1. Immunohematology
Dr. Julio Hilario Vargas
Department of Physiology
School of Medicine
National University of Trujillo

2. DEFINICION
Es la parte de la hematología que estudia los
procesos inmunitarios que tienen lugar en el
organismo en relación con los elementos
sanguíneos.
Uno de los aspectos más importantes de la
inmunohematología es el estudio y cuantificación
de los grupos sanguíneos eritrocitarios que son
componentes antigénicos presentes en la
superficie de los hematíes, ya que se relaciona
directamente con la terapéutica transfusional y la
prevención de accidentes hemolíticos graves.

3. En primer termino se identificaron sobre los hematíes,
pero luego se describieron determinantes antigénicos
plaquetarios, leucocitarios y séricos.
Los genes determinantes de los grupos sanguíneos
transmiten, generalmente, caracteres codominantes ( se
expresan en homocigotos y heterocigotos).
Existen genes “amorfos” que no generan productos que
puedan ser identificados como antígenos ( ej: gen d)
Todos los antígenos de los grupos sanguíneos han sido
definidos serológicamente por la presencia de sus
anticuerpos correspondientes.

4. 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
Primary immunological components: antigens &
antibodies  provides basis for blood bank testing
and reactions

5. CARDINAL RULE IN BLOOD BANK:
Antigens are found on the surface of
red blood cells and the antibodies
are found in serum or plasma

6. IMMUNOLOGIC PRINCIPLES

7. ANTIGENS
Substances that have the capability to stimulate the
production of an antibody
Characteristics:
1. Chemical nature – protein, CHO, nucleic acid or
lipopolysaccharide
2. Molecular weight > 10,000 daltons
3. Complexity – more complex, > antibody
stimulation
4. Stability – if unstable  degrade  less Ab
stimulation
5. Foreign

8. Chemical composition of antigens
1. Glycoproteins & lipoproteins – most potent
Glycolipids
2. Pure polysaccharides – not immunogenic except in
humans and mice
3. Pure lipids & nucleic acids – not immunogenic but
can be antigenic  serve as haptens

9. Grupos Eritrocitarios y sus Antígenos
ANTÍGENOS MAS
GRUPOS SANGUÍNEOS
IMPORTANTES
ABO A, B, AB, O
Rh D, C, c, E, e
MNS M, N, S, s, U
Lewis Lea, Leb
P P1, P2
Lutheran Lua, Lub
Kell K, k, Kpa, Kpb
Duffy Fya, Fyb
Kidd Jka, Jkb

10. SISTEMAS DE GRUPOS SANGUINEOS
Grispan S. Rev. Medica Hondur. 51:103-14. 1983

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

12. 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)

13. 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

14. 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)

15. ABO BLOOD GROUP SYSTEM

16. 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

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

18. ABO BLOOD GROUP SYSTEM
A and B genes do not directly produce
antigens  enzymatic reaction products of
enzymes called glycosyltransferases 
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

20. ABO BLOOD GROUP SYSTEM
Red blood cell precursor structure

21. ABO BLOOD GROUP SYSTEM
Antigen formation
H antigen B antigen B antigen

22. ABO Blood Group System
http://www.ncbi.nlm.nih.gov/gv/rbc/xslcgi.fcgi?cmd=bgmut/systems_info&system=abo

23. 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

24. 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

25. ABO BLOOD GROUP SYSTEM
Frequencies of ABO Blood Groups:
Blood Group Frequency
O 45%
A 41%
B 10%
AB 4%

26. ABO BLOOD GROUP SYSTEM
ABO Subtypes:
1. 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

27. ABO BLOOD GROUP SYSTEM
ABO Subtypes:
2. Weak A and weak B phenotypes
3. 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

28. 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

29. ABO ROUTINE TESTING
(slide or test tube method)
DIRECT 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

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

31. 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

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

33. 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

34. 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

35. 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 –
Roleaux
hypogammaglobulinemia, extreme
ages, immunosuppression

36. 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

37. FRECUENCIAS FENOTIPICAS DEL SISTEMA RH
ANTIGENO Rh Frecuencia ( %)
D (+) 85
D(-) 15
C 70
c 80
E 30
e 98

38. 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

39. 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

40. Comparación de las nomenclaturas para
los Ag del Sistema Rh
Wiener Fisher –Race Rosenfield
Rho D Rh1
rh` C Rh2
rh” E Rh3
h`r c Rh4
hr” e Rh5

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

42. 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

43. 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 (+)
Person who appear to be Rh (-) should be
proven to be Du (-) before they are considered
to be eligible to receive transfusion

44. 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)

45. Rh BLOOD GROUP SYSTEM
Rh Typing – slide or test tube method
False (+) results
a. Drying
b. Roleaux formation
c. Auto-agglutination
d. Patient’s red cells heavily coated with Ab’s
e. Presence of cold agglutinins

46. Rh BLOOD GROUP SYSTEM
Rh Typing
False (-) results
a. Use of old cells
b. Wrong cell concentration
c. Hemolysis
d. Inadequate mixing of cells
e. Inactive typing sera
f. Incorrect temperature
g. Existence of Du variant
h. High concentration of blocking antibodies

47. 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

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