blood is mixed with Anti B reagent, the individual is blood group B.
  3. If agglutination is observed when blood is mixed with both Anti A and Anti B reagents, the individual is blood group AB.
  4. If no agglutination is observed when blood is mixed with Anti A and Anti B reagents, the individual is blood group O.
  5. If agglutination is observed when blood is mixed with Anti RhD reagent, the individual is Rh positive.
  6. If no agglutination is observed when blood is mixed with Anti RhD reagent, the individual is Rh negative.

Blood Grouping Application

Blood grouping is used in many ways in medicine, such as:

  1. Blood transfusions: Blood group matching is very important for a successful blood transfusion because blood groups that aren’t compatible can cause serious problems like hemolysis (the destruction of red blood cells) and anaphylaxis (a severe allergic reaction).
  2. Pregnancy: Blood group testing is important during pregnancy because Rh incompatibility between a mother and her foetus can cause hemolytic disease of the newborn, a serious condition that can cause brain damage or death.
  3. Organ and tissue transplants: It is important to match blood groups for organ and tissue transplants because transplant rejection and other problems can happen if blood groups don’t match.
  4. In forensic science and crime investigation, blood grouping is also used to find suspects and link them to crime scenes.
  5. Blood grouping is also a very important part of a lot of scientific research and medical trials.
  6. Medical history: A person’s blood type is an important part of their medical history, and it can be used to tell important things about their health and possible risks in certain situations.

Blood grouping Limitations

There are several limitations of blood grouping, including:

  1. False negatives: Blood grouping can sometimes produce false negatives, which occur when an individual’s blood group is not detected correctly. This can occur due to technical errors, such as incorrect sample preparation or testing procedures.
  2. False positives: Blood grouping can also produce false positives, which occur when an individual’s blood group is incorrectly identified. This can also occur due to technical errors, as well as other factors such as the presence of antibodies that cross-react with the reagents used in the test.
  3. Limited accuracy: Blood grouping is not always 100% accurate and can produce results that are not completely reliable.
  4. Limited scope: Blood grouping only identifies the ABO and Rh blood groups, there are many more blood groups exist and can’t be identified by simple blood grouping test.
  5. Cost: Blood grouping can be costly, especially when multiple tests are needed to confirm results or when specialized reagents are required.
  6. Time-consuming: Blood grouping can be time-consuming, as it often requires multiple steps and can take several hours to complete.

FAQ

FAQs on ABO and Rh blood grouping procedure

The ABO blood type system classifies blood according to the presence or lack of certain antigens on the surface of red blood cells. The ABO system recognises four blood types: A, B, AB, and O.

The ABO blood type system was initially described in 1901 by the Austrian physician Karl Landsteiner, who is largely credited with its discovery.

Forward blood grouping refers to a method of determining the blood type of an individual by mixing their red blood cells with known antibodies and observing for agglutination, or clumping of the cells. This process is used to determine an individual’s ABO and Rh (Rhesus) blood type.

Blood grouping is important because it helps to ensure that blood transfusions are safe and effective.

The Rh group, also known as the Rhesus group, is a classification of blood types based on the presence or absence of a certain protein, called the Rh antigen, on the surface of red blood cells.

The Rh blood type is inherited genetically and is determined by the presence or absence of a specific gene, known as the RHD gene, which is located on chromosome 1. The gene codes for the production of the Rh antigen, which is found on the surface of red blood cells.