IMMUNOHEMATOLOGY - BB MIDTERM

Prepared by: Luis Dominick B. Antig, RMT, MSc.

Chapter 7: The Rh Blood Group System Fisher-Race Weiner Gene Agglutinogen
frequency
INTRODUCTION 1. Rh-positive
1. The Rh blood group system is comprised of some DCe R1 40% Rho, rh’, hr”
57 different antigens. Given are of particular DcE R2 16% Rho, hr’, rh”
importance: D, C, E, c and e. Dce Ro 2% Rho, hr’, hr”
2. There are three nomenclatures involved in the Rh DCE Rz 0.08% Rho, rh’, rh”
system. The Fisher-Race and Weiner are used 2. Rh-
interchangeably. negative r 38% - hr’, hr”
a. Fisher-Race terminology assumes that 3 closely dce r’ 1% - rh’, hr”
linked genes are inherited from each parent. dCe r” 1% - hr’, rh”
The most common alleles that occupy these dcE ry very rare - rh’, rh”
loci are described as: dCE
D and d
C and c C. THE D ANTIGEN (Rho Antigen)
E and e 1. The D antigen was discovered in 1939 by Levine
and Stetson. By the mid-1940, Cc and Ee antigens
Each gene with the exception of d codes for a were discovered.
specific antigen that can be detected on the red 2. The D antigen is the most immunogenic of all Rh
cell membrane. It is possible the d is a silent gene. antigens.
a. Rh positive individuals express the D antigen
b. Weiner nomenclature assumes the inheritance on RBC. Rh (+) individuals constitute 85% of the
of a single gene from each parent. Each gene is population.
a mosaic structure comprising a variable b. Rh negative individuals do not express the D
number of blood factors (agglutinogen). For antigen on RBC (i.e. dCe/Dce). Rh (-) individuals
example, R1 gene codes for factors that constitute 15% of the population.
correspond to D, C, and e. The r gene produces 3. Approximately 70% of Rh negative individuals
the c and e antigen but no d. produce anti-D if given Rh positive blood. Routine
R1 = DCe typing of C and E antigens is not performed
R2 = DcE because C and E antigens are not as immunogenic
R0 = Dce as D.
Rz = DCE 4. The order of immunogenicity of Rh antigens:
D>c>E>C>e
c. Rosenfield system assigns a number to each of
the antigens: D. RH GENES
D = Rh: 1 1. There are two genes that control the expression of
C = Rh: 2 Rh antigens
E = Rh: 3 RHD gene
c = Rh: 4 RHCE gene
e = Rh: 5 2. These two genes are located on chromosome 1.
3. RHD gene encodes for the presence or absence of
A. RH SYSTEM NOMENCLATURES RhD protein.
4. RHCE gene encodes either RhCe, RhcE, Rhce or
Fisher-Race Weiner Rosenfield RhCE proteins.
D Rho Rh : 1 5. RhAG gene resides in chromosome 6 and it
C rh’ (called rh prime) Rh : 2 encodes Rh-associated glycoprotein (RhAG).
E rh’’ Rh : 3 6. RhAG is a coexpressor protein found within the
c hr’ Rh : 4 RBC membrane and must be present for successful
e hr’’ Rh : 5 expression of Rh antigens.

E. WEAK D (Du)
B. POSSIBLE GENE COMBINATIONS
1. Du is a weak form of D antigen rarely found among
Caucasians but common among in Blacks (22%).
Traditionally, Fisher-Race terminology is used to
2. Du red cells give weak or negative reactions with
describe Rh antigens whereas Weiner terminology is used
anti-D.
when describing Rh phenotype.
 FAR EASTERN UNIVERSITY – MANILA
3. Du is detected by performing an indirect
antiglobulin test (IAT).
4. Categories of Du
Du red cells can be classified into 3 catergories:
a. Acquired Du. The inheritance of C in the
transposition to D (dCe/DcE results in the
weakened expression of the D antigen on red
cells). These individuals do not produce anti-D
if exposed to Rh-positive blood.
Depressed D
Genotype 1 Genotype 2
Dce/dce Dce/dCe
C in cis position to D C in transposition to D
Normal expression of D Du phenotype
b. Hereditary Du. The reason for the weakened
expression of the D antigen in these individuals
is not known.
c. Du variant. The D antigen normally consists of
at least 4 parts. If one or more parts of the
antigen can be weakly expressed.
5. Du variants
Du may also be divided into 2 grades:
a. High grade Du
 This is not passed on to future
generations since development
depends upon the rare r’ (Cde) or (CdE)
chromosomal arrangement.
 It reacts with either complete or
incomplete anti-D and only rarely
requires the use of antiglobulin test for
its detection.
b. Low grade Du
 It is a direct product of inherited gene
and can be passed on to future
generation.
 It is usually detected only by
antiglobulin test after first sensitizing
the cells with anti-D sera.
6. Significance of Du
a. Du individual should be considered as Rho (D)
positive blood donor and in an emergency can
be transfused with Rh positive or negative
blood when a recipient.
b. Many workers believe that patients do not
have to be typed, and that the safest, most
efficient and the easiest way to handle the
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issue is to routinely transfuse Rh negative
blood to all patients who are negative when
tested with anti-sera.
c. Du is common among Negroes.
F. RH ANTIBODIES
1. Rh antibodies are not natural antibodies. They are
classified as immune antibodies because they are
present only after exposure to Rh antigens through
ingestion, injection or pregnancy.
2. Rh antibodies do not ordinarily react in saline
medium since they are mostly of the IgG class.
3. Varieties of Rh antibodies
a. First order Rh antibodies (saline agglutinins,
bivalent antibodies, complete antibodies).
They react strongly with specific Rho antigens
in saline medium and react less strongly in a
protein medium.
b. Second order Rh antibodies (albumin-reacting,
incomplete, monovalent antibodies). They
react visibly with specific antigen in a protein
medium. In saline medium, they weakly
combine with the antigen but do not produce a
visible reaction.
c. Third order Rh antibodies (typical Rh
antibodies, antiglobulin Rh antibodies). They
react visibly with specific antigens in an
antiglobulin medium only.
G. LABORATORY PROCEDURES
1. Rh typing
Rh typing is performed with the commercial anti-
Rho (D) antibodies which are produced from the
serum of Rh-negative person who have been
immunized of the Rho (D) factor.
a. Slide Test
 It is a rapid method employing very potent
anti-D (Rho) antiserum.
 The cell-antiserum mixture is heated on an
illuminated Rh view box with a temperature
of about 45C.
 Agglutination if positive should be visible
within 2 minutes to avoid drying and
rouleaux formation.
b. Tube Test
 The cell-antiserum mixture is incubated at
37C for half hour.
 Agglutination should be achieved if positive
after the incubation period. Agglutination
can be made stronger through
 FAR EASTERN UNIVERSITY – MANILA
centrifugation at 1,500 rpm for 1 minute or
at 3,000 rpm for 15 secs.
c. Interpretation of Rh typing
 Where there is agglutination, the person
Rh-positive. When there is no agglutination,
the person is either Rh-negative or Du
positive.
 Whenever there is no agglutination, a test
for Du variant should follow. If the Du test is
negative, then the person is Rh-negative.
d. Unlike the ABO system, the Rh antigens are
found only on the RBC surface.
2. Detection of Du
a. In order to determine whether the patient’s
cells are Rho (D negative) or possible Du
positive, the patient’s cells are incubated with
anti-Rho (D) typing serum to coat the cells.
After incubation, the cells are centrifuged and
examined for agglutination. If no agglutination
is observed, the cells are developed into an
antiglobulin test by adding anti-human globulin
(Coomb’s reagent).
b. Interpretation
 Agglutination indicates the patient is Du
positive and is classified as Rh (D) positive
patient.
 No agglutination indicates the patient is Rh
(D) negative and Du negative.
H. RH DEFICIENCY SYNDROME
1. Individuals have Rh deficiency or Rhnull syndrome.
2. These individuals fail to express any Rh antigens on
the RBC surface.
3. There are two ways to inherit Rhnull syndrome:
a. Regulator type Rhnull syndrome
 A mutation occurs in the RHAG gene.
 This results in no RhAG protein expression
and subsequently no RhD or RhCE protein
expression on the RBCs.
 This mutation can be passed from parent
to offspring.
b. Amorphic type Rhnull syndrome
 There is a mutation in RHCE genes
inherited from each parent and the
common deletion of the RHD gene found
in most D-negative (Rh-negative)
individuals.
 The RHAG gene is normal.
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4. Symptoms include:
 Mild compensated hemolytic anemia
 Stomatocytosis
 ↑ A slight-to-moderate decrease in Hgb and
Hct levels
 ↑↑↑ Increase in Hgb F
 ↓↓↓ Decreased serum haptoglobin
 ↑↑ Elevated bilirubin
I. MEDICO-LEGAL USE OF BLOOD GROUPS
1. Using the common blood group system (ABO, Rh,
MNSs) it is possible to exonerate approximately
50% of wrongly accused males from being the
father of the infant in question.
2. Blood typing can never prove paternity and only
either disprove it or result in equivocation.
Chapter 8: Other Blood Group System
A. THE LEWIS SYSTEM AND SECRETOR
STATUS
1. The Lewis Antigen
a. The Lewis antigen differs from all other blood
groups in that it is soluble and found in saliva
and plasma. Hence, Lewis antigens are called
plasma antigens.
b. The Lewis antigen is not true blood group
antigen. The antigen is mainly acquired from
the body tissues and attaches itself onto the
red cell surface later in life. Most infants are
Le(a-b-) since the antigen is fully developed at
age six.
c. The expression of Lewis antigens is influenced
by the presence of Hh and Sese genes.
 Se gene determines secretor status. It is a
gene that produces the ability to secrete
water-soluble blood group specific
substances in the tissues.
 H gene produces the ability to secrete H
antigen (the basic matrix of the ABO
system).
 When H and Se genes are present
together, they convert the Lea substance to
Leb substance but a small residue of the Lea
substance remains unconverted.
d. The Lewis antigen is inherited by means of 2
genes: Le, le
 The Lewis positive (Le) gene converts a
precursor material to Lea substance.
 The Lewis negative (le) gene cannot
convert a precursor material to Lea
substance.
 FAR EASTERN UNIVERSITY – MANILA

e. Lewis antigens become weaker during b. Lewis antibodies are found in individuals who
pregnancy and females of Lewis-type Le (a-b+) have never been transfused or received other
during this period. antigenic stimuli.
c. Lewis antibodies are not known to cause
2. Lewis Pheontypes hemolytic disease of the newborn because of 2
a. There are 3 Lewis Phenotypes: reasons:
 Le (a-b+) – present in secretors  Most infants do not take up the soluble
 Le (a+b-) – present in non-secretors Lewis antigen from the plasma to the red
 Le (a-b-) – usually found in secretors cells by birth.
b. Rules regarding Le antigen expression  Lewis antibodies are invariably IgM in form
 A lele individual will not produce any and too large to cross the placental barrier.
antigen i.e. Le (a-b-).
 A person who inherits at least one Le gene B. MNSs
at least one Se gene will be Leb positive i.e. 1. This was discovered in 1927 by Landsteiner and
Le (a-b+). Levine.
 A person with at least one Le gene and 2. An individual is either MM, MN or NN.
sese genes will be Le (a+b-). 3. MNSs system demonstrated dosage effect i.e. MM
or NN reacts stronger than MN with anti-M and
Adult anti-N.
Levels 4. M and N are co-dominant alleles in paternity
Genes Reaction testing. The antigens are fully developed at birth.
Phenotype Secretors 5. The MNSs system differs from the ABO antigen in
inherited with
anti-Lea three ways:
- Leb a. Antigens found on red cells and some tissues
Le, H, Se Le (a-b+) 0 + A, B H Lea but not secretions.
70% and Leb b. Anti-M and anti-N are usually a result of a
substances transfusion or pregnancy.
Le, H, se Le (a+b-) + 0 Only Lea c. Anti-M and anti-N rarely occur naturally.
20% substances
Le, H, Se Le (a-b-) 0 0 10% of le 6. MNS antibodies
10% (a-b-) are a. Anti-M and anti-N are IgM and react best at 4C.
secretors b. The clinical importance of this system is in
Le, H, se Le (a-b-) 0 0  Transfusion reactions due to anti-S and
10% anti-s.
 Medico legal cases (paternity)
c. Se controls ABH secretions, but has no control  HDN (due to anti-S, anti-s and anti-U)
over Le secretion.
7. Problem (Paternity Testing)
3. Lewis antibodies Father: MM
a. At least four antibodies can be differentiated Mother: NN
 Anti-Lea is most commonly found. It is Baby: MN
usally accompanied by anti-Leb especially Question: Is the alleged father excluded
in Blacks possessing Lewis type Le (a-b-).
 Anti-Leb exists in two forms: anti-Lebh and Reactions:
anti-Lebl. The difference are as follows: Anti-M Anti-N
Cells Reactions Father MM 4+ Negative
O Le(b+) III Mother NN Negative 3+
A 2 Le(b+) III Baby MN 3+ 3+
Anti-Lebh
A1 Le(b+) o/w
H substance Neutralized Answer: The alleged father is not excluded.

Cells Reactions
Anti-Lebl O Le(b+) III
A2 Le(b+) III
A1 Le(b+) o/w
H substance Neutralized

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 FAR EASTERN UNIVERSITY – MANILA
C. Ii SYSTEM
1. I, i antigens
a. I is a public antigen i.e. most adults possess the
I antigen. I antigen is found on cord blood cells.
By the time the child is 1 ½ to 2 years old,
he/she will have mostly I.
b. It is transitional between I and i.
2. Anti-I
a. It is a cold agglutinin that is present in low
titers in healthy adults.
b. High titers are seen during and following
infections with Mycoplasma pneumoniae, in
elderly with autoimmune hemolytic anemia
and patients with cancer of the RES
(reticuloendothelial system).
c. Anti-I is an IgM and has never caused HDN.
d. Anti-I may cause problem because it can
agglutinate all adult cells including patient’s
own cells at room temperature. Such problem
may be resolved by auto absorption prior to
compatibility tests and by performing a pre-
warmed cross-match. Blood should be warmed
when given to a recipient who has anti-I.
D. P SYSTEMS
1. The antigens formed are P1, Pk and P.
2. The P1 antigen is present on the red cells of 79% of
the population. Individuals who lack P1 are termed
P2.
3. Individuals who lack P1P1 or Pk antigens are termed
p.
4. Antibodies
a. Anti-P1 is an IgM and has never caused HDN
but has occasionally caused a transfusion
reaction. In transfusion, recipients with anti-P1
must be given P1 negative units.
b. Anti-P has the specificity of the biphasic
Donath-Landsteiner antibody present in
Paroxysmal Cold Hemoglobinuria.
E. LUTHERAN BLOOD GROUP SYSTEM
1. The antibodies usually associated with this system
are:
a. The anti-Lua is a rare IgM antibody which does
not cause HDN and HTR.
b. Anti-Lub is an IgA which may cause HDN and
HTR.
c. Anti-LuaLub is IgM or IgA, and is active at 37C in
AHG or enzyme.
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F. XG BLOOD GROUP SYSTEM (SEX-LINKED
BLOOD GROUP SYSTEM)
1. Xga antigen is found more frequently in females, as
it is associated with its mode of inheritance on the
X chromosome.
2. Clinically, anti-Xga has not been implicated in HTR
or HDN but it possesses the ability to bind
complement.
G. KELL BLOOD GROUP SYSTEM
1. Kell Antigens
a. Kell antigens are produced from a precursor
substance Kx that is coded by the geneXK on
the X chromosome.
b. The popular antigens under this system are the
K antigen and k antigen.
 The K antigen is immunogenic and
therefore exposure to K+ red cells results
to production of anti-K antibodies. The
frequency of K is low (9%), hence it is not
difficult to find compatible blood for
patients with anti-K.
 The k antigen has a frequency of 99.9% and
for this reason anti-k is rarely encountered.
2. Kx substance
a. Kx (precursor substance of Kell antigens) is
present on the WBC and RBC of most
individuals.
b. Kx is lacking from red cells, the cells have an
abnormal shape (acanthocytes) and a reduced
in vivo survival. Such individuals are said to
have McLeod phenotype.
c. The absence of Kx from leukocyte has been
described by individuals with chronic
granulomatous disease. The leukocytes are
able to phagocytose but not kill bacteria.
Patients with CGD have recurrent bacterial
infection.
3. Kell antibodies
a. Kell antibodies are IgG and are stimulated by
transfusion or pregnancy. They can cause HDN
and HTR.
H. THE DUFFY (Fy) BLOOD GROUP SYSTEM
1. Duffy Antigens
a. The most important antigens are Fya and Fyb.
Fya is more immunogenic than Fyb.
b. The four phenotypes are:
 Fy (a+b+)
 Fy (a+b-)
 Fy (a-b+)
 Fy (a-b-)
 FAR EASTERN UNIVERSITY – MANILA

 Red cells that are Fy (a-b-) are resistant to 4. Cartwright Yta, Ytb Yta is poorly
penetration from Plasmodium vivax and developed at birth
Plasmodium knowlesi but Fy (a+) or Fy (b+)
cells are susceptible to this parasite since Yta is more
they invade the red cells at the Duffy common in
antigens. Chinese, Japanese
and North
2. Duffy Antibodies American
a. Most antibodies are IgG and can cause HTR 5. Diego Dia, Dib
and HDN. 6. Scianna Sm (Sc: 1)
Bua (Sc: 2)
I. KIDD BLOOD GROUP SYSTEM 7. Dombrock Doa, Dob
1. Kidd Antigens 8. Colton Coa, Cob
a. The antigen Jka and Jkb are weakly 9. Others
immunogenic.
b. The phenotypes are: a. Chido Cha Found on the C4d
 Jk (a+b-) fragment of
 Jk (a+b+) complement
 Jk (a-b+)
 Jk (a-b-) This is more commonly found in b. Rodgers Rga Adsorbed onto the
Orientals particularly Filipinos. RBC membrane

2. Kidd Antibodies Bga, Bgb, Bgc HLA antigens on

a. This blood group system is particularly the red cells
notorious for its danger in causing severe and
often fatal delayed hemolytic transfusion Bga = HLA-B7
reaction. It is also known to cause to cause Bgb = HLA-B17
HDN. Bgc = HLA-A28

J. ANTIGENS OF HIGH FREQUENCY, HIGH Sda Sda antigen also

TITER, LOW AVIDITY ANTIBODIES (HTLA) found in saliva and
1. Chido and Rodgers antigens wine
a. These are part of the C4 molecule
(complement) and are not part of the red cell Anti-Sda shows
membrane. mixed field
agglutination
2. Sid (Sda)
a. These are distributed in mammalian tissues K. STUDY GUIDE FOR BLOOD GROUP
and body fluids. SYSTEMS
1. IgM antibodies are “naturally” occurring and react
3. Bg antigens best in saline at room temperatures. Systems with
a. These antigens are expressed on both IgM antibodies include: ABO, Lewis, MNSs, Ii, P and
leukocytes and red cells. Lutheran.
b. The antigen Bga is believed to HLA-B7; Bgb to 2. IgG antibodies are “immune” antibodies and are
HLA-B17; and Bgc to HLA-A28. usually produced as a result of pregnancy or a
transfusion of a foreign antigen. These react best
Blood Group Antigens Comments at 37C in AHG and in albumin. Because IgG
System antibodies cross the placenta, they can cause HDN.
1. Lutheran Lua, Lub Anti-Lua gives a 3. Only Rh antibodies do not bind complement. ABO,
mixed field Lewis, and Kidd antibodies do bind complement.
reaction The others bind complement.
2. Xga Xga Xga locus is on the 4. All antibodies are clinically significant except anti-N
X chromosome and most cases of anti-I.
3. Wright Wra, Wrb Wra is found only in
Caucasians

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 FAR EASTERN UNIVERSITY – MANILA
Chapter 9: Antibody Detection & Identification
A. UNEXPECTED ANTIBODIES
1. Clinically unexpected antibodies occur in less than
3% of the population. Multiple antibodies are more
frequently seen in:
a. Women than men because of possible
sensitization during pregnancy.
b. Among patients older than 60 years old who
have undergone transfusion multiple times.
B. ANTIBODY SCREEN
1. Defintion
Antibody screen involves the reaction between
patient serum or plasma with 2 or 3 reagent
phenotyped for multiple antigens. It is also called
antihuman globulin test.
2. Purpose of Antibody Screen
The purpose of antibody screen is to detect red
blood cell antibodies other than expected anti-A
and anti-B antibodies. These antibodies are called
“unexpected antibodies” since they are present
only in 0.3 to 2% of the general population.
3. Importance of Antibody Screen
a. Selection of appropriate blood for transfusion
b. Investigation of HDN, immune hemolytic
anemia, and transfusion reactions.
C. ANTIBODY IDENTIFICATION
1. If the antibody screen is positive, and antibodies
are present in the serum, a more definitive test
called antibody identification or panel is
performed.
2. Another test to detect and identify antibodies is
the direct antiglobulin test (DAT).
 DAT is a test to detect antibodies coating the
surface of red cells in vivo. An antibody
detection method or panel also identifies these
antibodies.
 These antibodies may be removed from red
cells by process known as elution and then
identified by a panel.
3. Flow Chart of Antibody Identification
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4. Requirements for Antibody Detection and
Identification
a. Reagent Red Cells (Antibody Screening Cells)
 Reagent red cells are commercially
prepared Group O red cell suspensions
obtained from individual donors that are
phenotyped for the most commonly
encountered and clinically important RBC
antigens.
 Group O cells are used so that naturally
occurring expected anti-A or anti-B will not
interfere with detection of unexpected
antibodies.
 Screening cells are detected so that the
following antigens are present on at least
one of the RBC samples.
D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Fya,
Fyb, Jka, Jkb
 The reagent red cells are provided in either
two or three vials. Each vial contains cells
from a single, different donor. The vials are
provided with a description of the antigen
content of each of the cell. This description
is provided in a chart known as an
antigram.
b. Enhancement Reagents
 Enhancement reagents are solutions added
to serum and cell mixtures to promote
antigen-antibody binding or agglutination.
 The enhancement reagents are:
o Low ionic strength saline (LISS)
o Polyethylene glycol (PEG)
o Bovine albumin
c. Antihuman globulin reagents (AHG)
 AHG is used to promote agglutination of
RBC sensitized with immunoglobulin G
(IgG) or complement molecules.
d. Coombs Control Red Blood Cells
 Coombs Control cells are RBC coated with
human IgG. They are prepared by
incubating D positive RBC with potent anti-
D.
 They are used to ensure that AHG tests
with negative results are not false
negatives because of the inactivation of
the AHG reagent.
 In a negative AHG test, there is a free AHG
reagent in the test tube. When Coombs
control cells are added, the free AHG in the
test should cause agglutination of the
sensitized RBC.
 FAR EASTERN UNIVERSITY – MANILA
o Omission in the addition of AHG
o Neutralization of the AHG reagent
D. LABORATORY METHOD
1. Principle:
a. When the test is performed, each vial of
reagent red cells is tested with the serum of
the patient or donor.
b. The media of reactivity include the following
 Saline (immediate spin at room
temperature)
 Enhancement medium at 37C
o Albumin
o LISS
o PEG
 AHG sera (after incubated cells are washed
with saline)
c. Results from each phase are recorded and
evaluated at the end of testing. By using the
antigrams and knowing temperature and
media or reactivity, the potential antibodies
may be narrowed and a more definitive
diagnosis is provided by the use of the
antibody detection panel.
2. Interpretation of Results
a. IgM antibodies usually react on immediate spin
and include:
 M, N, Lea, Leb, and P1
b. IgG antibodies usually react in the AHG phase:
 Kell, Rh, Kidd and Duffy
c. A negative autocontrol (using patient cells and
patient serum) effectively rules out the
presence of an antibody.
E. LABORATORY METHOD (ANTIBODY
INDENTIFICATION)
1. Principle:
a. The patient’s serum constitutes the unknown
with the potential for presence of antibody;
whereas the cells represent known antigens.
Most commonly, there are between eight and
sixteen different cells included in the panel.
b. Interpretation of results
 Enhance the reaction of some antibodies:
Rh and Kidd
 Denature other antibodies: M, N, S and
Duffy
c. Hemolysis should be noted because some
antibodies can cause hemolysis.
ABO, P, Le, Jk and Kell systems
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F. TROUBLESHOOTING ANTIBODY PANELS
1. If all panel cells and autocontrol agglutinate
strongly at room temperature but less strongly at
37C and in the AHG phase.
The causes are:
 Cold reacting antibodies such as anti-I
 Rouleaux
2. If panel cells and autocontrol agglutinate only in
the AHG phase. The cause may be a warm
antibody.
3. If the panel cells agglutinate but autocontrol is
negative.
The causes are:
 Multiple antibodies
 Antibody against a high frequency antigen
found on all panel cells
4. If all panel cells are negative or give variable
positive reactions and autocontrol is positive in
AHG. The cause may be a warm AIHA.
G. OTHER BLOOD BANKING TECHNIQUES
1. Absorption of Antibodies
a. It is the process of removing antibody from the
serum.
b. Non-specific antibodies can be removed by
absorbing them temperature of the antibody.
c. Cold antibodies (IgM) are absorbed at 4C while
warm antibodies (IgG) at 37C.
d. Absorption Techniques
Absorption techniques are helpful in the
following situations:
 Remove non-specific antibodies from the
serum
 Separate mixtures of antibodies to aid in
their identification
 Determine the presence of a specific
isoantibody as well as a non-specific
autoantibody in the serum of a patient
with acquired hemolytic anemia.
e. Application of Absorption
 It is the removal of antibody from the
cells.
 Elution Techniques
Elution is done by:
o Heating the washed cell
suspension at 56C water bath for
10 minutes and agitating the
mixture frequently (heat elution).
This is done to elute ABO
antibodies from RBC.
o By the addition of ether, low pH
acid, digotonin acid, cold acid,
 FAR EASTERN UNIVERSITY – MANILA

chloroform, xylene or methylene enzymes) either before the

chloride. This is done to elute non- antiserum is added or at the same
ABO antibodies. time it is added.
o The enzymes used in blood bank
 Elution application are trypsin, papain, bromelin and
ficin. Papain is obtained from
Elution is done to: papaya while bromelin from
o Demonstrate and identify the pineapple.
antibody on the RBC of infants or o The enzymes digest away some of
cord blood in case of HDN. the RBC surface, thus making RBC
o Identify the antibody absorbed on more readily accessible to
the RBC in acquired hemolytic agglutination by certain antibodies.
anemia. o Excessive enzyme treatment must
o Identify the antibody absorbed on be avoided since it causes red cells
the RBC of recipient in transfusion to agglutinate spontaneously in the
reactions. absence of antibodies.
o Separate and identify antibodies in o Enzyme treatment destroys MN
a mixture of antibodies. and Duffy and may lower activity
of Ss, K, Jka and U antigens.
 Titration of Antibodies
o This is done to determine the
relative amount of antibody in the
serum.
o It is determined by making serial
two-fold dilutions of the unknown
serum and testing them against
saline and albumin suspended cells
of the appropriate type. The
amount of these cells added to
each tube is consistent.
o The titer is expressed as the
reciprocal of the highest dilution in
which agglutination is observed
macroscopically i.e. saline
agglutination end at 1:256 is
reported as a saline agglutionation
titer of 256.

 Typing of Immunoglobulin
o The typing of immunoglobulin is
accompanied by the use of
dithiothreitol (DTT) and 2-
mercaptoethanol (2-ME).
o DTT and 2-ME are used to remove
or inactivate IgM, leaving the IgG
intact. They are sometimes useful
to remove or break up red cell
agglutination by strong IgM cold
autoantibodies.

 Enzyme Testing
o Enzyme testing is done to facilitate
the agglutination of red cells in the
presence of certain antibodies.
o In the procedure, RBC are exposed
to proteolytic (or protein-digesting

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