BLOOD-BANKING ➢ R.A.C.E.

Che Marie A. Gelacio, RMT, MSMLS, MLS(ASCPi)

▪ Rescue—rescue anyone in immediate danger.
▪ Alarm—activate the institutional fire alarm
Blood Banking Procedures system.
▪ Contain—close all doors to potentially affected
➢ LABORATORY SAFETY AND QC OF REAGENTS areas.
and EQUIPMENT ▪ Extinguish—attempt to extinguish the fire, if.
- Chain of infection and safety practices related to
the biohazard symbol.
➢ Universal Precaution
- Consider ALL patient’s blood & body fluids to be
biohazardous regardless of the diagnosis.

Primary focus of safety: biohazardous materials

Biohazard materials
- all body fluids & tissue in the lab & the reagents
used that are of human origin

Factors affecting the accuracy of the test:

1. Reagents’ stability
2. Equipment

Reagents used for testing samples in BB originate in

HUMANS:
1. Antisera
2. Cell products

➢ QUALITY CONTROL of REAGENT and

➢ FIRE SAFETY EQUIPMENT

Class of Fire Type of Extinguisher Quality Assurance Program

Class A ▪ Pressured water - Evaluate test procedures being performed &
Ordinary combustibles ▪ Dry chemical correct problem that arise in the test or w/ the
✓ Wood equipment.
✓ Paper
✓ Clothe Method of ensuring ERROR-FREE performance
Class B ▪ Dry chemical Comprehensive program that monitors & evaluate all
Flammable liquid ▪ Carbon Dioxide Aspects of the performance
✓ Grease - encompass the categories of personnel,
✓ Gasoline policies & procedures and techniques
✓ Paints Includes testing kit, ( + )& ( - ) controls
✓ Oils
Class C ▪ Carbon Dioxide
A. Licensed for clinical use only (meet SPECIFICITY &
Electrical equipment ▪ Halon
▪ Dry Chemical POTENCY by FOOD & DRUGS ADMINISTRATION):
Motors
1. Commercial reagent antiserum
Switches
2. Reagent RBC
Class D ▪ Metal X
B. Manufacturer’s direction and specifications
Flammable Metals
- followed against loss of specificity & potency
✓ Magnesium

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C. Reagents, Materials and Equipment: ➢ Donor Screening Test
- 12X75 mm Disposable glass test tube
- Normal Saline Solution (0.85%) ✓ ABO & Rh typing
- Centrifuge, water bath or Hot block, Rh viewing ✓ Serologic screening (syphilis-uses serum / result:
Box reactive/nonreactive)
✓ Viral testing (Human Immunodeficiency Virus
➢ Records of Daily Reagent Controls Serum)
✓ HsB Ag
REAGENT SOURCE COLOR o Result: Reactive/non-reactive
ABO TYPING ANTI-A & ANTI-B: Anti-A: o HCV
SERA - Supernatants of BLUE o Malarial smear (test kit)
mouse hybridoma Anti- B: o 5 μL whole blood; 2 drop diluents (result
cell culture. YELLOW +/-)
Rh TYPING - Supernatants of GRAY ✓ Transfusion-transmitted viruses
SERUM cell cultures w/Ab
producing B
lymphocytes ➢ Donor Selection Process
obtained through Registration Information and General Requirements
EBV
transformation.
ANTI-HUMAN - Serum of rabbits GREEN Medical History through interview (Uniform Donor
GLOBULIN immunized w/ History Questionnaire)
SERUM purified human
/Coombs test IgG and mucine Physical Examination – Weight ( 110 lbs./ 50 kg) ;BP
monoclonal Ab (Upper limit: 140/90, Lower limit: 100/60); Pulse(60-
specific for an 100 beats /minute ); temperature ( 35- 37.5°C)
epitope on the
human C3d
molecule. Vein check (2 phleb sites)
ALBUMIN, 22% - Raw bovine PINK w/
serum black top
Pre-screening – Blood typing/ABO &Rh typing (Slide
typing); Hgb &Hct (Capillet) –Hgb (12.5-16.5);
Hct – F: 36-46 vol%, M:41-50 %
➢ Blood Bank-Philippine Red Cross
Category: Blood Center
➢ Blood Components and Anticoagulant
Services: ✓ Whole blood
✓ Recruitment of Voluntary Blood Donors ✓ Packed RBC 1-6 °C
✓ Health Education & Counseling ✓ Washed RBC
✓ Donor Screening & Selection ✓ Fresh frozen plasma - < -18°C
✓ Blood Collection ✓ Platelet concentrate – RT (22-24°C) w/ constant
✓ Blood Screening & Testing for Blood agitation
Transmittable Diseases
✓ Provision of Whole blood, RBC & all blood ANTICOAGULANT:
products • CPDA-1 (Citrate Phosphate Dextrose with
✓ Storage of Blood & Blood Products adenine)
✓ Issuance, Transport & Distribution of Whole - 35 days(expire)
blood, RBC & all blood products - stored: 2-8°C

Ian Khyndric ὢ
LABELING requirements for Blood products: Present:
• Unit ID - FIBRINOGEN (soluble plasma protein).
• Expiration date - Fibrin – Insoluble (difficult to distinguish
• Product type from real agglutination).
• Production site
• ABO-Rh Type B. PLASMA
• Type of Anticoagulant/preservative solution - Fluid portion of a blood sample that has
used clotted
- No anticoagulant
➢ Indications and Storage
Preferred specimen in Blood bank
1. Doesn’t contain fibrinogen.
A. WHOLE BLOOD (7:1) – blood to AC 2. Ab demonstratable through
- 4°C 35 days complement activation.
- 21 days (CPD) 3. Doesn’t use AC such as EDTA or citrate,
- Treatment cardiopulmonary bypass px which chelate Calcium and Magnesium
B. PACKED RBC thus preventing complement activation.
- 1-6°C EDTA NON-
- 35 days Evacuated
ANTICOAGULATED
- Symptomatic anemia Tube
(PLAIN RED)
✓ Plasma ✓ Serum
C. WASHED RBC
✓ Buffy Coat ✓ Clotted Blood
- 1-6°C (WBC)
- 24 hrs. (open system) Layers
✓ Packed
- Paroxysmal nocturnal hemoglobinuria Mature
(PNH) RBC
- Anaphylactoid rxn (type1) Time to clot N/A 7-15 minutes
Centrifugation 5 minutes 5 minutes
D. PLATELET CONCENTRATE Time
- Room Temp. with constant agitation
- 5 days Labelling: Crucial Step in every Laboratory Procedure
- 24 hrs. (open system)
- Dengue ➢ Factors that affect the degree of packed RBC:
- Bleeding
1. speed & time of centrifugation
E. FRESH FROZEN PLASMA 2. radius of centrifuge
- < -18°C 3. height of the blood column
- 1 year
- Multiple coagulation factor deficiency • Amount plasma trapped between red cells VARY w/
- Massive transfusion Height of cell column and shape of cell
• Hemolyzed samples free RBC Hb that mask Ab
F. CRYOPRECIPITATE induced hemolysis
- < -18°C • HEMOLYZED, EXCESSIVELY LIPEMIC, BACTERIALLY
- 1 year CONTAMINATED PLASMA: not used
- Correction of Factor VII deficiency
➢ Preparation of 2% RBC Suspension
➢ Serum vs Plasma
A. SERUM Washed RBC (3X) – RBC remain AFTER washing w/ a
- Liquid portion of blood sample volume of comparable solution to remove PLASMA.
- WITH Anticoagulant (EDTA) RESUSPENDED = 2-4 % NSS; Low Ionic Strength
Saline (0.2%)

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USES:
• Serologic test for IM (washed sheep RBC’s – act
as in vitro Ag)
• ASO (Antistreptolysin O test –Human
erythrocytes)- HEMOLYSIS
• Blood bank: blood typing (forward /reverse),
compatibility testing (Red cells of donor & px)
• COOMBs test –Direct/Indirect -Hemolysis
EFFICIENCY of washing depends: AMOUNT of saline,
METHOD used
➢ Purpose of Washing RBC
1. Remove plasma or serum to prevent formation
of fibrin clots.
2. Remove serum factors that inhibit complement
reactivity.
3. Removes soluble Ag-Ab complexes that compete
w/ the target cell for complement.
4. Removes unbound globulin by dilution.
2% cell suspension – gives the best result (weaker
suspension = greater agglutination)
1. more sensitive for agglutination.
2. avoid zoning phenomenon.
- (Postzone- excess Ag; Prozone-excess Ab)
- serum to cell ratio: 2:1
- INCREASE the serum to RBC ratio.
- INCREASE the demonstration of weakly
reactive Ab (degree of Ab coating).
3. it is best to use the weakest cell suspension that
can observe easily for agglutination.
➢ Formula Used
➢ Washing Steps
After the removal of plasma from EDTA, Forcefully
ADD NSS (0.9%) - 1/2 INCH from Top
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Cover w/ PARAFILM (invert to mix) – prevent
contamination (introduce GLOBULINS) & to protect
the worker’s health
Centrifuge – balance tube (1 minute @ 3400
rpm/revolutions per minute)
Discard parafilm (biohazardous waste)
Remove supernatant (use PASTEUR pipet) – discard
in beaker w/ DISINFECTANT. Repeat Step 1-5 one
more time. Total 2 washes
5ml serologic pipet (4.85 ml NSS in a test tube)
+ 1 mL serologic pipet (0.15ml washed RBC)
Mix using parafilm (exact 3% Red Cell suspension)
Discard in PUNCTURE-PROOF biohazardous waste
container
False-Positive Errors
1. Addition of the wrong antiserum to a test
tube.
2. Over centrifugation of a serum-cell mixture.
3. Dirty glassware
4. Hemolyzed patient serum or reagent RBCs.
5. Inadequate dispersal of centrifuged serum
cell mixture.
False-Negative errors
1. Omitting patient or donor serum from the test
mixture.
2. Omitting antiserum from the test mixture.
3. Failure to identify hemolysis of red blood cells
by the patient's serum as a positive reaction.
4. Inappropriately warming a cell-serum
mixture.
5. Under centrifugation of a serum-cell mixture.
6. Vigorous shaking of a centrifuged serum-cell
mixture.
False-Negative or False Positive Errors
1. Incorrect labeling of test tubes.
2. Addition of the wrong antiserum.
3. Erroneous reading or interpretation of
results.
4. Inaccurate recording of results.
5. Contamination of antiserum or reagent red
cells.
 6. An inaccurate serum-cell ratio in a test
mixture.
➢ GRADING AGGLUTINATION REACTIONS
Agglutination
- the clumping of particles with Ag on their surface
such as erythrocytes, by antibody (Ab) molecules
that form bridges between antigenic
determinants of adjacent cells.
- endpoint for most in vitro tests involving
erythrocyte antigens and blood group antibodies
Can be observed either through:
1. direct techniques such as ABO testing & Rh
typing or
2. Indirect techniques such as Antiglobulin test/ Ab
screening
➢ Stages of Agglutination
➢ Pseudo agglutination
- erythrocytes microscopically resemble rolls of
stacked of coins, rouleaux (translucent &
refractile) formation.
- to differentiate from true agglutination;
a. examine using microscopic appearance
b. use saline replacement technique (never
done before initial testing protocol →
dil. effect → false (-)
Ways to ELIMINATE rouleaux formation:
1. Wash RBC (4x) in NSS –remove proteins
2. use saline replacement technique (1-3 drops
saline)-rouleaux DISPERSE; Ab mediated
agglutination REMAINS
➢ Grading
- Determine strength of reaction (AMERICAN
ASSOCIATION OF BLOOD BANK: standard
system)

SENSITIZATION LATTICE FORMATION Procedure:

st
1 phase 2nd phase
(1) 1 drop serum + 2 drops RBC susp’n in a 12x75
- Physical attachment - Establishment of
mm test tube
of Ab to Ags on the crosslinks between
(2) Mix & centrifuge for 1 min @ 1000 rpm
RBC membrane when sensitized particles
Ab combines with Ag, such as RBC and Ab (3) Examine HEMOLYSIS after centrifugation
sensitizing the Ag. resulting in (red/pink supernatant (+))
aggregation. (4) Examine AGGLUTINATION (agglutination viewer)
REVERSIBLE IRREVERSIBLE (5) SHAKEN GENTLY (disrupt rbc button) & tilted –
FASTER SLOWER (Agglutination) CLUMPS (+)
(No agglutination: ONLY
ATTACHMENT)

➢ METHODS OF ENHANCING AGGLUTINATION

1. CENTRIFUGATION
2. TREATMENT with proteolytic enzymes (eg:
papain, ficin, Bromelin) which also eliminates the
Ag-binding sites for other Antibodies such as
Anti-M, Anti-S, Anti-Fya, anti-Fyb.
3. The use of COLLOIDS –Bovine Albumin 22%
protein concentration, pH 7.2 with 0.1% sodium
azide as preservative and acacia.
4. Addition of anti-human globulin (AHG) reagent.
5. The use of LISS
6. Polybrene
4+
7. POLYETHYLENE GLYCOL (PEG) – the more
Grade: “COMPLETE”
effective in detecting weak antibodies than LISS
Score: 12
manual polybrene.
Appearance: A single agglutinate. No free RBCs detected

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 3+ - use known A & B cells to det. the presence
Grade: 3+ of Ab(unknown) in the serum.
Score: 10 - can’t be used for newborn babies, and
Appearance: Strong reaction. A number of large infants less than 6 months old
agglutinates - uses:
a. to validate the results for the Forward
2+ typing
Grade: 2+ b. to check the reactivity of the anti-sera
Score: 8
Appearance: Large agglutinates in a set of smaller ➢ Characteristics of Antibody of the ABO blood
clumps, no free RBCs Group System
1. Agglutination of saline suspended RBC (they are
1+ IgM class Ab)
Grade: 1+ 2. Reactivity temp is @ 4-22°C
Score: 5 3. Produces hemolysis when binds w/ complement
Appearance: Many small agglutinates & a background of 4. Inactivation of IgM anti-A & ant-B w/ 2-
free RBCs mercaptoethanol (2-ME)

½+ or (-) Rule: Serum 1st before RBC (Clear solution 1st, RBC 2nd,
Grade: + / - Macro make sure to have added both source of Ab and Ag.
Score: 3
Appearance: Weak granularity in the suspension. A few ➢ Reagents
macroscopic agglutinates but numerous agglutinates
microscopically. Anti-A Anti-B
Source Monoclonal Ab (mouse)
Trace or Micro Grouping Highly specific IgM
Grade: (+) Micro Color Blue Yellow
Score: 2 Dye --- Acriflavine
Appearance: Appears negative macroscopically. A few Reaction 3+ to 4+
agglutinates of 6-8 RBC in most fields Drops 1-2 drops

0 A1 Cells B Cells
Grade: 0 Source Human source
Score: 0 RC Suspension 4-5 %
Appearance: An even RBC suspension. No agglutinates Reaction 2+ to 4+
detected Drop 1

➢ ABO BLOOD GROUP ➢ Procedure

2 Types of tests: Forward Typing/Cell Reverse Typing/Serum

1. Forward typing Grouping Typing
- use known Ab (serum) to determine the 1 drop Anti – A (left) 1 drop patient’s serum
1 drop Anti –B (right)
presence of Ag(unknown) present on the +
surface of the RBC + 1 drop A cells(left)
- suitable for newborn & infants less than 6 1 drop whole blood-50% 1 drop B cells(right)
months old (Ab are synthesize starting in cell suspension (to EACH
6th month & are well dev on the 5th or 6th antisera)
yr.)
2. Reverse typing

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 Mix w/ an applicator stick and rock the slide GENTLY - immunization of Rh (–) recipients
for about 2 mins (maximum time to observe for
agglutination) Levine & Stetson – Ab detected
+ - AGGLUTINATION Rh-hr Blood group system
0 - NEGATIVE - complex of all erythrocytes blood group
system (>40 Rh Ag)
➢ Results Additional gene: C, c, E, e

➢ Rh Antigen and Antibody

Rh Ag /D
- On surface of red cells, transmembrane
polypeptides, found on RBC.
- PRIMARY antigen
- RBC containing Ag were agglutinated with
antiserum produced by injecting rabbits w/
washed RBC of Macacus rhesus.
➢ ABO Grouping- Tube Method
Rh Ab
More sensitive because: - NO anti-Rh Ab resent in normal individual
a. the use of 5% RBC suspension - Acquired, immuneAb; production by
b. centrifugation – enhances agglutination Transfusion / Pregnancy.
- majority are IgG (IgG1 & IgG3-activate
Tubes used:
complement); cross placenta
a. RBC suspension – 12x75mm
b. Reaction tube – 10x75mm Detect IgM
- Saline Test system; IgG –Antiglobulin phase
➢ Procedure (enhanced by enzyme methods)
Forward Typing Reverse Typing
Rarely activate complement EXCEPT anti-D & anti-C
1 drop Anti – A (tube 1) 2 drops of px SERUM (to
1 drop Anti –B (tube 2) EACH CLINICALLY SIGNIFICANT & cause HDN
tube)
+ RhIgG (RhoGam)
1 drop 5 % RBC + - HDN due to anti-D not frequently seen
suspension (to EACH 1 drop A rgt red cell
tube) (tube1)
1 drop B rgt red cell
➢ RH Typing
(tube2)
Antisera “anti-D”
- Mix & Centrifuge for 15 sec @ 3400 rpm - Ab contained in Antisera will attach to D Ag on
- Gently resuspend each tube patient/donor red cells.
- Look for HEMOLYSIS & AGGLUTINATION - AGGLUTINATION
- from Human source, potential biohazard.

➢ RH Blood Group System Tube test

- 2-5% washed red cell suspension
Landsteiner & Wiener (Rh factors) – 1941 - 1:1 ratio w/ antisera
- human erythrocytes were agglutinated by this
Ab to Ag, common to all RHESUS MONKEY and
85% of humans.
FORMS of anti-D sera:
Rh0(D) factor

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 1. High protein anti-D; High protein rgt.; Low
protein rgt.
2. Chemically modified anti-D
3. Monoclonal anti-D
4. IgM anti-D
1. High protein media reduces zeta potential; cell
agglutinate on immediate spin.
2. Chemical modification of Immunoglobulin
molecules allows HINGE region of molecule to span
GREATER DISTANCE to attach to Antigens and
agglutinate.
3. Origination of Ab is from one Ab-producing clone
and is very SPECIFIC
4. Rh CONTROL
- high protein antisera used in parallel
w/control (assures agglutination obtained
represents a TRUE POSITIVE reaction).
- Chemically modified antisera are readily
available.
- may include ALBUMIN control / prepacked Rh
control.
“Rh Control, is composed of contents identical to
anti-D sera, but with NO antibody”
False POSITIVE
✓ Positive Direct Antiglobulin test
✓ “Coated red cells – in vivo”
✓ Rouleaux
✓ Reagent contamination
✓ Antibody to low-incidence- antigen
✓ Human error
False NEGATIVE
✓ HUMAN error
✓ RBC suspension –too heavy
✓ Failure to add anti-sera/wrong anti-sera
✓ Failure to follow manufacturer’s direction
✓ Under centrifugation
✓ Failure to incubate slide test for specified time
✓ Deterioration of Rh Ag (Inappropriate storage)
✓ Inappropriate technique, vigorous shaking
➢ RH Typing Procedure
Ian Khyndric ὢ
SLIDE TEST
- Reagents and samples to ROOM TEMP. before
testing.
Procedure: (Rh control not performed)
1. Label slide (3 “X 5”) with “anti-D”
2. Add 1 drop Anti-D
3. Add 2 drops 40-50 % suspension of red cells in
serum/plasma
4. Mix: area about 2cm x 4cm
5. Tilt slowly back and forth. Observe
AGGLUTINATION w/in 2 minutes
TUBE TEST
- CLOTTED specimen –most often used for
ROUTINE testing
- Use CHEMICALLY MODIFIED ANTISERA
Procedure:
1. Prepare 2-5% RC suspension (5% rc in 3 ml
used in lab. = 0.15 ml Red cell + 2.85 ml NSS
2. Label (crucial step) tube w/ px.
3. Identification & anti-D
4. 1 drop antisera
5. 1 drop px. cell suspension
6. CENTRIFUGE (15-30 SECONDS)
7. 6. Resuspend by gentle agitation
➢ Reading and Interpretation
READING & INTERPRETATION
Absence of agglutination (NEAGATIVE reaction)
Presence of agglutination (POSITIVE reaction)
- agglutination absent in albumin control.
If albumin control is positive, REPEAT using SALINE-
AGGLUTINATING ANTISERUM and Do DAT on red cells.
➢ Anti- Human Globulin Test
- Uses Ag-Ab reaction to detect SENSITIZATION/
Ab coating the red cells
- Anti-human globulin (AHG) Sera – Bridging effect
2 categories: Direct, Indirect
Principles of AHG test:
1. Ab molecule and complement components are
GLOBULINS.
2. Injecting an animal w/ human globulin, either
purified or in whole human serum, stimulates
the animal to produce Ab to foreign protein
(AHG).
*Hybridoma Technology – prepare AHG reagents.
*Polyspecific Gts – anti-IgG, anti- C3d
3. AHG will react with human globulin molecules
either bound to red cells or free in serum.
Unbound globulins may react and neutralize
AHG containing both coated RBC and free
globulin molecules. Unless RBCs are washed free
of unbound globulin BEFORE testing with AHG,
Unbound Globulin may neutralize AHG and
cause false-NEGATIVE result.
4. Washed RBCs coated with human globulin are:
- AGGLUTINATED by AHG.
- *Fab – AHG molecule
- *Fc - human ANITBODY
DIRECT antiglobulin test (DAT)
- Demonstrate In-coating / sensitization of RBCs
with globulins (IgG and C3d).
PROCEDURE:
1. Prepare 2% RC suspension
2. 2 DROPS prepared RC suspension
3. 2 drops patients’ serum
4. Incubate 37°C – 15 MINUTES (WATERBATH)
5. Add Normal saline solution (2/3). Mix using
parafilm
6. Centrifuge-15 SECONDS @ 3400 rpm
7. Wash (3-4x)
8. 1-2 drops AHG. spin 15 SECONDS @ 3400 rpm
9. Gently dislodge. Observe agglutination
microscopically and macroscopically.
➢ Results Interpretation
Left (DAT): 4+ (Presence of in vivo sensitization)
Right (IAT): 0 (Absence of in vitro sensitization)

- Washed RBCs from px/donor are tested directly

with AHG rgt.
- “incomplete” Ab present-agglutination occurs.

INDIRECT antiglobulin test (IAT)

- In vitro coating of red cells with
antibody/complement, achieved by incubating
serum with red cells, which are then washed to
remove unbound globulin.

- Antibody specificity may be known, as blood

grouping tests with coating Ab such as anti- Fya, or
antigenic composition of red cells may be known,
as in antibody detection and identification tests.
➢ Compatibility Testing
• Knowledge of DONOR PROCEDURES and
➢ AHG Procedure COMPONENTS available for transfusion are
Direct Antiglobulin Testing important for the technician
PROCEDURE: (tube: 12 x 75 mm) • COMPATIBILITY TESTING- series of procedures
1. 1 drop 2% Red cell suspension of Px. Cell designed to ensure the safety of blood for
(PROVIDED) transfusion
2. 1drop POLYSPECIFIC AHG • AABB Technical manual and AABB Standards
3. Mix. Centrifuge 1 min @ 1000 rpm / NOT over outline acceptable procedures for compatibility
20 sec @3500 rpm testing
4. Dislodge cell button to resuspend packed cells
• “CROSSMATCH” –performed prior to transfusion
5. *Manner in which RBC’S are dislodged from
of whole blood or packed red cells
bottom of tube is CRITICAL.
6. *Tube gently tilted back and forth • Method must DEMONSTRATE ABO
7. Examine AGGLUTINATION both incompatibility and clinically significant
microscopically and macroscopically. Antibodies to red cell antigens

Indirect Antiglobulin Testing

Ian Khyndric ὢ
 • Crossmatch Testing-incorporating testing of
both patient’s cell and plasma against the 3. Mix & spin: 15 SECONDS
reciprocal elements of the donor unit 4. Read, Interpret, and record result as “Immediate
• (2) tests: Major Crossmatch, Minor Crossmatch Spin”

MAJOR CROSSMATCH Antiglobulin Phase (Indirect)

Patient’s Serum ✓ Wash 3-4x ideally (lab: 2x) with NORMAL SALINE.
Donor’s red cells DECANT all trace of saline to obtain a dry cell
- Using Antiglobulin technique, mixed cells button.
suspended in saline from the originally attached
✓ 2 drops AHG sera
donor segment with the patient’s serum/plasma.
✓ Mix, CENTRIFUGE 15 SECONDS AT 3 400 rpm
- Testing continues to ensure COMPATIBILITY when ✓ Read, Interpret, and record result as “AHG”
px has history of/currently exhibits a clinically
significant antibody. ➢ Reading of Results
MINOR CROSSMATCH - “Immediate spin”
Donor’s Serum - “37°C Albumin”
Patient’s red cells “AHG”
- Involves testing the saline-suspended red cells of - GRADE (4+,3+,2+,1+) – If POSITVE
the px with the plasma of the donor unit.
- 0 - NEGATIVE
- Has been ELIMINATED because donor units are
*Stop if the phase shows HEMOLYSIS/AGGLUTINATION
screened for the presence of significant
(+)
Antibodies and are not included in general
inventory if an antibody is identified.
➢ Interpretation
• COMPATIBLE (ALL phases are NEGATIVE).
Complete Crossmatch Phases: • INCOMPATIBLE (1 or more phase shows
1. Immediate Spin/Saline phase/ Abbreviated HEMOLYSIS / AGGLUTINATION).
Crossmatch • *Cause incompatibility must be investigated.
2. Incubation at 37°C/High protein phase
3. AHG and Check cells
*Ab react at AHG phase are Ab that, if present in the
recipient, are most likely to cause IN VIVO.
*MIXED-FIELD Agglutination

*TYPE and SCREEN – Alternative for Compatibility

testing.

PROCEDURE:
MAJOR CROSSMATCH
Immediate Spin/Saline Phase
1. 2 drops PATIENT’S serum
2. 1 drop 2-5% DONOR’S Red cell • Reactions are graded from 0 to 4+.
(1) 4+ reaction is indicated by a solid band of
3. Mix & spin: 15 SECONDS red cells on the top of the gel;
4. Read, Interpret, and record result as “Immediate
(2) 3+ reaction displays agglutinated red cells
Spin”
in the upper half of the gel column;
MINOR CROSSMATCH (3) 2+ reaction is characterized by red cell
1. 2 drops DONOR’S serum agglutinates through the length of the
2. 1 drop 2-5% PATIENT’S Red cell column;

Ian Khyndric ὢ
 (4) 1+ reaction is indicated by red cell
agglutinates mainly in the lower half of the
gel column with some unagglutinated red
cells pelleted at the bottom;
(5) Negative reactions display a pellet at the
bottom and no agglutinates in the matrix of
the gel column. A mixed field reaction may
be observed.

Ian Khyndric ὢ