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IMMUNOHEMATOLOGY. Fe A. Bartolome, M.D. Dept. of Pathology & Laboratory Diagnosis. IMMUNOHEMATOLOGY. merges aspects of hematology, immunology & genetics

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IMMUNOHEMATOLOGY

Fe A. Bartolome, M.D.

Dept. of Pathology & Laboratory Diagnosis


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IMMUNOHEMATOLOGY

  • merges aspects of hematology, immunology & genetics

  • serologic, genetic, biochemical and molecular study of antigens associated with membrane structures on the cellular constituents of the blood

  • immunologic reactions involving all blood components and constituents


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IMMUNOLOGIC PRINCIPLES

  • primary immunological components: antigens & antibodies provides basis for blood bank testing and reactions

CARDINAL RULE IN BLOOD BANK:

The antigens are found on the surface of red blood cells and the antibodies are found in serum or plasma


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IMMUNOLOGIC PRINCIPLES

  • ANTIGENS

  • substances that have the capability to stimulate the production of an antibody

  • characteristics:

  • 1. Chemical nature – protein, CHO, lipopolysaccharide or nucleic acid

  • 2. Molecular weight > 10,000 daltons

  • 3. Complexity – more complex, > antibody stimulation

  • 4. Stability – if unstable  degrade  less Ab stimulation

  • 5. Foreign


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IMMUNOLOGIC PRINCIPLES

  • Chemical composition of antigens:

  • Glycoproteins & lipoproteins – most potent

  • Glycolipids

  • Pure polysaccharides – not immunogenic except in humans and mice

  • Pure lipids & nucleic acids – not immunogenic but can be antigenic  serve as haptens


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IMMUNOLOGIC PRINCIPLES

Immunogenicity of Blood Group Antigens

A, B and D (Rho) – most immunogenic

Kell (K)

Duffy: Fya

Fyb

Kidd: Jka

Jkb


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IMMUNOLOGIC PRINCIPLES

  • ANTIBODIES

  • also called immunoglobulins

  • characteristics:

  • 1. Protein

  • 2. Produced in response to stimulation by an antigen

  • 3. Specific for the stimulating antigen

  • consists of 2 heavy chains & 2 light chains held together by disulfide bonds

  • produce 3 fragments when cleaved by enzymes  2 Ag-binding fragments (Fab) & 1 crystallizable fragment (Fc)


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IMMUNOLOGIC PRINCIPLES

  • Classification of Blood Group Antibodies:

  • Alloantibodies

    • Reacts with foreign Ag not present on patient’s own RBC

    • Most produced as result of immune stimulation via transfusion or pregnancy (usually during delivery)

  • Autoantibodies

    • Reacts with an Ag on patient’s own cells & with that same Ag on the cells of other individuals


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ABO BLOOD GROUP SYSTEM

  • discovered by Karl Landsteiner; locus on chr 9

  • single most important blood group for the selection and transfusion of blood

  • widely expressed  tissues & body fluids including red cells, platelets & endothelial cells

  • three antigens: A, B, H

  • two major antibodies: anti-A and anti-B

  • four phenotypes: A, B, AB, O  A & B Ag’s autosomal co-dominant (expressed on grp A, B and AB red cells; O phenotype autosomal recessive (most frequent)


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ABO BLOOD GROUP SYSTEM

  • ABO Antigens

  • present on the surface of red cells as well as tissue and endothelial cells in the body

  • found in soluble form in plasma & other body secretions in people known as secretors

  • inherited in simple Mendelian fashion from an individual’s parents

  • 3 possible genes that can be inherited: A, B, O

  • A and B genes produce a detectable product

  • O gene does not produce a detectable product



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ABO BLOOD GROUP SYSTEM

  • A and B genes do not directly produce antigens  produce an enzyme called transferase  attaches a sugar molecule to the chemical structure of the antigen  sugar molecule responsible for specificity

  • O antigen  no transferase  no antigen produced

  • A and B antigens on surface of RBC  protrude from outermost layer of cell membrane






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ABO BLOOD GROUP SYSTEM

  • H Antigen

  • required to produce either A or B antigens

  • possible genetic combinations: HH, Hh, or hh

  • HH or Hh (+)  produce H Ag  99.99% of Caucasians

  • hh  does not produce H Ag  Bombay phenotype (Oh)

  • anti-H antibodies rare – found only in individuals with Bombay phenotype


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ABO BLOOD GROUP SYSTEM

Example of determining offspring blood types from known or suspected genotypes:

Genotype parent #1 (AO)

A O

Genotype parent AAAAO

#2 (AB) B ABBO

Phenotypes of possible offsprings: A, AB, B


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ABO BLOOD GROUP SYSTEM

Frequencies of ABO Blood Groups:

Blood Group Frequency

O 45%

A 41%

B 10%

AB 4%


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ABO BLOOD GROUP SYSTEM

  • ABO Subtypes:

  • A variants (A1, A2)

    • A1 most common (80%) & most antigenic

    • A1 and A2 differentiated using antisera specific for A1 Ag (anti-A1 lectin) prepared from seed known as Dolichos biflorus (+) reaction with A1 but not A2

    • Anti-A  reacts with both A1 & A2 but more strongly with A2


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ABO BLOOD GROUP SYSTEM

  • ABO Subtypes:

  • Weak A and weak B phenotypes

  • Null phenotypes:

  • (a) Bombay (Oh)

    • No A, B or H Ag on red cells & secretions

    • With anti-A, anti-B & anti-H in their sera

  • (b) para-Bombay

    • Absent or only trace A,B & H Ag’s detected on rbc w/ normal expression in secretions & body fluids


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ABO BLOOD GROUP SYSTEM

  • ABO Antibodies

  • Natural antibodies  antigenic stimulus is environmental  exposure occurs from birth

  • Newborns  without ABO antibodies of their own; begin to produce Ab with detectable titer at 6 months of age

  • Other characteristics of ABO antibodies:

    • IgM

    • Reacts at room temp. after an immediate spin


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ABO ROUTINE TESTING

(slide or test tube method)

  • DIRECT OR FORWARD TYPING

  • test for antigens

  • patient’s cells containing unknown antigens tested with known antisera

  • antisera manufactured from human sera

  • antisera used:

  • AntiseraColorSource

  • Anti-A Blue Group B donor

  • Anti-B Yellow Group A donor

  • Anti-A,B Clear Group O donor


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ABO ROUTINE TESTING

  • Anti-A,B

  • not a mixture of anti-A and anti-B

  • separate Ab that reacts with both A and B antigens

  • used in forward grouping for two purposes:

    • confirms the results of the anti-A and anti-B

    • will show a (+) reaction with weak subgroups of A and B that do not react with the anti-A and anti-B


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ABO ROUTINE TESTING

Reaction Patterns for ABO Groups


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ABO ROUTINE TESTING

  • INDIRECT/REVERSE TYPING

  • known antigen (cell) vs. unknown antibody (patient’s serum)

  • serum is combined with cells having known Ag content in a 2:1 ratio

  • uses commercially prepared reagents containing saline-suspended A1 and B cells


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ABO ROUTINE TESTING

Reaction Patterns for ABO Groups


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ABO ROUTINE TESTING

  • Stages of Hemagglutination

  • First Stage:

    • red cell sensitization

    • Ag and Ab held by non-covalent interactions

  • Second Stage:

    • formation of stable latticework  basis of visible reaction


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ABO ROUTINE TESTING

Grading of Agglutination:

Negative (0) No clumps or aggregates

Weak (+/-) Tiny clumps or aggregates barely visible macroscopically or to the naked eye

1+ Few small aggregates visible macroscopically

2+ Medium-sized aggregates

3+ Several large aggregates

4+ One solid aggregate


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ABO ROUTINE TESTING

  • Causes of Discrepancies in ABO Testing:

  • Technical

  • 1. Incorrect ID/recording

  • 2. Patient/donor serum not added

  • 3. Reagent contamination

  • 4. Under-/over-centrifugation

  • 5. Hemolysis

  • 6. Warming of test mixture


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ABO ROUTINE TESTING

Causes of Discrepancies in ABO Testing:

B. Red Blood Cells

1. Missing or weak A/B antigen

2. Acquired B Ag – colon or gastric CA, intestinal obstruction

3. Polyagglutinable RBC

4. Ab-coated RBC – post-transfusion incompatibility; autoimmune hemolytic anemia

5. Maternal-fetal agglutination – mismatched transfusion


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ABO ROUTINE TESTING

Causes of Discrepancies in ABO Testing:

C. Serum

1. Roleaux formation – presence of plasma expanders, monoclonal gamma globulins

2. Anti-A1

3. Unexpected alloantibodies

4. Expected antibody absent – hypogammaglobulinemia, extreme ages, immunosuppression


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ABO ROUTINE TESTING

  • WHAT TO DO?

  • Wash cells with saline 3-4x and repeat all tests and test for antibodies

  • Test for subgroups of A using anti-A1 and anti-A

  • Use cell panels to detect the specificity of abnormal antibodies


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Rh BLOOD GROUP SYSTEM

  • discovered in 1940 by Landsteiner & Wiener

  • most complex erythrocyte antigen system; located on chromosome 1

  • found exclusively on surface of rbc  integral part of red cell membrane

  • primary antigen  if present, consider Rh (+)

  • lack corresponding naturally-occurring antibodies in serum


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Rh BLOOD GROUP SYSTEM

  • CLASSIFICATION/NOMENCLATURE SYSTEM

  • Wiener

  • Multiple allele hypothesis

  • 5 antigens: Rho, rh’, rh”, hr’, hr”

  • Single locus inheritance system with 8 alternate common alleles coding for agglutinogens  1 individual produces 2 agglutinogens inherited from both parents


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Rh BLOOD GROUP SYSTEM

  • CLASSIFICATION/NOMENCLATURE SYSTEM

  • Fischer & Race

  • Three alleles: D/d, C/c and E/e

  • Five antigens: D, C, E, c, e

  • d  no D locus  no antigenic products

  • Rosenfeld

  • Numerical system

  • Rh1 to Rh5


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Rh BLOOD GROUP SYSTEM

  • Rh Antigens

  • with three integral membrane proteins

    • RhD

    • RhCcEe

    • Rh-associated glycoprotein (Rh50, RhAG)

  • D antigen resides in RhD protein  most immunogenic followed by c, E, C and e


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Rh BLOOD GROUP SYSTEM

  • Weak D Antigen (Du)

  • Rho variant

  • weak or absent red cell agglutination by anti-D  detected only with use of anti-human globulin reagent  use bovine anti-D

  • weakened form caused by 1 of 3 situations:

    • a piece of the D antigen is missing

    • D gene is on a chromosome opposite a C gene  (+) steric hindrance

    • Inheritance of a gene coding for less D antigen


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Rh BLOOD GROUP SYSTEM

  • Presence of D = presence of Rho factor  Rh (+)

  • Absence of D  Rh (-)


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Rh BLOOD GROUP SYSTEM

  • Testing for Rho (D) Antigen:

    • use antisera originating from human source

    • antisera with different constituents  use of high protein media necessary to produce agglutination since antigens are an integral part of the red cell membrane  less numerous than ABO antigens


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Rh BLOOD GROUP SYSTEM

  • Testing for Du Variant:

    • use bovine or albumin-suspended anti-D reagent

    • incubate at 37oC for 15-60 minutes to facilitate formation of Ag-Ab complex

    • interpretation: (+) Du consider Rh (+)

    • women who appear to be Rh (-) should be proven to be Du (-) before they are considered to be eligible to receive transfusion


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Rh BLOOD GROUP SYSTEM

  • Rh Antibodies

  • not naturally-occurring  immune antibodies  produced upon sensitization  IgG isotype

  • reactive at 37oC  enhanced with enzyme-treated red cells

  • can cross the placenta

  • associated with hemolytic transfusion reaction and hemolytic disease of the newborn (HDN)


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Rh BLOOD GROUP SYSTEM

  • Rh Typing – slide or test tube method

  • False (+) results:

    • Drying

    • Roleaux formation

    • Auto-agglutination

    • Patient’s red cells heavily coated with Ab’s

    • Presence of cold agglutinins


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Rh BLOOD GROUP SYSTEM

  • Rh Typing

  • False (-) results:

    • Use of old cells

    • Wrong cell concentration

    • Hemolysis

    • Inadequate mixing of cells

    • Inactive typing sera

    • Incorrent temperature

    • Existence of Du variant

    • High concentration of blocking antibodies


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MINOR BLOOD GROUP SYSTEMS

  • Significance:

  • For medico-legal parenthood studies

  • May cause transfusion reaction or HDN


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MINOR BLOOD GROUP SYSTEMS

  • Systems with cold-reacting antibodies

  • Antibodies formed react at temperatures 250C or colder

  • Not considered clinically significant since any reaction seen in the test tube will not be seen in the warmer temperatures of the body

  • Not likely to cause a transfusion-related accident


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MINOR BLOOD GROUP SYSTEMS

  • Systems with cold-reacting antibodies

  • Lewis (Le) System

    • Antigens: Lea and Leb formed in secretions & absorbed onto surface of rbc later

    • Antibodies – often encountered in individuals with no antigens; may be present at certain times (e.g. pregnancy) and then disappear

  • MNS System

    • Antigens are weakly antigenic

    • Antibodies: naturally-occurring or stimulated by direct exposure


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MINOR BLOOD GROUP SYSTEMS

  • Systems with cold-reacting antibodies

  • P-p System

    • P1 antigen most antigenic present on cells of 79% of whites & 94% of African-Americans

  • Ii system

    • Antigens: I and i both present in all individuals

    • I antigen – present in large quantities in adults

    • i antigen – present in large quantities on cells taken from the umbilical cord

    • Anti-I  freq. seen in serum of patient’s with recent infectious mononucleosis


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MINOR BLOOD GROUP SYSTEMS

  • Systems with warm-reacting antibodies

  • reactive at 370C in anti-human globulin medium

  • Clinically significant  most likely to cause HDN and HTR

  • Kell (K) – Cellano (k) System

    • k Ag present in 98% of the white population

    • antibodies primarily IgG

  • Kidd System

    • Antigens: Jka & Jkb – not very antigenic

    • Antibodies stimulated by direct exposure via either pregnancy or transfusion


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MINOR BLOOD GROUP SYSTEMS

  • Systems with warm-reacting antibodies

  • Duffy System

    • Antigens: Fya & Fyb

    • Antibodies stimulated through direct exposure  capable of causing HDN and HTR


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HEMOLYTIC DISEASE OF THE NEWBORN

  • involves hemolysis of red cells in the fetus and neonate

  • antibody is present in the mother that corresponds to an antigen on the surface of the red cells of the fetus  Ab crosses placenta  attaches to fetal Ag  hemolyze red cells of fetus

  • Differential diagnosis: physiologic jaundice, septicemia, CID, toxoplasmosis, congenital syphilis


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HEMOLYTIC DISEASE OF THE NEWBORN

  • ABO Disease

  • Most common type

  • Most cases are mild & do not require exchange transfusion

  • Most common scenario: mother is group O and infant is group A

  • Even first baby is affected


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HEMOLYTIC DISEASE OF THE NEWBORN

  • ABO Disease

  • Features:

  • Spherocytosis

  • Increased reticulocyte count

  • Increased indirect bilirubin in 1st 72 hours of life

  • Jaundice appearing during first 24 hrs of life

  • Good evidence for ABO disease is detection of immune anti-A or anti-B in the cord blood of the newborn.


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HEMOLYTIC DISEASE OF THE NEWBORN

  • Rh Disease

  • most severe; Rh (+) fetus & Rh (-) mother

  • FIRST PREGNANCY

  • Rh (+) baby  Ag enters maternal circulation  sensitize Rh (-) mother  anti-Rh production (IgG)  cross placenta  enter fetal circulation  baby not affected

  • SUBSEQUENT PREGNANCIES

  • Ab already present in mother  enter fetal circulation  (+) intravascular hemolysis accumulation of rbc destruction products  jaundice or kernicterus (erythroblastosis fetalis)



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HEMOLYTIC DISEASE OF THE NEWBORN

  • Rh Disease

  • first baby usually unaffected since it is the first time the mother is exposed to the antigen

  • occasionally, firstborns are affected either because of:

    • previous maternal exposure (e.g. previous aborted pregnancy)

    • unusually great maternal susceptibility to Rh stimulus during normal pregnancy


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HEMOLYTIC DISEASE OF THE NEWBORN

  • Rh Disease

  • Characteristics of Erythroblastosis Fetalis:

  • Increased number of circulating nucleated red cells

  • Increased osmotic fragility of cells

  • Increased amount of indirect/unconjugated bilirubin

  • Main Clinical Findings:

  • Anemia - < 15 gm/100 ml or 150 gm/L

  • Rapidly developing jaundice


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HEMOLYTIC DISEASE OF THE NEWBORN

  • Rh Disease

  • Management:

  • For the mother

  • RhoGam (Rh Immune Globulin)

    • concentrated anti-D

    • coats Rh (+) fetal cells in maternal circulation  recognized by mother’s system as abnormal & removed from circulation  prevents maternal immune system from processing the Ag on surface of fetal cells  no antibody formed



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HEMOLYTIC DISEASE OF THE NEWBORN

  • For the mother

  • RhoGam (Rh Immune Globulin)

    • Dose: routinely administered 2x – at 28 wks AOG & within 72 hrs after birth of an Rh (+) infant

    • Also administered following termination of any pregnancy, after amniocentesis in an Rh (-) mother & following accidental transfusion with Rh (+) red cells


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HEMOLYTIC DISEASE OF THE NEWBORN

  • For the baby: EXCHANGE TRANSFUSION

  • Indications:

    • Infant serum indirect bilirubin > 20mg/100 ml (342 mol/L) for fullterm infants OR >15mg/100 ml (257 mol/L) for premature infants

    • Cord blood indirect bilirubin > 3 mg/100 ml (51 mol/L)

    • Cord blood hemoglobin < 13 gm/dL (130 g/L)

    • Maternal Rh antibody titer of 1:64 or more


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HEMOLYTIC DISEASE OF THE NEWBORN

  • Beneficial Effects of Exchange Transfusion:

  • Removal of bilirubin

  • Removal of sensitized RBCs

  • Removal of incompatible antibody

  • Replacement of incompatible RBCs with compatible RBCs

  • Suppression of erythropoeisis (reduced production of incompatible RBCs


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HEMOLYTIC DISEASE OF THE NEWBORN

Comparison of ABO versus Rh HDN