Blood Banking

Tests and Procedures

Winona Mei A. Reyes, RMT

 ● Red cell suspension (RCS)
● ABO forward typing
● ABO reverse typing
● Direct AHG test (DAT)

Blood Banking
● Indirect AHG test (IAT)
● Crossmatching/Compatibility
testing
Tests & Procedures ● Antibody screening
○ Tube method
○ Gel method
Solid-phase adherence method
Topics covered:
○
● Enzyme tx
● Neutralization
● Adsorption
● Elution
● Antibody titration
ABO Forward Typing
What are the antigens and antibodies found in each ABO blood type?
What are the antigens and antibodies found in each ABO blood type?
 Forward Typing

● Commercial antisera (anti-A, anti-B) are used to

detect antigens on an individual’s RBCs
ABO Forward Typing Reagents
ABO Forward Typing Procedure (slide method)
ABO Forward Typing Procedure (tube method)
ABO Blood Typing
ABO Forward Typing
Results
ABO Forward Typing Results
ABO Forward Typing Results
2-5% Red Cell Suspension
2-5% Red Cell Suspension (RCS) Preparation
ABO Reverse Typing
 Reverse typing

● Detecting ABO antibodies in the patient’s serum by using known reagent

RBCs, namely A1 and B cells

● There is always an inverse relationship between the forward and reverse

type; thus, one serves as a check on the other.

● “Naturally occurring” antibodies are present in the serum without any

exposure of foreign RBCs. Examples are the ABO antibodies.
ABO Reverse Typing Procedure (tube method)
Agglutination Grade Reaction
Agglutination Grade Reaction
Agglutination Grade Reaction
Agglutination Grade Reaction
IMMUNOGLOBULINS
IMMUNOGLOBULINS
IgM vs IgG
Anti-Human Globulin Test
 History
● DR. ROBIN COOMBS (1945):
ALONG WITH HIS ASSOCIATES,
DESCRIBED THE USE OF THE
ANTIGLOBULIN TEST FOR THE
DETECTION OF WEAK AND
NON-AGGLUTINATING RH
ANTIBODIES IN SERUM
 What is the
Antihuman Globulin test (AHG test) ?
● ALSO KNOWN AS COOMBS’ TEST

● PRINCIPLE:
ANTIHUMAN GLOBULINS OBTAINED FROM IMMUNIZED NONHUMAN SPECIES BIND
TO HUMAN GLOBULINS SUCH AS IGG OR COMPLEMENT

● AN ANTIBODY TARGETED AGAINST HUMAN PROTEINS WILL BIND TO THOSE PROTEINS

ALREADY ATTACHED TO RBCS AND CAUSE VISIBLE AGGLUTINATION

● MAKES USE OF ANTI-ANTIBODY TO OBSERVE THE FORMATION OF AN AG/AB COMPLEX

THAT WOULD OTHERWISE GO UNDETECTED
 AHG Reagents
● POLYSPECIFIC AHG
■ ANTI-IGG & ANTI-C3 IN ONE
REAGENT
■ TEST TUBE METHOD

● MONOSPECIFIC AHG
■ SEPARATE REAGENTS

● ANTI-IGG:
GAMMA-CHAIN SPECIFIC,
MAY CONTAIN
ANTI-LIGHT CHAIN
SPECIFICITY: IGM, IGA,
IGG
● ANTI-C3
 Preparation of AHG Reagents

● INJECTION OF HUMAN SERUM OR PURIFIED GLOBULIN INTO LABORATORY ANIMALS

● THE RABBIT’S IMMUNE RESPONSE IS TRIGGERED PRODUCING AN ANTIBODY TO HUMAN GLOBULIN

● POLYCLONAL:
MIXTURE OF ANTIBODIES FROM DIFFERENT PLASMA CELL CLONES;
RECOGNIZING DIFFERENT EPITOPES

● MONOCLONAL:
DERIVED FROM ONE CLONE OF PLASMA CELLS;
RECOGNIZING A SINGLE EPITOPE’ ACHIEVED THROUGH HYBRIDOMA TECHNOLOGY
Polyclonal AHG Production
 Preparation of AHG Reagents
POLYCLONAL AHG PRODUCTION
● RABBITS, SHEEP, GOATS

1. USE SERUM FROM MANY DONORS THAT ARE INJECTED TO RABBITS

2. POOLING OF ANTI-IGG FROM MANY IMMUNIZED RABBITS

3. BOTH COLONIES OF ANIMALS ARE HYPERIMMUNIZED TO PRODUCE HIGH-TITER,

HIGH AVIDITY IGG ANTIBODIES
4. *** ABSORBED WITH A1, B, AND O CELLS ***

5. DILUTION TO PREVENT PROZONE PHENOMENON THAT MAY LEAD TO FALSE (-)

RESULTS
 Preparation of AHG Reagents
MONOCLONAL AHG PRODUCTION
○ By KOHLER AND MILSTEIN
○ HIGH-TITER ANTIBODIES WITH WELL DEFINED SPECIFICITIES EITHER TO IGG
AND FRAGMENTS OF C3
■ IGG1 AND IGG3
■ C3B AND/OR C3D
Monoclonal AHG
Production
 Preparation of AHG Reagents
MONOCLONAL AHG PRODUCTION
● HYBRIDOMA TECHNIQUE

1. MICE INJECTED WITH PURIFIED HUMAN GLOBULIN

2. MOUSE SPLEEN CELLS CONTAINING AB-SECRETING LYMPHOCYTES ARE FUSED

WITH MYELOMA CELLS
3. AB IS COLLECTED AS ASCITES

4. REAGENT CONTAINS BUFFERS, STABILIZER, BACTERIOSTATIC AGENTS, AND GREEN

DYE FOR IDENTIFICATION
Types of Antihuman Globulin Test

● DIRECT ANTIGLOBULIN TEST (DAT)

● INDIRECT ANTIGLOBULIN TEST (IAT)
 AHG Results

(+) result = macroagglutination or microagglutination

(-) result = no reaction; must be confirmed by Coomb’s check cells

Macroagglutination
Macroagglutination
Rouleaux Formation
Rouleaux Formation
Rouleaux formation is solved by…!
 Coomb’s Check Cells
● An unofficial term for the IgG antibody-coated red cells used as a quality control measure for negative indirect or
direct antiglobulin tests performed in test tubes.

● When a IAT or DAT is negative, the anti-human globulin (AHG) reagent is free in the tube (it did find any coated RBCs with
which to bind).

● RBCs already coated with antibody are added.

● The free AHG should attach to the IgG-coated check cells and give visible agglutination after centrifugation. This
required step ensures that the AHG reagent was added and is working properly.

● Check cells get their name from the fact that when they are added and agglutination is seen as expected, workers indicate
that fact by placing a “checkmark” next to the “0” that they wrote down for the negative reaction.
Retrieved from:
https://www.bbguy.org/education/glossary/glc07/#:~:text=An%20unofficial%20term%20for%20the,IgG%2Dcoated%20RBCs%E2%80%9D).

TRUE NEG:
(+) agglutination

FALSE NEG:
no agglutination
 Factors Affecting the AHG Test

● RATIO OF SERUM TO CELLS

● REACTION MEDIUM
● TEMPERATURE
● INCUBATION TIME
● WASHING OF RBCS
● SALINE FOR WASHING
● ADDITION OF AHG
● CENTRIFUGATION FOR READING
 Factors Affecting the AHG Test

RATIO OF SERUM TO CELLS

● MINIMUM RATIO OF 40:1

● ACHIEVED BY USING 2 DROPS OF SERUM: 1 DROP OF 5% SUSPENSION OF WPRBC (2:1)
● IF CELLS ARE IN SALINE, IT IS ADVANTAGEOUS TO INCREASE RATIO OF SERUM TO
CELLS TO DETECT WEAK ANTIBODIES.
 Factors Affecting the AHG Test
REACTION MEDIUM
Ratio of serum to
Reaction medium Principle Incubation time reaction medium
to cells
Allows antibody
coated cells to
Albumin come into closer 30 min 2:2:1
contact with each
other
Reduces zeta
LISS 10 to 15 min 2:2
potential
Removes water
molecules
PEG surrounding RBC 30 min
to increase
antibody uptake
 Factors Affecting the AHG Test

TEMPERATURE
○ 37ᵒC
 Factors Affecting the AHG Test
INCUBATION TIME

○ 30 TO 120 MINUTES
○ MAJORITY OF SIGNIFICANT ANTIBODIES CAN BE DETECTED AFTER 30 MIN OF
INCUBATION TIME
○ LISS:
10 TO 15 MIN.
INCREASING SERUM-TO-CELL RATIO WILL DECREASE SENSITIVITY OF TEST
 Factors Affecting the AHG Test

WASHING OF RBCS
● MINIMUM OF 3 TIMES BEFORE ADDITION OF AHG REAGENT

● INADEQUATE WASHING MAY RESULT IN A FALSE (-) BECAUSE OF NEUTRALIZATION OF AHG

REAGENT BY RESIDUAL UNBOUND SERUM GLOBULINS

● SHOULD BE DONE IMMEDIATELY TO MINIMIZE ELUTION OF LOW-AFFINITY ANTIBODIES

● CELL PELLET SHOULD BE COMPLETELY RESUSPENDED TO AVOID DESENSITIZATION OF

THE TEST

● CENTRIFUGATION AT EACH WASH SHOULD BE SUFFICIENT TO PROVIDE A FIRM CELL

PELLET TO MINIMIZE LOSS OF CELLS AT EACH DISCARD OF SALINE
 Factors Affecting the AHG Test
SALINE FOR WASHING

● 7.2 – 7.4 PH
● DECREASE IN PH 🡪
INCREASES THE RATE OF ANTIBODY ELUTION 🡪
FALSE (-)
 Factors Affecting the AHG Test
ADDITION OF AHG

● IMMEDIATELY ADDED AFTER WASHING TO MINIMIZE ANTIBODY ELUTION

AND NEUTRALIZING AHG REAGENT
 Factors Affecting the AHG Test

CENTRIFUGATION
● CBER RECOMMENDATION:
1,000 RCF FOR 20 SECONDS
● 500 RCFS FOR 15-20 SECONDS
● WHAT WILL HAPPEN IN THE FF? Will it
cause a false (+) or a false (-) result?
○ SHORT CENTRIFUGATION TIME
○ LONG CENTRIFUGATION TIME
○ HIGH RCF
○ LOW RCF
Crossmatching
 Compatibility Testing

1. Immediate Spin phase (IS phase):

● Detects IgM antibodies, such as ABO, Lewis, P, I, etc
● Conducted at room temperature (20-24℃; avg. 22℃)
● Confirms ABO compatibility
● Not usually performed, since it detects clinically insignificant antibodies or antibodies mainly
composed of carbohydrates

What to do:

1. Mix serum and 2-5% RCS - (2 drops:1 drop)

Note: serum and red cells used depends whether Major/Minor cross matching is conducted
2. Centrifuge at 1000 RCF for 20 seconds
3. Gently dislodge the red blood cell button
4. Check for agglutination. If there is no reaction, proceed to next phase.
 Compatibility Testing

2. Incubation phase (37℃ phase):

● Detects IgG antibodies, such as Rh, Kidd, Kell, Duffy, etc
● Incubated at body temperature (37℃); use water bath
● Detects clinically significant antibodies or antibodies mainly composed of proteins.

What to do:

1. Add enhancement media/reaction medium (LISS, albumin, or PEG) and mix - 2 to 3 drops
2. Incubate using water bath (37℃); incubate for recommended no. of min according to enhancement
media used:
i. LISS - 10 to 15 min incubation time
ii. Bovine Albumin - 30 to 60 min incubation time
iii. PEG - 30 to 60 min incubation time
3. Centrifuge at 1000 RCF for 20 seconds
4. Gently dislodge the red blood cell button
5. Check for agglutination. If there is no reaction, proceed to next phase.
 Compatibility Testing

3. Anti-human Globulin phase (AHG/IAT phase):

● Detects IgG antibodies, such as Rh, Kidd, Kell, Duffy, etc
● Confirms presence of IgG antibodies that have failed to achieve lattice formation leading to no
visible agglutination; AHG reagent is required to solve this problem
● Detects clinically significant antibodies or antibodies mainly composed of proteins

What to do:

1. Wash the red blood cells 3-5 times with isotonic saline. Decant supernatant saline completely.
2. Add Anti-Human Globulin (Polyspecific: Anti-IgG, C3d) to the packed red blood cells and mix. - 2 drops
3. Centrifuge at 1000 RCF for 20 seconds
4. Gently dislodge the red blood cell button
5. Check for agglutination. If there is no reaction, proceed to next phase.
 Compatibility Testing
4. Coomb’s Check Cells:
● Quality control measure to confirm a TRUE NEGATIVE result in the AHG phase
● Coomb’s check cells are composed of IgG-coated O-red cells that will bind to the free AHG
molecules in the mixture.
● This is to ensure the following:
1. There was enough AHG reagent used in the procedure
2. AHG is not deteriorated or expired

What to do:

1. Add Coomb’s Check Cells and mix. - 1 drop

2. Centrifuge at 1000 RCF for 20 seconds
3. Gently dislodge the red blood cell button
4. Check for agglutination.
i. TRUE NEGATIVE = (+) agglutination
ii. FALSE NEGATIVE = NO agglutination
 Compatibility Testing

● No agglutination = Compatible

● Agglutination = Incompatible
○ Proceed to antibody identification :(
 References
● Biotest Medical Diagnostics. (2008). Anti-Human Globulin Anti-IgG, -C3d; Polyspecific (Rabbit/ Murine Monoclonal).
Retrieved 7 April 2021, from https://www.fda.gov/media/72503/download
● Chaffin, J. (2021). Glossary: Check Cells - Blood Bank Guy Glossary. Retrieved 7 April 2021, from
https://www.bbguy.org/education/glossary/glc07/
● Letendre, P. University of Alberta (2010). Bovine Albumin. Retrieved 7 April 2021, from
https://sites.ualberta.ca/~pletendr/tm-modules/methods/70met-alb.html
● Young, N. A. F. (1957). Direct Compatibility Testing. Retrieved 7 April 2021, from
https://jcp.bmj.com/content/jclinpath/11/4/311.full.pdf
Antibody Screening
 Why perform
Antibody detection and Identification?
•Critical in pre-compatibility testing
•Principle tool in investigating potential hemolytic
transfusion reactions and immune hemolytic anemia
American Association of Blood Banks
(AABB)
•AABB is an international, not-for-profit Association representing
individuals and institutions involved in the fields of transfusion medicine
and biotherapies. The Association is committed to improving health
through the development and delivery of standards, accreditation and
educational programs that focus on optimizing patient and donor care
and safety.
•Requires the use of an antibody screen to detect clinically significant
antibodies in both the blood donor and the intended recipient as part of
pre-transfusion compatibility testing.
•RBC units, hematopoietic stem cells (HSC), bone marrow donors
 Antibody Detection Methods
•Focuses on detecting “irregular” or “unexpected” antibodies

•Immune alloantibodies
•Naturally occurring antibodies
•Passively acquired antibodies
•Clinically significant antibodies
•Autoantibodies
Antibody Detection Methods

•ANTIBODY SCREENING •ADSORPTION

•ANTIBODY IDENTIFICATION •ELUTION

•SELECTED CELL PANEL •AB TITRATION

•ENZYMES •PROVIDING
COMPATIBLE BLOOD
•NEUTRALIZATION
PRODUCTS
Antibody Screening (IAT)

•Tube method
•Gel method
•Solid phase adherence method
Ab Screening – Tube Mtd.
 Ab Screening – Tube Mtd.
Patient’s serum/plasma is mixed with
RBCs of known antigen content
•IS phase: not required, detection of clinically insignificant cold Ab
•37ᵒC phase: enhancement media may be added.
Observe for…
•Hemolysis: supernatant is pink or red; clarity
•Agglutination: Gently tilt tube to dislodge cell button
*judge only after all RBCs have been dislodged
 Ab Screening – Tube Mtd.
•AHG phase: wash with 0.9% saline and add AHG reagent.
Observe for…
•Hemolysis: loss of cell button mass
•Agglutination: Macroscopic or microscopic agglutination

•Confirm negative tests will Coomb’s control cells (Check cells)

 Ab Screening – Tube Mtd.
•RBC reagents
•Group O with most significant, RBC antigens
•Suspended at 2% - 5% diluent:
maintains integrity of antigens and prevents hemolysis
•Screen cells are packaged in sets of two or three cell suspensions
each with unique combination of antigen; accompanied with an
antigen profile; lot-specific
 Ab Screening – Tube Mtd.
•RBC reagents
•Homozygous expression of antigens:
allows detection of Ab that show dosage

•Commercially prepared screen cells vs pooled screen cells

•Clinically significant antigen content
•Homozygous expression = more reliable
 Ab Screening – Tube Mtd.
•Enhancememnt media (Potentiators)
•22% Albumin:
albumin disperses charges in the zeta potential
•LISS:
Glycine and albumin + increases Ab uptake
•PEG:
Removes water concentrating antibodies present; more sensitive
than LISS; not appr with elevated plasma protein content
 Ab Screening – Tube Mtd.
•AHG reagents
•Polyspecific AHG reagent
•Monospecific AHG reagent
•(+) anti-complement may lead to detecting clinically insignificant
Ab
 Ab Screening – Tube Mtd.
•Coomb’s control cells
•RH (+) RBCs coated with anti-D
•Reacts with anti-IgG in the AHG reagent that remains free 🡪
Visible agglutination

•Visible agglutination = true negative ☺

•No agglutination = false negative ☹
(Inadequate washing, deteriorated rgnt, REPEAT TEST)
Ab Screening – Gel Mtd.
 Ab Screening – Gel Mtd.
•Uses a microtubule filled with dextran acrylamide gel
•6 microtubules in a plastic card with a size of a credit card
•Screen cells are suspended in LISS of 0.8%
•Specimen and screen cells are added to reaction chamber that sits
above the gel
 Ab Screening – Gel Mtd.
•Incubate at 37ᵒC for 15 min to 1 hr.
•Centrifuge for 10 min

•No agglutination = RBC pellet at the bottom of microtubule

•Agglutination = anti-IgG with react with Ab-coated RBCs

(Sensitized RBCs) 🡪 trap within gel because agglutinates

are TOO LARGE to pass through the gel particles
Ab Screening – Solid Phase Adh. Mtd.
Ab Screening – Solid Phase Adh. Mtd.
•Example: Immucor’s Capture-R, Erythrocyte magnetized
technology (EMT)
•RBC antigens coat microtiter wells rather than being present on
intact RBCs
•Add patient’s serum or plasma to each well in the screen cell set
with LISS
Ab Screening – Solid Phase Adh. Mtd.
•Incubation at 37ᵒC
•Wells are washed
•Indicator RBC cells coated with anti-IgG are added. Centrifuge.

•No sensitization = Indicator cells form pellet in the bottom of well

•Sensitization = Diffuse pattern.

Indicator cells react with antibodies bound

to the antigens in microtiter well
 Interpreting
Antibody Screening
Antibody Screening - Interpretation
•In what phase(s) did the reaction occur?
• IS phase: IgM
• AHG phase: IgG
• Both: Lewis, M
•Autologous control?
• POSITIVE (+): autoantibodies
antibodies to medications
alloantibody coating circulating donor RBCs
(recently transfused previous 3 mo)
 In what phase(s) did the reaction occur?

1. Immediate Spin phase (IS phase):

● Detects IgM antibodies, such as ABO, Lewis, P, I, etc
● Conducted at room temperature (20-24℃; avg. 22℃)
● Confirms ABO compatibility
● Not usually performed, since it detects clinically insignificant antibodies or
antibodies mainly composed of carbohydrates
 In what phase(s) did the reaction occur?

2. Incubation phase (37℃ phase):

● Detects IgG antibodies, such as Rh, Kidd, Kell, Duffy, etc
● Incubated at body temperature (37℃); use water bath
● Detects clinically significant antibodies or antibodies mainly composed of
proteins.
 In what phase(s) did the reaction occur?

3. Anti-human Globulin phase (AHG/IAT phase):

● Detects IgG antibodies, such as Rh, Kidd, Kell, Duffy, etc
● Confirms presence of IgG antibodies that have failed to achieve lattice formation
leading to no visible agglutination; AHG reagent is required to solve this problem
● Detects clinically significant antibodies or antibodies mainly composed of proteins
 Antibody Screening - Interpretation
Did more than 1 screen cell sample react?
React at same strength and phase?

•Single Ab = (+) reaction at same strength and phase

(+) reaction of similar strength and phase 🡪 Ab show dosage

•Multiple Ab = (+) reaction at different phases or strengths

•AutoAb = (+) reaction at autologous control

Antibody Screening - Interpretation
Is hemolysis or mixed-field agglutination present?

•Hemolysis = anti-Lea, anti-Leb, anti-PP1Pk, anti-Vel

•Mixed-field = anti-Sda, anti-Lu
Antibody Screening - Interpretation
•True agglutination or rouleaux?
•Rouleaux = “Stacked coin” appearance
observed in ALL tests containing patient’s serum incl.
autologous and reverse ABO
altered albumin-to-globulin ratio, EXCEPT AHG TESTING
received plasma expanders (e.g. dextran)

RESOLVED BY: Adding 1-3 drops of saline to test tube

Rouleaux Formation
Antibody Screening - Limitations
Limitations Resolution

Antibody titer less than the level of

sensitivity of the screening method Perform antibody titer
employed

2/3 of Ab no longer detectable after 5 yrs of Check patient’s history

formation Perform antibody titer

Ab directed against low-prevalent Ab that

Perform selected screen cell panel
are not present in the RBC screen cell set

Ab showing dosage if screen cells do not

Use commercially prepared
have homozygous expression of target Ag
Antibody Screening - Limitations
•Cell-to-Serum ratio: prozone; postzone
•Temp & Phase reactivity: cold Ab (4ᵒC)
• Cold alloantibodies
• Anti-Lea • Cold autoantibodies
• Anti-Leb • Anti-I
• Anti-M • Anti-i
• Anti-N
• Anti-P

•Length of Incubation

•pH: pH of 6.5 → anti-M

Step 1
Starting to exclude
antigens without
reaction in all 3 phases;
When both pairs are +
(heterozygous cases),
they are both excluded
(here marked by X),
except for C/c, E/e,
Duffy, Kidd and MNS
antigens (where
antibodies of the patient
may still react towards
blood cells with
homozygous antigen
expression, because
homozygous expression
results in a higher
dosage of the antigen).
Step 2
Going to the next
reference cell row with a
negative reaction (in this
case cell donor 4), and
repeating for each antigen
type that is not already
excluded.
Step 3
Repeating the same for
each reference cell row
with negative reaction.
Step 4
Discounting antigens that
were absent in all or
almost all reactive cases
(here marked with \).
These are often antigens
with low prevalence, and
while there is a possibility
of such antibodies being
produced, they are
generally not the type that
is responsible for the
reactivity at hand.
Step 5
Comparing the remaining
possible antigens for a
most likely culprit (in this
case Fya), and selectively
ruling out significant
differential antigens, such
as with the shown
additional donor cell type
that is known to not
contain Fya but contains C
and Jka
Conclusion

Anti-Fya is the culprit!

If you need more
help in solving
antibody
screening,
search the link
below

http://learnserology.ca/
LearnSerology_content/
LS4_Appraoch_to_the_A
ntibody_Panel.html
 Antibody Identification
• Patient history
• Reagents
• Exclusion
• Evaluation of Panel Results
 Antibody Identification
•Patient history
•Age, sex, race, diagnosis, transfusion and pregnancy history, medications, IV
solutions
•African descent: anti-U, Fy(a-b-)
•Medications (IVIG, RhIG, antilymphocyte globulin):
passively transfer Anti-A, Anti-B, anti-D, etc
•Infectious and autoimmune disorders:
RBC autoantibodies
•Delayed HTR:
transfusion w/in past 3 mo
•Donor RBCs rxn:
mixed-field agglutination
 Antibody Identification
•Reagents
• Antibody identification panel consists of 11 to 20 group O RBCs
with various Ag expression
• Ag expression should be diverse
• Homozygous expression of Rh, Duffy, Kidd, and MNSs Ags
• Profile sheet
• Lot-specific and should not be interchanged with another panel
 Antibody Identification
•Exclusion
•Also known as “Ruling Out”
•Usually low prevalence antigens are excluded (K, Kpa, Jsa, Lua)
 Antibody Identification
•Evaluation of Panel Results
•Eliminate (+) reactions with antigens in screen cells with a (+)
reaction with Check Cells (CC). This means this a TRUE (-) Result.
•In what phase/s and at what strength/s did the positive reactions
occur?
•Do all of the (+) cells react at the same phase or at different
multiple phases?
 Antibody Identification
•Does the serum reactivity match any of the remaining
specificities?
•Are all commonly encountered RBC antibodies excluded?
(anti-E, anti-Kpa, anti-Jsa are not ruled out)
•Is the autologous control (+) or (-) ?
•Is there sufficient evidence to prove the suspected antibody?
•3 and 3 rule: 3 antigen positive and 3 antigen negative cells
 Antibody Identification
•Does the patient lack the antigen corresponding to the antibody?
•False-typing. (e.g. R1R1 can not have anti-C)
•(+) DAT 🡪 ELUTION is necessary chloroquine diphosphate (30min to 2 hrs)
or acid glycine/EDTA
•Kell Ag are denatured
•ABSORPTION is done if there is resistance to elution. Diluted with antiserum
and supernatant is harvested
•Mixed field: perform reticulocyte typing. Microhematocrit tubes are
centrifuged and are found at the top of the RBC layer
Resolving Antibody Identification
• Selected Cell Panel
• Enzymes
• Neutralization
• Adsorption
Selected Cell Panel
 Selected Cell Panel
•Specially used to eliminate the possibility of any reaction towards
low-prevalent antigens

Anti-E
Anti-Kpa
Anti-Jsa
Anti-Fya
Anti-Jkb
Enzymes
 Enzymes
•Ficin, papain, bromelin, trypsin
•Removes sialic acid residues on the RBC surface and denatures or
removes glycoproteins.
•Destroys certain antigens and enhances expression of others
•More sensitive if enzyme treatment is performed first then Ab ID
panel will be performed next.
Why are some antigens
destroyed/enhanced??
ENZYME TREATMENT
• Enhanced by enzyme treatment:
Some red blood cell antigen expressions
are enhanced, because they are
located deeper within the red blood Reaction
cell as compared to the RBC antigens Reaction after
before enzyme
that are destroyed by enzymes. Not all enzyme tx
tx
of them will react both to ficin and
papain. The red blood cell antigen 1+ 3+
expression that will be ENHANCED upon
the addition of enzymes are the
following:

ABO, Rh, Kidd, P, Lewis, I

 ENZYME TREATMENT
• Destroyed by enzyme treatment:
Some red blood cell antigens are
Reaction
destroyed, because they are before enzyme
Reaction after
located superficially on the red enzyme tx
tx
blood cell membrane. The red blood
cell antigens that will be DESTROYED 2+ 0
upon the addition of enzymes are
the following:
Fya, Fyb, M, N, S, Lua, Lub, Xg
ENZYME TREATMENT
• Enzymes are used primarily in antibody
identification workups, usually as a
confirmation of antibody specificity
(e.g., weakening reactions after enzyme
incubation would support the
identification of anti-Fya and would not
support identification of anti-D).
• Blood bank enzymes modify antigens,
not antibodies! We see the impact of
antibody reaction strength changes,
but the effect is on the antigen.
Neutralization
 Neutralization
•Other substances found in the body and are structurally similar to
RBC antigens are used to neutralize antibodies in serum. Allowing
separation or confirmation that a particular antibody is present
•This substance inhibits reactions between Ab and panel RBCs
•Use of control is necessary
Adsorption
 Adsorption
•Antibodies are removed from serum by adding target antigen and
allowing antibody to bind to the antigen
•After complex is formed, the solid precipitate is removed

•Commercial reagent:
• human platelet concentrate: Bg-like Ab
•Rabbit erythrocyte stroma: Cold reacting autoAb (I, H, IH)
 Adsorption
•Autoadsorption
•It is performed to reveal any alloAb that had been previously
masked by the AutoAb
•Homologous
•Px is anemic. Phenotypically matched with px RBCs
•Differential
•(+) DAT or recent transfusion of px
•Px serum sample is divided into minimum of 3 aliquots. Each
adsorbed a different cell (R1R1, R2R2, rr)
Autoadsorption
Elution
 Elution
•Used to release or dissociate, concentrate, and purify Ab
•Ab freed into solution is known as “ELUATE”
•The eluate may be tested against an RBC panel to identify the Ab

•Total elution: Ab released, RBC Ag are destroyed

•Partial elution: Ab released, RBC Ag are intact
•Chloroquine diphosphate, EGA, ZZAP
Elution
•EGA
• EDTA Glycine-Acid
• Remove IgG antibodies from the surface of red blood cells
• Useful in settings where the RBCs are coated by autoantibodies or alloatibodies
• Damages Kell antigens on RBC surface
•ZZAP
• Mixture of a proteolytic enzyme (papain) and a sulfhydryl reagent (dithiothreitol, or
“DTT“)
• Removes immunoglobulins and complement from the surface of DAT + RBCs
• Deactivates a multitude of red cell antigens on the red cell surface.
• Kell
•M
•N
• Two main Duffy antigens (Fya and Fyb)
 Elution
•Temperature-Dependent methods:
•Partial Elution: Gentle heat method (45ᵒC)
•Total Elution: 56ᵒC
•Lui freeze method: -18ᵒC 🡪 thawed rapidly 🡪 RBCs burst
•pH
•Glycine acid: ph 3
•Citric acid, digitonin acid
 Elution
•Organic solvents
•Dichloromethane, xylene, ether
•Acts on lipids found on the RBC membrane that will reduce the
surface tension and reverse van der Waals forces that hold Ag
and Ab together
•Best for detecting non-ABO Ab
•Original washing is most critical step
Antibody Titration
 Antibody Titration
•Quantify amounts of antibodies
• Titration is a semiquantitative method used to determine the
concentration of antibody in a serum sample or to compare the
strength of antigen expression on different red cell samples.
•Score of 10 or more is considered to be significant
• When the titer (eg, >16) and the antibody specificity have been
associated with HDFN, it is recommended that repeat titration
studies be performed every 2 to 4 weeks, beginning at 18 weeks’
gestation
 Antibody Titration
•Application:
• Estimating antibody activity in alloimmunized pregnant women to determine whether and
when to perform more complex invasive investigation of the fetal condition.
• Confirms the presence of Ab that are known as
HTLA (High Titer, Low Avidity)
• anti-Ch, anti-Rg, anti-Csa, anti-Yka, anti-Kna, anti-McCa, and anti-JMH

•Prepare:
• Patient serum
• 2-5% RCS group O
(Important that the laboratory be consistent and use red cells of the same phenotype for future titrations to test the same patient’s serum.)

• IgG-coated red cells

 Antibody Titration
Procedure:
1. Using 0.5-mL volumes, prepare serial twofold dilutions of serum in saline. The initial tube should
contain undiluted serum and the doubling dilution range should be from 1 in 2 to 1 in 2048 (total of 12
tubes).
2. Place 0.1 mL of each dilution into appropriately labeled test tubes.
3. Add 0.1 mL of the 2% suspension of red cells to each dilution.
4. Gently agitate the contents of each tube; incubate at 37 C for 1 hour.
5. Wash the red cells four times with saline; completely decant the final wash supernatant.
6. To the dry red cell buttons thus obtained, add anti-IgG according to the manufacturer’s directions.
7. Centrifuge as for hemagglutination tests.
8. Examine the red cells macroscopically; grade and record the reactions.
9. Add IgG-coated red cells to all negative tests; recentrifuge and examine the tests for macroscopic
agglutination; repeat the testing if the tests with IgG-coated red cells are nonreactive .
 Antibody Titration
Notes:
• DO NOT use enhancement techniques [albumin, polyethylene glycol, low
ionic strength saline (LISS)] or enzyme-treated red cells because falsely
elevated titers may be obtained. Gel testing is not recommended.
• Save an appropriately labeled aliquot of the serum (frozen at –20 C or
colder) for comparative studies.
 Antibody Titration
Reporting:
• The titer is reported as the reciprocal of the highest dilution of serum at
which 1+ agglutination is observed.
# of Cells Clumped Together
Agglutination Grade Reaction
Reported under Scoring

4+ 12

3+ 10

2+ 8

1+ 5

w or +/- 2 or 3
 Antibody Titration

Reference:
Jaber, M. A. Antibody Titration. Retrieved from: https://slideplayer.com/slide/5011822/