IMMUNOHEMATOLOGIC TESTS AND PROCEDURES

BASIC PRINCIPLE: HEMLAGGLUTINATION (Antibody binds with antigen present in the surface of RBC)
• It is the single most important in vitro immunologic reaction in blood banking because it is the
endpoint of almost all test systems designed to detect RBC antigens and antibodies.
HEMEAGGLUTINATION REACTIONS occur in two stages:
RBC SENSITIZATION 1st stage
• Combination of single paratope and single epitope in a reversible reaction that follows the law of
mass action and has an associated equilibrium constant.
• Antigen and antibody are held together by noncovalent attractions.
LATTICE FORMATION 2nd stage
• Multiple RBCs with bound antibody form a stable latticework through antigen-antibody
bridges formed between adjacent cells.
• Basis of all visible agglutination
NOTES: FACTORS IMPORTANT IN MAKING RBCS APART
#ZETA POTENTIAL
• Formation of the latticework during the second stage of hemagglutination is naturally prevented by the
fact that RBCs in solution normally repel each other. → This is due to the net negative charge
of the RBC membrane created BY SIALIC ACID.
• If RBCs are suspended in an ionic medium such as normal saline
• → cations arrange themselves around each cell to form an ionic cloud
• → Cations closest to the cell membrane are firmly bound and move with the RBC, while the outer cations
move freely.
• The difference in charge at the surface between the inner and outer cation layers, called the surface of
shear, creates an electrical potential named the zeta potential
• The potential keeps RBCs in solution about 25 nm apart
ROLEUAX FORMATION / "PSEDUOAGGLUTINATION
• A phenomenon where zeta potential is avoided
• Patients with multiple myeloma
• Waldenström'smacroglobulinemia, and hyperviscosity syndromes have high concentrations of
abnormal serum proteins that change the net surface charge on the RBC membrane. "stacked of coin
appearance
• Plasma expanders, such as dextran and hydroxyethyl starch, as well as some intravenous X-
ray contrast materials can also cause rouleaux formation
• Also seen in cord samples contaminated with Wharton's jelly (due hyaluronic acid and albumin)
WATER HYDRATION
• Hydrophilic polar heads of lipid molecules making up the outer cell membrane bilayer attract water
molecules.
• The water thus creates a surface tension that helps keep the cells apart.

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FACTORS AFFECTING THE HEMAGGLUTINATION REACTION

GRADING OF HEMEAGGLUTINATION REACTIONS

• Hemagglutination in blood bank testing is observed and graded according to the strength of the reaction
• Currently, three methods are available for evaluating visual hemagglutination reactions.
• Tube testing
o Positive result: 1 solid agglutinate at the bottom of the tube (not easily disperse
o Negative result: No solid agglutinate at the bottom of the tube (easily disperse)
• Column agglutination (gel technology)
• Positive result: There is an agglutination at the top of the gel
• Negative result: Agglutination at the bottom of the gel
• Solid-phase technology
• Positive result: Diffused / Homogenous appearance
• Negative result: solid button
NOTES:
TUBE REACTIONS
• Hemolysis is considered the strongest
positive reaction that can occur and indicates
the presence of a potent, complement-fixing
antibody.
• When reading for agglutination:
o The tube should be shaken, using a
gentle wrist action, until all cells are
dislodged. This is most easily
accomplished by gently holding the test
tube at the top of the tube in a vertical
fashion and using the wrist to gently
dislodge the cell button, while watching
closely for agglutinates. As the cell
button begins to resuspend, tip the
tube gently to observe for agglutinates.
o Proper illumination with a concave
mirror is an invaluable aid for GRADING AND SCORNG OF HEMAGGLUTINATION
macroscopic reading. By placing the
tube about 2.5 inches above a 3-inch concave mirror aggregates can be differentiated easily from
the free cells by looking at the mirror, not the tube.

ANTIGLOBULIN TESTING

ANTIGLOBULIN/ANTIHUMAN-GLOBULIN TEST
• also called COOMB'S TEST
• Principle: antihuman globulins (AHGs) obtained from immunized nonhuman species bind to
human globulins such as lgG or complement, either free in serum or attached to antigens on red
blood cells (RBCs).
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 • Detect IgG and/or complement-sensitized RBCs.
• There is an RBC attached in the antibody and complement
• IgG antibodies → are termed nonagglutinating because their monomer structure is too small to
agglutinate sensitized RBCs directly. Therefore, the addition of AHG containing anti-IgG to RBCs
sensitized with IgG antibodies allows for hemagglutination of these sensitized cells.
HISTORY
• Before the discovery of the antiglobulin test, only IgM antibodies had been detected. The introduction
of the antiglobulin test permitted the detection of nonaggalutinating IgG antibodies and led to the
discovery and characterization of many new blood group systems.
• 1945: Coombs and associates described the use of the antiglobulin test for the detection of weak and
nonagglutinating Rh antibodies in serum.
• 1946: Coombs and coworkers described the use of AHG to detect in vivo sensitization of the RBCs of
babies suffering from hemolytic disease of the newborn (HDN).
• Although the test was initially of great value in the investigation of Rh HDN, its versatility for the detection
of other lo blood group antibodies soon became evident. Although Coombs and associates were
instrumental in introducing the antiglobulin test to blood group serology, the principle of the test had in
fact been described by Moreschi in 1908.
o Moreschis studies involved the use of rabbit antigoat serum to agglutinate rabbit RBCs that
were sensitized with low nonagglutinating doses of goat anti-rabbit RBC serum.
ANTIHUMAN GLOBULIN REAGENTS
1) POLYSPECIFIC ANTIHUMAN GLOBULIN
a) Contains antibody to human IgG and to the C3d component of human complement.
i) Other anticomplement antibodies, such as anti-c3b, anti-c4b, and anti-c4d, may also be present.
b) Notes:
i) Commercially prepared polyspecific AHG→ contains little, if any, activity against IgA and IgM
heavy chains. However, the polyspecific mixture may contain antibody activity to kappa and
lambda light chains common to all immunoglobulin classes, thus reacting with IgA or IgM
molecules.
CLASSIC/CONVENTIONAL METHOD
a) Involves injecting human serum or purified globulin into rabbits (sheep, goat), and an immune
stimulus triggers production of antibody to human serum.
b) Antigen→ Ab → Harvest→ Absorbed with A1, B and O cell→ to remove heterospecific Ab
i. Note: Without removal: PROZONE (Ab in excess→ false negative)
c) Production of POLYCLONAL ANTIBODIES.
i. Polyclonal antibodies are a mixture of antibodies from different plasma cell clones. The
resulting polyclonal antibodies recognize different antigenic determinants (epitopes), or
the same portion of the antigen but with different affinities.
2) MONOSPECIFIC ANTIHUMAN GLOBULIN
a) Contain only ONE ANTIBODY SPECIFICITY: either anti-IgG or antibody to specific
complement components such as C3b or c3d.
b) Does not react with IgG4
i) ANTI-IgG
• Contain no anticomplement activity.
• Contain antibodies specific for the Fc fragment of the gamma heavy chain of the loG
molecule.
• If not labeled "gamma heavy chain-specific, anti-IgG may contain anti-light chain specificity and
therefore react with cells sensitized with IgM and IgA as well as with IgG.
ii) ANTI-COMPLEMENT
• contain no activity against human immunoglobulins.
• Anti-complement reagents, such as anti-C3b-C3d reagents, are reactive against the
designated complement components only.

HYBRIDOMA TECHNOLOGY (MONOCLONAL ANTIBODY TECHNIQUE)

a) Introduced by Kohler and Milstein

b) Monoclonal antibody production begins with the immunization of laboratory animals, usually MICE, with
purified human globulin.
c) After a suitable immune response, mouse spleen cells containing antibody-secreting lymphocytes are
fused with myeloma cells.
d) The resulting "hybridomas" are screened for antibodies with the required specificity and affinity. e.
e) The antibody-secreting clones may then be propagated in tissue culture (HAT medium) or by
inoculation into mice, in which case the antibody is collected as ascites.
f) All antibody produced by a clone of hybridoma cells are identical in terms of antibody structure and
antigen specificity → MONOCLONAL ANTIBODIES

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 DIFFERENTIATION OF POLYCLONAL VS. MONOCLONAL ANTIBODIES POLYCLONAL
MONOCLONAL
POLYCLONAL MONOCLONAL
Heterogenous Homogenous
Not Antigen specific Antigen specific
Inexpensive Expensive
3 months (viability of reagent) 6 months (viability of reagent)
Rabbit, Sheep, Goat Mice
High cross-reactivity Low/No cross-reactivity
ANTIBODIES REQUIRED IN AHG TEST

1) Anti-IgG
a) The majority of these antibodies are a mixture of lgG1 and lgG2 subclass.
b) Notes:
i) IgM antibodies may be found; however, they have always been shown to fix complement and may
be detected by anticomplement
ii) IgA antibodies with Rh specificity have been reported however, IgG antibody activity has always been
present as well
iii) The ONLY RBC ALLOANTIBODIES THAT HAVE BEEN REPORTED AS BEING SOLELY IGA
HAVE BEEN Binding Complement EXAMPLES OF ANTI-Pr, and those antibodies were
agglutinating. IgA Most autoantibodies have been reported although very rarely.
iv) Anti-IgM and anti-IgA activity may be present, but neither is essential. The presence of anti-light-
chain activity allows detection of all immunoglobulin classes.
2) Anti-Complement
a) Some antibodies "fix" complement components to the RBC membrane after complexing of the antibody
with its corresponding antigen
b) In 1957, Dacie and coworkers published data showing that the reactivity of AHG to cells sensitized
with “warm" antibodies resulted from anti-gamma globulin activity, whereas anti-nongamma globulin
activity was responsible for the activity of cells sensitized by "cold" antibodies. The nongamma globulin
component was shown to be beta globulin and had specificity for complement. Later studies, revealed
that the complement activity was a result of C3 and C4
c) Presence of anticomplement would enhance reactions of some clinically significant antibodies → Anti
FY3. Anti K
d) Some clinically significant antibodies are detected with anti-complement component of AHG
but not with Anti-IgG→ Anti Jk
e) Notes:
i) Anti C3c → most important anticomplement because of limited capacity to cause NON
SPECIFIC REACTIONS
ii) RBCs incubated for > 15 minutes→ # of C3c determinants decreased rapidly because they split off
to C3bi
iii) C3b to C3d degradation can occur in vitro- if incubation is >1 hour

PRINCIPLE OF AHG TEST

AHG test is based on the following principles:

1) Antibody molecules and complement components are globulins
2) Injecting an animal with human globulin stimulates the animal to produce antibody to the foreign protein i...
AHG). Serologic tests employ a variety of AHG reagents reactive with various human globulins including anti-
ag, antibody to the C3d component of human complement, and polyspecific reagents that contain both anti-
IgG and anti-C3d activity.
3) AHG reacts with human globulin molecules, either bound to RBCs or free in serum
4) Washed RBCs coated with human globulin are agglutinated by AHG.
A. DIRECT ANTIGLOBULIN TEST (DAT) •
• Detects IN-VIVO SENSITIZATION OR RBC with IgG and/or complement components
• Clinical conditions that can result in in vivo coating of RBCs with antibody and/or complement are:
o HDN
o HTR
o AUTOIMMUNE HEMOLYTIC ANEMIA
o DRUG INDUCED HEMOLYTIC ANEMIA
TABLE 5-3 Direct Antiglobulin Test Principle: Detects In-Vivo RBC Sensitization
Application In-Vivo Sensitization
HDN Maternal antibody coating fetal RBCS
HTR Recipient antibody coating donor RBCs
AIHA Autoantibody coaling individual's RBCS
• The DAT IS NOT A REQUIRED test in routine pretransfusion protocols.
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 • Initial DATs Include testing one drop ola 3 to 5 percent suspension of washed RBCs with polyspecific
(anti-IgG, anti-C3d) reagent. Positive results are monitored by a DAT panel using monospecific
anti4gG and anti-C3d to determine the specific type of protein sensitizing the cell.
• Some institutions choose to run polyspecific and monospecific reagents at one time as well as a saline
control
o SALINE CONTROL → serves to detect spontaneous agglutination of cells or reactions
occurring without the addition of AHG reagents
• In warm AIHA, including drug-induced hemchicanemia, the RBCs may be coated with IgG or C3d, or
both
• PHENOTYPING can be complicated with a positive DAT
• Can be resolved by elution using: .
o Choloroquinediphosphate
o EDTA-glycine
o Murine monoclonalabs
• Interpreting the significance of a positive DAT requires
knowledge of the
o patient's diagnosis
o drug therapy
o recent transfusion history.
• **A positive DAT may occur without clinical
manifestations of immune-mediated hemolysis
IN VIVO PHENOMENA ASSOCIATED WITH A POSITIVE

DAT
• Answering the following questions BEFORE investigating a positive DAT for patients other than neonates
will help determine what further testing is appropriate.
• Note: ACCORDING TO AABB, A POSITIVE DAT ALONE IS NOT DIAGNOSTIC.
o is there evidence of in-vivo hemolysis?
o Has the patient been transfused recently?
o Does the patient ‘s serum contain unexpected antibodies?
o Is the patient receiving any drugs?
o Has the patient received blood products or components containing ABO-incompatible plasma?
o Is the patient receiving antilymphocyte globulin or antithymocyte globulin?

B. INDIRECT ANTIGLOBULIN TEST (IAT)

• The IAT is performed to detect IN-VITRO SENSITIZATION and is used in the following situations:
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 • PRINCIPLE: DETECTS IN-VITRO SENSITIZATION
▪ Detection of incomplete (nonagglutinating) antibodies to potential donor RBCs→ (compatible
testing)
▪ Detection of incomplete (nonagglutinating) antibodies to screening cells in serum → (antibody
screen)
▪ Determination of RBC phenotype using known antisera (e.g., Kell typing, weak D testing)→ (RBC
phenotyping)
▪ Titration of incomplete antibodies → (titration studies)
• REQUIRED INPRETRANSFUSION TESTING (CROSSMARCHING. AB SCREEN AND AB
IDENTIFICATION)
• MINOR- DONOR SERUM MIXED WITH RECIPIENT CELL
• MAJOR- DONOR RBC IS MIXED WITH RECIPIENTS SERUM

CROSSMATING- The recipient antibody is reacting with the donor cell.

Direct antiglobulin test (DAT)

1) Prepare a 3%-5% suspensionin normal saline of the red cells to be tested.

2) Add 1 drop of red cell suspension to a tube and wash three to four times with saline.
3) Blot the tube after the last wash.
4) Add 2 drops of polyspecific AHG (anti-IgG+ anticomplement).
5) Centrifuge and examine for agglutination.
6) Add check cells to all negative tubes (see Quality Control of the Antihuman Globulin Test section).

Note: For DAT, blood samples should be collected in EDTA to prevent fixation of complement in vitro by clinically
insignificant cold autoagglutinins.

Indirect antiglobulin test (IAT)

1) Add 2-3 drops of patient serum or the recommended number of drops of commercially prepared antisera to
each tube.
2) Place 1 drop of the 3%-5% red cell suspension to be tested in a tube (e.g., donor cells, reagent screening
cells).
3) Add recommended number of drops of enhancement media (e.g., LISS, PEG) If indicated.
4) Incubate at 37° C for the time period indicated for the assay being performed (from 15-60 minutes).
5) If indicated in the procedure, centrifuge and examine the serum for hemolysis and the cells for agglutination.
6) Whether step 5 is performed or not, continue by washing the tube(s) three to four times with normal saline.
7) Blot tubes dry after the last wash.
8) Add 2 drops of antiglobulin sera (polyspecific or monospecific anti-IgG) to all tubes.
9) Centrifuge and examine for agglutination.
10) Add check cells to all negative tubes,

ANTIHUMAN GLOBULIN CONTROL

• Anytime you are using AHG reagent and get a negative test result (by the tube method). YOU MUST
ADD COOMB'S CONTROL CELLS AND SHOULD GET A POSITIVE RESULT (VALID)
o Note: NO ADDITION OF COOMB'S CONTROL CELLS ON THE GEL AND SPRA METHOD
OF AHG TESTING
• A negative result after addition of Coomb's control cell INVALIDATES the antiglobulin test. The test
would need to be completely repeated.
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 • COOMB'S CONTROL CELLS/ CHECK CELLS
o Composition: 4% or 0.8% suspension of single donor group O red cells
o Coomb's control cells will react with AHG reagent
o It will prove that:
▪ AHG was added
▪ AHG was working properly
▪ The WASHING STEP was adequate enough to remove any unbound globulins
o Sources of error Resulting in NEGATIVE CHECK CELL RESULTS:
▪ Inadequate washing of cells. MOST COMMON!!!
▪ Omission of AHG sera from test
▪ Omission of check cells from test
▪ Contaminated AHG
▪ Contaminated saline

FACTORS AFFECTING THE ANTIGLOBULIN TEST

• DAT SENSITIVITY
o Can detect 100-500 IgG/RBC and 400-1,000 C3d/RBC
• IAT SENSITIVITY
o Can detect 100-200 IgG or C3d RBC
A. RATIO OF SERUM TO CELLS.
• Increased ratio of Ab:Ag = Increase sensitivity of test
• 2 drops of serum and 1 drop ofa 5% RCS
o Ratio: 40:1
• 4 drops of serum and 1 drop of a 3 % RCS
o Ratio: 133:1
o Used when cells suspended in saline to detect weak antibodies
B. REACTION MEDIUM (potentiators) → enhanced detection of antibodies
• ALBUMIN (22%, BOVINE ALBUMIN)
o A high MW protein
o The macromolecules of albumin allow antibody-coated cells to come into closer contact with each
other so that aggregation occurs.
o INCREASES Dielectric constant
o DECREASES Zeta potential - by dispersing some of the cations surrounding each
negatively charged red cell
• Note: ZETA POTENTIAL
o RBCs are surrounded by negative ions when they are suspended in ISOTONIC SALINE
o Cloud of negative charge surrounding the red cells causes them to REPEL each other
o Zeta potential is defined as the DIFFERENCE IN ELECTRICAL POTENTIAL BETWEEN THE
SURFACE OF THE RBC AND OUTER LAYER OF THE IONIC CLOUD
o Reduction of zeta potential allows the RBCs to approach each other and aids in lattice formation
• LOW IONIC STRENGTH SOLUTION (LISS)
o These enhance antibody uptake and allow incubation time to be decreased.
o Optimum reaction conditions: 2 drops of serum and 2 drops of a 3 percent RCS In LISS.
o Increasing the serum-to-cell ratio Increased the lonic strength of the reaction mixture, leading to
a decrease in sensitivity, thus counteracting the shortened incubation time of the test.
• POLYETHYLENE GLYCOL (PEG)
o Is a water-soluble linear polymer and is used as an additive to increase antibody uptake.
o Action: Remove water molecule – to increase reaction
o Anti-IgG is the AHG reagent of choice when using PEG to avoid false positive reactions
o Because PEG may cause aggregation of RBCs, reading for agglutination following 37’C
incubation in the IAT is OMITTED
o MORE SENSITIVE than LISS, ALBUMIN, SALINE SYSTEMS
o NOT RECOMMENDED IN PATIENTS WITH
▪ Multiple myeloma
▪ Waldenstrom’s macroglobinemia
▪ Hyperviscosity syndrome (have high serum protein)
• TEMPERATURE
o IgG and Complement activation→ 37°C and IAT Phase
o IgM→ Immediate spin phase or Room temperature Phase
• INCUBATION TIME
• For cells suspended in:
o SALINE(NSS) - 30-120 mins

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 ▪ The mainly of clinically significant antibodies can be detected after 30 minutes of
incubation, and extended incubation times are usually not necessary
o LISS = 5-15 mins
▪ With these shortened times, is essential that tubes be incubated at a temperature 37°C.
Extended incubation (e. a to 40 minutes in the LISS technique has been shown to cause
antibody to elute from the RBCs causing a decrease in the sensitivity of the
test.
o ALBUMIN= 15 – 60 mins
o PEG=16-30 mins
• WASHING OF RBCS
o When both the DAT and IAT are performed. RBCS must be saline-washed a minimum of
three times BEFORE the addition of AHG reagent.
o PURPOSE: Remove excess/ free globulin molecules
o INADEQUATE WASHING may result in a false-negative reaction because of
neutralization of AHG reagent by residual unbound serum globulins,
• pH of SALINE FOR WASHING
o Ideally, the saline used for washing should be FRESH or alternatively, buttered to a pH of 7.2
to 7.4
o Saline stored for long periods in plastic containers has been shown to decrease in pH, which may
Increase the rate of antibody elution during the washing process
o Significant levels of bacterial contamination in saline have been reported45: this situation can
contribute to false-positive results.
• Addition of AHG
o AHG should be added to the cells immediately after washing to minimize the chance of antibody
eluting from the cell and subsequently neutralizing the AHG reagent.
• CENTRIFUGATION FOR READING
• The CBER-recommended method for the evaluation of AHG uses 1000 relative centrifugal forces
(RCFs) for 20 seconds, although the technique described in this chapter suggests 500 RCFs for 15
to 20 seconds.
• The higher the RCF = the more sensitive the result

SOURCES OF ERROR IN ANTIHUMAN GLOBULIN TESTING

FALSE-POSITIVE RESULTS FALSE-NEGATIVE RESULTS
• Improper specimen (refrigerated, clotted) may • Inadequate or improper washing of cells
cause in vitro complement attachment • Failure to wash additional times when
• Over centrifugation and overreading increased serum volumes are used
• Centrifugation after the incubation phase • Contamination of AHG by extraneous protein
when PEG or other positively charged (i.e., glove, wrong dropper)
polymers are used as an enhancement • High concentration of IgG paraproteins in test
medium serum
• Bacterial contamination of cells or saline used • Early dissociation of bound IgG from RBCs due
in washing to interruption in testing
• Dirty glassware • Early dissociation of bound IgG from RBCs due
• Presence of fibrin in the test tube may mimic to improper testing temperature (i.e., saline or
agglutination. AHG too cold or hot)
• Cells with a positive DAT will yield a positive • AHG reagent nonreactive because of
IAT. deterioration or neutralization (improper
• Polyagglutinable cells reagent storage)
• Saline contaminated by heavy metals or • Excessive heat or repeated freezing and
colloidal silica thawing of test serum
• Using a serum sample for a DAT (use EDTA, • Serum nonreactive because of deterioration of
ACD, or CPD anticoagulated blood) complement
• Samples collected in gel separator tubes may • AHG reagent, test serum, or enhancement
have unauthentic complement attachment medium not added
• Complement attachment when specimens are • Under centrifuged or over centrifuged
collected from infusion lines infusing dextrose • Cell suspension either too weak or too heavy
solutions • Serum-to-cell ratios are not ideal.
• Preservative-dependent antibody directed • Rare antibodies are present that are only
against reagents detectable with polyspecific AHG and when
active complement is present.
• Low pH of saline
• Inadequate incubation conditions in the IAT
• Poor reading technique

MODIFIED AND AUTOMATED ANTIGLOBULIN TEST TECHNIQUES

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1. LOW IONIC POLYBRENE TECHNIQUE

• The technique relies on low ionic conditions to rapidly sensitize cells with antibody
• POLYBRENE→ a potent rouleaux forming reagent to allow the sensitized cells to approach each other
to permit cross-linking by the attached antibody.
o Has low sensitivity for the detection of Kidd ant bodies
• HIGH IONIC STRENGTH SOLUTION → added to reverse the rouleaux however, if agglutination
present, it will remain
• If this is performed, a monospecific anti lgG reagent must be used because the low ionic conditions
cause considerable amounts of C4 and C3 to coat the cells and would give false positive reactions
polyspecific reagent were used.

2. ENZYME-LINKED ANTIGLOBULIN TEST

• In the enzyme-linked antiglobulin test (ELAT). an RBC suspension is added to a microtiter well and
washed with saline
• AHG, which has been labelled with an enzyme is added.
• The enzyme-labeled AHG will bind to IgG-sensitized RBCs.
• Excess antibody is removed, and enzyme substrate is added
• The amount of color produced is directly proportional to the amount of antibody present. It is
measured spectrophotometrically.
• The optical density is usually measured at 405 nm,
• The number of IgG molecules per RBC can also be determined from this procedure.

3. SOLID PHASE TECHONOLOGY

• Solid-phase technology may be used for the performance of antiglobulin tests.

• Several different techniques have been reported using either test tubes or microplates
• With the availability of microplate readers, this modification lends itself to the introduction of
semiautomation

4. GEL TECHNOLOGY

• Invented by Dr. Yves Lapierre of France

• Used to minimize problems associated with conventional techniques (TUBE METHOD)
• Introduced in circa in 1988 for routine use
• NOW USED WORLDWIDE
• MAIN ADVANTAGE: ______
• It is a process to detect RBC antigen-antibody reactions by means of using a chamber filled
with polyacrylamide gel.
i. GEL acts as a trap
ii. FREE UNAGGLUTINATED RBCS form pellets in the bottom of the tube
→ NEGATIVE REACTION
iii. AGGLUTINATED RBCS are trapped in the tube for hours. → POSITIVE
REACTION
• There are three different types of gel tests:
o NEUTRAL GEL TEST
▪ Does not contain any specific reagent and acts only by its property of trapping
agglutinates.
▪ main applications:
• a. Antibody screening and identification
• b. reverse ABO typing.
o SPECIFIC GEL TEST
▪ use a specific reagent incorporated into the gel and are useful for antigen
determination.
o LOW IONIC ANTIGLOBULIN TEST (GLIAT) IAHG GEL TEST
▪ Is a valuable application of the gel test and may be used for the IAT or the DAT.

PROBLEMS WITH CONVENTIONAL TEST ADVANTAGE OF GEL TECHNIQUE

• Labour intensive • STANDARDIZATION
• Skilled reading required • Simple and rapid
• Instability of completed tests • Stable reactions
• Poor wash phase: falsely weak negative • NO WASHING
• Inappropriate handling of completed tests, • Small sample volumes
results downgraded /negative • Enhanced Lab safety
• Addition of sensitized control cells to check • Long shelf life
negative reaction • Decrease waste
• Time consuming • Automated
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 AGGLUTINATION REACTION IN GEL TECHNOLOGY

4+ SOLID BAND OF AGGLUTINATED RED CELLS AT THE TOP OF THE GEL COLUMN
Usually NO RED CELLS ARE VISIBLE IN THE BOTTOM of the microtube
3+ Predominant amount of agglutinated red cells towards the top of the gel column with
a few agglutinates staggered below the thicker band.
The majority of the agglutinates are observed in the top half of the gel column
2+ Red cell agglutinate dispersed throughout the gel column with few agglutinates at the
bottom of the microtubes.
Agglutinates should be distributed through the upper and lower halves of gel.
1+ Red cell agglutinate predominantly observed in the lower half of the gel column with red
cells also in the bottom.
These reactions may be weak, with few agglutinates remaining in the gel area just above
the red cell pellet in the bottom of the microtube.
NEGATIVE Red cells forming a well delineated pellet at the bottom of the microtube. The gel
above the red cell pellet is clear and free of agglutinates.
MIXED-FIELD Layer of red cell agglutination at the top of gel column accompanied by a pellet of
unagglutinated cells in the bottom of the microtube.
SUMMARY OF ROUTINE PROCEDURES IN IMMUNOHEMATOLOGY OR BLOOD BANK SECTION

PROCEDURE PURPOSE SOURCE OF ANTIGEN SOURCE OF

ANTIBODY
ABO/D typing forward) Detects A, B, D antigens
ABO typing (reverse) Detects ABO antibodies
Detects antibodies with
Antibody screen specificity to red cell
antigens
Identifies the antibody
Antibody identification with specificity to red cell
antigens
Determined SEROLOGIC
COMPATIBILITY between
Crossmatch (major) DONOR RED CELLS and
RECIPIENTS SERUM
BEFORE TRANSFUSION
REGULATION OF REAGENT MANUFACTURE

• Licensed by the CENTER FOR BIOLOGICS EVALUATION AND RESEARCH of the FOOD AND DRUG
ADMINISTRATION.
• The publication CODE OF FEDERAL REGULATION outlines the FDA's criteria for the licensure of reagents
in conunction with other regulations for the manufacture of blood and blood components
o SPECIFICITY → unique recognition of an antigenic determinant and its corresponding antibody
molecule
o POTENCY → strength of Ag-Ab reaction
• ISSUES ON EXPIRATIONS
• According to FDA regulations routine blood banking reagents cannot be used in testing after the
expiration date.
• Exceptions to this rule can be made for rare antisera and red cells if the reagent demonstrate acceptable
quality control results.
• If reagents are produced for in-house use (within the facility), a license is not required.
• Each manufacturer provides a package or product insert to the consumer that details and reagents
description, the procedure for the proper use, the specific performance characteristics, and the reagent's
limitations

ESSENTIALS OF PRETRANSFUSION

• ANTIBODY SCREEN AND IDENTIFICATION

• COMPATIBILITY TESTING -crossmatching

SEROLOGIC SYSTEMS USED IN TRADITIONAL LABORATORY METHODS FOR RED CELL ANTIBODY
DETECTION
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 TYPE OF
IMMUNOGLOBULIN PURPOSE AND TESTS THAT
REACTION ANTIBODIES
COMMONLY MECHANISM OF USE SEROLOGIC
PHASE COMMONLY
DETECTED REACTION SYSTEM
DETECTED
IgM antibodies react best
ABO reverse
at cold temp.- Expected ABO
Blood typing
IMMEDIATE pentameric alloantibodies
- Indirect
SPIN/ROOM
agglutination test
TEMPERATURE IgM IgM is an Unexpected
- Ab screening
PHASE agglutinating antibody cold reacting
- Ab identification
IS/RT that has the ability to alloantibodies or
- Crossmatching
easily bridge the distance autoantibodies
-Autocontrol
between red cells.
IgG antibodies react best
at warm temp.

No visible
agglutination
commonly seen.
IAT:
37'C - Ab screening
IgG is sensitizing antibody
INCUBATION IgG - Ab identification
with fewer antigen sites
PHASE - Crossmatching
than IgM and cannot
-Autocontrol
undergo lattice formation.

Complement may be
bound during reactivity,
which may or may not
result in visible hemolysis.
AHG has specificity for the
Fc portion of the heavy IAT:
chain of the human IgG - Ab screening
molecule and or - Ab identification Unexpected
ANTIGLOBULIN
complement components - Crossmatching warm reacting
AHG IgG
-Autocontrol alloantibodies or
(IAT)
AHG acts as a bridge autoantibodies
crosslinking red cells IS/RT → 37’C →
sensitized with IgG or AHG
complement.
IMPORTANT TERMS TO REMEMBER:

ALLOANTIRODY→ antibody produced against the antigen of a DIFFERENT individual of THE SAME
SPECIES.
AUTOANTIBODY→ antibody produced against the antigen of a person OWN red cell antigen

ANTIBODY SCREEN

• It determines whether an antibody to a red cell antigen has been made.

• AIM: detect as many clinically significant antibodies as possible.
• Clinically significant Abs refers to Abs that are reactive at 37'C or in the DAT or both and are known to
wave caused a transfusion reaction or unacceptably short survival of the transfused red blood cells
• It is performed to detect antibodies in the following people:
1) Patients requiring transfusion
2) Women who are pregnant or following delivery
3) Patients with suspected transfusion reactions
4) Blood and Plasma donors

METHODS:

1. TUBE TECHNIQUE (Traditional)

• Method:
o The traditional method of detecting antibodies is an indirect antiglobulin test performed in
a test tube
• Use of the tube test remains popular, due to the flexibility of the test system, use of commonly
available laboratory equipment, and relative inexpensiveness.

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• Disadvantages:
o instability of the reactions
o subjective nature of grading by the technologist
o amount of hands-on time for the technologist
o problems related to the failure of the washing phase to remove all unbound antibody.
o RBC REAGENTS(SCREENING CELLS):
o Composition: GROUP O RED BLOOD CELLS
o Cells are suspended at a concentration between 2 and 5 percent in a preservative diluent
which maintains the antigens and prevents hemolysis.
o 2-3 cells with varied antigen expression.

• ENHANCEMENT REAGENTS:
o LISS
o 22% ALBUMIN
o PEG-Poly ethylene glycol used in 37°C .
• AHG REAGENTS:
o addition of AHG reagent allows for the agglutination of incomplete antibodies

2. GEL TECHNIQUE - Same sensitivity with PEG Tube technique

• Ab screen can also be used with Gel technology using a microtubule filled with a dextran acrylamide
gel
• SCREENING CELLS: same criteria as for the tube test but are suspended in LISS to a concentration
of 0.8 percent.
• It is reported to be as sensitive as the PEG tube test method.
• Advantages:
o The omission of the washing and Coombs' control steps results in fewer hands-on steps
for the technologist to perform.
o Reactions are stable for up to 24 hours and may be captured electronically, leading to
standardized grading of reactions, and easier review by a supervisor.
o Mixed field reactions may be more readily detected with the gel technique.
o Ability to automate many of the pipetting and reading steps, thereby allowing Increased
productivity.
• Disadvantages:
• The need for incubators and centrifuges that can accommodate the gel cards.

POSITIVE REACTION → CELLS TRAPPED IN GEL

NEGATIVE REACTION→ CELLS IN PELLET AT BOTTOM OF THE MICROTUBE

3. SOLID PHASE TECHNIQUE

• With this method, RBC antigens coat microtiter wells instead of being present on intact RBCs.
• The patient's serum or plasma is added to each well in the screen cell set along with LISS.
• ADVANTAGES:
o Automated

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 o Smaller sample size (when compared with the tube test), making it ideal in a pediatric
setting and a LISS reagent that changes color when added to serum or plasma.
• DISADVANTAGES:
• With such a small sample and reagent volume, careful pipetting is necessary when performing
the test manually.
• An inadequate volume of indicator cells may result in a pattern similar to that of a weak positive
reaction.
• Staff should be carefully trained to visually interpret results if automation is not used.
• Staff members who have primarily used the tube test previously may interpret the diffuse positive
pattern as a negative reaction and the dense pellet of the negative reaction as a positive (4+)
reaction.
• Incubators, washers, and centrifuges that can hold the wells are among the special equipment
needed for this method.
• A final disadvantage is the need to run a positive and negative control with each batch of
tests, which adds to the expense of this method.

INTEPRETATION:

• Agglutination or hemolysis at any stage of testing is a positive test result, indicating the need for
antibody identification studies.
• However, evaluation of the antibody screen results (and autologous control, if run at this time) can
provide CLUES and GIVE DIRECTION for the IDENTFICATION AND RESOLUTION of the
antibody or antibodies.
• The Investigator should consider the following questions:

IS/RT - IgM
1. In what phase(s) did the reaction(s) occur?
37°C /AHG( IAT)-IgG
Autocontrol positive – Autoantibody
2. Is the autologous control negative or positive?
Autocontrol negative - Alloantibody
React at SAME PHASE and STRENGTH → SINGLE
3. Did more than one screening cell sample react; if Antibody
so, did they react at the same strength and phase? React to DIFFERENT PHASE and STRENGTR→
MULTIPLE AB / SINGLE AB that exhibit dosage
IN-VITRO HEMOLYSIS - Anti PP1PK and Anti-vel
4. Is hemolysis or mixed-field agglutination present?
Mf - anti-Sda (Refractile tiny) Anti-Lu ( Loose)
5. Are the cells truly agglutinated, or is rouleaux Stack coin appearance. Rouleaux formation
present? Use saline to check the rouleaux formation

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NOTES:

• ADVANTAGES OF ANTIBODY
SCREENING OVER DIRECT
CROSSMATCH FOR
DETECTION OF ANTIBODIES
o Testing is performed using
selected group O red cells
o Testing can be performed
well in advance of the
anticipated transfusion,
allowing ample time for
identification of
unexpected antibody and
location of suitable donor
units lacking the
corresponding antigen
• LIMITATIONS
o Screening reagents and
methods is designed to
detect as many clinically
significant antibodies as
possible but not to detect
those antibodies that are
insignificant

ANTIBODY IDENTIFICATION

• Once an antibody has been detected, additional testing is necessary to identify the antibody and
determine. Its clinical significance. The method used should be as sensitive as that used for
detection
• REAGENT CELLS (PANEL CELLS)
• Composition: GROUP O RBCS
• 11-20 cells
• The pattern of antigen expression should be diverse enough that it will be possible to distinguish one
antibody from another and should include cells with homozygous expression of Rh, Duffy, Kidd, and
MNSs antigens

CONSIDERATIONS/ GUIDELINES FOR INTERPRETATION OF PANEL

1. PATIENT HISTORY

• Information concerning the patient's age, sex, race, diagnosis, transfusion and pregnancy
history, medications, and intravenous solutions may provide valuable clues in antibody
identification studies, especially with complex cases.
• Examples:
• Anti D in pregnant patients
• Recent bacterial infections
• Recent viral illness
• Recent transfusion
• Race
• The patient's history is especially important when the autologous control or DAT is positive.
• In a patient transfused within the past 3 months, a positive DAT result may indicate a delayed
hemolytic transfusion reaction.

2. AUTOCONTROL

• It determine whether an ALLOANTIBODY or AUTOANTIBODY exists.

• Autocontrol is a suspension of patient's red cells with the patient's serum.
PAGOD NA AKO BEH GRRRR
 • It is included at the end of panel or antigram indicated as PC (patient's cells).
• Notes:
o AUTOCONTROL (+) and DAT (-) → potentiator being used may be causing the false positive
results
o AUTOCONTROL (+) and DAT (+) → indicate the presence of autoantibodies

3. PHASES OR REACTION

• It is an indication if an antibody is an IgM or IgG

o IMMEDIATE SPIN/RT PHASE-Cold or IgM
o 37'C PHASE
▪ may be cold (IgM) if reactions started at RT phase.
▪ May be warm (IgG) if reactions are not seen at RT but noticed at AHG
o AHG
▪ Warm (IgG): clinically significant

4. REACTION STRENGTH:

• It is a clue for the NUMBERS OF ANTIBODY PRESENT

o SINGLE STRENGTH (reaction of cells in only one phase): one antibody specificity
o VARYING STRENGTHS (reaction of cells in only one phases): more than one antibody
specificity or one antibody showing dosage
• Antibodies such as Anti K. Anti-D. Anti-E, Anti-e, Anti-c, Anti-Care commonly stronger than Anti-Fya Anti-
Fyb. Anti-Jka Anti-Jkb. Anti-S, Anti-s.
• The strength of the reaction also varies with antigen dosage.
• A stronger reaction may be due to dosage ("homozygous cells reacting more strongly than
"heterozygous" cells).
o If a panel is HOMOZYGOUS, a stronger reaction may be noticed.
o If panel is HETEROZYGOUS, a weaker reaction may be noticed: DO NOT RULE OUT!!! → tavolo
excluding a weak antibody that is showing dosage.

5. RULING OUT (EXCLUSION)

• Exclude antibodies BNs that could not be responsible for the reactivity seen
• To do this, the cells that gave a negative reaction in all phases of testing are examined.
• The antigens on these negatively reacting cells will probably not be the target of the antibody.
• Generally, it is advisable to perform this rule out technique ONLY IF THE ANTIGEN IS
HOMOZYGOUSLY EXPRESSED ON THE CELL.
o NEGATIVE REACTIONS
▪ if no reaction was observed, the antibody to the antigen on the panel was probably not
present.
o POSITIVE REACTIONS
▪ Never rule out
• NOTES:
o LOW FREQUENCY ANTIGENS
▪
Lua, CW, Kpa, Wra, V. Jsa, VS, Cob
▪ Because these antigens are rare in the population, the probability of producing antibody
to them is low,
▪ Antibodies to these antigens are typically being RULED OUT but may be considered
if the patient has been MULTIPLY TRANSFUSED or has an INCOMPATIBLE
CROSSMATCH

6. MATCHING THE PATTERN

• Look at the reactions that are positive and match the column.
o SINGLE ANTIBODY: If the specificity is a single antibody, the patterns matches one of the
antigen columns,
o MULTIPLE ANTIBODIES: When more than one antibody is present it is difficult to match a
pattern unless the phases of reaction strength are unique.

7. RULE OF THREE

• Identifying antibodies involves performing tests and making a conclusion based on reaction patterns.
• Testing the patient's serum with at least three antigen-positive and three antigennegative cells (also
known as the "3 and 3 rule") will result in a probability (P) value of 0.05
• A P value is a statistical measure of the probability that a certain set of events will happen by random
chance.

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 • A P value of 0.05 or less is required for identification results to be considered valid, and it means the
interpretation of the data will be correct 95 percent of the time

8. PATIENTS PHENOTYPE

• Individuals do not make alloantibodies to the antigen they possess

• This is a test of patient's red cells to ensure they are negative for the antigen corresponding
to the identified antibody.
• DONE ONLY IF NO RECENT TRANSFUSION HAVE OCCURRED.

ADDITIONAL TECHNIQUES FOR RESOLVING ANTIBODY IDENTIFICATION

• Used when patients have MORE THAN ONE ANTIBODY (MULTIPLE ANTIBODIES)
• There may be times when the first antibody identification panel performed does not produce a clear
Specificity. When multiple specificities remain following exclusion/inclusion techniques, additional testing
is necessary.

1. SELECTED CELL PANELS

SELECTED CELLS

• Cells chosen from another panel to confirm or eliminate the possibility of an antibody
• The cells selected for testing should have minimal overlap in the antigen they possess.

• Also used when a patient has a known antibody and the technologist is attempting to determine if
additional antibodies are present.

2. ENZYME TREATMENT

• Used to eliminate or enhance antibody activity

• Ficin, papain, trypsin, and bromelin are all commonly used to treat RBCs.
• Enzymes modify the RBC surface by removing sialic acid residues and by denaturing or
removing glycoproteins. The effect is to destroy certain antigens and enhance expression of others.
• Two procedures can be used:
o ONE STAGE ENZYME TECHNIQUE
▪ SIMULTANEOUS incubation of test serum, enzyme (papain) and red cells is performed

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 o TWO STAGE ENZYME TECHNIQUE
▪ Panel or screening cells are PRETREATED WITH ENZYMES (ficin or papain), washed,
and use without other enhancement media in the AHG test
▪ After enzyme treatment of red cells, these cells are retested with the serum of patient to
determine whether the antibody (or mixture of antibodies) is still reacting.
• Because enzymes destroy some antigens, the exclusion technique cannot be used on the enzyme
panel alone. The enzyme panel should be compared with a panel using the same cells untreated
3. DTT TREATMENT

• It is a reagent that can be used to denature KELL SYSTEM ANTIGENS on red cells.
• It works by disrupting the tertiary structure of proteins which makes them unable to bind with specific
antibody.
• The use of DTT is not a routine procedure in most transfusion services. It is most useful when antibodies
to the high frequency Kell system antigens are suspected.
• Can also be used in pretreating the patients red cells in adsorption technique

4. NEUTRALIZATION

• Useful procedures for working with certain antibodies to DETERMINE IF CLINICALLY SIGNIFICANT
ANTIBODIES ARE BEING MASKED BY THEIR REACTIONS.
• Soluble forms of Lewis, P1, Sda, Ch and Rg antigens can be added to the serum to inhibit the reactivity
of an antibody
• It is important when performing a neutralization procedure that controls must be tested along with the
neutralized serum. The control indicates that the negative reactions after neutralization are due to the
elimination reactivity and not simply dilution
• Sources of substances for neutralization:
o Anti P1= Hydatia cyst fluid, pigeon droppings, turtledove’s egg white
o Anti Lewis=Plasma / serum saliva
o Anti Ch, Rg= Serum (Contains component)
o Anti-Sda=Urine
o Anti I= Human breast milk
• Ex. NEUTRALIZATION OF Anti Leb S
SELECTED CONTROL: NEUTRALIZATION:
CELLS USED Serum + Serum + Lewis INTERPRETATION
FOR TESTING Saline substance
Le (a-b+) 1+ 0 Lewis antibody was neutralized
Lewis antibody was not neutralized or
Le (a-b+) 1+ 1+
diluted
Le (a-b+) 0 0 Antibody was probably diluted

5. ADSORPTION
• Basic definition: removal of AB directly in the serum
• Used to remove autoantibodies from serum to determine whether an underlying alloantibody
exists
• Antibodies may be removed from serum by adding the target antigen and allowing the
antibody to bind to the antigen (similar to the neutralization technique).
• In the adsorption method, the antigen/antibody complex is composed of solid precipitates
and is removed from the test system by CENTRIFUGATION.
• The absorbed/adsorbed serum is tested against an RBC panel.

PAGOD NA AKO BEH GRRRR

DIFFERENT TECHNIQUES:

ADSORPTION TECHNIQUE DESCRIPTION LIMITATION

Removed cold (IgM)
RABBIT ERTHROCYTE May adsorb Anti-B and other IgM
antibodies particularly Antii I
STROMA (RESt) antibodies
specificities
Patients red cells are used to
remove COLD
Do not use if recently transfused
COLD AUTOADSORPTION AUTOANTIBODIES to
within the last 3 months
determine if allotransfused
antibodies are present
Patients red cells are used to
remove COLD
Do not use if recently transfused
WARM AUTOADSORPTION AUTOANTIBODIES to
within the last 3 months
determine if allotransfused
antibodies are present
Uses known phenotyped red cells
to separate specificities:
• Warm autoantibodies
DIFFERENTIAL/ALLOGENEIC from alloantibodies May adsorb a high frequency
ADSORPTION • Alloantibodies from alloantibody
several specificities
Used for patients who have been
transfused for the last 3 months.
6. DAT and ELUTION TECHNIQUES

• DAT
o detect in-vivo sensitization of RBCS
o Patient's cells are washed thoroughly to remove any unbound antibody, and then AHG reagent is
added.
o When IgG antibodies are detected, the next step is to dissociate the antibodies from the surface
to allow for identification.
• ELUTION
o Basic definition: A process whereby cells that are coated with antibody are treated in such a
manner as to disrupt the bonds between the antigen and antibody
o This is helpful to identify the specificity of IgG that is attached to red cells when the DAT is
positive.
o used to release, concentrate, and purify antibodies.
o To identify the IgG specificity
o IgG attached to red cells is dissociated and place into a solution to test the specificity.
o The recovered antibody is called the eluate.

DIFFERENT ELUTION METHODS:

METHOD ANTIBODY REMOVAL BENEFITS LIMITATIONS

GLYCINE ACID (pH of 3.0)
The antigen/antibody bond is Lower pH
Rapid and sensitive disrupted, and the
antibody is Commercially released into the
acidic available supernatant. The Rapid Not sensitive
supernatant is harvested, and the pH is Lower pH Commercially -
neutralized so that antibody identification available
testing can take place.

Citric acid and digitonin acid are also

used in similar methods.
HEAT
45'C→ Allows for Not sensitive
Rapid
56'C→ total elution method, allowing for for other
Effective for ABO
antibody identification Physical antibodies
antibodies
FREEZE-THAW other than
Inexpensive
Lui freeze method → also produces total ABO
elution: frozen at 18’C
ETHER
METHYLENE CHLORIDE
Hazardous,
DICHOLOROMETHANE Sensitive and
Organic Solvent carcinogenic.
CHLOROFORM inexpensive
Flammable
Act on the lipids in RBC membrane to
reduce surface tension and lead to the
PAGOD NA AKO BEH GRRRR
reversal of the van der Waal forces that
hold antigens and antibodies together
More potent

7. ANTIBODY TITRATION

• Once identified, it is sometimes useful to quantify the amount of antibody present

• Performing an antibody titration can help determine antibody concentration levels.
• twofold serial dilations of serum containing an antibody are prepared and tested against a
suspension of RBCs that possesses the target antigen.
• The titer level is the reciprocal of the greatest dilution in which agglutination is observed.
• Contamination from a tube with a higher antibody concentration can lead to falsely elevated
titer level results.
o Remedy: Changing pipette tips between each tube when preparing the dilutions and
working/reading from the most diluted tube to the least diluted can help avoid this problem

COMPATIBILITY TESTING

• Compatibility testing is also called as PRETRANSFUSION TESTING

• Compatibility testing is considered synonymous with CROSSMATCHING, but remember that cross
matching is only a part of compatibility testing,
• Compatibility testing includes RECIPIENT ID, SAMPLE COLLECTION AND HANDLING, and REQUIRED
PRETRANSFUSION TESTING
• Basically, the process begins with transfusion request and ends with the transfusion of blood product to
the patient.
• These are series of procedures designed to ENSURE THE SAFETY OF BLOOD TANSFUSION
• PURPOSE:
o TO SELECT THE BLOOD COMPONENTS THAT WILL NOT CAUSE TO HARM THE RECIPIENT AND
WILL HAVE ACCEPTABLE SURVIVAL WHEN TRANSEUSED.
o ENSURES THE GREATEST COMPATIBILITY BETWEEN THE DONOR AND RECIPIENT DURING
BLOOD TRANSEUSION.

• COMPONENTS OF COMPATIBILITY TESTING:

1. IDENTIFICATION OF THE PATIENT AND DONOR and COLLECTION OF APPROPRIATE SAMPLE
FOR TESTING
2. TESTING THE PATIENT SAMPLE and REVIEW OF PAST BLOOD BANK RECORDS
3. SELECTION of APPROPROATE BLOOD DONOR UNITS
4. TESTING OF DONOR SAMPLE
5. CROSSMATCHING
6. REIDENTIFICATION OF THE PATIENT BEFORE INFUSION OF BLOOD.

1. IDENTFICATION, COLLECTION, AND PREPARATION OF SAMPLES

• POSITIVE PATIENT IDENTIFICATION

o The major cause of transfusion-associated fatalities → clerical error resulting in incorrect
ABO groupings.
▪ Clerical error → greatest threat to safe transfusion therapy
▪ The most common cause of error → MISIDENTIFICATION OF THE RECIPIENT
• To prevent collection of samples from the wrong patient
o The recipient's wristband identification must always be compared with the requisition
form (blood request form).
o The request form must state the intended recipient's full name and unique hospital Identification
number.
▪ Other information such as age, date of birth, address, sex, and Damen requesting
physician can be used to further verify patient identity but is not required on the form
o Printing must be legible, and indelible nameplate impressions or computer printouts are preferable
to handwritten forms.
o In some transfusing facilities, if the patient does not have a wristband and is coherent, it is
missible to ask the patient to state his or her full name and to spell it out.
o If the date of birth or home address is printed on the requisition form, the patient might be asked
to state this information
o The phlebotomist should never offer a name and ask the patient to confirm that correct
o If the patient is very young or is incoherent some other reliable professional individual who knows
the patient must confirm the identity and document this on the requisition form

PAGOD NA AKO BEH GRRRR

 • COLLECTING PATIENT SAMPLES
o Alter positive identification has been accomplished blood samples should be drawn, using
careful technique to avoid hemolyzing the sample.
▪ In-vitro hemolysis of recipient samples for pretransfusion testing cannot be used
because it can mask hemolysis caused by antigen-antibody complexes that
activate complement to completion.
• Serum or plasma may be used for pretransfusion testing
o MOST BLOOD BANKS PREFER SERUM over plasma
▪ plasma may cause small fibrin clots to form which may be difficult to distinguish from
true agglutination.
▪ plasma may inactivate complement so that some antibodies may not be detected.
o About 10 ml of blood is usually sufficient for all testing procedures if there are no known
serologic problems.
o To avoid contamination with materials that may cause confusing serologic results, blood
samples should not be taken from intravenous (IV) tubing lines.
▪ Venous samples are not to be drawn from above an infusion site but are to be drawn
from below the site.
o If a sample must be taken from an IV line, the line should be disconnected for 5 to 10 minutes
the first 10 ml of blood drawn should be discarded, and then the sample for testing should
be obtained.
o Recipient serum should also be separated from the patient's RBCs as soon as possible after the
sample has clotted. If testing cannot be performed immediately, samples should be kept
at 1 to 6C
• DONOR SAMPLES
• Samples for donor testing must be collected at the same time as the full donor unit.
• RBCs for donor pretransfusion compatibility testing can be prepared from the segmented tubing
through which the donor blood was collected.
• The tubing or segment is attached to the collection bag and each segment is imprinted with the same
number. These numbers are different from the donor unit number but nonetheless area positive means
of sampling a given unit of blood.
• NOTE: Both donor and recipient samples must be STORED FOR A MINIMUM OF 7 DAYS
following transfusion at 1-6°C

2. COMPATIBI COMPATIBILITY TESTING PROTOCOLS

• TESTING OF THE PATIENT SAMPLE

o A record must be maintained of all results obtained in testing patient samples.
o Some large transfusion services keep this information on a computerized retrieval system for
ready access.
o Any discrepancies between previous and current results must be resolved before transfusion is
initiated.
o A new sample should be collected from the patient, if necessary to resolve the problem, ABO and
Rh grouping must be included in the file.
o Also, notations concerning unusual serologic reactions and the identity of unexpected antibodies
in the patients serum should be included.
o ABO and RH grouping and AB screening of the patient's serum can be performed
advance or at the same time as the crossmatch
o If the patients has had a transfusion or has been pregnant within the LAST 3 MONTHS
Or if the history is unavailable or uncertain the sample must be obtained from the
patient WITHIN 3 DAYS OF SCHEDULED TRANSFUSION.
▪ ABO GROUPING
▪ The MOST CRITICAL pre-transfusion serologic test
▪ If the cell and serum grouping results do not agree, additional testing must be conducted
to resolve the discrepancy.
▪ If the patient's ABO group cannot be satisfactory determined and immediate transfusion
is essential, GROUP O RH negative PRBC should be used.
o Rh GROUPING
▪ Control must be run in parallel with Rh grouping tests performed on patient's samples, to
avoid incorrect designation of Rh negative patient as Rh positive.
▪ Testing for weak D (D) is unnecessary when testing Individuals typing as Rh-negative
in direct testing should receive Rh-negative blood, and those typing as Rh-positive in direct
testing should receive Rh-positive blood.

PAGOD NA AKO BEH GRRRR

 o DAT
▪ Should be performed on the patient's RBCs to determine whether uptake of autoantibody,
alloantibodies (if the patient has been recently transfused) is responsible for the positive
control result
o AB SCREEN
▪ The objective of the antibody screening test is to detect as many clinically significant
unexpected antibodies as possible.
▪ If positive → Abid.
• SELECTION OF APPROPRIATE DONOR UNITS
o In almost all cases, the FIRST CHOICE FOR TRANSFUSION is blood and blood components
of the patient's own ABO and Rh group → should be ABO group-specific
o When blood and blood components of the patient's ABO blood group are not available or some
other reason precludes their use, units selected must lack any antigen against which the
recipient has a clinically significant antibody.
o It is completely acceptable, however, to use blood and blood components that do not
contain all of the antigens carried on the patient's own RBCS
▪ e.g., group A-or-packed RBCS can be safely given to a group AB recipient
o When a recipient must be given blood of a different ABO group, only packs RBCs can be given
Why nor WHOLE BLOOD? → Whole blood cannot be administered in these situations because
incompatible preformed ABO antibodies are present in the whole blood plasma
o For example, group A whole blood cannot be transfused into a group AB recipient because the
plasma of the group A whole blood has anti-antibodies present

ABO GROUP SELECTION ORDER FOR TRANSFUSION OF RBCs

• TESTING OF THE DONOR SAMPLE

o COLLECTION FACILITY
▪ According to the Code of Federal Regulations and the American Association of
Blood Banks (AABB) Standards, ABO and Rh grouping Uncluding a test for weak
and tests intended to prevent discase transmission MUST BE PERFORMED ON
A SAMPLE OF BLOOD TAKEN AT THE TIME OF COLLECTION FROM THE DONOR.
▪ A SCREENING TEST FOR UNEXPECTED ANTIBODIES to RBC antigens is required by
AABB Standards on samples from donors who reveal history of prior transfusion or
pregnancy
▪ Testing is performed by the facility collecting the donor unit and results must be
clearly indicated on all product labels appearing on the unit
o TRANSFUSING FACILITY
▪ They are required by AABB Standards to CONFIRM THE ABO CELL GROUPING ON
ALL UNITS AND RH TYPING ON UNITS LABELED RH-NEGATIVE ONLY
▪ Repeat weak-D testing is NOT REQUIRED

3. CROSSMATCH TESTING

• Has traditionally meant the testing of the patient's serum with the donor rbcs including an antiglobulin
phase or simply an immediate spin phase to CONFIRM ABO COMPATIBILITY
• The terms "compatibility test and crossmatch" are sometimes used interchangeably, they should be
clearly differentiated. A crossmatch is only part of pretransfusion compatibility testing
• It routinely performed ONLY ON DONOR PRODUCTS CONTAINING RED CELLS
• A crossmatch MUST BE PERFORMED FOR ALL RED CELL TRANSFUSIONS. the exception is
situations requiring URGENT NEED FOR THE BLOOD.
• PURPOSES of CROSSMATCH:
o Prevent life threatening or uncomfortable transfusion reactions
o Maximize In vivo survival of transfused red cells
o Double checks for ABO errors
PAGOD NA AKO BEH GRRRR
 o Another method of detecting antibodies Red
• TYPE OF CROSSMATCH:
o MAJOR CROSSMATCH
▪ Routinely performed in laboratories
▪ Mix (DC+RS)
o MINOR CROSSMATCH
▪ Not required by AABB since 1976
▪ Mix (DS+RC)
• Notes:
• WHY IS THE MINOR CROSSMATCH UNNECESSARY?

STANDARDS and REGULATIONS COVERNING THE CROSSMATCH

• The AABB and FDA athe the two principal organization applying standards to the blood banking
and transfusion service Communities in the USA.
• FDA→ governing body for the BLOOD BANK INSPECTIONS EVERY YEAR.
• The College of American Pathologists and The Joint Commission also inspect blood establishments.
• AABBS STANDARDS:
1) Requires that a XM procedure always be performed using the RECIPIENT'S SERUM OF PLASMA
and a sample of DONOR CELLS taken from a segment originally attached to the blood
product bag.
• Exception: seen in urgent/emergency situations in which blood may be released for
transfusion before crossmatch.
2) The crossmatch is defined by the AABB standards that it is a technique that shall use methods that
demonstrate ABO incompatibility and clinically significant antibodies to red cell antigens
and shall include an antiglobulin test.
• Exception: If no clinically significant antibodies were detected in the current sample or in
the patient's past records an immediate spin crossmatch is permitted to fulfil the
requirement of detecting ABO incompatibility.
3) 3. Computer crossmatch-an alternative for all the requirements
CROSSMATCH PROCEDURES:

1. SEROLOGIC CROSSMATCH
2. COMPUTER CROSSMATCH

A. SEROLOGIC CROSSMATCH

a) Originally the serologic crossmatch preceded antibody screening as part of pretransfusion compatibility
testing to check for unexpected alloantibodies.
b) Considering that over 99 percent of clinically significant unexpected antibodies in patients' sera can be
detected by adequate antibody screening procedures, many blood bankers abbreviate er altogether
eliminate the serologic crossmatch.
c) What, then is the value of performing serologic crossmatching between patient and donor samples? Two
Main functions of the serologic crossmatch test can be cited:
i. It is a final check of ABO Compatibility between donor and patient
ii. It may detect the presence of an antibody in the patient's serum that will react
with antigens on the donor RBC but that was not detected in antibody
screening because corresponding antigen was lacking from the screening cells.
d) Crossmatching will NOT:
i. Guarantee normal survival of RBCS
ii. Prevent patient from developing an antibody
iii. Detect all antibodies
iv. Prevent delayed transfusion reactions
e) The current AABB Standards states that tests to detect ABO incompatibility are sufficient if:
i. No clinically significant antibodies were detected in the antibody screening DERES
ii. No historical record exists of the detection of clinically significant unexpected
antibodies
f) The SEROLOGIC CROSSMATCH PROCEDURE tests the recipient's serum er plasma with the RBCs from
the donor unit in an immediate spin XM and Antiglobulin XM.
g) Serologic crossmatch can be performed in using many methods, including the TUBE TESTING. SOLID
PHASE. AND GEL This discussion illustrates more on TUBE TESTING.

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 IMMEDIATE SPIN CROSSMATCH Done when:
- There is NO clinically significant unexpected
antibodies are detected in the current sample and
there are no previous records of such antibodies
- Mixing the recipient's serum with donor RBCs and
centrifuging immediately (l.e, immediate spin).
Notes:
However, the immediate spin does not detect all ABO
incompatibilities. False reactions may be seen in:
- Presence of immediate spin-reactive antibodies (eg,
autoanti-I)
- In patients with hyperimmune ABO antibodies
- When the procedure is not performed correctly (le,
delay in centrifugation or reading)
- Rouleaux is observed
- Infants specimens are tested
Adding EDTA to the test system has been reported
to eliminate some of the false positive reactions
thus improving the sensitivity of the immediate
spim crossmatch.
ANTIGLOBULIN CROSSMATCH Done when:
- There is EVIDENCE of clinically significant
unexpected antibodies in the current sample and
historical record of patient.
- Begins in the same manner as the immediate spin
crossmatch, continues to a 37°C incubation, and
finishes with an antiglobulin test.
- Potentiators may be added
- For greatest sensitivity, an antihuman globulin (AHC)
reagent containing both anti-IgG and anticomplement
may be selected for the final phase of this crossmatch
method. However, many laboratories routinely use
mono specific anti-IG AHG reagents
INTERPRETATION OF RESULTS FOR CROSSMATCHING in TUBE TESTING:

APPEARANCE INTERPRETATION
NO AGGLUTINATION /HEMOLYSIS COMPATIBILE
AGGLUTINATION OR HEMOLYSIS INCOMPATIBLE
INCOMAPTIBLE CROSSMATCHES

ANTIBODY
CROSSMATCH CAUSE RESOLUTION
SCREEN
Antibody is directed against antigen on Proceed to Ab id.
+ - screening cell Select antigen negative
blood
Antibody is directed against antigen on Proceed to Ab id.
donor cell which may not be on screening Select antigen negative
- +
cell OR Select antigen negative donor unit blood or;
may have IgG previously attached Perform DAT
Antibodies directed against both Antibody Id.
+ + SCREENING CELLS and DONOR CCELLS Select antigen negative
blood
Notes:

Donor and recipient must be stored for a minimum of ________ following transfusion

The samples should be stoppered and refrigerated at ______________,carefully labelled, and adequate in
volume so that they can be reevaluated if the patient experiences an adverse response to the transfusion.

B. COMPUTER CROSSMATCH

a) It uses a COMPUTER to make FINAL CHECK OF ARO COMPATIBILITY in the selection of appropriate units
for transfusion.
b) Same prerequisites pertains to the computer crossmatch and Immediate spin crossmatch
c) The computer crossmatch compares recent ABO serologic results and interpretations on file for both the
donor and the recipient being matched and determines compatibility based on this comparison
d) The AABB Standards specifies that the computer crossmatch can be used ONLY FOR THE PURPOSE
OF DETECTING AN ABO INCOMPATIBILITY RETWEEN THE DONOR UNIT AND THE SAMPLE
THAT WAS SUBMITTED FOR PRETRANSFUSION TESTING.
i. CURRENT SAMPLE SHOULD BE NEGATIVE FOR UNEXPECTED ANTIBODIES
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 ii. NO HISTORY OS SUCH ANTYIBODIES
iii. Also, AT LEAST TWO CONCORDANT PATIENT ABO/RH TYPES MUST BE OND FILE

CROSSMATCHING FOR PLATELETS AND PLASMA

• NO COMPATIBILITY TESTING REQUIRED

• For FRESH FROZEN PLASMA
o NO NEED FOR COMPATIBILITY TESTING
o ABO matching only is required
• For PLATELET CONCENTRATES and CRYOPRECIPITATE
o NO NEED FOR COMPATIBILITY TESTING an ABO and RH matching is required
• Exceptions:
o Neonates, alloimmunized patients-preferably ABO and RH matched platelets
o IF FRESH FROZEN PLASMA, PLATELET CONCENTRATES, CRYOPRECIPITATE is contaminated
with > 2ml of RBCS → DO COMPATIBILITY TESTING!

COMPATIBILITY TESTING IN SPECIAL CIRCUMSTANCES

A. EMERGENCIES

• Some laboratories use an emergency compatibility testing procedure that employs a shortened
incubation time, often with addition of LISS, to accelerate antigen-antibody reactions.
• Others maintain that regular procedures should be used in all circumstances and that blood should be
issued before completion of the standard compatibility testing procedure, if necessary.
• Blood to be issued in case of EMERGENCY:
o Do ABO and RH typing → so that group-compatible blood can be given
• In EXTREME EMERGENCIES, when there is no time to obtain and to test a sample:
o Give Group O Rh-negative PRRCS
• Accurate records of all units issued in the emergency must be maintained.
o A conspicuous tie tag or label must be placed on each unit indicating that compatibility
testing was not completed before release of the unit and the physician must sign a

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 release authorizing and accepting responsibility for using blood products prior to
completion of pretransfusion testing, according to the Code of Federal Regulations

B. TRANSFUSION OF NON-GROUP-SPECFIC BLOOD

• When donor units of an ABO group other than the recipient's own type have been transfused, such as
giving a group A recipient large volumes of group O donor RBCs, testing the recipient's serum
in a freshly drawn sample for the presence of unexpected anti-A and/or anti-B must be performed
prior to giving any additional RBC transfusions.

C. CENTRALITERINE TRANSFUSIONS

• Blood for intrauterine transfusion must be compatible with maternal antibodies capable of crossing the
placenta.
• If the ABO and Rh round of the fetus have been determined following amniocentesis. Chorionic villus
sampling, or percutaneous umbilical blood sampling → GIVE GROUP-SPECFIC BLOOD
• If the ABO and Rh groups of the fetus are not known → GIVE GROUP O RH-NEGATIVE PRO
• Crossmatch testing is performed using the mother's serum sample.

D. NEONATALT TRANSFUSIONS (<4 months)

• Only ARO and RH tuning: No serum typing

• Antibody detection/screening testing can be performed using the maternal serum or, alternatively using
the infant's serum (e. cord serum) and/or an eluate prepared from the infant's RRCS.
• For both intrauterine and infant (younger than 4 months transfusions, blood should be as fresh as
possible and no older than 7 days

SELECTING BLOOD FOR EXCHANGE TRANSFUSION IN NEONATES

E. TRANSFUSION IN AIHA

• Avoid transfusion as far as possible in patients with Warm AIHA

• There could be problems in blood grouping
o Spontaneous agglutination of red cells on addition of antisera in warm AIHA
o Non-specific agglutination of reagent cells during serum grouping in Cold AIHA
• Difficult to find absolutely compatible blood for such patients
• In emergency consider the least incompatible blood.
o Blood unit showing minimum strength of reaction in terms of titer designated as the least
incompatible
o Blood unit must be compatible with the patient's auto-absorbed serum.
• Transfusion should be done under strict medical supervision

F. MASSIVE TRANSFUSIONS

• When the amount of whole blood or packed cell components infused within 24 hours
approaches or exceeds the patient's total blood volume, the compatibility testing procedure may
be shortened or eliminated at the discretion of the transfusion service physician following written policy
guidelines.
• The original patient's sample no longer represents the circulating blood of the patient.
• The physician should make a policy in place dictating what is done under these circumstances and what
point a new "recipient" sample is required:
o Some policies indicate that the serologic immediate spin-crossmatch is eliminated for a time, and
ABO-identical donor units are simply tagged and issued.
o When clinically significant antibodies are involved, all units must be negative for antigens, but an
immediate spin-crossmatch still could be used
o Can used Group O Rh negative PRBCS iv. When the blood group is established-give blood group
specific unit
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G. SPECIMENS WITH PROLONGED CLOTTING

• Testing difficulties may be observed in blood samples from patients who have prolonged clotting times
caused by copulation abnormalities associated with disease or medications (such as heparin)
• Complete coagulation of these samples can often be accelerated by addition of thrombin
o One drop of thrombin 50 U/ml, to 1 mL of plasma (or the amount of dry thrombin that will
adhere to the end of an applicator stick) is usually sufficiently to induce clotting
• A small amount of protamine sulfate can be added to counteract the effects of heparin in samples of
blood collected from patients on this anticoagulant

SPECIAL TOPIC: DANGEROUS GROUP "O" DONOR

• In emergency situations Group O donor blood is used as a universal donor where group identical blood
is not available.
• This is already outdated concept in major blood banks. Certain donors possess in their plasma POTENT
ABO ANTIBODIES, which are dangerous to the recipient’s red cells.
• These are Anti-A and Anti-B hemolysins titer of which is >32
• Such donors are-called DANGEROUS O DONORS
• Therefore, if group O blood is to be used as universal donor, It should always be PLASMA DEPLETED

DIFFERENT PROBLEMS ENCOUNTERED IN GROUPING AND CROSSMATCHING

1. ROLEAUX FORMATION

• Causes:
• Diseases:
• Synthetic plasma expanders:
• Fibrinogen

2. PAN-AGGLUTINATION

• Spontaneous clumping of cells against a given serum

• Causes:
o Bacteriogenic pan-agglutination called:
▪ Reaction takes place at 20’C not at 37’C
o Non-bacteriologic pan-agglutination caused by acquired hemolytic anemia or by rare specific
antibody.
▪ Reaction takes place at 37’c and DAT is always positive.

3. Polyagglutination

• Condition wherein RBC agglutinates with ABO compatible blood due to ALTERED MEMABRANE
COMPONENT.
o ACQUIRED POLYAGGLUTINATION .
▪ MICROBIALLY ASSOCIATED
• T polyagglutination
o Cryptic antigen, under N-acetyl neuraminic acid
o Organisms with neuraminidase
▪ V. cholera
▪ Pneumococci
▪ E.coli
▪ Clostridium
▪ Influenza virus
• b. Tk-polyagglutination
o Organisms with Beta-endogalactosidase
▪ Bacteroides fragilis
▪ Serratia marcescens
▪ Candida albicans
• Acquired B phenomenon
o Organisms with D acetylase
▪ E.coli 086
▪ P. vulgaris 0519
▪ Clostridium tertium
• Th polyagglutination
• Tx-palyagelutination
• VA-olyagglutination .
o NON-MICROBIALLY ASSOCIATED
o Tn-polyagglutination- Acquired A phenomenon

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 • INHERITED POLYAGGLUTINATION
• CAD (CAZAL AUTOSOMAL DOMINANCE)
o Acquired A phenomenon
o Associated with SID antigen
• HEMOGLOBIN M-hyde park
• HEMPAS/CDA II
• NOR-associated with P1 antigens

PANEL OF LECTIN

Tn CAD T Tk
Arachis hypogaea - - + +
Salvia sclarea + - - -
Salvia horminum + + - -
Glycine soja + + + -
4. Cold agglutinins

5. Wharton’s Jelly

6. Prozone

7. AG-AB deterioration

ABO COMPATIBILITY FOR WHOLE BLOOD, RED BLOOD CELLS, PLASMA TRANSFUSION

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