Pre-Transfusion

Testing
JOED T. TICSE, MD
Objectives

 Describe proper identification, collection, and

preparation of samples.
 Outline the procedure for testing donor and patient
specimens.
 Give an overview of antibody screening and
identification.
 Explain compatibility testing procedures.
Pre-Transfusion Testing

STEPS IN PRETRANSFUSION TESTING

1. Request for transfusion
2. Identification of transfusion recipient and blood specimen
collected
3. Testing of transfusion recipient’s blood specimen:
• Blood specimen acceptability
• ABO group and Rh type
• Antibody detection testing
• Antibody identification
• Comparison of current and previous test results
4. Donor RBC unit testing:
• ABO group confirmation and Rh type confirmation for Rh
negative RBC units
Pre-Transfusion Testing

STEPS IN PRETRANSFUSION TESTING

5. Donor red cell unit selection:
• Selection of components of ABO group and Rh type that
are compatible with the transfusion recipient and with any
unexpected allogeneic antibodies
6. Compatibility testing (crossmatch):
• Serologic
• Computer or electronic
7. Labeling of blood or blood components with the recipient’s
identifying information and issue
Pre-Transfusion Testing Process
Map
Required Components of
Pretransfusion Testing
Identification, Collection, and
Preparation of Samples

 Proper ID and collection of the patient sample.

 Clerical error is a major cause of transfusion-associated
fatalities .
 incorrectABO groupings and transfusion of ABO
incompatible blood
 Recipient’s ID must always be compared with the blood
request form.
 recipient’s full name and hospital ID number.
 age, DOB, address, sex, &name of requesting
physician
Collection of Patient Samples

 Avoid hemolyzing the sample.

 Serum or plasma may be used for pretransfusion testing.
 About 10 mL of blood is usually sufficient for all testing
procedures.
 Samples should not be taken from IV tubing lines.
 The phlebotomist must initial or sign the label.
Collection of Patient Samples

 When a specimen is received in the laboratory, blood

bank personnel must confirm that the information.
 Receipt of an unlabelled specimen requires that a new
sample be obtained.
 Recipient samples should be tested as soon as possible
after collection.
 Iftesting cannot be performed immediately,
samples should be kept at 1°C to 6°C.
Collection of Donor Samples

 RBCs for donor pretransfusion testing can be prepared

from the segmented tubing through which the donor
blood was collected.
 One technique is to cut the RBC end of the segment
tubing with scissors and use an applicator stick to
remove cells or squeeze the tubing to express a drop.
Testing the Donor Sample

 ABO grouping and Rh typing (including a test for weak

D) must be performed on a sample of donor blood
taken at the time of collection.
 Weak D donors are labeled as Rh positive
 AABB Standards requires a screening test for
unexpected antibodies to RBC antigens on samples
from donors who have a history of transfusion or
pregnancy.
 Confirm the ABO cell grouping on all units and Rh typing
on units labeled Rh-negative. Repeat weak-D testing is
not required.
Testing the Patient Sample

ABO Grouping
 ABO grouping can be performed on slides or in tubes,
using solid-phase RBC adherence or column gel
technology.
 Forward and reverse typing.
 If the patient’s ABO group cannot be satisfactorily
determined and immediate transfusion is required,
group O–packed RBCs should be used.
Testing the Patient Sample

Rh Typing
 The test for weak D is unnecessary when testing
transfusion recipients.
 Weak D recipients are considered Rh negative
 If the Rh type of the recipient cannot be determined
and transfusion is essential, Rh-negative blood should be
given.
 Female patients whose RBCs type as weak D are
considered Rh-positive and may receive Rh-positive
blood during transfusion.
Testing the Patient Sample

Antibody Screening
 Test for so-called unexpected antibodies.
 Detect as many clinically significant antibodies as
possible
 HDN – anti-AB, anti-D, anti-kell, anti-c
 IHTR – anti-A, anti-Kell, anti-Jka, anti-Fya
 DHTR – anti-Jka, anti-E, anti-D, anti-C.
 Alloantibodies are almost always acquired (i.e.
transfusion and pregnancy).
Testing the Patient Sample

Antibody Screening
 Performed with 2 or 3 test cells, all type O negative.
 Only IAT is required.
 To each test cell, patient’s serum is added followed by
AHG.
 An AC is run parallel (but not required) with the test cells
(AHG is added to patient’s RBC and serum).
 The antibody screen may be carried out at room temp
and 37°C, or at 37°C only.
Testing the Patient Sample
Testing the Patient Sample
Testing the Patient Sample
Testing the Patient Sample
Testing the Patient Sample

Antibody Screening
 If no reactions take place with any of the test cells, then
the antibody screen is negative and one may proceed
to crossmatch.
 If any of the cells react in the antibody screen, then the
responsible antibody must be identified.
Testing the Patient Sample

Antibody Identification
 In blood banking, we test “knowns” with “unknowns”

Known: Unknown:
Reagent RBCs + patient serum
Reagent antisera + patient RBCs

 When detecting and/or identifying antibodies, we test

patient serum (unknown) with reagent RBCs (known)
Testing the Patient Sample

Antibody Identification
 ID Panel is basically an extended screening panel

Screening cells Panel cells

• Antibody
• Antibody detection
identification
• Sets of 2 or 3 vials • 11 to 20
Testing the Patient Sample
(Antibody Identification)
 Testing the Patient Sample
(Antibody Identification)

Example: Panel Cell #10 has 9 antigens present: c, e, f, M, s,

Leb, k, Fya, and Jka
Testing the Patient Sample
(Antibody Identification)

Autocontrol
Patient RBCs + Patient serum
Testing the Patient Sample
(Antibody Identification)
Testing the Patient Sample
(Antibody Identification)
Immediate Spin (IS) phase

2+
0
0

Last
tube
Testing the Patient Sample
(Antibody Identification)
LISS and 37°C

2+ 0
0 0
0 0
2+
0
0
2+
0
2+
0
0
Testing the Patient Sample
(Antibody Identification)
AHG phase

2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
0 0 0
And don’t forget….

….add “check” cells

to any negative AHG !
IS LISS AHG CC
37°
2+ 0 0  All cells are
negative at
0 0 0 
AHG, so
0 0 0  add
2+ 0 0  “Check”
0 0 0  Cells

0 0 0 
2+ 0 0 
0 0 0 
2+ 0 0 
0 0 0 
0 0 0 
Testing the Patient Sample
(Antibody Identification)
You have agglutination…now what?
CC

2+ 0 0 
0 0 0 

0 0 0 

2+ 0 0 
0 0 0 
0 0 0 
2+ 0 0 
0 0 0 

2+ 0 0 
0 0 0 
0 0 0 
0 0 0 

??
Testing the Patient Sample
(Antibody Identification)

Interpreting Antibody Panels

 There are a few basic steps to follow when interpreting
panels
1. “Ruling out” means crossing out antigens that did
not react
2. Circle the antigens that are not crossed out
3. Consider antibody’s usual reactivity
4. Look for a matching pattern
Always remember:

An antibody will only react with

cells that have the corresponding
antigen; antibodies will not react
with cells that do not have the
antigen
Here’s an
example:
 1) Ruling Out

2+ 0 0 
0 0 0 

0 0 0 

2+ 0 0 
0 0 0 
0 0 0 
2+ 0 0 
0 0 0 

2+ 0 0 
0 0 0 
0 0 0 
0 0 0 

Cross out antigens that show NO REACTION in any phase; do

NOT cross out heterozygous antigens that show dosage.
2. Circle antigens not crossed
out

2+ 0 0 
0 0 0 

0 0 0 

2+ 0 0 
0 0 0 
0 0 0 
2+ 0 0 
0 0 0 

2+ 0 0 
0 0 0 
0 0 0 
0 0 0 
 3. Consider antibody’s usual
reactivity

2+ 0 0 
0 0 0 

0 0 0 

2+ 0 0 
0 0 0 
0 0 0 
2+ 0 0 
0 0 0 

2+ 0 0 
0 0 0 
0 0 0 
0 0 0 

 Lea is normally a Cold-Reacting antibody (IgM), so it makes

sense that we see the reaction in the IS phase of testing;
 The E antigen will usually react at warmer temperatures
 4. Look for a matching pattern

2+ 0 0 
0 0 0 

0 0 0 

2+ 0 0 
0 0 0 
0 0 0 
2+ 0 0 
0 0 0 

2+ 0 0 
0 0 0 
0 0 0 
E doesn’t match and it’s a warmer rx Ab 0 0 0 

…Yes, there is a matching pattern!

Interpretation

anti-
Lea
Testing the Patient Sample
(Antibody Identification)

Guidelines
 Autocontrol
 Negative – alloantibody
 Positive – autoantibody or DTR (i.e.,alloantibodies)
 Phases
 IS – cold (IgM)
 37° - cold (some have higher thermal range) or
warm reacting
 AHG – warm (IgG)…significant!!
Testing the Patient Sample
(Antibody Identification)

Guidelines
 Reaction strength
 1 consistent strength – one antibody
 Different strengths – multiple antibodies or dosage
 Homozygous = strong reaction
 Heterozygous = weak or even non-reactive
 Panel cells that are heterozygous should not be
crossed out because antibody may be too weak to
react
Testing the Patient Sample
(Antibody Identification)

Guidelines
 Matching the pattern
 Single antibodies usually shows a pattern that
matches one of the antigens (see previous panel
example)
 Multiple antibodies are more difficult to match
because they often show mixed reaction strengths
Testing the Patient Sample
(Antibody Identification)

Guidelines
 Rule of 3
 Positive with 3 cells with the antigen
 Negative with 3 cells without the antigen
 The rule of three must be met to confirm the presence
of the antibody
 Gives a 95% confidence interval
 When multiple antibodies are present, the 3 and 3 rule
must be applied to each specificity
 previous example fulfills the
“rule of three”

2+ 0 0 
0 0 0 
3 Positive 0 0 0 
cells
2+ 0 0 
0 0 0 
0 0 0 
2+ 0 0 
0 0 0 
3 Negative 2+ 0 0 
cells 0 0 0 
0 0 0 
0 0 0 

 Panel Cells 1, 4, and 7 are positive for the antigen and gave a reaction at immediate
spin
 Panel Cells 8, 10, and 11 are negative for the antigen and did not give a reaction at
immediate spin
Testing the Patient Sample
(Antibody Identification)

Guidelines
 Rule of 3
 What if the “rule of three” is not fulfilled?
 If there are not enough cells in the panel to fulfill
the rule, then additional cells from another panel
could be used
Crossmatching

 “Crossmatch” we usually mean “Major” crossmatch,

showing compatibility between recipient serum and
donor RBCs
 Two main functions:
1. It is a final check of ABO compatibility between
donor and patient.
2. It may detect the presence of an antibody in the
patient’s serum that was not detected in the
screening.
Crossmatching

Required before transfusion of any product that contains

at least 2 mL of RBCs
 NEEDED for transfusion of:
 Whole blood
 Red blood cells
 Granulocyte concentrate
 But are NOT needed for transfusion of:
 Plasma (FFP or FP24)
 Platelets (unless heavily contaminated with RBCs)
 Cryoprecipitate
Crossmatching

1. Serologic crossmatch
a. Full (AHG) crossmatch
 IS - 37° - AHG
b. Immediate-spin (abbreviated) crossmatch
 may ONLY be performed if antibody screen is
negative
 99.9% effective in preventing occurrence of
an incompatible transfusion
2. Electronic crossmatch
Crossmatching

Causes of Positive Results in the Serologic

Crossmatch
1. Incorrect ABO grouping of the patient or donor.
2. An alloantibody in the patient’s serum reacting with the
corresponding antigen on donor RBCs.
3. An autoantibody in the patient’s serum reacting with
the corresponding antigen on donor RBCs.
4. Prior coating of the donor RBCs with protein, resulting in
a positive antihuman globulin test.
5. Abnormalities in the patient’s serum.
6. Contaminants in the test system.
Crossmatching

Final clerical checks.

At issue
 Recipient information:
 Two independent identifiers
 ABO group & RhD type
 Donor/product information:
 Donor identification number
 ABO group & RhD type (if required)
 Compatibility testing results (if applicable)
 Special requirements (irradiation, leukocyte reduction, washing,
etc.)
 Issuance and Expiration date/time
Crossmatching

Final clerical checks.

At bedside
 Recipient information:
 Two independent identifiers
 ABO group & RhD type
 Donor/product information:
 Donor identification number
 ABO group & RhD type (if required)
 Compatibility testing results (if applicable)
 Special requirements (irradiation, leukocyte reduction, washing,
etc.)
 Expiration date/time
References

1) Modern Blood Banking & Transfusion Practices 6e by

Denise Harmening, PhD, MT(ASCP)
2) Pretransfusion Testing (Basic Immunohematology Part 2)
Podcast and Lecture by Joe Chaffin, MD
3) Antibody Identification lecture by Renee Wilkins, PhD,
MLS(ASCP)cm
THANK YOU!