Transcript Document

Fe A. Bartolome, M.D.
Dept. of Pathology & Laboratory Diagnosis
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
components and constituents
all
blood
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
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
IMMUNOLOGIC PRINCIPLES
Chemical composition of antigens:
1. Glycoproteins & lipoproteins – most potent
2. Glycolipids
3. Pure polysaccharides – not immunogenic except
in humans and mice
4. Pure lipids & nucleic acids – not immunogenic but
can be antigenic  serve as haptens
IMMUNOLOGIC PRINCIPLES
Immunogenicity of Blood Group Antigens
A, B and D (Rho) – most immunogenic
Kell (K)
Duffy: Fya
Fyb
Kidd: Jka
Jkb
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 Agbinding fragments (Fab) & 1 crystallizable fragment (Fc)
IMMUNOLOGIC PRINCIPLES
Classification of Blood Group Antibodies:
1. 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)
2. Autoantibodies
•
Reacts with an Ag on patient’s own cells & with
that same Ag on the cells of other individuals
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)
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
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
AB
O
None
Anti-A,
Anti-B
OO
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
ABO BLOOD GROUP SYSTEM
ABO BLOOD GROUP SYSTEM
ABO BLOOD GROUP SYSTEM
ABO BLOOD GROUP SYSTEM
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
ABO BLOOD GROUP SYSTEM
Example of determining offspring blood types from
known or suspected genotypes:
Genotype parent #1 (AO)
Genotype parent
#2 (AB)
A
O
A
AA
AO
B
AB
BO
Phenotypes of possible offsprings: A, AB, B
ABO BLOOD GROUP SYSTEM
Frequencies of ABO Blood Groups:
Blood Group
Frequency
O
45%
A
41%
B
10%
AB
4%
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
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
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:
1. IgM
2. Reacts at room temp. after an immediate
spin
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
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:
1. confirms the results of the anti-A and anti-B
2. will show a (+) reaction with weak subgroups
of A and B that do not react with the anti-A
and anti-B
ABO ROUTINE TESTING
Reaction Patterns for ABO Groups
Blood group
Agglutination
with Anti-A
Agglutination
with Anti-B
A
+
-
B
-
+
AB
+
+
O
-
-
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
ABO ROUTINE TESTING
Reaction Patterns for ABO Groups
Blood Group
Agglutination
with A cells
Agglutination
with B cells
A
-
+
B
+
-
AB
-
-
O
+
+
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
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
ABO ROUTINE TESTING
Causes of Discrepancies in ABO Testing:
A. 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
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
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
ABO ROUTINE TESTING
WHAT TO DO?
1. Wash cells with saline 3-4x and repeat all tests
and test for antibodies
2. Test for subgroups of A using anti-A1 and anti-A
3. Use cell panels to detect the specificity of
abnormal antibodies
Rh BLOOD GROUP SYSTEM
•
discovered in 1940 by Landsteiner & Wiener
•
most complex erythrocyte
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
antigen
system;
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
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
Rh BLOOD GROUP SYSTEM
Rh Antigens
•
with three integral membrane proteins
1. RhD
2. RhCcEe
3. Rh-associated glycoprotein (Rh50, RhAG)
•
D antigen  resides in RhD protein  most
immunogenic followed by c, E, C and e
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:
1. a piece of the D antigen is missing
2. D gene is on a chromosome opposite a C gene 
(+) steric hindrance
3. Inheritance of a gene coding for less D antigen
Rh BLOOD GROUP SYSTEM
• Presence of D = presence of Rho
factor  Rh (+)
• Absence of D  Rh (-)
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
Rh BLOOD GROUP SYSTEM
Testing for Du Variant:
• use bovine
reagent
or
albumin-suspended
anti-D
• 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
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)
Rh BLOOD GROUP SYSTEM
Rh Typing – slide or test tube method
•
False (+) results:
1. Drying
2. Roleaux formation
3. Auto-agglutination
4. Patient’s red cells heavily coated with Ab’s
5. Presence of cold agglutinins
Rh BLOOD GROUP SYSTEM
Rh Typing
•
False (-) results:
1. Use of old cells
2. Wrong cell concentration
3. Hemolysis
4. Inadequate mixing of cells
5. Inactive typing sera
6. Incorrent temperature
7. Existence of Du variant
8. High concentration of blocking antibodies
MINOR BLOOD GROUP SYSTEMS
Significance:
1. For medico-legal parenthood studies
2. May cause transfusion reaction or HDN
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
MINOR BLOOD GROUP SYSTEMS
Systems with cold-reacting antibodies
1. 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
2. MNS System
•
Antigens are weakly antigenic
•
Antibodies: naturally-occurring or stimulated by
direct exposure
MINOR BLOOD GROUP SYSTEMS
Systems with cold-reacting antibodies
3. P-p System
•
P1 antigen most antigenic  present on cells of
79% of whites & 94% of African-Americans
4. 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
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
1. Kell (K) – Cellano (k) System
•
k Ag present in 98% of the white population
•
antibodies primarily IgG
2. Kidd System
•
Antigens: Jka & Jkb – not very antigenic
•
Antibodies stimulated by direct exposure via
either pregnancy or transfusion
MINOR BLOOD GROUP SYSTEMS
Systems with warm-reacting antibodies
3. Duffy System
•
Antigens: Fya & Fyb
•
Antibodies stimulated through direct exposure
 capable of causing HDN and HTR
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
septicemia,
syphilis
diagnosis: physiologic jaundice,
CID, toxoplasmosis, congenital
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
HEMOLYTIC DISEASE OF THE NEWBORN
ABO Disease
Features:
1.Spherocytosis
2.Increased reticulocyte count
3.Increased indirect bilirubin in 1st 72 hours of life
4.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.
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)
HEMOLYTIC DISEASE OF THE NEWBORN
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,
because of:
firstborns
are
affected
either
1. previous maternal exposure (e.g. previous
aborted pregnancy)
2. unusually great maternal susceptibility to
Rh stimulus during normal pregnancy
HEMOLYTIC DISEASE OF THE NEWBORN
Rh Disease
Characteristics of Erythroblastosis Fetalis:
1. Increased number of circulating nucleated red cells
2. Increased osmotic fragility of cells
3. Increased amount of indirect/unconjugated bilirubin
Main Clinical Findings:
1. Anemia - < 15 gm/100 ml or 150 gm/L
2. Rapidly developing jaundice
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
HEMOLYTIC DISEASE OF THE NEWBORN
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
HEMOLYTIC DISEASE OF THE NEWBORN
For the baby: EXCHANGE TRANSFUSION
•
Indications:
1. Infant serum indirect bilirubin > 20mg/100 ml
(342 mol/L) for fullterm infants OR
>15mg/100 ml (257 mol/L) for premature
infants
2. Cord blood indirect bilirubin > 3 mg/100 ml
(51 mol/L)
3. Cord blood hemoglobin < 13 gm/dL (130 g/L)
4. Maternal Rh antibody titer of 1:64 or more
HEMOLYTIC DISEASE OF THE NEWBORN
Beneficial Effects of Exchange Transfusion:
1. Removal of bilirubin
2. Removal of sensitized RBCs
3. Removal of incompatible antibody
4. Replacement
of
compatible RBCs
incompatible
5. Suppression
of
erythropoeisis
production of incompatible RBCs
RBCs
with
(reduced
HEMOLYTIC DISEASE OF THE NEWBORN
Comparison of ABO versus Rh HDN
Characteristic
First pregnancy
Disease predicted by titers
Antibody IgG
Bilirubin at birth
Anemia at birth
Phototherapy
Exchange transfusion
Intrauterine transfusion
Spherocytosis
ABO
HDN
Yes
No
Yes (anti-A,B)
Normal range
No
Yes
Rare
None
Yes
Rare
Yes
Yes (anti-D)
Elevated
Yes
Yes
Common
Sometimes
Rare