Immuno Lab Finals

IMMUNOHEMATOLOGY
(LAB)- Indirect Coomb's Test

2nd SEM, 2022
INDIRECT COOMBS TEST examine for agglutination.

INDIRECT ANTIHUMAN GLOBULIN TEST 10. Interpret and record results.
(IAT) - Absence of agglutination it will confirm that the
blood group is Rh negative but if after dislodge
● Detects: in vitro sensitization of RBC’s with there is a presence of agglutination that
IgG or complement components (require means there is a weak reacting D antigen or
incubation at 37C) weak D antigen.
- Monospecific- it has only 1 either IgG or 11. For each negative tube, add 1 drop of
complement components well mixed check cells.
- Polyspecific- it has the 2 the IgG and - Check cells or coombs cells- are RBCs coated
complement components with immunoglobulin G
The difference between direct antiglobulin test - A positive result is expected after the addition
(DAT) and indirect antihuman globulin test of check cell, this implies that the test has
(IAT): been properly perform
- DAT- in vivo sensitization - If negative result with the cells after the
- IAT- in vitro sensitization, it requires addition of check cells there is an improperly
incubation at 37°C procedure or performed test (kailangan ulitin)
● Materials: after the addition of Red cells
Test tube
Water bath at 37 °C GUIDE IN THE INTERPRETATION OF TEST
Centrifuge FOR WEAK D ANTIGEN
Anti-D antisera Anti-D (slide or Manner of Additional
Antihuman globulin reagent/Coomb’s tube Reporting Comment
reagent method)
● Procedure:
1. Prepare 2-5% red cell suspensions to + (with Rh POSITIVE Positive
be tested. agglutination) reaction
2. Label another set of tubes as U and indicates
NC. presence of D
3. Follow the table: Antigen
CONTENT UNKNOWN NEGATIVE
CONTROL 0 (no Rh NEGATIVE Negative
agglutination) (initial) reaction should
Anti-D 1 drop - be further
tested for the
presence of
22% Bovine Serum - 1 drop weak D with
Albumin the use of
AHG.
2-5% RCS 1 drop 1 drop
4. Mix gently and cover all tubes with
parafilm. Incubate both tubes for 15
minutes at 37 degrees Celsius water AHG TEST for WEAK D: (Indirect Anti-Human
bath. Du Reaction Globulin Test)
Manner of Additional
5. Centifuge tubes at 3400 rpm for 15 Reporting Comment
seconds. Unknown tube with
agglutination is regarded as Rh 0 (no Du Rh (-) Du (-):
POSITIVE. Unknown tube without agglutination) NEGATIVE negative
agglutination goes to the next Reactions
procedure. confirm that the
6. Wash the cells 3 times with NSS. person is Rh
7. Decant the saline completely after the NEGATIVE.
final washing. (Patient or
8. Add 2 drops of Anti-Human Globulin donor.)
reagent to all tubes and mix gently.
Cover with parafilm.
- AHG reagent is color GREEN
9. Gently dislodge each cell button and
1
ARAULLO, ASGARE, BAIS, BALATBAT, BANAWA, BRIONES, DE CASTRO, DE LEON, DELOS TRINOS, DURAN, GALANG, MENDOZA, MUZADA, OLBES, ORDONA, OSDON, PUNZALAN, RASING, SALVO, RODRIQUEZ, TOLENTINO, VENTURA
IMMUNOHEMATOLOGY
(LAB)- Indirect Coomb's Test
2nd SEM, 2022
+ (with Du POSITIVE Rh (-) Du (+): Using serum as a AHG reagent, test

agglutination) should be given sample for DAT serum, enhancement
proper media is not added
classification:
False Negative
If patient: Rh
● Rare antibodies are present that are only
NEGATIVE
detectable with polyspecific AHG when active
complement is present
If donor: Rh
● Undercentrifugation
POSITIVE
● Serum:cell ratio not ideal
● Low pH of saline
SOURCES OF ERRORS IN THE ● Poor reading technique
ANTIHUMAN GLOBULIN TESTING ● Inadequate incubation in IAT
False positive results False negative results
Improper specimen Inadequate or improper

(refrigerated, clotted washing of cells
samples)
Overcentrifugation and Failure to wash

overreading additional times when
increased volume of
serum is used
Centrifugation after Contamination of AHG

incubation (when PEG is by extraneous protein
used)
Bacterial contamination High concentration of

of saline used for IgG paraproteins in test
washing serum
Dirty glasswares Elution of antibodies

from RBC’s due to
interruption
in testing
Presence of fibrin Elution of antibodies

(pseudoagglutination) from RBC’s due to
improper
testing temperatures
Cells with positive DAT Improper reagent

will yield positive IAT storage (deterioration or
neutralization of reagent)
Polyagglutinable cells Excessive heat,

repeated freezing and
thawing of test serum
Saline contaminated by Non-reactive serum

heavy metals because of deterioration
of complement
2
IMMUNOHEMATOLOGY
(LAB)- Direct Coomb's Test
2nd SEM, 2022
RECALL ●
AHG reagent is added on patients sample
ANTIHUMAN GLOBULIN TEST (Washed RBCs).
(IAT) / ANTIGLOBULIN TEST o If Patients RBC is sensitized with IgG
● Commonly known as Coomb’s test and/or C3b/C3d, AHG will bind to
● It is based on the principle that antihuman these components and will agglutinate
the red blood cells.
globulins (AHGs) obtained from immunized
ADDED NOTES: AHG REAGENT IS COLOR GREEN
nonhuman species bind to human globulins
such as IgG or complement, either free in
serum or attached to antigens on red blood
cells (RBCs).
● There are two major types of blood group
antibodies, IgM and IgG.
o Because of their large pentamer
structure, IgM antibodies bind to
corresponding antigen and directly
agglutinate RBCs suspended in saline.
o IgG antibodies are termed non-
agglutinating because their monomer
structure is too small to agglutinate PROCEDURE
1. Prepare a test tube and label it with
sensitized RBCs directly.
patient’sname
DETECTION OF IN-VIVO OR IN-VITRO
2. Deliver 2 drops of well-mixed anticoagulated
SENSIZATION OF RBCs
blood into the tube.
● The use of AHG to detect in-vitro sensitization 3. Wash the anticoagulated blood three time with
of RBCs is a two-stage technique referred to as NSS.
the indirect antiglobulin test (IAT) 4. Decant completely the supernatant after the
● In-vivo sensitization is detected by a one-stage last washing
procedure, the direct antiglobulin test (DAT). 5. Add 2 drops of AHG reagent
AHG REAGENT 6. Centrifuge for 15seconds at 3400 rpm
● Polyspecific AHG: anti-IgG and anti-C3b or 7. Gently dislodge the cell button and examine
for hemolysis or agglutination.
anti-C3d (reacts with Fc region of the gamma o Note: Coomb’s check cells are also
rays heavy chain of the IgG molecule) added to validate a negative reaction
● Monospecific AHG: anti-IgG or anti-C3b or and confirm AHG was previously
anti-C3d added. If AHG was not added, no
(if positive with polyspecific AHG use agglutination will be observed after the
monospecific AHG to determine which addition of check cells. It is suggested
that the procedure should be
component reacts with the red cell) repeated.
DIRECT AHG TEST 8. INTERPRETATION:
● The DAT detects in-vivo sensitization of RBCs ● With agglutination: DAT POSITIVE
with IgG and/or complement components. ● No agglutination: DAT NEGATIVE
● Clinical conditions that can result in in-vivo FOR TEST TO BE VALID FOR NEGATIVE AHG
coating of RBCs with antibody and/or TEST
complement are: ● Group O RBCs sensitized with IgG are added.
1. Hemolytic disease of the newborn ● Valid if: there is agglutination
(HDN) ● Invalid if: there’s no agglutination
2. Hemolytic transfusion reaction (HTR)
3. Autoimmune and drug-induced AIHA.
● The DAT does not require the incubation
phase because of the antigen-antibody
complexes formed in vivo.
AHG PRINCIPLE
● Detects in-vivo sensitization of RBCs.
(Sensitized with IgG and/or C3b/C3d)
1
IMMUNOHEMATOLOGY
(LAB)- Direct Coomb's Test
2nd SEM, 2022
2
IMMUNOHEMATOLOGY
(LAB)- Crossmatching
2nd SEM, 2022
CROSSMATCHING
Donor’s serum - 2 drops -
● The test between a prospective recipient of a
blood transfusion and his proposed donor (or
2-5% donor’s RCS 1 drop - -
donors) is known as the crossmatch or
compatibility test (able to know if blood is safe
2-5% patient’s RCS - 1 drop 1 drop
to transfuse). This test is performed to show
any possible incompatibility between the
recipient's serum and the donor's red cells (the 3. Mix the contents and cover with
major crossmatch); infrequently, the plasma parafilm.
of the donor is also tested against the red cells 4. Centrifuge for 15 seconds at 3400
of the recipient (the minor crossmatch). rpm.
● Compatible blood- blood containing 5. Gently dislodge the cell button and
erythrocytes which have been tested in vitro observe for agglutination or hemolysis.
against the patient's serum and, which is 6. If no agglutination is observed,
thought, will survive normally if administered to proceed to the THERMO PHASE.
that patient. o Interpretation of a positive reaction in the
● Compatibilty test- a test carried out between protein phase:
serum and erythrocytes to ensure that they are 1. Incompatibility in the ABO system will
not antagonistic. Usually this term refers to the be detected at this point. Hemolysis
direct crossmatch between the patient's serum especially may indicate the presence
and donor's red cells. of an immune anti-A and/or anti-B;
● Crossmatch – to test a patient and a 2. An incompatibility due to cold
prospective donor for compatibility. antibodies such as anti-M, anti-P1, or
● Major Crossmatch – recipient's serum tested anti-Lea will be detected; the latter
with donor cells. may hemolyze incompatible red cells.
● Minor Crossmatch – recipient's cells tested B. Thermo Phase / Incubation Phase (37.0
with donor's serum. C)
PRE-ANALYTICAL PHASE 1. Add 2 drops of 22% Bovine Serum
● Set-up of waterbath or dry bath at 37 °C Albumin on all tubes. (M, N, and AC).
● Collection of blood through venipuncture 2. Incubate all of the tubes for 30
● Preparation of 2% to 5% red blood cell minutes at 370C water bath.
suspension (approximate) NOTE: LISS can be used instead of
ANALYTICAL PHASE albumin, in this case, incubate the tubes
● Materials needed: for 15 minutes only.
o Water bath 3. Centrifuge for 15 seconds at 3400
o Test tubes rpm.
o 5% cell suspension of donor's cells 4. Gently dislodge the cell button and
o 22% bovine albumin observe for agglutination or hemolysis.
o Patient's serum 5. If no agglutination or hemolysis is
o Anti-human globulin reagent observed in all of the three tubes,
o Normal saline solution (NSS)- maintain the proceed to Antihuman GlobulinPhase.
integrity of Red cells o Interpretation of a positive reaction in the
● Procedure: thermo phase:
A. Protein / Albumin / Room Temperature 1. Incompatibility in this phase is usually due
Phase (Immediate Spin Phase) to the presence of a lower-titered anti-Rh
1. Label 3 test tubes: M, N, AC antibody that does not react on immediate
o Legend: centrifugation.
M: Major Crossmatch 2. Certain Rh antibodies (anti-c, anti-E, and
N: Minor Crossmatch some anti-D) occasionally react only in an
AC: Auto-control albumin medium and are non-reactive in
(cereal code) the antiglobulin test
2. Follow the table below: C. Antihuman Globulin Phase / Coomb’s
Phase (AHG Phase)
CONTENT M N AC
1. Wash cells of all the three tubes with
NSS 3 times.
Patient’s serum 2 drops - 2 drops
2. Decant NSS COMPLETELY after the
last washing.
1
IMMUNOHEMATOLOGY
(LAB)- Crossmatching
2nd SEM, 2022
3. Add 2 drops of AHG antisera.

4. Centrifuge for 15 seconds at 3400 The PPEs must be removed properly and be disposed
rpm. as appropriate. These cannot be exposed outside the
5. Dislodge cell button and examine for laboratory premises.
agglutination or hemolysis.
6. Add 1 drop of Coomb’s check cells to
tubes showing no agglutination.
o Interpretation of a positive reaction in the AHG
phase:
1. Antibodies are detected here which
usually react only in this test, e.g., anti-
Fya, anti-Jka, anti-K etc.
2. Those antibodies in the Rh system which
react only in the antiglobulin test are so-
called “third order” or “cryoagglutinoid
antibodies”.
3. Antibodies present in acquired hemolytic
anemia will be found.
NOTE: All the three phases must be free from
agglutination and/or hemolysis before the donor's
blood could be transfused to the recipient.
Agglutination or hemolysis in any or all of the three
phases will mean an incompatible blood and donor's
blood cannot be transfused to the recipient.
Interpretation:
o COMPATIBLE: if there’s no agglutination or
hemolysis in all tubes in all phases
o INCOMPATIBLE: if there’s agglutination or
hemolysis in all tubes in all phases
POST-ANALYTICAL PHASE
● Observation and Results:
o (+) = with agglutination
o (0) = no agglutination
PROCEDURAL TUBE INTERPRE
PHASE REACTIONS TATION
A. PROTEIN Major XM:

PHASE Minor XM:
Autocontrol
B. THERMO Major XM:

PHASE Minor XM:
Autocontrol
C. AHG Major XM:

PHASE Minor XM:
Autocontrol
REMINDERS:
All contaminated consumable
devices/materials should be disinfected with 10%
bleach or 10% Lysol before disposal in trash bags for
infective wastes. All reusable devices must be
disinfected as well. The working area should be
cleansed with disinfectant after the experiment.
2
IMMUNOHEMATOLOGY
WEEK 4&5: DIRECT ABO BLOOD GROUPING
(LAB) 2nd SEM, 2022
FORWARD AND REVERSE TYPING

BLOOD REACTION REACTION REACTION
GROUP
HISTORICAL PERSPECTIVE WITH WITH WITH ANTI-
● 1901- ABO discovered (detection of ANTI-A ANTI-B
● Karl Landsteiner (1868-1943) AB
○ Discovery of the first human blood antigen)
group system.
○ First to conduct reverse and Forward A + - +
typing
○ ABO Blood group
○ In 1902, Sturle and Von Descatello B - + +
discovered the fourth blood group in
the system (Group AB)
AB + + +
THE LANDSTEINER LAW
● The antigen is present on the RBC surface
O - - -
and determines the blood group/type
● The corresponding antibody is NEVER
FOUND in the individual’s serum (an antigen is
specific to antibody) ● Reverse Blood Typing
● The OPPOSITE naturally occurring antibody is ○ Detects ABO antibodies in the
always present in the individual’s serum patient’s serum by using known
reagent RBCs (A and B cells)
○ Only unique to the ABO blood group
Blood Antigens on the Naturally occurring system; checks results of forward
typing
groups RBC surface Antibody in serum ○ Specimen: patient’s serum/plasma
▪ Serum- non-additive tube
A A Anti-B ▪ Plasma- anticoagulant tube
○ Reagent: Known Red cells (antibody)
B B Anti-A BLOOD GROUP REACTION WITH REACTION WITH

(detection of A B
AB A and B NONE antibody) CELLS CELLS
O O Anti-A and Anti-B A - +
B + -
THE ABO FORWARD AND REVERSE
GROUPING/TYPING
AB - -
● Forward Blood Typing
○ Uses known sources of commercial
anti-sera (anti-A, anti-B) to detect O + +
antigens on an patient’s RBC
○ Specimen: patient’s Red cells
○ Reagent: anti-sera
❖ Blue- A (asul) CHARACTERISTIC OF ABO ANTIGENS
❖ Yellow- B (banana)
● They can be demonstrated as early as the
second month of fetal life. The A antigen
appears to be weak at birth but at the age of
one year, agglutinogens reach final strength
1
IMMUNOHEMATOLOGY
(LAB) 2nd SEM, 2022
● They persist throughout life. However, each drop of the typing antiserum
abnormal antigens may be found as acquired (the suspension may be prepared
characteristics in leukemia (weak A antigen) by adding 20 parts of red cells to
and cancer (abnormal secretion of ABH 80 part of normal saline).
substance) 3. Mix the cells and reagent using a
● They may be found in saliva, pancreatic clean applicator stick. Spread
secretions, gastric secretions of people who each mixture evenly on the slide
are secretors over an area of 10-15 mm
● They may be found in bacteria and other diameter.
species 4. Tilt the slide for 2 minutes at room
temperature (22°C – 24°C) and
CHARACTERISTICS OF ABO ANTIBODIES observe for agglutination. Do not
● Not normally present at birth. If present at read results after 2 minutes.
birth, they originated from the mother through 5. Read and Record the result.
placental leakage during delivery 6. Report as “+” for agglutination and
● They develop 3-6 months after birth “0” for no agglutination.
● Predominantly IgM and react at room 7. Cal the instructor to check results.
temperature or colder 8. Dispose all biohazardous waste in
● Occur in two forms: a puncture-proof waste container.
o Naturally occurring antibodies
o Immune antibodies produced during DIRECT/FORWARD ABO BLOOD GROUPING
incompatible transfusion or (TUBE METHOD) PROCEDURE-2
incompatible pregnancies (acquired) ● Materials:
● They are present in some animals and plants o Test tube
as lectins. Lectins are plants or seed extracts o Centrifuge
diluted to agglutinate specific human blood o Glass marker or an alternative
group antigens labelling device
o Dolichos biflorus: agglutinates A1 or o Typing sera A (anti-A antisera)
A1B cells (anti-A1 lectin) o Typing sera B (anti-B antisera)
o Bandeiraea simplicifolia: agglutinates o Group “O” type serum
B cells (anti-B lectin) ● Procedure:
o Ulex europaeus: agglutinates O cells 1. Prepare three clean test tubes and
(H specificity) and other ABO blood label as follows: anti-A, anti-B, and
groups depending on the amount of H anti-A, B
antigen (anti-H lectin) NOTE: Labeling should be done with
● Present in low titer or even absent in cases of care since clerical errors are the most
acquired and congenital frequent errors in the blood bank.
hypogammaglobulinemia and agamma 2. Place two drops of the appropriate
globuleniemia reagent (anti-A, Anti-B and O serum).
NOTE: Use a free falling drop. Do not
DIRECT ABO BLOOD GROUPING (SLIDE METHOD) touch the dropper to the side of the
PROCEDURE-2 tube. Always add antisera before cells.
● Materials: Always check for the clarity and
o Applicator sticks expiration of the antisera.
o Disposable blood lancet or pricker 3. Add one drop of 2% to 5% suspension
o Glass marker or labelling material of red blood cells to be tested to each
o Typing Sera A (anti-A antisera) tube.
o Typing Sera B (anti-B antisera) NOTE: Use a free falling drop. Do not
● Procedure: touch the dropper to the side of the
1. Place 1 drop of anti-A and 1 drop tube.
of Anti-B reagent separately on a 4. Mix the reagent and RBC suspension
labeled slide. and centrifuge at 3400 rpm for 15
2. Add 1 drop of 20% test red cell seconds.
suspension or a drop of whole NOTE: Time may vary with each
blood from a capillary puncture to centrifuge. Check the calibration
2
IMMUNOHEMATOLOGY
(LAB) 2nd SEM, 2022
information for each individual

centrifuge. Never open the lid of the
centrifuge before the spinning motion
stops.
5. Gently resuspend the RBC button and
then observe for agglutination or
hemolysis macroscopically.
6. Observe suspected weakly reacting
results by viewing the mixture under
the low power objective of the
compound microscope.
7. Grade reactions and record the
results.
8. Have the instructor check your work.
9. Dispose all the biohazardous waste in
the puncture –proof container.
3

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Immuno Lab Finals

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IMMUNOHEMATOLOGY

(LAB)- Indirect Coomb's Test

INDIRECT COOMBS TEST examine for agglutination.

+ (with Du POSITIVE Rh (-) Du (+): Using serum as a AHG reagent, test

False positive results False negative results

Improper specimen Inadequate or improper

Overcentrifugation and Failure to wash

Centrifugation after Contamination of AHG

Bacterial contamination High concentration of

Dirty glasswares Elution of antibodies

Presence of fibrin Elution of antibodies

Cells with positive DAT Improper reagent

Polyagglutinable cells Excessive heat,

Saline contaminated by Non-reactive serum

3. Add 2 drops of AHG antisera.

A. PROTEIN Major XM:

B. THERMO Major XM:

C. AHG Major XM:

FORWARD AND REVERSE TYPING

B B Anti-A BLOOD GROUP REACTION WITH REACTION WITH

O O Anti-A and Anti-B A - +

information for each individual

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