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Blood Typing Lab


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Background Information


The blood typing test determines what surface antigens are present on an individual’s erythrocytes (RBCs). Identified thus far are more than 50 RBC surface antigens. They can be carbohydrates or proteins in structure and have designated names or abbreviations such as ABO, Rh, Kell, Duffy, Kidd, and Lewis. The ABO and Rh blood groups are clinically significant because they may cause the most harm if transfused into an incompatible recipient.

ABO Blood Group Antigens

Although the ABO blood group name consists of three letters, there are only two antigens involved, called A and B. The antigen(s) that appear on the surface of an individual’s RBCs determine their ABO blood type.

  • A type: A antigen only.
  • B type: B antigen only.
  • AB type: both A and B antigens.
  • O type: neither A nor B antigens.

Both A and B surface antigens are oligosaccharides (short sugar chains), differing in structure only at their terminal (last) sugars. They are also categorized as glycoproteins or glycolipids because they link to proteins or lipids in the RBC plasma membrane. A third ABO oligosaccharide, designated as H, appears on all RBCs but does not have a terminal sugar, nor is it antigenic.

Anti-A and anti-B antibodies structure

A single gene (the ABO gene) controls an individual’s ABO blood type, and there are three alleles (or versions) of the gene: A, B, and O. An individual inherits an ABO allele from each parent, and the pair of alleles determine the individual’s blood type.

The A and B alleles are codominant. Thus, if both alleles are present, the RBCs express both antigens. The O allele is recessive, and either A or B alleles will dominate.

ABO blood group inheritance chart

The body makes antibodies to RBC surface antigens that it considers to be foreign. Typically, the body must first be exposed to a foreign antigen before antibody production begins. However, for ABO blood group antigens, prior exposure to incompatible blood is not required. Instead, the generation of antibodies occurs naturally. Blood type A individuals make anti-B antibodies, and blood type B individuals make anti-A antibodies. Blood type AB individuals do not make anti-A or anti-B antibodies. Individuals with type O blood, who lack antigens A and B, make anti-A and anti-B antibodies. The natural production of anti-A and anti-B antibodies begins a few months after birth, peaking around 5 to 10 years of age.

Antibodies to ABO blood group

Anti-A and anti-B antibodies are both IgM class in structure, and each has ten binding sites. These relatively large antibodies cause rapid agglutination (or clumping) when encountering the opposing RBC surface antigens.

ABO blood group agglutination

Rh Blood Group Antigens

Another RBC surface antigen group, the Rh group, can also cause great harm. Researchers applied the Rh designation after initially discovering the antigen group in rhesus macaque primates. 

Unlike the ABO antigen, Rh antigens are transmembrane proteins that coil and loop along the RBC plasma membrane. The arrangement of amino acids on the extracellular loops determines the Rh type. Over 49 Rh antigens are now recognized, but the D antigen is the most clinically significant.

Rh antigen structure

The Rh+ allele is dominant over the Rh- allele, and about 85 percent of Americans are Rh-positive (Rh+). The rest of the American population is Rh-negative (Rh−).  

Note that the Rh group is distinct from the ABO group, so any individual may have or lack this Rh antigen, no matter their ABO blood type. When identifying a person’s blood type, a positive or negative is added to the ABO type to designate the Rh group. For example, A positive (A+) means ABO group A blood with the Rh antigen present, and AB negative (AB−) means ABO group AB blood without the Rh antigen.

Blood types by ethnicity in America

Antibodies to the Rh antigen are produced only in Rh− individuals after exposure to incompatible Rh+ blood. The antibodies produced are mostly IgG class, each having only two antigen-binding sites.

Rh blood group agglutination reaction

Sensitization is the term given to exposure-activated antibody production. Rh sensitization most frequently happens with the birth of an Rh+ baby to an Rh− mother. Problems are rare in a first pregnancy because the baby’s Rh+ cells rarely cross the placenta (the organ of gas and nutrient exchange between the baby and the mother). However, the mother may become exposed to the baby’s Rh+ cells during or immediately after birth. Research has shown that this occurs in about 13−14 percent of such pregnancies.

Rh blood group agglutination reaction

After exposure, the mother’s immune system begins to generate anti-Rh antibodies. If the mother should conceive another Rh+ baby, the small IgG Rh antibodies can cross the placenta and enter the fetal bloodstream, destroying the fetal RBCs. This condition is known as hemolytic disease of the newborn (HDN) or erythroblastosis fetalis.  The result may be mild anemia. However, agglutination and hemolysis can be so severe that the fetus may die without treatment in the womb or shortly after birth.

A drug called RhoGAM (Rh immune globulin) can temporarily prevent the development of Rh antibodies in the Rh− mother, thereby averting this potentially severe disease for the fetus. RhoGAM antibodies destroy any fetal Rh+ erythrocytes that may cross the placental barrier. The administration of RhoGAM typically occurs during weeks 26−28 of pregnancy and within 72 hours following birth. It has proven remarkably effective in decreasing the incidence of HDN. Earlier, we noted that the incidence of HDN in an Rh+ subsequent pregnancy to an Rh− mother is about 13–14 percent without preventive treatment. Since the introduction of RhoGAM in 1968, the incidence has dropped to about 0.1 percent in the United States.

ABO Transfusion Protocols

It is best to transfuse only matching blood types. A blood type B+ recipient should only receive RBCs from a blood type B+ donor, for example. However, when acute hemorrhage threatens a person’s life, there may not be time for proper crossmatching of blood types. In this case, RBCs from a donor with blood type O− blood can be transfused. Type O RBCs do not display A or B antigens, so the anti-A or anti-B antibodies in the recipient’s blood will not encounter any RBC surface antigens on the donated RBCs, and agglutination will not occur. For this reason, individuals with type O- blood are often called universal donors.

There is a potential problem with the universal donor designation. If an Rh− recipient had prior exposure to the Rh antigen, antibodies for this antigen would likely be present in the recipient’s blood and trigger agglutination to some degree. 

Recipients with blood type AB+ are known as the universal recipients. They can theoretically receive RBCs of any blood type because they do not produce anti-A, anti-B, anti-Rh antibodies.


Rh blood group agglutination reaction

Frank is married to Christy, who is pregnant. This will be the first child for both parents. They want to determine if their different blood types could cause any potential harm to the fetus or future offspring.

Test Procedures

Step 1

Partition a glass slide into three sections using a marking pen and label them as “A”, “B”, and “D”.

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Step 2

Use a lancet device to pierce the skin of the index finger. Allow a large drop of blood to accumulate on the finger surface.

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Step 3

Place a drop of blood in each of the labeled areas by gently touching the slide with the lanced finger.

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Step 4

Add a small drop of anti-A serum to the first blood sample, anti-B serum to the second sample, and anti-D (Rh) to the third sample. The anti-A and anti-B antisera contain IgM antibodies, and the anti-D antiserum typically contains a mixture of IgG and IgM antibodies.

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Step 5

Thoroughly mix the antisera and blood using a different stir stick for each sample. This will increase the contact between antigens and antibodies and accelerate the agglutination reactions.

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Test Results

O positive blood type

Frank’s blood type results.

A negative blood type

Christ’s blood type results.

Test Assessment

ABO Blood Group Questions.

O +

OO alleles

A –

AA or AO alleles

AO or OO

A or OO

If it is blood type A, it will make anti-B antibodies. If it is blood type O, it will make both anti-A and anti-B antibodies.

Rh Blood Group Questions.

Frank is Rh+ and Christy is Rh-.

No. Frank is Rh+ and would not produce antibodies to his own blood type. Christy, who is Rh-. would not produce anti-Rh antibodies except if she is exposed (sensitized) to incompatible Rh+ blood.

RH antigens are transmembrane proteins and ABO antigens are oligosaccharides.


Anti-Rh antibodies are much smaller (two antigen-binding sites) than ABO antibodies (10 antigen-binding sites).

Rh+, because the Rh+ allele is dominant.

If it is blood type A, it will make anti-B antibodies. If it is blood type O, it will make both anti-A and anti-B antibodies.

Not likely because fetal Rh+ cells rarely cross the placenta during pregnancy.

Rho-GAM (Rhₒ(D) immune globulin) is an injection of anti-D antibodies.

The injection should be administered to Christy shortly after the baby’s birth.

Christy may become exposed to the baby’s Rh+ cells during or immediately after birth. The injection would eliminate any fetal RBCs before they are recognized, and Christy’s immune system starts producing anti-Rh antibodies.

If Rho-GAM is not administered, Christy’s immune system may produce anti-Rh antibodies. These IgG antibodies are relatively small and can cross the placenta and enter the bloodstream of a future fetus. The anti-Rh antibodies would destroy the fetal RBCs, causing a condition is known as hemolytic disease of the newborn (HDN) or erythroblastosis fetalis.

Donor/Recipient Questions.


  • He can donate blood to recipients with type-O+ because it is his own blood type.
  • He cannot donate blood to recipients with type-A, type-B, or type-AB because his blood contains anti-A and anti-B antibodies.
  • He should not donate blood to recipients with type Rh- because they will develop anti-Rh antibodies to his Rh+ RBCs.

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Reference page: “Blood Typing