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The document discusses the Rh blood group system, including different nomenclatures (Fisher-Race, Weiner, Rosenfield), genes involved (RHD, RHCE), and relative immunogenicity of Rh antigens.

The three nomenclatures involved in the Rh system are Fisher-Race, Weiner, and Rosenfield nomenclatures.

The two genes that control the expression of Rh antigens are the RHD gene and the RHCE gene.

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 issue is to routinely transfuse Rh negative


antiglobulin test (IAT). blood to all patients who are negative when
4. Categories of Du tested with anti-sera.
c. Du is common among Negroes.
Du red cells can be classified into 3 catergories:
a. Acquired Du. The inheritance of C in the F. RH ANTIBODIES
transposition to D (dCe/DcE results in the 1. Rh antibodies are not natural antibodies. They are
weakened expression of the D antigen on red classified as immune antibodies because they are
cells). These individuals do not produce anti-D present only after exposure to Rh antigens through
if exposed to Rh-positive blood. ingestion, injection or pregnancy.

Depressed D 2. Rh antibodies do not ordinarily react in saline


Genotype 1 Genotype 2 medium since they are mostly of the IgG class.
Dce/dce Dce/dCe
3. Varieties of Rh antibodies
a. First order Rh antibodies (saline agglutinins,
C in cis position to D C in transposition to D bivalent antibodies, complete antibodies).
They react strongly with specific Rho antigens
in saline medium and react less strongly in a
Normal expression of D Du phenotype protein medium.
b. Second order Rh antibodies (albumin-reacting,
b. Hereditary Du. The reason for the weakened incomplete, monovalent antibodies). They
expression of the D antigen in these individuals react visibly with specific antigen in a protein
is not known. medium. In saline medium, they weakly
c. Du variant. The D antigen normally consists of combine with the antigen but do not produce a
at least 4 parts. If one or more parts of the visible reaction.
antigen can be weakly expressed. c. Third order Rh antibodies (typical Rh
5. Du variants antibodies, antiglobulin Rh antibodies). They
react visibly with specific antigens in an
Du may also be divided into 2 grades: antiglobulin medium only.
a. High grade Du
 This is not passed on to future G. LABORATORY PROCEDURES
generations since development 1. Rh typing
depends upon the rare r’ (Cde) or (CdE)
chromosomal arrangement. Rh typing is performed with the commercial anti-
 It reacts with either complete or Rho (D) antibodies which are produced from the
incomplete anti-D and only rarely serum of Rh-negative person who have been
requires the use of antiglobulin test for immunized of the Rho (D) factor.
its detection.
a. Slide Test
b. Low grade Du  It is a rapid method employing very potent
 It is a direct product of inherited gene anti-D (Rho) antiserum.
and can be passed on to future  The cell-antiserum mixture is heated on an
generation. illuminated Rh view box with a temperature
 It is usually detected only by of about 45C.
antiglobulin test after first sensitizing  Agglutination if positive should be visible
the cells with anti-D sera. within 2 minutes to avoid drying and
rouleaux formation.
6. Significance of Du
a. Du individual should be considered as Rho (D) b. Tube Test
positive blood donor and in an emergency can  The cell-antiserum mixture is incubated at
be transfused with Rh positive or negative 37C for half hour.
blood when a recipient.  Agglutination should be achieved if positive
b. Many workers believe that patients do not after the incubation period. Agglutination
have to be typed, and that the safest, most can be made stronger through
efficient and the easiest way to handle the

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centrifugation at 1,500 rpm for 1 minute or 4. Symptoms include:


at 3,000 rpm for 15 secs.  Mild compensated hemolytic anemia
 Stomatocytosis
c. Interpretation of Rh typing  ↑ A slight-to-moderate decrease in Hgb and
 Where there is agglutination, the person Hct levels
Rh-positive. When there is no agglutination,  ↑↑↑ Increase in Hgb F
the person is either Rh-negative or Du  ↓↓↓ Decreased serum haptoglobin
positive.  ↑↑ Elevated bilirubin
 Whenever there is no agglutination, a test
for Du variant should follow. If the Du test is I. MEDICO-LEGAL USE OF BLOOD GROUPS
negative, then the person is Rh-negative. 1. Using the common blood group system (ABO, Rh,
MNSs) it is possible to exonerate approximately
d. Unlike the ABO system, the Rh antigens are 50% of wrongly accused males from being the
found only on the RBC surface. father of the infant in question.
2. Blood typing can never prove paternity and only
2. Detection of Du either disprove it or result in equivocation.
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 Chapter 8: Other Blood Group System
anti-Rho (D) typing serum to coat the cells.
After incubation, the cells are centrifuged and A. THE LEWIS SYSTEM AND SECRETOR
examined for agglutination. If no agglutination
is observed, the cells are developed into an STATUS
antiglobulin test by adding anti-human globulin 1. The Lewis Antigen
(Coomb’s reagent). a. The Lewis antigen differs from all other blood
groups in that it is soluble and found in saliva
b. Interpretation and plasma. Hence, Lewis antigens are called
 Agglutination indicates the patient is Du plasma antigens.
positive and is classified as Rh (D) positive b. The Lewis antigen is not true blood group
patient. antigen. The antigen is mainly acquired from
 No agglutination indicates the patient is Rh the body tissues and attaches itself onto the
(D) negative and Du negative. red cell surface later in life. Most infants are
Le(a-b-) since the antigen is fully developed at
H. RH DEFICIENCY SYNDROME age six.
1. Individuals have Rh deficiency or Rhnull syndrome. c. The expression of Lewis antigens is influenced
2. These individuals fail to express any Rh antigens on by the presence of Hh and Sese genes.
the RBC surface.  Se gene determines secretor status. It is a
3. There are two ways to inherit Rhnull syndrome: gene that produces the ability to secrete
water-soluble blood group specific
a. Regulator type Rhnull syndrome substances in the tissues.
 A mutation occurs in the RHAG gene.  H gene produces the ability to secrete H
 This results in no RhAG protein expression antigen (the basic matrix of the ABO
and subsequently no RhD or RhCE protein system).
expression on the RBCs.  When H and Se genes are present
 This mutation can be passed from parent together, they convert the Lea substance to
to offspring. Leb substance but a small residue of the Lea
substance remains unconverted.
b. Amorphic type Rhnull syndrome d. The Lewis antigen is inherited by means of 2
 There is a mutation in RHCE genes genes: Le, le
inherited from each parent and the  The Lewis positive (Le) gene converts a
common deletion of the RHD gene found precursor material to Lea substance.
in most D-negative (Rh-negative)  The Lewis negative (le) gene cannot
individuals. convert a precursor material to Lea
 The RHAG gene is normal. substance.

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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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C. Ii SYSTEM F. XG BLOOD GROUP SYSTEM (SEX-LINKED


1. I, i antigens BLOOD GROUP SYSTEM)
a. I is a public antigen i.e. most adults possess the 1. Xga antigen is found more frequently in females, as
I antigen. I antigen is found on cord blood cells. it is associated with its mode of inheritance on the
By the time the child is 1 ½ to 2 years old, X chromosome.
he/she will have mostly I. 2. Clinically, anti-Xga has not been implicated in HTR
b. It is transitional between I and i. or HDN but it possesses the ability to bind
complement.
2. Anti-I
a. It is a cold agglutinin that is present in low G. KELL BLOOD GROUP SYSTEM
titers in healthy adults. 1. Kell Antigens
b. High titers are seen during and following a. Kell antigens are produced from a precursor
infections with Mycoplasma pneumoniae, in substance Kx that is coded by the geneXK on
elderly with autoimmune hemolytic anemia the X chromosome.
and patients with cancer of the RES b. The popular antigens under this system are the
(reticuloendothelial system). K antigen and k antigen.
c. Anti-I is an IgM and has never caused HDN.  The K antigen is immunogenic and
d. Anti-I may cause problem because it can therefore exposure to K+ red cells results
agglutinate all adult cells including patient’s to production of anti-K antibodies. The
own cells at room temperature. Such problem frequency of K is low (9%), hence it is not
may be resolved by auto absorption prior to difficult to find compatible blood for
compatibility tests and by performing a pre- patients with anti-K.
warmed cross-match. Blood should be warmed  The k antigen has a frequency of 99.9% and
when given to a recipient who has anti-I. for this reason anti-k is rarely encountered.

D. P SYSTEMS 2. Kx substance
1. The antigens formed are P1, Pk and P. a. Kx (precursor substance of Kell antigens) is
2. The P1 antigen is present on the red cells of 79% of present on the WBC and RBC of most
the population. Individuals who lack P1 are termed individuals.
P2. b. Kx is lacking from red cells, the cells have an
3. Individuals who lack P1P1 or Pk antigens are termed abnormal shape (acanthocytes) and a reduced
p. in vivo survival. Such individuals are said to
have McLeod phenotype.
4. Antibodies c. The absence of Kx from leukocyte has been
a. Anti-P1 is an IgM and has never caused HDN described by individuals with chronic
but has occasionally caused a transfusion granulomatous disease. The leukocytes are
reaction. In transfusion, recipients with anti-P1 able to phagocytose but not kill bacteria.
must be given P1 negative units. Patients with CGD have recurrent bacterial
b. Anti-P has the specificity of the biphasic infection.
Donath-Landsteiner antibody present in
Paroxysmal Cold Hemoglobinuria. 3. Kell antibodies
a. Kell antibodies are IgG and are stimulated by
E. LUTHERAN BLOOD GROUP SYSTEM transfusion or pregnancy. They can cause HDN
1. The antibodies usually associated with this system and HTR.
are:
a. The anti-Lua is a rare IgM antibody which does H. THE DUFFY (Fy) BLOOD GROUP SYSTEM
not cause HDN and HTR. 1. Duffy Antigens
b. Anti-Lub is an IgA which may cause HDN and a. The most important antigens are Fya and Fyb.
HTR. Fya is more immunogenic than Fyb.
c. Anti-LuaLub is IgM or IgA, and is active at 37C in b. The four phenotypes are:
AHG or enzyme.  Fy (a+b+)
 Fy (a+b-)
 Fy (a-b+)
 Fy (a-b-)

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 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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Chapter 9: Antibody Detection & Identification 4. Requirements for Antibody Detection and
Identification
A. UNEXPECTED ANTIBODIES a. Reagent Red Cells (Antibody Screening Cells)
1. Clinically unexpected antibodies occur in less than  Reagent red cells are commercially
3% of the population. Multiple antibodies are more prepared Group O red cell suspensions
frequently seen in: obtained from individual donors that are
a. Women than men because of possible phenotyped for the most commonly
sensitization during pregnancy. encountered and clinically important RBC
b. Among patients older than 60 years old who antigens.
have undergone transfusion multiple times.  Group O cells are used so that naturally
occurring expected anti-A or anti-B will not
B. ANTIBODY SCREEN interfere with detection of unexpected
1. Defintion antibodies.
 Screening cells are detected so that the
Antibody screen involves the reaction between following antigens are present on at least
patient serum or plasma with 2 or 3 reagent one of the RBC samples.
phenotyped for multiple antigens. It is also called
antihuman globulin test. D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Fya,
Fyb, Jka, Jkb
2. Purpose of Antibody Screen
 The reagent red cells are provided in either
The purpose of antibody screen is to detect red two or three vials. Each vial contains cells
blood cell antibodies other than expected anti-A from a single, different donor. The vials are
and anti-B antibodies. These antibodies are called provided with a description of the antigen
“unexpected antibodies” since they are present content of each of the cell. This description
only in 0.3 to 2% of the general population. is provided in a chart known as an
antigram.
3. Importance of Antibody Screen
a. Selection of appropriate blood for transfusion b. Enhancement Reagents
b. Investigation of HDN, immune hemolytic  Enhancement reagents are solutions added
anemia, and transfusion reactions. to serum and cell mixtures to promote
antigen-antibody binding or agglutination.
C. ANTIBODY IDENTIFICATION  The enhancement reagents are:
1. If the antibody screen is positive, and antibodies o Low ionic strength saline (LISS)
are present in the serum, a more definitive test o Polyethylene glycol (PEG)
called antibody identification or panel is o Bovine albumin
performed.
2. Another test to detect and identify antibodies is c. Antihuman globulin reagents (AHG)
the direct antiglobulin test (DAT).  AHG is used to promote agglutination of
 DAT is a test to detect antibodies coating the RBC sensitized with immunoglobulin G
surface of red cells in vivo. An antibody (IgG) or complement molecules.
detection method or panel also identifies these
antibodies. d. Coombs Control Red Blood Cells
 These antibodies may be removed from red  Coombs Control cells are RBC coated with
cells by process known as elution and then human IgG. They are prepared by
identified by a panel. incubating D positive RBC with potent anti-
3. Flow Chart of Antibody Identification 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.

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o Omission in the addition of AHG F. TROUBLESHOOTING ANTIBODY PANELS


o Neutralization of the AHG reagent 1. If all panel cells and autocontrol agglutinate
strongly at room temperature but less strongly at
D. LABORATORY METHOD 37C and in the AHG phase.
1. Principle:
a. When the test is performed, each vial of The causes are:
reagent red cells is tested with the serum of  Cold reacting antibodies such as anti-I
the patient or donor.  Rouleaux
b. The media of reactivity include the following 2. If panel cells and autocontrol agglutinate only in
 Saline (immediate spin at room the AHG phase. The cause may be a warm
temperature) antibody.
 Enhancement medium at 37C 3. If the panel cells agglutinate but autocontrol is
o Albumin negative.
o LISS
o PEG The causes are:
 AHG sera (after incubated cells are washed  Multiple antibodies
with saline)  Antibody against a high frequency antigen
c. Results from each phase are recorded and found on all panel cells
evaluated at the end of testing. By using the 4. If all panel cells are negative or give variable
antigrams and knowing temperature and positive reactions and autocontrol is positive in
media or reactivity, the potential antibodies AHG. The cause may be a warm AIHA.
may be narrowed and a more definitive
diagnosis is provided by the use of the G. OTHER BLOOD BANKING TECHNIQUES
antibody detection panel. 1. Absorption of Antibodies
a. It is the process of removing antibody from the
2. Interpretation of Results serum.
a. IgM antibodies usually react on immediate spin b. Non-specific antibodies can be removed by
and include: absorbing them temperature of the antibody.
 M, N, Lea, Leb, and P1 c. Cold antibodies (IgM) are absorbed at 4C while
b. IgG antibodies usually react in the AHG phase: warm antibodies (IgG) at 37C.
 Kell, Rh, Kidd and Duffy d. Absorption Techniques
c. A negative autocontrol (using patient cells and Absorption techniques are helpful in the
patient serum) effectively rules out the following situations:
presence of an antibody.  Remove non-specific antibodies from the
serum
E. LABORATORY METHOD (ANTIBODY  Separate mixtures of antibodies to aid in
INDENTIFICATION) their identification
1. Principle:  Determine the presence of a specific
a. The patient’s serum constitutes the unknown isoantibody as well as a non-specific
with the potential for presence of antibody; autoantibody in the serum of a patient
whereas the cells represent known antigens. with acquired hemolytic anemia.
Most commonly, there are between eight and
sixteen different cells included in the panel. e. Application of Absorption
 It is the removal of antibody from the
b. Interpretation of results cells.
 Enhance the reaction of some antibodies:
Rh and Kidd  Elution Techniques
 Denature other antibodies: M, N, S and Elution is done by:
Duffy o Heating the washed cell
suspension at 56C water bath for
c. Hemolysis should be noted because some 10 minutes and agitating the
antibodies can cause hemolysis. mixture frequently (heat elution).
This is done to elute ABO
ABO, P, Le, Jk and Kell systems antibodies from RBC.
o By the addition of ether, low pH
acid, digotonin acid, cold acid,

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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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