MEDICAL TECHNOLOGY ASSESSMENT PROGRAM

IMMUNOHEMATOLOGY – TRANSFUSION MEDICINE LECTURE NOTES

BLOOD TYPING/GROUPING ❑ New born – antibody is present 3-

6 months after birth; red cells are
already present in 37th day of fetal
Immunohematology – branch of science that deals with
life
the study of red cell antigens and antibodies that are
❑ Geriatric patient – they have weak
important in transfusion medicine
immune system, with that follows a
Antigens and antibody should be correctly identified so weak antibody formation. The titer
that the doctor will be able to request for a correct blood of the antibody in the serum is low,
type/component for patient use it will not be detected in indirect
typing
Blood typing/blood grouping – routine blood banking ❑ Patients with
procedure that is performed to detect unknown antigen or immunodeficiency/immunosupp
unknown antibody using known reagent ression – caused by infection;
patients taking
2 PROCEDURES: immunosuppressive drugs
➢ Abnormally active
1. DIRECT – also known as forward or cell typing; immune system – patient
considered as a routine screening procedure in has a hyperactive
blood typing immune system that is no
a. PRINCIPLE – is performed to detect longer beneficial for the
unknown antigen using commercially px.
prepared typing sera
b. SAMPLE – patient’s RBC/red cell METHODS
suspension
c. REAGENTS – commercially prepared 1. SLIDE – method of choice for rapid/bed side
typing sera typing; performed in direct/forward typing
❑ ABO typing – anti-A typing procedure
serum/anti-B typing serum & anti- • Reaction and interpretation should be
AB typing serum carried out within a minute
➢ Anti-A – used to detect A • If failed to interpret, there will be false
antigen; color blue positive interpretation → repeat
➢ Anti-B – used to detect B 2. TUBE – method of choice for routine typing; the
antigen; color yellow procedures performed are direct and indirect
➢ Anti-A, B – used as a • Reaction and interpretation should be
control in forward or direct carried out within 3 minutes
or cell typing; colorless • Enhanced reaction with centrifuge
2. INDIRECT – also known as 3. GEL – automated method in blood typing
reverse/backward/serum typing; considered as • 3 types:
the confirmatory procedure in blood typing i. Plain/neutral gel test – the gel
a. PRINCIPLE – to detect unknown used has no reagent; used in
antibodies in the serum using known reverse typing
red cell suspension ❑ In microtube, add known
b. SAMPLE – serum; plasma can be RCS + px serum
used but serum is preferred ii. Specific gel test – the reagent is
❑ Serum lacks factor I (Fibrinogen) already incorporated or added into
therefore it will prevent the gel
spontaneous fibrin clot in the ❑ Applicable to
sample forward/direct typing
❑ Plasma may cause false clumping procedures
of red cell suspension = false iii. Low ionic gel test – used in
positive Coomb’s test
c. REAGENTS – known red cell
suspension; laboratory prepared POSITIVE REACTIONS IN BLOOD TYPING
(prepared by med techs) – 5% red cell
suspension A. Heme agglutination reaction – commonly
❑ 5% RCS – tomato red color observed positive reaction RBC clumping;
d. Not qualified for indirect testing: RBCs form clumps – indicates lattice formation

PREPARED BY: MARK RODRIGO D. MENDROS 1

MEDICAL TECHNOLOGY ASSESSMENT PROGRAM
IMMUNOHEMATOLOGY – TRANSFUSION MEDICINE LECTURE NOTES
• Cross bridging/cross linking of antibodies • H antigen is present on the RBC of individuals
adjacent to antigen regardless of the blood type →
• Antigen on the RBC is where the Ab are prerequisite/precursor structure so
immunodominant sugars for A and B
sensitized → Ab can now link with the nearby
antigens to attach
antibodies
• BOMBAY PHENOTYPE – No H antigen
B. Hemolysis – red cell destruction; release of individual; absent
hemoglobin which would change the color of the • MUST NOT GIVE POSITIVE REACTION TO
serum/antisera into clear red/pink ANY BLOOD TYPE
• Due to the ff:
i. Complement protein
activation → cytolysis
ii. Strong agglutination reaction ABH SOLUBLE SUBSTANCE DETERMINATION
→ hemolytic reaction
ABH SOLUBLE SUBSTANCES
FORWARD AND REVERSE GROUPINGS
✓ GLYCOPROTEINS
BLO FORWARD OR REVERSE OR o soluble and therefore present in body
OD CELL TYPING SERUM TYPING fluids
TYPE ANT ANT ANT A B O o Macromolecules and unable to
I-A I-B I-A, CEL CEL CEL cross the blood brain barrier
B LS LS LS ✓ GENE REGULATORS
O - - - + + - o ABH – seen in body fluids except CSF
A + - + - + - o Se (SeSe/Sese) – secretor gene, can
B - + + + - - be homozygously and heterozygously
inherited
AB + + + - - -
▪ Secretor individuals –
Rule of Specificity
individual who has Se
✓ If the antigen is specific to the antibody, there is ✓ NON-SECRETORS – individuals who has
always a positive reaction (cell clumping/lysis) homozygous recessive secretor gene
✓ If the antigen is not specific to the antibody o sese
there is no reaction (negative) o No ABH soluble substance

Anti-A BLOOD GENE A B H

• Nonspecific to the H antigen present on the TYPE/SECRET INVOLVE SUB SUB SUB
blood type O red cells OR STATUS D S S S
• Specific to the antigen found on type A blood A SECRETOR A, H, Se  X 
• Nonspecific to the B antigen present on the B SECRETOR B, H, Se X  
RBC AB SECRETOR A, B, H,   
Anti-B
Se
• Nonspecific to the H antigen present on the
O SECRETOR H, Se X X 
RBC
NON- se X X X
• Nonspecific to the A antigen
SECRETOR
Anti-A. B *Double check – greater or higher concentration
• Control; to check reaction of Anti A & Anti B
• Used to detect A & B Ag; used in detecting TEST FOR ABH SOLUBLE SUBSTANCE
the subgroups of A & B Ags
• Nonspecific to H antigen SALIVA NEUTRALIZING TEST
A CELLS
• Detect anti A
✓ PRINCIPLE – heme agglutination inhibition
• Specific to anti A
reaction; RBC clumping is prevented through
• Nonspecific to anti B present in A serum
B CELLS neutralizing the sample
• Detect anti A o 1st step: soluble Ag in px sample +
• Nonspecific to the Anti A present in B serum known Ab rgt.
• B antigen is unable to react to any anti B o 2nd step: particulate Ag is added; once
serum neutralized, Ab will no longer react with
O cells the particulate Ag present on the
• Control; detects anti-H surface
✓ REAGENTS

PREPARED BY: MARK RODRIGO D. MENDROS 2

MEDICAL TECHNOLOGY ASSESSMENT PROGRAM
IMMUNOHEMATOLOGY – TRANSFUSION MEDICINE LECTURE NOTES
oTyping sera – Anti-A & Anti-B ✓ hard plastic w/ microtubes
oLectin – Anti-H; source: Ulex ✓ PLAIN GEL: the sample and reagent are added
europaeus – antibody-like substance ✓ SPECIFIC GEL: only add the sample
present in plant source that can react ✓ COLUMN: contains the gel (dextran
to specific Ag; highest level of lectin is polyacrylamide gel; Sephadex)
often extracted in seeds. ✓ GEL: acts as a sieve (used to capture/trap
o Known A, known B, known O red cell agglutinates when cells clump); allows
suspension unagglutinated cells to pass through
✓ RESULTS
o (+) absence of agglutination reaction Advantages Disadvantage
o (-) presence of agglutination • Standardizatio • Interferences
n of the must be
STEP 1 STEP 2 REACTION SOLUBLE procedure prevented
SUBSTANCE • Stable and because it can
PRESENT well-defined affect the quality
end point of the result
Saliva A RCS No A substance
reaction; o Hemolysi
+ Anti- agglutination
stable for up to s
A
3 days o Lipemia
Saliva B RCS No B substance
• Decreased o Ictericia
+ Anti agglutination sample
B volume
Saliva O RCS No H substance needed for
+ Anti agglutination testing →
H micro
sampling is
performed (<1
Patient: AB SECRETOR (A, B, H Substance) mL)
• There is
SALIVA PLUS A CELL B CELL O CELL enhanced
ANTI A 0 0 0 sensitivity &
ANTI B 0 0 0 specificity of
ANTI H 0 0 0 the result

Patient: O SECRETOR (H Substance) PROCEDURE

SALIVA PLUS A CELL B CELL O CELL 1. Addition of cells

ANTI A 4+ 0 0 2. Addition of plasma/serum
ANTI B 0 4+ 0 3. Incubation (Ag. – Ab. Rx)
ANTI H 0 0 0 4. Centrifugation (5 mins)
5. Result (>10mins)

GRADING OF REACTION
GEL TEST TECHNOLOGY
4+ One solid clump
• RBC are present on the
✓ Used in automated procedures topmost later
✓ Developed by a French Dr. Yves Lapierre in 3+ Presence of numerous large size
1985 who aims to standardize serologic clumps
reactions • RBC are present on the upper
✓ Cell washing is no longer performed portion of the column
2+ Presence of medium size clumps
PRINCIPLE • RBC are present at the upper &
lower part of the column
where most cells present at the
✓ Performed to trap agglutinates formed when
center
antigens react with antibodies; used to detect 1+ Presence of numerous small size
agglutination reaction clumps
• Found on the lower portion of
the gel
+/- Very small size clumps
COMPONENTS

PREPARED BY: MARK RODRIGO D. MENDROS 3

MEDICAL TECHNOLOGY ASSESSMENT PROGRAM
IMMUNOHEMATOLOGY – TRANSFUSION MEDICINE LECTURE NOTES
• RBC are found near the of Rabbit (classical/conventional
bottom part of the tube method)
Negative Absence of agglutination reaction 2. MONOSPECIFIC AHG REAGENTS – contains
• RBC button is formed at the only one antibody specificity.
bottom of the tube ✓ Either
i. Anti-IgG
ii. Anti-C3b or C3d
ANTIHUMAN GLOBULIN TEST (COOMBS TEST) ✓ METHOD OF PREPARATION –
prepared using Color and Kohler &
✓ PRINCIPLE: A technique for detecting cell- Milstein
bound immunoglobulin. It is used to detect o Responsible for the
incomplete antibodies (IgG). development of hybridoma
o IgG antibodies – sensitizing method
antibodies; incomplete antibodies; able o Utilizes laboratory mice
to sensitize the RBCs left because they o Hybridoma cell – a.k.a.
are monomers (small in size); immortalized ab forming cell;
▪ Agglutination reaction is not the product of the fusion of the
possible because they are mouse, plasma cell and the
short malignant myeloma cell
▪ The addition of anti-human ❑ The immortality of
globulin will promote the the hybridoma cell is
bridging or the cross-linking of due to the
the sensitized IgG on RBC → characteristic of the
lattice formation → myeloma cell
agglutination ❑ The ab producing
capability is due to
ANTIBODIES OF INTEREST the characteristic of
the plasma cell
IgM IgG
• Natural • Immune Ab STAGES OF ANTIGEN-ANTIBODY INTERACTION
• Complete – • Incomplete –
pentamer; smallest antibody; ✓ The first stage is sensitization. Sensitization
biggest monomer occurs when antibodies react with antigens on
• Agglutinating • Coating/sensitizing the cells and coat the cells.
Ab – best
o The fab region of the antibody interacts
agglutinating
ab with the epitope of the antigen present
• Cold-reacting • Warm-reacting on RBC
– reacts best o IgM & IgG are both capable of this
at low temp. stage
• Saline- • Albumin/AHG- ✓ The second stage of the reaction is
reactive (e.g., reactive (e.g., Rh agglutination. It occurs when antibodies on
ABO antibody) coated cells form cross-linkages between cells
antibody)
resulting in visible clumping.
• Complement • Complement
binding – binding (IgG3 → o There will be lattice formation – visible
most potent IgG1 → IgG2) agglutination/clumping
of all types of • IgG4 – cannot bind o Only IgM can proceed to this stage
Ab complement
Side notes:
➢ IgG can’t proceed with agglutination because
AHG REAGENTS (Commercially Prepared) of its size → addition of anti-human globulin
will cross link IgG to form lattice formation →
agglutination
1. POLYSPECIFIC AHG REAGENTS consists of
a pool of rabbit anti-human IgG and mouse
monoclonal anti-C3b and anti-C3d.
TYPES OF AHG PROCEDURES
✓ Also referred to as Broad Spectrum
Coombs Reagent
1. DIRECT AHG TEST (DAT)
✓ METHOD OF PREPARATION –
✓ Detects in vivo sensitization of red
prepared using Hyper Immunization
cells with IgG and/or complement.

PREPARED BY: MARK RODRIGO D. MENDROS 4

MEDICAL TECHNOLOGY ASSESSMENT PROGRAM
IMMUNOHEMATOLOGY – TRANSFUSION MEDICINE LECTURE NOTES
o In-vivo – reaction happens o EDTA & citrate are anticoagulants
intravascularly (in the blood that has anti-complementary activity
vessel, circulation) specifically to C1 protein
✓ Useful in the ff. situations: o C1 protein – trimolecular complex
composed of C1q, C1r and C1s –
i. Investigation of transfusion
these 3 subunits are held together in
reactions (e.g., HTR) the presence of calcium
❑ If during transfusion → stop o If there is calcium, C1 is active
agad o If the C1 becomes inactive the
❑ If pag tapos/delayed possible attachment of
reaction → observe px → complements in the RBCwill be
minimized → false positive DAT
collect blood spx to observe
➢ The direct antihuman globulin test (DAT) is
to know the cause/signs & needed to demonstrate antibodies in the
symptoms → test for in vivo event of in vivo erythrocyte sensitization.
sensitization ➢ Thus, antibodies or complement components
❑ Normally, ‘di nag- already fixed to the patient's erythrocytes are
sesensitize and red cell ng detected.
cells of antibody, pag ➢ Following a triple washing process with the
sensitized cells, the AHG serum is added.
nasalinan ng dugo there is
➢ Washing of the RBCs is performed to
a possibility that the eliminate unbound antibodies present in the
circulating Ab in the px sample. The unbound IgG antibodies when
blood can sensitize the not removed in the sample can cause
donor red cells when these neutralization of the reagent → after washing
cells are transfused to the add the reagent immediately → check for
px. agglutination reaction
❑ Fever, chills – common 2. INDIRECT AHG TEST (DAT)
immediate manifestations ✓ A two-step procedure: sensitization
ii. Diagnosis of HDN/HDFN (incubation step) and agglutination
❑ Baby’s red cell will be (AHG step) that determines in vitro
tested sensitization of red cells
❑ The Ab of the mother o In vitro – in test tube pinag
attacks the red cell Ag of halo yung red cell suspension
the baby because IgG can ng px tapos yung serum ng
cross the placenta isa pang px →aalamin kung
❑ Red cell or cord blood can yung antibody sa serum nung
be tested px can sensitize the red cell
❑ DAT – considered as the antigen nung other px
single most important o Incubation is critical because
procedure in the diagnosis IgG antibodies can sensitize
of HDFN red cells at warm temp.
iii. Diagnosis of autoimmune and ✓ Useful in the ff. situations:
drug-induced hemolytic i. Detection of incomplete antibodies
anemias in compatibility testing or to
❑ In autoimmune disorder the screening cells in antibody screen
immune system of the px ii. Identification of antigen specificity,
can’t discriminate cells from using a panel of red cells
non-cells → the immune iii. Determination of red cell
system will produce auto- phenotype using known antisera
ab against the auto-ag → in (e.g., Du testing)
circulation, auto-ab will iv. Titration of incomplete antibodies
sensitize the antigens of ✓ Alloantibody – ab nung ibang tao nag-
the red cells →cognitive in sensitize sa red cell ng px
vivo sensitization ✓ Autoantibody – serum ng px and red
cell ng px (same person) may
Side notes: sensitization
➢ Sample used for DAT is whole blood
➢ Cells used for DAT should be collected into FACTORS AFFECTING THE AHG TEST
either EDTA or citrate containing
anticoagulant to minimize the possibility of in 1. Ratio of serum to cells
vitro attachment of complement components.

PREPARED BY: MARK RODRIGO D. MENDROS 5

MEDICAL TECHNOLOGY ASSESSMENT PROGRAM
IMMUNOHEMATOLOGY – TRANSFUSION MEDICINE LECTURE NOTES
✓ Minimum ratio: 40:1 = 2 drops serum • Dirty • Postzone and
and 1 drop of 5%v/v cell suspension glasswares Prozone (cell
o Importance of ratio – to • Saline suspension either
prevent prozone and ozone contaminated too weak or too
with silica or heavy)
2. Temperature- Optimal: 37ºC
heavy metals • Undercentrifugatio
✓ If the temp is too low or too high, di *In false-positive n
mag-sensitize ang IgG ab to red cell results, the test • Poor reading
antigen specificity is technique
3. Incubation Time decreased/low* *In false-neg. results, the
✓ In saline suspension: 30-120 minutes test sensitivity is decreased
✓ LISS suspension: 10-15 minutes or low*
o Potentiator – increases the
affinity rxn between reactants
4. Reaction medium
✓ 60-minute saline test = 30-minute AUTOMATED AHG TECHNOQUE:
albumin techniqu
A. LOW IONIC POLYBRENE TECHNIQUE (LIP)
✓ 22% Albumin – 2 drops 22% albumin +
✓ Polybrene – a rouleaux promoting
2 drops serum + 1 drop 3-5% cell
reagent; a low ionic environment would
suspension
allow sensitized red cell to interact with
✓ Is said to reduce the zeta potential
one another
between RBCs thus increasing the rate
B. ENZYME-LINKED ANTIGLOBULIN TEST
of antibody uptake on the cell
(ELAT)
✓ LISS – 2 drops 3% RBC suspension in
✓ Enzyme test that utilizes red cell
LISS + 2 drops serum
sensitized with IgG – can be in vivo or
o also increases sensitivity and
in vitro
shortens incubation times
✓ AHG – has conjugate enzyme → if may
5. Washing of cells – minimum of three times
red cell na sensitization, the red cell will
✓ Only used for conventional tube
react → mag lalagay ng substrate, if
method of AHG
may positive rxn there will be a color
6. Saline for washing –should be fresh and
production → measured
buffered to a pH of 7.2-7.4
spectrophotometrically
7. Addition of AHG reagents should be added to
✓ Used to determine the quantity of the
washed cells immediately after washing.
antibody that is bound to red cell
✓ To prevent spontaneous elution
C. SOLID PHASE METHOD (DIRECT &
process → false negative
INDIRECT)
✓ AHG reagent – green
✓ In vivo and in vitro
8. Centrifugation- 1000 rcf for 15-20 seconds
✓ Small microwells are used for the
testing
SOURCES OF ERROR IN THE AHG TECHNIQUE
✓ Can be applied to detect auto and
FALSE-POSITIVE FALSE-NEGATIVE alloantibodies
RESULTS RESULTS
• Auto • Inadequate or ANTIBODY SCREENING PROCEDURE
agglutinable improper washing
cells of cells (most ✓ Antibody Screening – is the detection of all
• Bacterial common cause)
clinically significant antibodies outside the ABO
contaminatio • AHG reagent
n or other nonreactive owing system.
contaminatio to deterioration or o Clinically significant – IgG warm
n in cells or neutralization reacting antibodies
saline • AHG reagent not ✓ If the Antibody Screen is reactive, the antibody
• Cells with a added specificity must be determined.
positive dat • Serum not added in ✓ So safe blood can be administered to the
used for iat the indirect test Recipient.
• Over • Serum nonreactive
centrifugation ✓ 11 reagent panel cells are to be used for
owing to
and deterioration of identification.
overreading complement ✓ AHG – to detect sensitization; enzyme testing
• Poly • Inadequate – to eliminate other antibodies and to facilitate
agglutinable incubation identification of antibody interest
cells conditions

PREPARED BY: MARK RODRIGO D. MENDROS 6

MEDICAL TECHNOLOGY ASSESSMENT PROGRAM
IMMUNOHEMATOLOGY – TRANSFUSION MEDICINE LECTURE NOTES
Other techniques may be used to eliminate clinically a. Landsteiner-Miller Heat – the
insignificant reactions and make identification of sensitized RBC sample is heated along
significant antibodies easier. with albumin medium at 56 deg. C
o Heating process would
✓ Use of AET, DTT, and ZZAP which inactivates promote the detachment of
some antigens especially Kell. IgG on red cells
✓ Prewarm procedure. Clinically insignificant b. Lui-Freeze-Thaw – rapid freeze
cold antibodies may be removed by this thawing procedure can detach
technique. Patient serum, reagent red cells and antibodies from the RBCs
enhancement medium can be warmed 2. SECOND GENERATION
separately at 37°C for 5-10 minutes prior to a. Use of Organic solvents – ether
mixing 3. THIRD GENERATION
✓ Use of sulfhydryl or thiol reagents (DTT and a. Uses Non-Hazardous Chemical
2-ME) which denature IgM antibodies by Agents
breaking disulfide bonds. b. “Acid Elution”
✓ Use of adsorption and elution techniques to ✓ Another technique for facilitating antibody identification is
remove unwanted antibodies such as cold or NEUTRALIZATION
warm autoantibodies, or to help resolve multiple o Commercial substances are available to
antibodies neutralize or to inhibit reactivity of some
antibodies.
ANTIBODY IDENTIFICATION ✓ Sources of Substances for Neutralization of
Antibodies:
✓ If the antibody screen is reactive, the antibody o Hydatid cyst fluid – anti-P1
specificity must be determined. So safe blood o Plasma or serum w/ Le subs. – anti-Lea and
can be administered to the recipient anti-Leb
✓ 11 reagent panel cell are to be used for o Pooled serum or plasma – anti-Chido, anti-
identification Rogers
o Urine – anti-Sda
ADSORPTION & ELUTION TECHNIQUES o Saliva of “secretors” – anti-ABH
o Human milk – anti-I
✓ ADSORPTION- used to remove unwanted
antibodies from serum COMPATIBILITY TESTING
o If an autoantibody such as I, H, or IH
are defined, it can be adsorbed onto
COLLECTION AND PREPARATION OF SAMPLES
the patient’s enzyme pretreated cells at
4ºC. Rabbit cells may also be used as Compatibility Testing/Crossmatching – pre-
adsorbents for anti-I since they are rich transfusion procedure that is composed of series of test
in I antigen. to ensure the safety of the recipient during blood
✓ ELUTION - used to dissociate IgG Abs from transfusion; performed to select the appropriate donor
sensitized red cells unit for px transfusion
o the recovered antibody, eluate, can be
tested like serum to determine the 1. Patient Identification – very critical
antibody’s specificity 2. Collection. SERUM is the preferred specimen
o techniques include heat, freeze-thaw for compatibility testing. Hemolysis should be
process, use of organic solvent, acid avoided.
eluates, or by using ZZAP or o Why SERUM and NOT PLASMA?
chloroquine diphosphate ❑ Plasma may cause small fibrin
❑ ZZAP- mixture of DTT and papain that is used clots to form which may be
to remove Ab from sensitized red cells and to difficult to distinguish from true
enzyme treat them at the same time agglutination.
❑ Chloroquine diphosphate- reagent used to ❑ Plasma may inactivate
remove IgG Abs from the surface os sensitized complement so that antibodies
cells; inactivates Bg antigens may not be detected.
3. Age of Specimen. The freshest sample
TYPES OF ELUTION TECHNIQUES possible should be used for compatibility testing.
Specimens must be less than 3 days old if the
1. FIRST GENERATION patient has been transfused or pregnant within
the past 3 months.

PREPARED BY: MARK RODRIGO D. MENDROS 7

MEDICAL TECHNOLOGY ASSESSMENT PROGRAM
IMMUNOHEMATOLOGY – TRANSFUSION MEDICINE LECTURE NOTES
o This age of the spx can correctly ✓ Patient serum and donor red cells are
represent the current immunologic tested in saline medium
status of the px ✓ IgM antibodies are detected
4. Sample Storage. The AABB requires that 2. HIGH-PROTEIN/ALBUMIN TECHNIQUE
patient samples must be stored between 1-6ºC ✓ Patient serum and donor red cells are
for at least 7 days after transfusion tested in high protein media
✓ IgG antibodies are detected/Rh
COMPATIBILITY TESTING PROTOCOLS antibodies
3. AHG TECHNIQUE
1. ABO GROUPING. Most critical pretransfusion ✓ Patient serum and donor red cells are
serologic test. tested in AHG medium
✓ If the patient’s ABO group cannot be ✓ Non-agglutinating IgG antibodies are
satisfactorily determined and detected
immediate transfusion is essential, 4. BROADSPECTRUM TECHNIQUE
group O packed red cells should be ✓ Composed of three stages:
utilized. i. Immediate spin – IgM is
2. Rh TYPING detected
✓ *If Rh type of the recipient cannot be ii. Thermophase/incubation
determined and transfusion is space – IgG
essential, Rh-negative blood should be iii. AHG technique
given.
3. CROSSMATCHING 3 PHASES OF CROSSMATCHING
✓ MAJOR X-MATCH: Donor’s cells +
Recipient’s serum 1. Immediate Spin in saline at RT - Detects IgM
✓ MINOR X-MATCH: Donor’s serum + 2. Thermophase/37ºC incubation for 30 minutes
Recipient’s cells with enhancement medium (e.g., albumin, LISS,
o Purpose: PEG) – Detects IgG
a. Final check of ABO 3. AHG Phase after washing incubated cells with
compatibility saline
between patient and a. Check cells/Coombs control cells
donor to prevent (IgG sensitized cells) should be added
transfusion reaction. to tubes that demonstrate no
b. Detects presence of agglutination.
antibody in patient’s ❑ Used as control for AHG when
serum that will react AHG is tested at a negative
to donor’s RBC that ❑ Used to validate result if AHG is
is not detected n negative
antibody screen. ❑ Invalid result in check cell –
negative AHG, negative check
cell
▪ Possible causes: the med
tech failed to wash the
cells → could lead to
unbound ab actively
neutralizing the reagent
AHG & the reagent is
already non-reactive
(expired rgt.)
b. For results to be considered valid,
agglutination must occur
c. Reporting of Results.
❑ A compatible crossmatch is
indicated by absence of
agglutination and/or hemolysis at
4 CROSSMATCHING TECHNIQUES: any stage of the crossmatch. The
absence of agglutination
1. SALINE TECHNIQUE indicates that the patient has no
demonstrable antibodies with a

PREPARED BY: MARK RODRIGO D. MENDROS 8

MEDICAL TECHNOLOGY ASSESSMENT PROGRAM
IMMUNOHEMATOLOGY – TRANSFUSION MEDICINE LECTURE NOTES
specificity for any antigen on Advantages of SFHS Disadvantages of
donor’s RBC SFHS
Long shelf life Short intravascular half-
life
Very stable Possible toxicity
Not immunogenic High O2 affinity
No requirement for High oncotic effect
blood typing procedures
2. PERFLUOROCHEMICALS (PFCs)
a. E.g., Fluosol-DA-20, Oxygen
b. Excellent gas (O2 and CO2) solvents

Advantages of PFCs Disadvantages of

PFCs
Biologic inertness Adverse clinical effects
Lack of immunogenicity High O2 affinity
Easily synthesized Retention in tissues

Troubleshooting Incompatible Crossmatches

Ab AC Major Possible problem
Screen crossmat
ch
(-) (-) (+) ✓ ABO/Rh
typing
error
✓ Donor unit
w/ (+) DAT
✓ Patient w/
low
incidence
Ab
(+) (-) (+) ✓ Patient
alloantibod
y
(+) (+) (+) ✓ Patient
autoantibo
dy -
Rouleaux
The Future of Compatibility Testing

✓ Red cell/Blood substitutes***

✓ Biochemical modification of non-O blood
✓ Galvanic biosensor – energy measured
✓ Dipstick method of typing
o (ex. Eldoncard Blood typing kit)
✓ Dry plate method

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)
a. E.g., PHP, PEG-Hb, Hemolink,
Polyheme, HemAssist, Hemopure,
Optro)

PREPARED BY: MARK RODRIGO D. MENDROS 9