4immunohematologylab

Immunohematology Immunohematology

Antonio Rivas PA-C2008

Antonio Rivas PA-C2008

A 28-year-old woman receiving her first blood transfusion begins to complain of flushing, tightness in her chest, and difficulty breathing within minutes of the start of the infusion. Her blood pressure drops rapidly and the transfusion is immediately discontinued. Epinephrine is administered with good effect. Specimens sent to the lab show no evidence of hemolysis.

What is the most likely cause of such a reaction?

A 28-year-old woman receiving her first blood transfusion begins to complain of flushing, tightness in her chest, and difficulty breathing within minutes of the start of the infusion. Her blood pressure drops rapidly and the transfusion is immediately discontinued. Epinephrine is administered with good effect. Specimens sent to the lab show no evidence of hemolysis.

What is the most likely cause of such a reaction?

A. An ABO incompatibility B. Recipient antibodies against donor red cell antigens not detected on cross match C. Recipient antibodies against donor white cell or platelet antigens

D. Recipient antibodies against donor IgA

E. Donor antibodies against recipient white blood cells

A. An ABO incompatibility B. Recipient antibodies against donor red cell antigens not detected on cross match C. Recipient antibodies against donor white cell or platelet antigens


E. Donor antibodies against recipient white blood cells

Answer Answer D. Recipient antibodies against donor IgA

Anaphylactic reactions may occur in rare patients who are IgA-deficient and have developed anti-IgA antibodies. Reactions occur in response to IgA, which is normally present in donor blood. Symptoms develop rapidly (seconds to minutes after starting the transfusion) and may include bronchospasm, nausea, vomiting, flushing, urticaria, and hypotension. Immediate treatment including epinephrine and possible intubation is required. Subsequent transfusions must consist of red cells, which are washed to remove IgA and plasma from IgA-deficient donors.


Anaphylactic reactions may occur in rare patients who are IgA-deficient and have developed anti-IgA antibodies. Reactions occur in response to IgA, which is normally present in donor blood. Symptoms develop rapidly (seconds to minutes after starting the transfusion) and may include bronchospasm, nausea, vomiting, flushing, urticaria, and hypotension. Immediate treatment including epinephrine and possible intubation is required. Subsequent transfusions must consist of red cells, which are washed to remove IgA and plasma from IgA-deficient donors.

A 47-year-old woman presents to your office after developing a low-grade fever and mild jaundice over the past several days. Her recent history is significant for a total abdominal hysterectomy performed 12 days ago, at which time she received 2 units of packed red blood cells without incident. Although there is no evidence of bleeding at present, her hemoglobin level is only 10.2 g/dl. You believe this may be a delayed transfusion reaction. What would be the most likely cause of such a reaction?

A. An ABO incompatibility B. Recipient antibodies against donor red cell antigens not detected on cross match

C. Recipient antibodies against donor white cell or platelet antigens

D. Recipient antibodies against donor IgA E. Donor antibodies against recipient white blood cells

A 47-year-old woman presents to your office after developing a low-grade fever and mild jaundice over the past several days. Her recent history is significant for a total abdominal hysterectomy performed 12 days ago, at which time she received 2 units of packed red blood cells without incident. Although there is no evidence of bleeding at present, her hemoglobin level is only 10.2 g/dl. You believe this may be a delayed transfusion reaction. What would be the most likely cause of such a reaction?




AnswerAnswer

Recipient antibodies against donor red cell antigens not detected on cross match

Recipient antibodies against donor red cell antigens not detected on cross match

Delayed hemolytic reactions occur in patients previously sensitized (through transfusion or pregnancy) to red blood cell antigens other than ABO. Levels of these antibodies are not detectable by routine laboratory tests but quickly rise when the patient is challenged with donor cells displaying the appropriate antigen (an anamnestic response). These reactions are extra vascular in nature (hemolysis occurs mainly in the spleen). Clinical symptoms appear days to weeks after the transfusion, and may include fever and mild jaundice, along with an unexpected fall in hemoglobin. While rarely life threatening, these reactions are important to recognize because of the potential for more severe reactions with subsequent transfusions.

Delayed hemolytic reactions occur in patients previously sensitized (through transfusion or pregnancy) to red blood cell antigens other than ABO. Levels of these antibodies are not detectable by routine laboratory tests but quickly rise when the patient is challenged with donor cells displaying the appropriate antigen (an anamnestic response). These reactions are extra vascular in nature (hemolysis occurs mainly in the spleen). Clinical symptoms appear days to weeks after the transfusion, and may include fever and mild jaundice, along with an unexpected fall in hemoglobin. While rarely life threatening, these reactions are important to recognize because of the potential for more severe reactions with subsequent transfusions.

Immunohematology Immunohematology Study of the human blood groupsEvaluation of blood donorsCollection and processing of donor blood

Testing for blood group antigensMatching patient with compatible blood before transfusion

Antiglobulin testsIAT / DAT

Study of the human blood groupsEvaluation of blood donorsCollection and processing of donor blood

Testing for blood group antigensMatching patient with compatible blood before transfusion

Antiglobulin testsIAT / DAT

Terminology Terminology

Antibody : serum protein that is induced by, and reacts specifically with a foreign substance (Ag)

Antigen : foreign substance that induces an immune response by causing production of antibodies and or sensitized lymphocytes that react specifically with that substance

Antibody : serum protein that is induced by, and reacts specifically with a foreign substance (Ag)

Antigen : foreign substance that induces an immune response by causing production of antibodies and or sensitized lymphocytes that react specifically with that substance

Terminology Terminology

Primary Ab response: immune response occurring after the first exposure to the Ag

First Ab detected is IgM, 3-4 days after exposure

Peaks and drops in a few weeksIgG detectable after 1-2 weeks, peaks within weeks and return to normal within few months

Primary Ab response: immune response occurring after the first exposure to the Ag

First Ab detected is IgM, 3-4 days after exposure

Peaks and drops in a few weeksIgG detectable after 1-2 weeks, peaks within weeks and return to normal within few months

TerminologyTerminology

IgM increases reflects recent exposure

Changes in IgG titters within 2-3 weeks also means recent exposure

Seroconversion: when an antibody becomes detectable in serum/plasma of a patient who was previously tested as negative for the same Ab

IgM increases reflects recent exposure

Changes in IgG titters within 2-3 weeks also means recent exposure

Seroconversion: when an antibody becomes detectable in serum/plasma of a patient who was previously tested as negative for the same Ab

TerminologyTerminology

Secondary Ab response (anamnestic response): seen after reexposure to an Ag

Immune cells remember the Ag response is fast

IgM and IgG increase quickly within 2-3 days

Higher levels that stay for months to years

booster vaccination

Secondary Ab response (anamnestic response): seen after reexposure to an Ag

Immune cells remember the Ag response is fast

IgM and IgG increase quickly within 2-3 days

Higher levels that stay for months to years

booster vaccination

ImmunoglobulinsImmunoglobulins

ImmunohematologyImmunohematologyEarliest recorded Blood transfusion in 1628

Transfusing blood from animals to human Disastrous results

Prohibition of transfusion in the late 1600s

1900 discovered ABO blood group Patient blood mixed with donor blood before transfusion to look for reactions

Discover anticoagulants First Blood bank in US in 1937 in Chicago

Earliest recorded Blood transfusion in 1628

Transfusing blood from animals to human Disastrous results

Prohibition of transfusion in the late 1600s

1900 discovered ABO blood group Patient blood mixed with donor blood before transfusion to look for reactions

Discover anticoagulants First Blood bank in US in 1937 in Chicago

ImmunohematologyImmunohematology1940 US government established a nationwide blood collection program

Continued improvement next three decades

In 1980 transfusion medicine became a medical specialty

1984 the cause of AIDS was proven to be the HIV-transmissible by blood

Changes in blood donor screening Development of more sensitive tests to detect HIV and other infectious agents

1940 US government established a nationwide blood collection program

Continued improvement next three decades

In 1980 transfusion medicine became a medical specialty

1984 the cause of AIDS was proven to be the HIV-transmissible by blood

Changes in blood donor screening Development of more sensitive tests to detect HIV and other infectious agents

General health requirements for blood donors

General health requirements for blood donors

Age > 16 yoWeight 110 poundsTemperature < 99.5 FBlood pressure < 180/<100 mm Hg

Hemoglobin >11 g/dl

Age > 16 yoWeight 110 poundsTemperature < 99.5 FBlood pressure < 180/<100 mm Hg

Hemoglobin >11 g/dl

Tests for infectious diseases

performed on donated blood Tests for infectious diseases

performed on donated blood Antibody tests

Anti-HBc Anti-HCV Anti-HIV 1 and 2 Anti HTLV I and II

Syphilis test

Antibody tests

Anti-HBc Anti-HCV Anti-HIV 1 and 2 Anti HTLV I and II

Syphilis test

Antigen tests• HBsAg• HIV-1• Hepatitis C virus• West Nile virus

Blood componentsBlood componentsCollected as whole blood(RBC/WBC/Plts suspended in Plasma)

Separated into Red blood cells PlateletsPlasma (FFP)

Packed RBCs can be stored refrigerated up to 42 hrs

Other components: factor VIII, IX concentrate

Collected as whole blood(RBC/WBC/Plts suspended in Plasma)

Separated into Red blood cells PlateletsPlasma (FFP)

Packed RBCs can be stored refrigerated up to 42 hrs

Other components: factor VIII, IX concentrate

Pretransfusion testingPretransfusion testing

ABO forward testingRh testingABO reverse testingAntibody screenCrossmatch

ABO forward testingRh testingABO reverse testingAntibody screenCrossmatch

Procedures performed in the Blood Bank

Procedures performed in the Blood Bank

ABO grouping Rh typingCompatibility testing before transfusion

Typing of donor bloodUnusual blood group antibody testing

ABO grouping Rh typingCompatibility testing before transfusion

Typing of donor bloodUnusual blood group antibody testing

Blood group systemBlood group system

Major blood group system is the ABO system

Four major blood types A, B, AB, or OABO grouping tests based on principle of agglutination

45 % of the population of the US is group O, 40% is group A, 11% is B and 4 % is AB

Differs according to racial and ethnic group

Major blood group system is the ABO system

Four major blood types A, B, AB, or OABO grouping tests based on principle of agglutination

45 % of the population of the US is group O, 40% is group A, 11% is B and 4 % is AB

Differs according to racial and ethnic group

Blood group antigensBlood group antigensPresence or absence of group A or B antigens in RBCs, leukocytes and Plts.

Inherited One allele from each parent A and B are codominant with respect to each other

Group A person has A ag in blood cellsGroup B person has B ag in blood cellsGroup AB person has A and B antigen on blood cells

Group O person has neither A or B antigens on blood cells

Presence or absence of group A or B antigens in RBCs, leukocytes and Plts.

Inherited One allele from each parent A and B are codominant with respect to each other

Group A person has A ag in blood cellsGroup B person has B ag in blood cellsGroup AB person has A and B antigen on blood cells

Group O person has neither A or B antigens on blood cells

Testing Blood group Antigens

Testing Blood group Antigens

Patients RBCs are combined with known antiserum (antibodies) commercially available, observing for agglutination

If the Ag present on the cells correspond to the antibody in the reagent, Ab will bind to the Ag and cause clumping of the cells (agglutination)

Forward grouping

Patients RBCs are combined with known antiserum (antibodies) commercially available, observing for agglutination

If the Ag present on the cells correspond to the antibody in the reagent, Ab will bind to the Ag and cause clumping of the cells (agglutination)

Forward grouping

Forward groupingForward grouping

ABO antibodies (Ab)ABO antibodies (Ab)

Naturally occurringIgMAb is present if Ag is missingNot well developed in newbornsDetected by reverse grouping

Naturally occurringIgMAb is present if Ag is missingNot well developed in newbornsDetected by reverse grouping

ABO antibodies (Ab)ABO antibodies (Ab)

Reverse groupingDetects Abs present in patients serum, by reacting the serum/plasma with suspension of commercially available A or B cells suspension and looking for agglutination

Reverse groupingDetects Abs present in patients serum, by reacting the serum/plasma with suspension of commercially available A or B cells suspension and looking for agglutination

Importance of ABO grouping

Importance of ABO grouping

Must be done before blood transfusionPatient should be transfused with blood of the same ABO group

Severe blood transfusion reaction if not properly matched blood is transfused

“avoid giving the patient an Ag he does not already have”

In emergency O packed RBcells given-universal donor

Must be done before blood transfusionPatient should be transfused with blood of the same ABO group

Severe blood transfusion reaction if not properly matched blood is transfused

“avoid giving the patient an Ag he does not already have”

In emergency O packed RBcells given-universal donor

Tissue transplantsTissue transplants

Kidney, liver, cornea, skin, pancreas, BM, heart, lung, intestine and bone

Autologous//autografts: from the same individual

Homologous //allografts : from the same species

ABO and Rh matchedHistocompatibility testing

Kidney, liver, cornea, skin, pancreas, BM, heart, lung, intestine and bone

Autologous//autografts: from the same individual

Homologous //allografts : from the same species

ABO and Rh matchedHistocompatibility testing

Exercise Exercise

Rh blood groupRh blood group

Second most important blood group system(name from the Rhesus monkey)

Composed of many AgsMost important D Ag-most antigenicOnly test for D AgProduct of inherited genesOnly present in RBCs

Second most important blood group system(name from the Rhesus monkey)

Composed of many AgsMost important D Ag-most antigenicOnly test for D AgProduct of inherited genesOnly present in RBCs

Rh blood groupRh blood group

Antibodies to Rh system do not occur naturally

RBCs that posses the D antigen are called: Rh –positive or D-positive

Weak D antigen is possible needs xtra. Testing in order to detect

RBCs are incubated with commercially available anti-D

Antibodies to Rh system do not occur naturally

RBCs that posses the D antigen are called: Rh –positive or D-positive

Weak D antigen is possible needs xtra. Testing in order to detect

RBCs are incubated with commercially available anti-D

Rh antibodiesRh antibodies

Produced by a D negative person who has been sensitized or immunized to the D Ag

Can occur during pregnancy or after blood transfusion

Anti-D is an IgG

Produced by a D negative person who has been sensitized or immunized to the D Ag

Can occur during pregnancy or after blood transfusion

Anti-D is an IgG

Hemolytic Disease of the newborn

Hemolytic Disease of the newborn

Abs from the mother enters the fetal circulation and destroys fetal red blood cells

Caused primarily by maternal Anti-D recognizing D Ag in fetal RBCs

D-negative mother becomes pregnant with a D-positive fetus

During pregnancy or at birth (feto-maternal hemorrhage), mother becomes exposed to fetal RBCs and develops Anti-D

Abs from the mother enters the fetal circulation and destroys fetal red blood cells

Caused primarily by maternal Anti-D recognizing D Ag in fetal RBCs

D-negative mother becomes pregnant with a D-positive fetus

During pregnancy or at birth (feto-maternal hemorrhage), mother becomes exposed to fetal RBCs and develops Anti-D

Hemolytic Disease of the newborn (HDN)


Anti-D is a monomer can cross the placenta

Usually affects subsequent pregnancies

First pregnancy is not affectedPrenatal screening for ABO/Rh and possible antibodies already present in their first trimester

Father typing for Rh group

Anti-D is a monomer can cross the placenta

Usually affects subsequent pregnancies

First pregnancy is not affectedPrenatal screening for ABO/Rh and possible antibodies already present in their first trimester

Father typing for Rh group



Mild cases :mild anemia, jaundice, or breathing problems detected at birth

Severe cases : heart failure, brain damage, stillbirth or miscarriage

Prevention with Rh immune globulin (RhIG) given to the Rh negative mothers

Mild cases :mild anemia, jaundice, or breathing problems detected at birth

Severe cases : heart failure, brain damage, stillbirth or miscarriage

Prevention with Rh immune globulin (RhIG) given to the Rh negative mothers

RhIG (ROGHAM)RhIG (ROGHAM)

Concentrated solution of anti-D from human plasma

Given to D negative mothers at 28 Wks and 72hrs after delivery of a D-positive baby

Also given after miscarriage, abortion or amniocentesis

Repeated every pregnancy

Concentrated solution of anti-D from human plasma

Given to D negative mothers at 28 Wks and 72hrs after delivery of a D-positive baby

Also given after miscarriage, abortion or amniocentesis

Repeated every pregnancy

Other immunological tests

Other immunological tests

Antihuman globulin test (Coomb’s test)

Uses a commercial antibody against human globulin (AHG) to detect globulin coated RBCs

Also used for Hemolytic conditions

Antihuman globulin test (Coomb’s test)

Uses a commercial antibody against human globulin (AHG) to detect globulin coated RBCs

Also used for Hemolytic conditions

Hemolytic transfusion reactions


Most severe with ABO mismatches

Rapid intravascular hemolysisFree Hgb is released to the plasma

Severity also related to the amount of RBCs given

Acute or delayed

Most severe with ABO mismatches

Rapid intravascular hemolysisFree Hgb is released to the plasma

Severity also related to the amount of RBCs given

Acute or delayed



Major HTRFeverChillsBackacheHeadaches Apprehension Dyspnea Hypotension Vascular collapse

Major HTRFeverChillsBackacheHeadaches Apprehension Dyspnea Hypotension Vascular collapse



In very severe cases DIC Acute renal failure – from tubular necrosis

Patient under general anesthesia will not voice symptoms, first findings may be oliguria, generalized bleeding

Treatment : if suspected stop transfusion, draw sample of blood and look for Hemoglobenimia(pink plasma)

Vigorously hydrate the patient Force diuresis with Manitol may help prevent renal damage

In very severe cases DIC Acute renal failure – from tubular necrosis

Patient under general anesthesia will not voice symptoms, first findings may be oliguria, generalized bleeding

Treatment : if suspected stop transfusion, draw sample of blood and look for Hemoglobenimia(pink plasma)

Vigorously hydrate the patient Force diuresis with Manitol may help prevent renal damage

Other transfusion reactions


Leukoagglutinin rx: to Ags in WBCs sensitized by previous pregnancy or transfusions

Non hemolytic, Htc increases as expected Fever and chills within 12 hrs of transfusion

Cough, dyspnea, and lung infiltrates may develop

Treated with: Tylenol, Benadryl, and steroids

Leukopoor filters are used to prevent

Leukoagglutinin rx: to Ags in WBCs sensitized by previous pregnancy or transfusions

Non hemolytic, Htc increases as expected Fever and chills within 12 hrs of transfusion

Cough, dyspnea, and lung infiltrates may develop

Treated with: Tylenol, Benadryl, and steroids

Leukopoor filters are used to prevent



Anaphylactic ReactionsUrticaria or bronchospasmDue to plasma proteins most of the time

IgA deficient patients , may have anti-IgA and develop reactions to the IgA dissolved in the plasma(transfusion)

Anaphylactic ReactionsUrticaria or bronchospasmDue to plasma proteins most of the time

IgA deficient patients , may have anti-IgA and develop reactions to the IgA dissolved in the plasma(transfusion)



Contaminated bloodWith gram negative bacteriaRareSepticemia and shock from endotoxins

If suspected, culture the blood unit and treat patient with antibiotics

Contaminated bloodWith gram negative bacteriaRareSepticemia and shock from endotoxins

If suspected, culture the blood unit and treat patient with antibiotics

Blood ProductsBlood Products

Whole BloodPacked cellsPlatelets concentratesPlateletaphoresisGranulocytes Fresh Frozen plasmaCryoprecitate Clotting factors concentrates

Whole BloodPacked cellsPlatelets concentratesPlateletaphoresisGranulocytes Fresh Frozen plasmaCryoprecitate Clotting factors concentrates

A 55-year-old man complains of chills, and is noted to have a rise in temperature from 98.9 to 101.7° F, within 15 minutes of starting a transfusion of packed red blood cells. The transfusion is stopped and antipyretics are administered, lowering his temperature. Specimens sent to the blood bank show no evidence of hemolysis, and there are no clerical discrepancies. What is the most likely cause of his reaction?




A 55-year-old man complains of chills, and is noted to have a rise in temperature from 98.9 to 101.7° F, within 15 minutes of starting a transfusion of packed red blood cells. The transfusion is stopped and antipyretics are administered, lowering his temperature. Specimens sent to the blood bank show no evidence of hemolysis, and there are no clerical discrepancies. What is the most likely cause of his reaction?




Answer Answer



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