Major & Minor Blood Group System

Antiglobulin Test
Pretransfusion Testing
Automation in Blood Bank

ARCIAGA, FRANCES GELINE R.

 ABO Blood Group

Most well known & clinically important blood group

system.
Discovered by Karl Landsteiner (1901)
Von Decastello and Sturli (1902) - AB blood group.
Von Dungern and Hirszfeld (1911)- divided group A into 2
subgroups A1 and A2
Landsteiner Law:
Antigen on the RBC determines the blood group

The corresponding antibody is never found in the individuals

serum.
The opposite antibody is always present in the individuals serum.
Blood group Antigens on Naturally % Population
RBC occuring Abs
A A Anti-B 40%
B B Anti-A 10%
AB A, B - 5%
O - Anti-A, Anti-B 45%
 Forward typing Reverse typing
Reaction of Cells Tested Reaction of Serum Tested
ABO With Against
Group
Anti-A Anti-B A1 Cells B Cells
A + 0 0 +
B 0 + + 0
AB + + 0 0
O 0 0 + +
ABO Lectins - plant/animal extract that agglutinates
human red cell with specificity
A Dolichos biflorus
B Bandeiraea simplicifolia
O Ulex europeus
 Genotype
Found in genes; not observable; can be homozygous or heterozygous
Phenotype
Demonstrable antigen; observable; A, B, AB, O
Homozygous genotype
Identical alleles
Heterozygous
Different alleles
Amorph
Gene that does not produce any detectable trait
Dominant gene:
Always expressed
Recessive gene
Gene that in the presence of a dominant gene does not express itself
 ABH Antigen
H-gene - Chromosome 19
Formation of ABH antigen on RBC surface

ABO gene - Chromosome 9; A, B, O Ag

Se gene - Chromosome 19
Formation of ABH antigen on secretions

Gene Glcosyltransferase Immunodominant sugar Antigen

H L- fucosyl trnsferas L-fucose H

A N acetylgalactosaminyl N-acetyl-D-galactoseamine A
transferase

B D- galactosyl transferase D-galactose B

AB Both A & B Both A & B AB

 Bombay Phenotype

Discovered in 1952 by Bhende in Bombay, India

Bombay individuals lack all normal expression of the A, B,
or O genes they inherited.
Lacks H gene, making it incompatible with all ABO donors.

In routine forward grouping, using anti-A, anti-B, and anti-

AB. The Bombay would phenotype as an O blood group.
However, transfusing normal group O would cause
immediate cell lysis by the potent anti-H of the Bombay
individuals. Thus, only blood from another Bombay
individual can be transfused to a Bombay recipient.
Classification Genes Glycosyltransferase RBC Ag Presence of Abs present
present ABH
substance
Classic Bombay hh/sese None None None Anti-A
Anti-B
Anti-H
Para Bombay hh/Se A & / or B transferase Weak to none Yes Weak Anti-H
Anti-A/B
H-partially Weak A & / or B transferase Weak None Anti-H
deficient variant Anti-A/B
hh/se
 ABO Typing Discrepancies

Group I: Weak or Absence of Antibody Reaction

Serum Typing; Missing Reaction
Age: Too young and Too old
Agammaglobulinemia
Hypogammaglobulinemia
Immunodeficiency diseases
Group II: Weak or Absence of Antigen Reaction
Cell Typing; Missing Reaction
Subgroups of A & B (Lower antigen: No reaction/Weak reaction)
Blood group soluble substance tendency to neutralize the Anti-sera
Leukemia, Hodgkins
Antibodies to low incidence antigens
 Group III: Unexpected Antibody Reaction
Serum Typing
Autoantibodies
Increased globulin content and fibrinogen
Plasma expanders
Whartons Jelly
Group IV: Unexpected Antigen Reaction
Poly Agglutination
Cold reactive antibody
Unexpected ABO isoagglutinins
Antibodies against acriflavine
 RH Blood Group

Rhesus monkey (Macaca mulatta)

Discovered by Levine & Stetson
Clinically significant: D (Rh+)
Increasing immunogenicity: D>c>E>C>e

Rh system nomenclature:
Fisher-Race (CDE) Wiener (Rh-Hr) Rosenfield (Numerical) ISBT

D Rho Rh 1 004 001

C rh Rh 2 004 002

E rh Rh 3 004 003

c hr Rh 4 004 004

e hr Rh 5 004 005
RHD Gene RHCE Gene Gene Complex Shorthand Nomenclature
D ce Dce RO
D Ce DCe R1
D cE DcE R2
D CE DCE RZ

d ce dce r
d Ce dCe r
d cE dcE r
d CE dCE rY
 Rh Antibodies

Mostly IgG
Do not bind complement
Causes HDN
Pathogenesis: occurs when maternal IgG attaches to specific Ags of
fetal RBCs
Hemolysis occurs in babys spleen
Results to kernicterus
Diagnosis
Serologic tests
Intrauterine transfusion
Phototherapy
Exchange therapy
RhIg
 Other Blood Group

ISBT Blood Group

IgG producing:
001 ABO
Rh, Duffy, Kidd, Kell, Ss
002 MNSs
003 P
004 Rh Dosage Effect:
005 Lutheran MnSs, Duffy, Kidd, Rh, Lutheran
006 Kell
007 Lewis
008 Duffy
009 Kidd
010 Diego
 Lewis MnSs
Aka. Plasma antigen
Chromosome 4
Glycoprotein produced in
secretions but adsorbed in the Glycoprotein found only on
surface of red cells RBC
Le gene codes for the Determined by MN and Ss loci
production of
fucosyltransferase enzyme Important for paternity studies
Manufactured by tissue cells Antibodies
and not by RBC; can be
Anti-M, Anti-N -> IgM
neutralized by the plasma
Lewis Abs: IgM, naturally Anti-S, Anti-s -> IgG
occuring
Anti-N: Hemodialysis patient
Lewis Phenotypes
Le (a-b-) found in cord
Anti-M: Reacts best at pH 6.5
blood; cancer; pregnant (M-N-): Resistant in
women Plasmodium falciparum
Le (a+b-) Lea; present in infections
non-secretors
Le (a-b+) Leb; present in
secretors
 Phenotype Antigens Possible
present in Abs
RBC
P P1 P1, Pk, P None Ii
P2 P k, P Anti-P1 High incidence Ag
P1k P1, Pk Anti-P Related to ABH, Lewis and P
P2k Pk Anti-P1 & Ag
Anti-P
P None Anti-PP1k Natural transition from small i
Anti-P1 to big I
Weak IgM Anti-I: IM, HEMPAS,
Anti-PP1k (Anti-Tja) Alcoholic cirrhosis,
Associated to spontaneous reticulosis
abortion
Anti-I: Primary atypical
Anti-P (Donath Landsteiner pneumonia
Antibody)
Biphasic hemolysin
Seen in paroxysmal cold
hemoglobinuria
Disease associated
Pk Antigen receptor for Shiga
toxin and E.coli associated HUS
P Antigen receptor for
Parvovirus B19
 Kell Kidd
Immunogenic Function: urea transport
Well-developed at birth Jka, Jkb
Expression very weak on Not destroyed by enzyme
McLeod phenotype Anti- Jka, Anti-Jkb
Related to CGD Not easily detected
Duffy No. 1 cause of severe fatal
Destroyed by enzymes delayed hemolytic
Function: chemokine receptor transfusion
Fya, Fyb
Most common
Fy (a-b-)
African black
 Lutheran
Lu (a-b+) : most common
Anti-Lua
IgG, IgM, IGgA
Anti- Lub
IgG , immune antibody
Minor Blood Group
 Antihuman Globulin Reagent

Reagents
Monospecific made against specific Ig class or complement
Polyspecific made against a combination of Ig class or complement
but always contain anti-IgG and anti-C3d
Direct antiglobulin test
For in vivo coating of RBC with Ab/complement
Clinical condition: HDN, HTR, AIHA
Anticoagulant of choice: EDTA
False (+)
Septicemia
Overcentrifugation
False (-)
Inadequate washed RBC
Undercentrifugation
 Indirect antiglobulin
In vitro coating of RBCs with Ab/complement.
Uses
Antibody detection
Compatibility testing
Investigating HDN, HTR, AIHA
Enhancements
Bovine serum albumin
LISS
PEG
False (+)
Overcentrifugation
Polyagglutination from in vivo coated RBCs
False (-)
Inadequate washed RBC
Undercentrifugation
 Pretransfusion Compatibility Testing
For the safety of the patient
Positive identification of the patient: 1st and most
important step
Compatibility testing protocol
testing donor and patient sample -> ABO, Rh, Ab screen
Antibody screening involves reaction between patient serum
with 2 or 3 reagent phenotyped for multiple antigens; for detection
of unexpected antibodies
Selection of appropriate blood
1st 2nd 3rd 4th
Choice Choice Choice Choice
AB AB A B O
A A O
B B O
O O
 Crossmatch
Final check of ABO compatibility between donor and patient

Major crossmatch: PS + DR

Phases
Immediate spin
37oC
AHG

Sample: serum
 Automation in Routine Blood Bank Testing

Gel technology
Advantage: standardization of result
Does not require washing but is expensive
Never use hemolyzed, icteric or lipemic spx because it will give
you erroneous result
Uses: ABO & Rh testing, Ab screening, compatibility testing,
Ab identification
Solid Phase Technology
Advantage: standardization of result
Occurs in microplate well; requires washing
May use hemolyzed, icteric or lipemic spx
Uses: Ab screening, Ab identification, compatibility testing
 Solid-phase Protein A
Uses: antiglobulin testing, Ab identification and detection

Solid-phase Immunosorbent assay (ELISA)

Uses: compatibility testing

Luminex-based assay
Uses: test for platelet/HLA antibodies
 Questions:

The immunodominant sugar responsible for blood

group B specificity is:
A. L-fucose
B. N-acetyl-D-galactosamine
C. Uridine diphosphate-N-acetyl-D-galactose
D. D-galactose

This test is used to detect ABO antibodies in an

individual's serum.
A. Forward typing
B. Reverse typing
C. Either
D. Neither
 What group of ABO typing discrepancy is associated with
an increase in globulin and fibrinogen content?
A. Group I
B. Group II
C. Group III
D. Group IV
The answer above is also referred to as:
A. Unexpected Antigen Reaction
B. Unexpected Antibody Reaction
C. Weak or Absence of Antigen Reaction
D. Weak or Absence of Antibody Reaction
 In the direct antiglobulin test, the antiglobulin
reagent is used to:
A. Detect preexisting antibodies on erythrocytes
B. Precipitate anti-erythrocyte antibodies
C. Measure antibodies in a test serum by fixing complement
D. Mediate hemolysis of indicator red cells by providing
complement.
 Case study

An obstetrical patient has had 3 previous

pregnancies. Her 1st baby was healthy, the 2nd was
jaundiced at birth and required a transfusion, while
the 3rd was stillborn. Which of the following is the
most likely cause:
A. ABO incompatibility
B. Immune deficiency disease
C. Congenital spherocytic anemia
D. Rh incompatibility
 Answer

D. Rh incompatibility
HDFN is caused by maternal antibody crossing the placenta
and destroying fetal antigen-positive red cells. Unlike ABO
antibodies, which are naturally-occurring and can affect the
first pregnancy, Rh antibodies are not produced until the
mother has been exposed to Rh-positive red cells, usually
during delivery of the first Rh-positive child. Once immunized,
subsequent pregnancies with Rh-positive infants are affected,
usually with increasing severity.
References:

Hubbard, J. D., & Hubbard, J. D. (2010). A concise review of

clinical laboratory science (2nd ed.). Philadelphia: Wolters
Kluwer Health/Lippincott Williams & Wilkins.
Blood Groups Overview:
https://www.youtube.com/watch?v=ogr90JOvsI4&t=206s&i
ndex=8&list=PL4MaXmzlVM-YeWSyXBjaTU2NtpiDMp4Y_
http://www.bbguy.org/pdf/BB1Updated1211.pdf
Harmening, D., Baldwin, A. J., & Sohmer, P. R.
(1983). Modern blood banking and transfusion practices.
Philadelphia: F.A. Davis.
Blood Group: https://www.slideshare.net/moisture/blood-
group-24464268?qid=0a67c982-764c-43d2-bb8d-
9da98721935d&v=&b=&from_search=2