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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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slide1

IMMUNOHEMATOLOGY

Fe A. Bartolome, M.D.

Dept. of Pathology & Laboratory Diagnosis

slide2

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
slide3

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

slide4

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
slide5

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
slide6

IMMUNOLOGIC PRINCIPLES

Immunogenicity of Blood Group Antigens

A, B and D (Rho) – most immunogenic

Kell (K)

Duffy: Fya

Fyb

Kidd: Jka

Jkb

slide7

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)
slide8

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
slide9

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)
slide10

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
slide12

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
slide17

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
slide18

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

slide19

ABO BLOOD GROUP SYSTEM

Frequencies of ABO Blood Groups:

Blood Group Frequency

O 45%

A 41%

B 10%

AB 4%

slide20

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
slide21

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
slide22

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
slide23

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
slide24

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
slide25

ABO ROUTINE TESTING

Reaction Patterns for ABO Groups

slide26

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
slide27

ABO ROUTINE TESTING

Reaction Patterns for ABO Groups

slide28

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
slide29

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

slide30

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
slide31

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

slide32

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

slide33

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
slide34

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
slide35

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
slide36

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
slide37

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
slide38

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
slide39

Rh BLOOD GROUP SYSTEM

  • Presence of D = presence of Rho factor  Rh (+)
  • Absence of D  Rh (-)
slide40

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
slide41

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
slide42

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)
slide43

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
slide44

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
slide45

MINOR BLOOD GROUP SYSTEMS

  • Significance:
  • For medico-legal parenthood studies
  • May cause transfusion reaction or HDN
slide46

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
slide47

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
slide48

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
slide49

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
slide50

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
slide51

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
slide52

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
slide53

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.
slide54

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)
slide56

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
slide57

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
slide58

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
slide60

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
slide61

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
slide62

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
slide63

HEMOLYTIC DISEASE OF THE NEWBORN

Comparison of ABO versus Rh HDN

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