IMMUNOHEMATOLOGY-TRANSFUSION fibrin formation will be prevented in the

MEDICINE sample
o Plasma has Factor I = Fibrinogen
Immunohematology - branch of science deals with study of will undergo spontaenous conversion
red cell antigens and antibodies important in transfusion of to fibrin which may cause false
blood components clumping red blood cells)
• important that antigens are correctly identified for • REAGENTS: Known red cell suspension
doctor to be able to request for a correct blood type or (RCS must be laboratory prepared -
component. usually 5% is the average concentration)
o A 5% RCS - have tomato red color
A. BLOOD TYPING/GROUPING: routine blood banking (acceptable)
procedure performed to detect unknown antigens or unknown • There are cases where Indirect Typing is
antibodies using known reagent. not applicable therefore only screening
test is done
2 PROCEDURES: • Patients not qualified for indirect typing:
o Newborns - non-detectable because
1. DIRECT - also known as FORWARD or CELL
antibodies specific to ABO are
TYPING or CELL GROUPING; considered as the routine
produced 3-6 months after birth;
screening procedure in blood typing
unlike red blood cells antigens which
• PRINCIPLE: To detect unknown antigen using are already present starting on the
commercially prepared typing sera (patient red
37th day of the fetal development
blood cells or red cell suspension; antigen are
o Geriatric patients - weak immune
tested on red cell surface)
system follows a weak antibody
• SAMPLE: Red cell sample formation so the titer of antibody in
• REAGENTS: Commercially prepared the serum is low and is not readily
typing sera detected in indirect typing
o ABO Typing - Anti-A, Anti-B, Anti- o Patient with
A,B typing serum immunodeficiency/immunosuppre
▪ Anti-A - used to detect the ssion - weak immune system because
A antigen (color: BLUE) of infection (i.e., HIV patients, those
▪ Anti-B - used to detect the taking immunosuppressive drugs)
B antigen (color: ▪ Immunosuppressed patients
YELLOW) - have abnormally hyperactive
▪ Anti-AB - used as a immune system and is not
control in forward or healthy/beneficial anymore.)
direct typing (color: ▪ Hyperactive immune system
CLEAR/ COLORLESS) - autoimmune disorders;
antibody titer is low and will
2. INDIRECT - also known as the cause discrepancy in indirect
REVERSE/BACKWARD or SERUM TYPING typing; characterized in
or GROUPING; considered as the confirmatory immunosuppressed patients
procedure in blood typing
• PRINCIPLE: To detect unknown METHODS
antibodies in the serum using known red 1. SLIDE : the method of choice for rapid or bedside typing
cell suspension (mass blood donation, medical missions, etc.)
• SAMPLE: Serum (Plasma can be used • used in DIRECT/FORWARD Typing procedure
but serum is preferred because serum lacks • reaction time and reading time: limited to 1
Factor I or Fibrinogen so spontaneous minute because mixture tend to dry easily
HULLANA, IDMILAO, NARA, & PASCACIO
 o FALSE POSITIVE = if you fail to read it 2. Hemolysis
within 1 minute; subject to repeat procedure • red cell destruction; indication of hemolytic reaction is
if you doubt if it is a true agglutination the release of Hemoglobin which would change the
reaction/false positive because of drying up serum/anti-sera color into clear red or clear pink.
of mixture) o occur due to the activation of the complement
proteins. End result of complement activation:
2. TUBE: the method of choice for routine typing CYTOLYSIS
• procedures performed are: DIRECT and o due to strong agglutination reaction = lead to
INDIRECT HEMOLYTIC REACTION
• reaction and interpretation time: carried out within
3 minutes FORWARD AND REVERSE GROUPINGS
• with the aid of centrifuge, there is enhanced and Forward/ Reverse / Serum
faster reaction Cell Typing Typing
Blood (Specimen: Red (Specimen: Serum ;
3. GEL: the automated method in blood typing Type cell ; Rgt: RCS)
• 3 types of Gel Test Method: Rgt: serum)
o Plain or Neutral Gel test - the gel use has no Anti- Anti- Anti- A B O
reagent/ no reagent incorporated; used in A B A,B CELLS
REVERSE typing. O - - - + + -
▪ Gel is plain. In microtube, we put reagent A + - + - + -
(RCS) then add serum B - + + + - -
o Specific Gel Test - the reagent is already AB + + + - - -
incorporated or added to the gel
▪ We add patient sample in microtube. It is Rule of Specificity:
applicable to FORWARD/DIRECT typing • If the antigen is specific to the antibody, there is always
because the anti-sera is already on the gel. a positive reaction (either cell clumping or cell lysis)
We only need to add the patient RED CELL • If the antigen is non-specific to the antibody, there is no
SAMPLE. reaction/negative.
▪ has specific color indicating the typing sera
used - can be blue/yellow. Rationalization:
o Low Ionic Gel Test - used in AntihumanGlobulin • For Forward typing:
Test (Coomb’s test) o Blood type O & Anti-A = Neg (Anti-A - non-
specific to the H antigen present on blood type O
2 positive reactions in Blood Typing: red cells)
1. Hemagglutination reaction o Blood type A & Anti-A = Pos (Anti-A is specific
• red blood cell clumping; RBCs form clumps and to A antigen found on blood type A)
clumping indicates the presence of LATTICE o Blood type B & Anti-A = Neg (Anti-A is non-
formation specific to B antigen present on red blood cells)
o commonly observed positive reaction in blood o Blood type AB & Anti-A = Pos (Anti-A is specific
typing to A antigen found on AB red blood cells)
• Lattice formation - cross-bridging or cross-linking of o Anti-A,B - used as control to check reaction of
antibodies adjacent to the antigen. Antibodies sensitize Anti-A and Anti-B; detect A and B antigens as
the red cell antigens and when they sensitize the well as subgroups of A and B antigens
antigens, the Abs can now link with the nearby Ab. (red • For Reverse typing:
cell antigens is where Abs attach) o A cells to O serum = Pos (A antigen on A cell are

o Visible result: Clumping or Agglutination specific to Anti-A present on blood type O serum)

reaction o A cells to A serum = Neg (A antigen on A cell is

non-specific to Anti-B present on A serum)
HULLANA, IDMILAO, NARA, & PASCACIO
 o A cells to B serum = Pos (A antigen on A cell is homozygous recessive gene
specific to the Anti-A present on B serum) • sese
o A cells to AB serum = Neg (AB serum lacks Anti- o No ABH soluble substance
o yuA antibody) Blood
o B cells to AB serum = Neg (B antigen is unable to Type / Gene A B H
react with Anti-B in AB serum which lack Anti-B Secretor Involved subs subs subs
Ab) Status
o O cells - is used as control; detects anti-H antibody A A,H,Se ✓✓ Absent ✓
B B,H,Se Absent ✓✓ ✓
• Is anti-H commonly produced/formed by individuals AB A,B,H,Se ✓✓ ✓✓ ✓
on the ABO system? H antigen is present on the red cells
O H,Se Absent Absent ✓✓
of individuals regardless of blood type. It is a precursor
Non - se Absent Absent Absent
structure for immunodominant sugars to attach for A
Secretor
and B antigen (L-fucose and n-acetyl galactosamine will
not attach to paragloboside without H antigen)
• Double check - indicated greater or higher
o Basis: Karl Landsteiner - If you have the antigen
concentration
on the red blood cell, you should not produce. the
• One check - present
corresponding antibody in the serum
o Example:
Test for ABH Soluble Substance:
▪ If you have A antigen on red cell, you should
• SALIVA NEUTRALIZING TEST
not have Anti-A because it will have in-vivo
o Principle: Hemagglutination Inhibition
reaction. You have anti-B instead.
Reaction - prevention of RBC clumping through
▪ If you have B antigen, you should not have
neutralizing the sample
Anti-B. Instead, you have Anti-A (opposite
o Sample: Saliva
Ab)
o Reagents:
▪ If blood type O, you can produce Anti-A and
Anti-B but not Anti-H because you have H ▪ Typing sera (Anti-A and Anti-B)
antigen. ▪ Lectin (Anti-H lectin) - source: Ulex
- Those without H antigen = H- europaeus - plant source
null or Bombay phenotype are ▪ Lectin is an antibody-like substance
expected to produce Anti-H present in plant source that can react to
because H antigen are not present specific antigen (stem, bark, fruit, leaves or
in their body. whole plant but highest level of lectin is
extracted in SEEDS)
B. ABH SOLUBLE SUBSTANCE DETERMINATION ▪ Anti-H - is not Ulex europaeus (minsan
• ABH Soluble Substances: basically glycoproteins; are napagbabaliktad kasi)
seen in body fluids except CSF (cerebrospinal fluid) = o Known A, Known B, and Known O RCS (red
are unable to cross the blood brain barrier cell suspension)
(macromolecules); o RESULTS:
• Glycoproteins - soluble and present in body fluids and ✓ (+) Absence of Agglutination Reaction
are regulated by the following regulators ✓ (-) Presence of Agglutination Reaction
o Gene regulators o st
1 step: Soluble antigen (Ag) in Patient sample
▪ ABH gene saliva + known Antibody reagent (typing
▪ Se (SeSe/Sese) - Secretor gene sera/lectin)
(homozygously & heterozygously inherited) o 2nd step: Particulate Antigen is added (source of
particulate antigen: known red cell suspension)
• Secretor individuals - individuals with Secretor gene
• Non-secretors - individuals who inherit the
HULLANA, IDMILAO, NARA, & PASCACIO
 STEP 1 STEP 2 RXC Sol. Subs. Present agglutinates formed when antigens react to antibodies.
Saliva + No A substance – Used to detect agglutination reaction.
Anti - A A RCS Aggltn. neutralized the
Anti-A
Saliva + A RCS No B substance –
Anti - B Aggltn. neutralized Anti-B
so Anti-B will no
longer react with B
RCS
Saliva + O RCS No H substance –
Anti - H Aggltn. neutralized Anti-H

Application:
• COMPONENTS: Hard plastic is used with microtube.
• Patient is AB secretor: (A, B, H substance)
o Microtube - composed of upper reaction chamber
Saliva Plus A Cell B Cell O Cell
(where sample and reagent is added) which is
Anti-A 0 0 0 wider than the lower reaction chamber.
Anti-B 0 0 0 o Column - narrow portion; contains the gel
Anti-H 0 0 0 (dextran polyacrylamide gel or brand name:
o Anti-A will never react with B-cell with or Sephadex)
o Gel - acts as a sieve - used to capture or trap
without saliva (Applicable to Anti-A and O cell
agglutinates when cells clump
too)
• GRADING of REACTION:

• O secretor: (H substance)
Saliva Plus A Cell B Cell O Cell
Anti-A 4+ 0 0
Anti-B 0 4+ 0
Anti-H 0 0 0

• Patient is B Non-Secretor (-A, -B, -H substance)

Saliva Plus A Cell B Cell O Cell
Anti-A 4+ 0 0 o 4+ = one solid clump; 100% reaction; strongest;
RBC are present on the top most layer of the
Anti-B 0 4+ 0
column
Anti-H W+ W+ 4+
o 3+ = when the red blood cells are present on the
upper portion of the column; numerous large size
• Grading of reaction:
clumps
o 4+
o 2+ = red blood cells are present at the upper and
o Weak +,
lower part of the column with most cells present at
o 0 (negative)
the center; presence of medium-sized clumps
o 1+ = presence of numerous small size clumps;
C. GEL TEST TECHNOLOGY
situated or found in the lower part of gel; weak
• used in automated procedures; standardize
reaction/variable
serologic (Ag-Ab) reactions
o +/- or variable weak = very small sized clumps;
• History: Developed by a French doctor (Dr. Yves
near the bottom part of the tube
Lapierre) in 1985
o Negative/0 = absence of agglutination rxn; red
• Principle of the Test: Performed to capture or trap
HULLANA, IDMILAO, NARA, & PASCACIO
 cell button is formed at the bottom of the tube and then magkaka roon ng lattice formation and
• No trapped red cell = no agglutination reaction agglutination will follow
• Gel - semi-solid; molecules are slightly apart; structure • 3 antibodies capable to bind complement (most-least
of the gel would allow unagglutinated RBCs to pass potent)
through o IgG3-IgG1-IgG2
o Note: IgG4 cannot bind complement
Advantages:
• Standardization of the procedure (reading,
IgM IgG
grading, interpretation)
Natural Immune
• Stable and well-defined end-point reaction
(reaction is stable for up to 3 days) Complete Incomplete

• Decrease sample volume needed for testing Best Agglutinating

Coating/Sensitizing
(microsampling - less than 1 mL) antibody
• Enhanced sensitivity and specificity of the Cold-reacting Warm-reacting
result Saline-reactive Albumin/AHG-reactive
• Cell washing is no longer performed.
Ex. ABO antibody Ex. Rh antibody
Complement binding
Disadvantage: Complement binding
(more potent)
• Interferences from the sample must be prevented
because it can affect the quality of the result Additional notes: Additional notes:

o Interferences: Hemolysis, Lipemia, Ictericia -Biggest antibody -Smallest antibody, the

-A Pentamer only antibody capable of
PROCEDURE: -The antibody that can transplacental movement
1. Addition of cells - source of Ag activate the classical - Reacts to tests at body
2. Addition of plasma/serum - source of pathway temperature, considered as
Ab clinically significant
3. Incubation (Antigen-Antibody
Reaction) - at warm temperature if IgG,
if IgM room temperature
AHG reagents (commercially prepared)
4. Centrifugation - specialized type of
centrifuge 1. Polyspecific AHG Reagents

5. Results (> 10 min) o Consists of a pool of rabbit anti-human IgG and

mouse monoclonal anti-C3b and anti-C3d

ANTIHUMAN GLOBULIN TEST (COOMBS TEST) o Also referred to as Broad Spectrum Coombs

• Principle: A technique for detecting cell-bound Reagent.

immunoglobulin. It is used to detect incomplete o Method of Preparation: Hyper-immunization of

antibodies (IgG) rabbit (Classical/Conventional method)

o Cell bound immunoglobulin (IgG) sensitizing

antibodies/ incomplete antibodies are able to 2. Monospecific AHG Reagents

sensitize the RBC membrane o Contains only one antibody specificity

• Since IgG are small and monomers, they are only o Either:

capable of sensitizing RBC ▪ Anti-IgG

o The agglutination reaction is not capable because ▪ Anti-C3b or C3d
they are small. o Method of Preparation: Kohler Milstein technique
o Addition of antihuman globulin would promote the ▪ It uses laboratory mice

bridging/ cross-linking of sensitized IgG on RBC

HULLANA, IDMILAO, NARA, & PASCACIO
 ▪ Köhler and César Milstein described the
hybridoma technique
▪ Hybridoma cell/Immortalized antibody
forming cells is the product of the fusion of
mouse plasma cell and malignant myeloma cell
▪ The immortality of hybridoma cells is due to
the characteristics of the myeloma cell
▪ The antibody(monospecific) producing
capability of hybridoma cells is due to the
characteristics of the plasma cell
STAGES OF ANTIGEN-ANTIBODY INTERACTION
• 1ST stage is Sensitization/Coating
o Sensitization occurs when antibodies react with
antigens on the cells and coat the cells
o The Fab region of the antibody interacts with the
epitope of the antigen
o IgM and IgG are both capable of this stage of
interaction
• 2nd stage is Agglutination reaction
o Occurs when antibodies on coated cells form
cross-linkages between cell with the adjacent
antigens resulting development of lattice
formation/visible agglutination or clumping
• Indirect AHG test (DAT)
o Antibody that proceeds to this stage is IgM
TYPES OF AHG PROCEDURE
• Direct AHG test (DAT)
o Detects in vivo sensitization of red cells with IgG
and/or complement proteins
o Useful in the following situations:
▪ Investigation of transfusion reaction (e.g HTR)
▪ Diagnosis of Hemolytic disease of the newborn
(HDN)
❖ Single most
❖ The newborns red cell is tested/ Cord
sample
❖ Single most important procedure in the
diagnosis of HDFN
❖ Babies only have Maternal antibodies (IgG)
▪ Diagnosis of autoimmune and drug-induced
hemolytic anemias
• Cells (whole blood) used for DAT should be
collected into either EDTA or Citrate containing
anticoagulant to minimize the possibility of in vitro
attachment off complement components
1. EDTA and Citrate have anti-complementary
activity specifically to C1 protein
2. EDTA and citrate is use to prevent False
positive DAT
3. C1 complementary protein is a trimolecular
complex
▪ C1q, C1r, C1s subunits
▪ These subunits are held together by
calcium
• Procedure
1. Washed (3x’s) patient red cells (sensitized)
▪ Importance of washing it 3x to eliminated
all unbound antibodies present in the
sample
▪ If unbound antibodies are not removed in
the sample, it can cause neutralization of
the reagent
▪ Unbound antibodies must be remove
using isotonic saline solution
2. Add immediately (Coomb’s Sera) AHG
Reagent
3. (+) Agglutiination
o A 2-step procedure (sensitization /Incubation step
and agglutination/ AHG step) that determines in
vitro sensitization of red cells
▪ Incubation is critical because IgG can only be
sensitized red cells in warm temperature
o Useful in the following situations:
• Detection of incomplete antibodies in
compatibility testing or to screening cells
in antibody screen
• Identification of antigen specificity, using
panel of red cells
• Determination of red cell phenotype
using known antisera (e.g Du testing)
HULLANA, IDMILAO, NARA, & PASCACIO
 • Titration of incomplete antibodies (IgG
sample in the serum)
Human Red Cells (from different person) + Serum of the
patient = Sensitization
o Mix
o Incubate
o After incubation, Identify if there is sensitization
(immediately add reagent)
o If tube method, centrifuge and check for
agglutination
o (+) Agglutination = sensitization
o (-) No clumping
o In this example, we can detect alloantibody and
autoantibody sensitization
o Alloantibody – yung antibody galing sa ibang tao
nag sensitized sa red cell ng patient
o Autoantibody – yung serum nung patient kapag
hinalo sa red cell ng patient then nag karon ng
sensitization
Weak D Determination
• Rule
o If nag negative sa Anti-D testing, don’t report it
as negative. You need to do a confirmatory
procedure which is Indirect Anti hematology
test
o You need to do confirmatory testing to know the
cause of negative anti-D result. Need to do
Indirect Anti Hematology testing to know if the
antigen of the patient is low grade weak D
▪ Low grade weak D – doesn’t react with
anti-D reagent
o Gagawin mo yung Indirect Coomb’s test kapag
nag negative ang Anti-D test nung patient.
▪ If nag positive sa Indirect Coomb’s test yung
red cell na nag negative sa Anti-D that is
considered Low Grade weak D.
▪ Low Grade weak D – considered as Rh
positive
▪ NOTE: we do not report weak D in the
laboratory
▪ If doesn’t have low grade weak D antigen,
that is when you reported as the patient is
Rh negative.
Factors affecting the AHG Test:
1. Ratio of serum to cells.
o Minimum ratio 40:1 = 2 drops serum and 1 drop
of 5%v/v cell suspension
o Important is the ratio to prevent prozone and
postzone effect
2. Temperature - Optimal: 37ºC
o If the temperature is too low or too high, the
antibodies will not sensitize.
3. Incubation Time
o In saline suspension: 30-120 minutes
o LISS suspension: 10-15minutes
o LISS just like bovine albumin is considered as
potentiators
o Potentiators – it increases the reaction affinity
between reactants to antigen to antibody ; to
reduce zeta potential between RBCs thus
increasing the rate of antibody uptake on the
cell ;
o NOTE: if mababa ang zeta potential, cells can
interact readily. Unlike kapag mataas ang zeta
potential, cells will just stick from each other
therefore mas matagal ang reaction
4. Reaction medium
o 60-minute saline test = 30-minute albumin
HULLANA, IDMILAO, NARA, & PASCACIO
 technique
o 22% Albumin – 2 drops 22% albumin + 2 drops
serum + 1 drop 3-5% cell suspension
▪ is said to reduce the zeta potential between
o RBCs thus increasing the rate of antibody uptake
on the cell
o LISS – 2 drops 3% RBC suspension in LISS + 2
drops serum
▪ - also increases sensitivity and shortens
incubation times ; if it reduced the zeta
potential, mas maiksi ang incubation time and
there is also increase sensitivity.
5. Washing of cells – minimum of three times
o Note: Washing is only for conventional tube
method
o If you will performed AHG test in Gel Test
Technology, washing of a cell is not a requirement.
6. Saline for washing – should be fresh and buffered to a pH
of 7.2-7.4
o It must be buffered near plasma pH. If it too much
acidic or basic the wash solution, It can affect the
performance of the test because antibodies are
proteins and proteins can be denatured when the
pH is too acidic or too basic.
7. Addition of AHG reagents should be added to washed
cells immediately after washing.
o It is to prevent spontaneous elusion process
o Elusion process – the sensitized antibodies in red
cells will detached / dissociate
▪ It will cause False Negative
o AHG Reagent – Green
8. Centrifugation - 1000 rcf for 15-20 seconds
o Over centrifugation can cause false positive
o Under centrifugation can cause false negative
SOURCES OF ERROR IN THE AHG TECHNIQUE
• False-Positive Results – if there is False positive result,
the test specificity is decreased or low.
o Autoaaglutinable cells
o Bacterial contamination or other contamination in
cells or saline
o Cells with a POSITIVE DAT used for IAT
o Overcentrifugation and overreading
o Polyagglutinable cells
o Dirty glasswares
o Saline contaminated with silica or heavy metals
• False Negative Results – the test sensitivity is
decreased or low
o Inadequate or improper washing of cells (most
common cause)
o AHG reagent nonreactive owing to deterioration or
neutralization
o AHG reagent not added
o serum not added in the indirect test
o serum nonreactive owing to deterioration of
complement
o inadequate incubation conditions
o Postzone and Prozone (cell suspension either too
weak or too heavy)
o Undercentrifugation
o Poor reading technique
Example of false negative:
o The patient red cells and donor serum = in vitro
mixing of the antibody in the serum of the donor
and red cell antigen of the patient.
o The medtech forgot to wash the red cell so the
unbound antibodies are still present and they added
AHG reagent = The unbound antibodies
neutralized the AHG reagent. Instead of
reacting the sensitized IgG, the AHG is already
neutralized by the unbound antibodies
▪ This can lead to False negative reaction,
AHG is neutralized by the unbound
immunoglobulins
HULLANA, IDMILAO, NARA, & PASCACIO
Example of positive reaction: Antibody Screening procedure

o The medtech washed the red cells (the unbound

antibodies are not present) so the reagent will
bound to the sensitized red cells = True Positive
Reaction

• Antibody screening is the detection of all clinically

Automated AHG Technique
significant antibodies outside the ABO system
A. Low Ionic Polybrene Technique (LIP)
o Clinically significant – this means IgG warmed
• Polybrene – aerolou promoting agent ; it promotes
reacting antibodies (Antibodies in Kell’s, Duffy,
further interaction
Kidd, Rh antibodies)
• Low Ionic Environment would allow sensitized
• AHG and Enzyme Testing is included
red cells to interact with one another in the
o AHG – to detect sensitization
presence of polybrene
o Enzyme Testing – to eliminate the other
B. Enzyme-Linked Antiglobulin Test (ELAT)
antibodies and to facilitate the identification of the
• Test that utilizes red cell sensitized with IgG
antibodies of interest
• In can be in vivo or in vitro
• One-cell pool (donors)
• Antihuman IgG will be added and it has label or
• Two cells pool
conjugate enzyme
• Three cells (recommended for patient)
• If the red cell has sensitization, AHG will react
• Six cells
with an enzyme and will put substrate
• Procedure:
o If positive reaction the sensitized red cell
o Patient Serum is tested – in search for clinically
with AHG = color production / development
significant antibodies outside of ABO antibodies
of the substrate
o Add the screening cell (Group O Cells with
▪ It is measured spectrophotometrically
known antigens) - Group O cells contains any of
• It is performed to determined the quantity of the
red blood cells including Red cell antigens, D, C,
antibody that is bound to the red cell
e, k, Fya, Jkb, Lea, P, M, N, S
C. Solid Phase Method (Direct and Indirect)
◆ Why blood type O cells – to avoid / prevent
• Small microwells are used in the testing
the interference of the high titer ABO
• The antigen are bound to the bottom of the well
antibodies
and after that, the patient plasma is incubated in the
o If agglutination of patient antibody with
well.
corresponding red cell antigen(s) = Positive in
• Can be applied to detect autoantibody and
serum
alloantibody

HULLANA, IDMILAO, NARA, & PASCACIO


• This procedure is specifically used for alloantibodies
screening
o Why alloantibody? – because the screening cell is
came from the different donors (pooled cell of
blood type O individuals)
• How to screen autoantibody? – the patient serum will
test on the patient red cell. If will react, there’s
autoantibody in the patients serum.
• NOTE: In the antibody screening, patient serum is not
only tested but also the donor serum is tested
• When antibody screening was introduced as part of the
pre-transfusion testing of donor unit. Minor
crossmatching was no longer routinely done in pre-
transfusion testing because it was replace by
antibody screening procedure
EXAMPLE OF AHG:
◼ In other words, if the auto control is positive
there is a presence of autoantibody
◼ If the screening cell 1 or 2 is positive, there is a
presence of alloantibody
◼ Result in patient number 1: NO
AUTOANTIBODY IS PRESENT AND ALSO
NO ALLO ANTIBODY IS PRESENT
THEREFORE IT IS NEGATIVE ANTIBODY
SCREEN
• Patient 2:
◼ Screening cell 1 – positive (3+)
◼ Result: There is a presence of Alloantibody
therefore it is POSITIVE ANTIBODY SCREEN
◼ Additional Notes: In transfusion medicine, the
most clinically significant antibodies that are
commonly tested / detected in the blood bank
laboratory is the alloantibodies.
• If the test is positive, the next step you will need to do
is identification of antibody specificity . So you will
need to undergo Antibody Identification
Antibody Identification
➢ If the Antibody Screen is reactive, the antibody
specificity must be determined.
✓ So safe blood can be administered to the Recipient.
✓ 11 reagent panel cell are to be used for
identification.
EXAMPLE OF GEL TEST TECHNOLOGY:

• Patient 1:
◼ Autocontrol tube contains patients red cell and • For ABO type:
serum o Anti – A – positive (4+)
◼ Screening cell 1 – contains patient serum and o Anti – B – negative
screening cell reagent o Anti – D – positive (4+)
◼ Screening cell 2 - contains patient serum and
• For antibody screening:
screening cell reagent
• AHG – positive (3+)
HULLANA, IDMILAO, NARA, & PASCACIO
• RESULT: A positive, Antibody screen positive
• The sample of the patient will need to undergo antibody
identification
o Prewarming the sample, inactivates IgM
• c.3 Use of sulfhydryl or thiol reagents (DTT and 2-
ME) which denature IgM antibodies by breaking
disulfide bonds.
o DTT- Dithiothreitol
⚫ 2-ME- 2-Mercaptoethanol
• c.4 Use of adsorption and elution techniques to
remove unwanted antibodies such as cold or warm
autoantibodies, or help to resolve multiple antibodies.
o Adsorption- removal of Ab from serum thru
adsorbent agents
o Elution-removal of Ab from red cell surface.

• Donor Cell Number 1-10 = for alloantibody ADSORPTION & ELUTION TECHNIQUES
identification 1. Adsorption- used to remove unwanted Ab from serum
• AC – autocontrol for autoantibodies • If an autoantibody such as I, H, or IH are defines, it
• Upper part of the table – antigens from different
can be absorbed onto the patient;s enzyme pretreated
blood group outside the ABO
cells at 4 deg C. Rabbit cells may also be used as
• Below the blood group antigens
adsorbents for anti-I since they are stitch in I
o + = antigen is present
antigens.
o -
o 0 = antigen is absent
2. ELUTION- used to dissociate IgA from sensitized red
• Last three columns
o IS = immediate spin for IgM detection therefore it cells

must be negative in antibody identification (IgG

ang target dapat)
o AHG = IgG antibodies
o CC = Checked cells for the coomb’s control cells
▪ Checked cells is tested when AHG is
negative
▪ NOTE: If AHG is negative, Checked Cells
MUST BE POSITIVE

Other techniques may be used to eliminate clinically

• They recovered antibody, eluate, can be tested like serum
insignificant reactions and make identification of
to determine the antibody’s specificity
significant antibodies easier.
• Techniques include heat, freeze-thaw process, use of
• c.1 Use of AET, DTT, and ZZAP which inactivates
organic solvent, acid elevated, or by using ZZAP or
some antigens especially Kell.
chloroquine diphosphate
• c.2 Prewarm procedure. Clinically insignificant cold
o Eluate- is the elution product. It contains detached
antibodies maybe removed by this technique. Patient
Ab from red blood cells and can be treated as serum.
serum, reagent red cells and enhancement medium can
be warmed separately at 37 deg C for 5-10 minutes prior
to mixing
HULLANA, IDMILAO, NARA, & PASCACIO
 a. ZZAP- mixture of DTT and papain that is
used to remove Ab from sensitized red cells
and to enzyme treat them at the same time
b. Chloroquine diphosphate- reagent used to
remove IgG Abs from the surface of
sensitized cells, inactivates Bg antigens
TYPES OF ELUTION TECHNIQUES
A. FIRST GENERATION
1. Landsteiner-Miller Heat
• Sensitized rbc sample/ cell suspension is
heated with albumin medium at 56 degC
prompting detachment of IgG on red cells
2. Lui-Freeze-Thaw
• Rapid freezing thawing procedure. Can
detach Ab from rbc
B. SECOND GENERATION
1. Use of Organic Solvents (i.e. ether)
C. THIRD GENERATION
The one being used today BB facility
1. Use of Non Hazardous Chemical Agents (acid
agents)
2. “acid elution”- latest technology for elution
• *common are end result- ELUATE containing
detached Ab
c.5 Neutralization
• Commercial substances are available to neutralize or
to inhibit reactivity of some antibodies. The target
are the clinically insignificant and non-target Ab to
prevent them from interfering
• Sources of Substances for Neutralization of
Antibodies:
o Hydatid cyst fluid- anti-P1
o Plasma or serum w/Le subs- anti-Lea &
anti-Leb
o Pooled serum or plasma- anti-Chido, anti-
Rogers
o Urine- anti-SDA
o Saliva of “secretors”- anti-ABH
o Human milk- anti-I
COMPATIBILITY TESTING
• “crossmatching”
• A pre-transfusion procedure that is composed of a series
of test to ensure the safety of the recipient during blood
transfusion
• Performed to select the appropriate donor/ blood unit for
patient transfusion
Collection and preparation of samples:
1. Patient Identification. This is very critical. Always check
the label of the tube. Also when you release the donor unit.
2. Collection. SERUM is the preferred specimen for
compatibility testing. Hemolysis should be avoided.
Why SERUM and NOT PLASMA?
• Plasma may cause small fibrin clots to form which
may be difficult to distinguish from true
agglutination
• Plasma may inactivate complement so that
antibodies may not be detected. Plasma has
anticoagulants leading to anti-complementary.
3. Age of specimen. The freshest sample possible should be
used for compatibility testing. Specimens must be less than 3
days old if the patient has been transfused or pregnant within
the past 3 months.
• The fresher, the better.
- Less than 3 days old because this age of specimen
can correctly represent the current immunologic
status of the patient.
4. Sample Storage. The AABB requires that patients samples
must be stored between 1-6 degC (ref temp) for at least 7 days
after transfusion.
HULLANA, IDMILAO, NARA, & PASCACIO
 - Samples are kept for investigation of transfusion
reaction
- At least 7 days for delayed symptoms
COMPATIBILITY TESTING PROTOCOLS
1. ABO GROUPING. Most critical pretransfusion serologic
test.
*If the patient’s ABO group cannot be satisfactorily
determined and immediate transfusion is essential, group O
packed red cells should be utilized.
2. RH TYPING.
*If the Rh type of the recipient cannot be determined and
transfusion is essential, Rh-negative blood should be given.
Rh-negative is uncommon in our country.
2 positive reaction in crossmatching:
1. Agglutination
2. Hemolysis
Presence of these are (+) result which indicates
incompatibility
4. CROSSMATCHING
• MAJOR X-MATCH: Donor’s cells + Recipient’s serum
• MINOR X-MATCH: Donor’s serum + Recipient’s cells
o *In the pretransfusion procedure, major x match is
routinely done. Minor is not routinely done and is
replaced by antibody screening of donor sample-
o Why? If you perform Ab screening in the donor's
serum and give negative results. Minor crossmatch is
unnecessary to do because it is in fact presumed to be
negative because of absence of Ab in donor’s serum
Purpose:
1. Final check of ABO compatibility between patient and
donor to prevent transfusion reaction
2. Detects presence of antibody in patient;s serum that will
react to donor’s RBC that is not detected in the antibody
screen
Crossmatching methods
1. Saline Test/ Technique- patients' serum and
donor’s red cells are tested in saline medium. IgM
antibodies are detected.
2. High Protein Crossmatching Technique/
Albumin Technique- patients serum and donor red
cells are tested in high protein media. IgG antibodies
are detected (i.e. Rh antibodies)
3. AHG technique- patients' serum and donor red cells
are tested in AHG medium. Non-agglutinating IgG
antibodies are detected.
4. Broad Spectrum Technique- IgM & IgG are
detected. In BB lab, it is done. composed of 3 stages:
1. Immediate spin at RT- IgM are detected
2. Thermophase/ 37degC incubation phase- for
IgG detection. Done for about 30 minutes with
enhancement medium to shorten incubation period
and sensitivity enhancement (e.g. albumin, LISS,
PEG)
3. AHG Phase after washing incubated cells with
saline.
o *Check cells/ Coombs control cells (IgG
sensitized cells) should be added to tubes that
demonstrate no agglutination. It used for AHG
HULLANA, IDMILAO, NARA, & PASCACIO
 control. Done when AHG is tested and
nagnegative, you need to test for check cell to • Blood type O- universal donor of rbs, universal
validate result. Check cell is (+) for recipient of plasma
agglutination, if AHG is negative • Blood type AB- universal recipient, universal donor of
o For results to be considered valid, plasma
agglutination must occur. Invaild result are ◼ major and minor crossmatch
AHG and check cell are both negative causes ➔ If major is compatible, (-) and minor is
includes: compatible, (-) = RELEASE DONOR
▪ Failure to wash cell- leading to unbound UNIT
Ab actively neutralizing reagent AHG ➔ If major crossmatch is (-) and minor
▪ Reagent is deteriorating or non-reacting- crossmatch is (+) = RELEASE DONOR
expired. UNIT AS PACKED RBC
➔ If major crossmatch is incompatible (+)
and minor crossmatch (-) compatible=
DO NOT RELEASE THE DONOR
UNIT DUE TO TRANSFUSION
REACTION RISK
➔ If major and minor are both (+)
incompatible= NEVER RELEASE
DONOR UNIT

Reporting of Result:
• A compatible crossmatch is indicated by absence of
agglutination and/or hemolysis at any stage of the
crossmatch. The absence of agglutination indicates that
the patient has no demonstrable antibodies with a
specificity for any antigen on donor’s RBC

Troubleshooting Incompatible Crossmatches

Ab AC Major Possible pattern

screen (autocontrol) X
match

- - + -ABO/ Rh typing error

-Donor unit w/ (+) DAT
-Patient w/low
incidence Ab

+ - + Patient alloantibody

+ + + -Patient autoantibody
-Rouleaux (stock-up
rbc)

HULLANA, IDMILAO, NARA, & PASCACIO

Which of the following would likely be responsible for an B. Biochemical modification of non-O blood
incompatible major crossmatch? C. Galvanic biosensor- energy measured
• MAJOR= PSDR D. Dipstick method of typing
a. Recipient’s red cell possess a low-frequency antigen (ex Eldoncard Blood typing kit)
b. Anti-K (Kell) antibody in donor serum E. Dry Plate mETHOD
c. Recipients red cells are polyagglutinable
d. Donor red cells have a positive direct antiglobulin
test

The Future of Compatibility Testing

A. Red cell/Blood substitutes***
• Blood Substitutes- substances that are able to carry
oxygen in the absence of intact red cells
1. Stroma-free Hb solns/ Hemoglobin-Based Oxygen
Carriers (HBOCs) (e.g. PHP, PEG-Hb, Hemolink,
Polyheme, Hem Assist, Hemopure, Optro)
Stroma- cytoskeleton of cell-membrane -> no
intact cell
• Advantage of SFHS
o long shelf life
o very stable
o not immunogenic
o no reqt for blood typing procedures
• Disadvantages of SFHS
o short intravascular halflife
o possible toxicity
o high O2 affinity
o high oncotic effect

2. Perfluorochemicals (PFCs)
(examples: Fluosol-DA-20), Oxygent)
• excellent gas (O2 AND CO2) solvents. It can
dissolve gas better than the donor whole blood
unit. Greater capacity to carry O2 and CO2
• Advantages of PFCs
o -biologic inertness
o -lack of immunogenicity
o -easily synthesized
• Disadvantages of PFCs
o -Adverse clinical effects
o -high o2 affinity
o -retention in tissues
HULLANA, IDMILAO, NARA, & PASCACIO