1. Dept. of Pathology & Laboratory Diagnosis
IMMUNOHEMATOLOGY
Fe A. Bartolome, M.D.
Dept. of Pathology & Laboratory Diagnosis

2. 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

3. 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

4. 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

5. 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

6. IMMUNOLOGIC PRINCIPLES
Immunogenicity of Blood Group Antigens
A, B and D (Rho) – most immunogenic
Kell (K)
Duffy: Fya
Fyb
Kidd: Jka
Jkb

7. 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)

8. 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

9. 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)

10. 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

11. ABO BLOOD GROUP SYSTEM ABO System Phenotype Antigen Natural antibody
Genotype A
A only
Anti-B
AA or AO B
B only
Anti-A
BB or BO AB
A and B
None O
Anti-A, OO

12. 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

13. ABO BLOOD GROUP SYSTEM

14. ABO BLOOD GROUP SYSTEM

15. ABO BLOOD GROUP SYSTEM

16. ABO BLOOD GROUP SYSTEM

17. 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

18. ABO BLOOD GROUP SYSTEM
Example of determining offspring blood types from known or suspected genotypes:
Genotype parent #1 (AO)
A O
Genotype parent A AA AO
#2 (AB) B AB BO
Phenotypes of possible offsprings: A, AB, B

19. ABO BLOOD GROUP SYSTEM Frequencies of ABO Blood Groups:
Blood Group Frequency
O %
A %
B %
AB %

20. 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

21. 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

22. 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

23. 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:
Antisera Color Source
Anti-A Blue Group B donor
Anti-B Yellow Group A donor
Anti-A,B Clear Group O donor

24. 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

25. Agglutination with Anti-A Agglutination with Anti-B
ABO ROUTINE TESTING
Reaction Patterns for ABO Groups
Blood group
Agglutination with Anti-A
Agglutination with Anti-B A + - B AB O

26. 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

27. Agglutination with A cells Agglutination with B cells
ABO ROUTINE TESTING
Reaction Patterns for ABO Groups
Blood Group
Agglutination with A cells
Agglutination with B cells A - + B AB O

28. 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

29. 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

30. 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

31. 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

32. 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

33. 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

34. 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

35. 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

36. 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

37. 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

38. 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

39. Presence of D = presence of Rho factor  Rh (+) Absence of D  Rh (-)
Rh BLOOD GROUP SYSTEM
Presence of D = presence of Rho factor  Rh (+)
Absence of D  Rh (-)

40. 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

41. Rh BLOOD GROUP SYSTEM Testing for Du Variant:
use bovine or albumin-suspended anti-D reagent
incubate at 37oC for 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

42. 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)

43. 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

44. 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

45. MINOR BLOOD GROUP SYSTEMS
Significance:
For medico-legal parenthood studies
May cause transfusion reaction or HDN

46. 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

47. 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

48. 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

49. 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

50. 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

51. 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

52. 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

53. 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.

54. 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)

55. HEMOLYTIC DISEASE OF THE NEWBORN

56. 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

57. 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

58. 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

59. HEMOLYTIC DISEASE OF THE NEWBORN

60. 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

61. 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

62. 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

63. HEMOLYTIC DISEASE OF THE NEWBORN
Comparison of ABO versus Rh HDN
Characteristic
ABO
HDN
First pregnancy
Disease predicted by titers
Antibody IgG
Bilirubin at birth
Anemia at birth
Phototherapy
Exchange transfusion
Intrauterine transfusion
Spherocytosis
Yes No
Yes (anti-A,B)
Normal range
Rare
None
Yes (anti-D)
Elevated
Common
Sometimes

64. Thank you!