Transcript Document 7154409

Practical
Blood Bank
Compatibility Testing
Blood Transfusion Process
Pre-transfusion
 Transfusion
 Post-transfusion

What is compatibility testing?

Also called pretransfusion testing

Purpose:


To select blood components that will not cause harm to the
recipient and will have acceptable survival when transfused
If properly performed, compatibility tests will confirm
ABO compatibility between the component and the
recipient and will detect the most clinically significant
unexpected antibodies
Compatibility testing?

There are several components of compatibility testing
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Proper specimen collection
Reviewing patient transfusion history
ABO, Rh, and antibody testing (screen/ID)
Crossmatching
Actual transfusion
Compatibility testing

Can be divided into 3 categories:


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Preanalytical procedures
Serological testing
Postanalytical procedures
Pre-analytical phases
Patient identification
 Specimen collection
 Review of patient history

Patient Identification
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Must confirm recipient’s
ID from bracelet ON the
patient
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Full patient name and
hospital number
Name of physician
Sample Identification
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The sample should also
have the full patient
name, hospital number,
and physician
Date and time of
collection,
phlebotomist’s initials
All of this should be on
the request form and
the sample
Specimen Tubes
Pink Top - EDTA
Red Top – no additives
Specimen Collection
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Collected in tube with EDTA or no additives
If the venipuncture causes hemolysis, the sample may
be rejected
True hemolysis in the patient is the result of
complement activation
Samples are labeled at the bedside (pre-labeling is not
recommended)
A record of individuals who collect (or test) the
specimens should be documented in order to
“backtrack” in case of an error
Specimen Collection

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If the sample is drawn from an IV line,
the IV infusion should be stopped 5-10
minutes prior to blood drawing and the
first 10 mL discarded
Testing should be performed on samples
less than 72 hours or else complement
dependent antibodies may be missed
(complement can become unstable)
Getting the history


Look at recipient’s records for any
prior unexpected antibodies
Previous transfusion reactions
Serological Testing

3 tests:
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ABO/Rh
Antibody detection/identification
Crossmatch
ABO/Rh Typing
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In the ABO typing, the forward and reverse
MUST match
In the Rh typing, the control must be
negative
Both of these will indicate what type of
blood should be given
Antibody screen and/or ID
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The antibody screen will detect the
presence of any unexpected antibodies
in patient serum
If antibodies are detected,
identification should be performed
using panel cells (with an autocontrol)
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IS
37° (LISS)
AHG
If an antibody is present, units
negative for the antigen must be given
Proceed to the crossmatch…
Crossmatching
 Purpose:
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Prevent transfusion reactions
Increase in vivo survival of red
cells
Double checks for ABO errors
Another method of detecting
antibodies
Crossmatch
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Two types of crossmatches
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Major – routinely performed in labs
Minor – not required by AABB since 1976
Major vs Minor Crossmatch

Why is the minor
crossmatch
unnecessary?
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Donated units are
tested for antibodies
Most blood is
transfused as packed
cells, having little
antibodies
The plasma volume is
small, and Abs will be
diluted in recipient
circulation
Crossmatches
The crossmatch “shall use methods that demonstrate
ABO incompatibility and clinically significant antibodies to
red cell antigens and shall include an antiglobulin phase”
Crossmatch
No agglutination ~ compatible
Donor RBCs
(washed)
Patient serum
Agglutination ~ incompatible
The procedure
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Donor cells are
taken from
segments that are
attached to the
unit itself
Segments are a
sampling of the
blood and
eliminate having
to open the actual
unit
Units of whole blood with segments
attached
Procedure
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ABO/Rh typing is FIRST performed
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Antibody Screen is performed next….
Crossmatch Procedure
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If antibodies are NOT detected:
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Only immediate spin (IS) is performed
using patient serum and donor blood
suspension
This fulfills the AABB standard for ABO
incompatibility
This is an INCOMPLETE CROSSMATCH
If antibodies ARE detected:
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Antigen negative units found and Xmatched
All phases are tested: IS, 37°, AHG
This is a COMPLETE CROSSMATCH
Crossmatches…
 Will
 Will
Not

Verify donor cell ABO
compatibility
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Garantee normal survival of
RBCs

Detect most antibodies
against donor cells
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Prevent patient from
developing an antibody
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Detect all antibodies
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Prevent delayed transfusion
reactions

Detect ABO/Rh errors
Incompatible crossmatches
Antibody
screen
Positive
Negative
Positive
Crossmatch
Cause
Resolution
Negative
Antibody directed
against antigen on
screening cell
ID antibody,
select antigen
negative blood
Positive
Antibody directed
against antigen on
donor cell which may
not be on screening
cell OR donor unit
may have IgG
previously attached
ID antibody,
select antigen
negative blood
OR perform DAT
on donor unit
Positive
Antibodies directed
against both
screening and donor
cells
Antibody ID,
select antigen
negative blood
Additional Information on Types of
Compatibility Tests
Manual (IS and IAT)
Gel Technology
Electronic (Computerized) Cross match
Red cell Affinity Column Technology (ReACT)
Solid Phase Adherence Assays (SPAA)
Manual (IS and IAT)
IS detect RT reactive antibodies (Auto,
Alloantibody, Naturally occuring)
IAT detect IgG antibodies (Auto & alloantibody)
Gel Technology
Patient serum, and 1% of suspended RBCs in LIM are
dispensed
into the microtube and incubated at
o
37 C for 15 minutes.
The card containing the microtubes is then
centrifuged at a controlled speed for 10 minutes.
At the start of centrifugation the cells are separated
from the serum; then they meet the AHG contained
in the microtube.
Finally the cells are trapped by the gel (if
agglutinated) or pellet to the bottom of the tube.
New Technologies…
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The electronic crossmatch
According to the AABB, the following must be fulfilled:
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Critical elements of the information system have been
validated on-site.
No clinically significant antibodies are detected in the current
blood sample and there is no record of clinically significant
antibodies in the past
Computer crossmatch (cont’d)
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The patient's ABO group and Rh type has been
done twice and entered in the computer
The donor ABO/Rh have been confirmed and
entered in the computer. The donor unit
identification number, component name, and
ABO/Rh type must also be entered in the
computer
The computer system will alert the technologist
to ABO & Rh discrepancies between information
on the donor label and results of donor
confirmatory testing
Red Cell Affinity Column Technology
(ReACT)
Based on affinity adherence of coated red cells
in an immunologically active matrix.
Antibody- sensitized red cells bind or adsorbed to
ligands attached to an agarose matrix.
The main ligand is Protein G (prepared from
Group C or G Streptococcus or by recombinant
technology), which has high affinity for all four
IgG subclasses.
Another ReACT ligand is Protein A (from Group A
Staphlococcus), which binds to IgG 1, 2, and 4.
Red Cell Affinity Column Technology
(ReACT)
Positive reaction: the coated red blood
cells with IgG are bound to
immunoreactive gel particles, occurs
mostly at the top of the gel column.
Negative reaction: the red blood cells
are not coated with antibody and pass
through to the bottom of the gel
column.
Solid Phase Adherence Assays (SPAA)
Uses red cell membrane bound to the surfaces of
polystyrene microtitration strip wells, capturing IgG
antibodies (if present) in patient sera.
Patient serum is added to wells coated with screen
cells
Incubated at 37oC for 15 min.
Washing
anti-IgG-coated indicator red cells are added.
centrifuge
SPAA
Post-analytical phase
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Involves labeling, inspecting, and issuing the
blood unit
Labeling form includes patient’s full name, ID
number, Location, ABO/Rh(D) of patient and
unit, donor #, compatibility results, and tech ID
Form is attached to the donor unit and only
released for the recipient
The unit is visually inspected for abnormalities,
such as bacterial contamination, clots, etc
Issuing blood
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When it’s time to release a blood product to the
nurse or physician, a few “checks” must be
done
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Requisition form
Comparing requisition form  donor unit tag 
blood product label
Name of persons issuing and picking up blood
Date and time of release
Expiration date
What if the unit is unused?
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Blood can be returned to the blood bank if it is
not needed for transfusion
Unit closure has to remain unopened
Storage temperature must have remained in the
required range (1° to 10°C for RBCs)
If not at correct temp, unit must be returned
within 30 minutes of issue
Special Circumstances
Emergency Release
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In an emergency, there may not be enough time
to test the recipient’s sample
In this case, blood is released only when signed
by the physician (O negative)
The tag must indicate it is not crossmatched
Segments from the released units should be
retained for X-matching
Every detail is documented (names, dates..)
Emergency Release
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Once the specimen is received, ABO/Rh typing
and antibody screening should be performed
Crossmatching the segments from the released
unit should be tested
In addition, the lab may crossmatch additional
units as a precaution if more blood is needed
If death should occur, testing should be complete
enough to show that the death was unrelated to
an incompatibility
What can be given in an emergency?
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Group O Rh(D)-negative red cells or AB plasma
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Emergency release
Women below or of childbearing age
Group O Rh(D)-positive red cells
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Used as a substitution if O negative is not available
Male or elderly females
Massive transfusion
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Defined as a transfusion approaching or exceeding
the recipient’s own blood volume (about 5 liters or
10-12 units in an adult male) within 24 hour period
The original sample no longer represents the
patient’s condition
Complete Crossmatch not necessary (if no
antibodies were detected originally)
Give ABO identical units
 If antibodies were originally ID’s, continue to give
antigen negative units
Donor Selection: Appropriate donor units to give
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ABO specific blood should always be given first.
When ABO-specific blood is not available or is in less
than adequate supply, alternative blood groups are
chosen as summarized in the following table; (must
be administered as red blood cells).
Patient’s Type
O
A
1st Choice
O
A
Other Choices
None
O
B
B
O
AB
AB
A, O, B only one of the three
should be used for a given patient
Selection of Appropriate Donor Units.
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Rh-negative blood can be given to Rh-positive
patients, however, good inventory
management should conserve this limited
resource for use in Rh-neg recipients.
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If Rh-neg units is near expiration, the unit should
be given rather than wasted.
Selection of Appropriate Donor Units.
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Rh-pos blood should not be given to Rh(D) -neg
women of childbearing age.
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Transfusion of Rh-neg male patients and female
patients beyond menopause with Rh-pos blood
is acceptable as long as no performed anti-D is
demonstrable in the sera.
Major Crossmatch Tests
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It is done both for IgM and IgG antibodies
Requirement:
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Recipient’s serum.
Donor’s red cells taken from the tube attached to
the bag.
A-Saline technique
Saline technique is designed to detect compatibility of
IgM antibody(ies) in patient’s serum against antigens on
donor’s red cells.
Method:
1. Label 1 tube for each donor sample to be tested.
2. Put 2 drop of patient’s serum in labeled tube.
3. Add 1 drop of 2-5% saline suspended red cells of
donor
4. Mix and incubate for 5-10 min. (spin method) or
incubate for 30-60 min (sedimentation method) at
RT.
5. Centrifuge at 1000 rpm for 1 min. in spin method
(after 5-10 min. incubation);centrifugation is
optional in sedimentation method.
6. Read the result, observe for hemolysis and
agglutination.
7. Negative result should be confirmed under
microscope.
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Interpretation
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Agglutination or hemolysis indicates a positive result
(incompatible)
Note: In emergency spin technique is acceptable.
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Saline technique is inadequate as a complete
compatibility test because it is inadequate to detect
clinically significant IgG antibodies.
B- Anti -Human Globulin Test (IAT)
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Indirect anti human globulin test (IAT)
is the most important and widely used
serological procedure
in modern blood banking to test the IgG
compatibility between recipient’s serum
and donor’s cells. The majority of
incomplete antibodies are IgG and are
detected by AHG test.
Method
1. Put 2 drops of patient’s serum in a
labeled tube.
2. Add 1 drop of 2-5 % saline suspended
red cells of donor.
3. Incubate for 30-60 min at 37° C
4. Centrifuge at 1000 rpm for 1 min,
check for hemolysis/agglutination
5. If there is no hemolysis
/agglutination, wash the cells three
times with normal saline.
6. Perform IAT test
•
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•
Add 2 drops of polyspecific AHG serum to
washed cells
Centrifuge at 1000 rpm for 1 minute
See for agglutination
7. Add IgG coated red cells to negative
AHG test.
8. Centrifuge and check for agglutination
- if there is no agglutination test is
invalid.
Interpretation
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Hemeolysis or agglutination at any stage
indicates incompatibility.
Note: Cross-match can be done by two tubes
technique for IgM and IgG separately as
described above or by one tubes in which
donor’ cell and the patient’s serum after
step 5 in saline technique is incubated at
37°C for 20-30 minutes and then do IAT.
In major-cross for IgG antibodies albumin
or enzyme or LISS can be used with IAT to
increase sensitivity. For techniques see
chapter on Antiglobulin Test.
Cross Match – Major Compatibility Test:
1.
2.
3.
4.
5.
6.
Label 3 tubes S1, S2 (Saline) and A1 (Albumin).
To each tube add 2 drops of fresh serum from
recipient.
To each Tube add 2 drops of 5% saline suspension of
donor's Cells.
To tube A1 add 2 drops of Bovine Albumin (22%).
Centrifuge both tubes S1 and A1 for 15 seconds at
3400 rpm.
Read Macroscopically for Haemolysis and/or
agglutination and record results.
ABO incompatibility may be detected in this phase.
7.
8.
9.
10.
11.
12.
13.
14.
Incubate the Tube S1 at room temperature for 15
min (Optional).
Incubate the Tube S2 and A1 in the water bath for
30 min at 37o C.
When the incubation time finished centrifuge the
tube/tubes for 15 second at 3400 rpm.
Read the tube/tubes macroscopically for Haemolysis
and/or agglutination and record results.
Wash Tube A1 with saline 3 times.
Add drops of Anti Human Globulin serum and mix
well.
Centrifuge tube A for 15 second at 3400.
Read for agglutination and record the results.
Interpretation:
If no agglutination of Haemolysis is present in
corssmatch procedure, the blood is regarded
compatible and reported as crossmatch
Negative.
Cross Match – Minor Compatibility Test:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Label a test tube with donor number and recipient's initials.
Add one drop of 2-5% suspension Recipient cells.
Add 2 drops of Donor serum and 1 drop of 22% bovine albumin to
the tube.
Centrifuge immediately 1 min at 1000 rpm.
Read macroscopically for Haemolysis and agglutination.
Incubate at 37o C for 30 minutes.
Centrifuge 1 min at 1000 rpm.
Read macroscopically for Haemolysis and agglutination.
Wash the tube 3 times with saline.
Add 2 drops of anti human globulin serum to the dry cell button.
Centrifuge 1 min at 1000 rpm.
Read macroscopically for Haemolysis and agglutination.
Add Check Cells to all negative tests; spin, read and record results.
Interpretation:
If no agglutination of Haemolysis is present in
corssmatch procedure, the blood is regarded
as serological compatible and reported as
crossmatch Negative.
The Incompatible Crossmatch:
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Although the majority crossmatches will indicates compatibility, problems
still occur. Even if an incompatible is detected before crossmatch has been
carried to the anti-globin stage, the procedure should be completed for
investigational purpose. If blood is urgently needed, additional donor blood
should be crossmatched before starting to investigate the problem.
Rather than continuing to crossmatch blindly, it is always advisable to try to
determine the cause of the incompatibility. However, in emergency
situations, it may be necessary to crossmatch many units of blood of
appropriate ABO group and Rh Type, in the hope that a compatible unit will
be found. In addition, the patient's blood relatives should be tested for
compatibility since there is an increased chance of finding suitable donors
among them.
The antibody should be identified, not only for the present transfusion, but
also to protect the patient in any future transfusions when the antibody
titer may have decreased or even disappeared.
The following Questions and Answers will
help guide subsequent investigations:
1.
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2.
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3.
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4.
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Identification:
Is the "Patient's Blood Specimen" really from the intended recipient?
Was a unit of blood with the correct ABO group and Rh Type Selected?
Does both the blood unit pilot tube have the same identification?
ABO grouping:
Recheck recipient and donor from original specimens using freshly prepared red cell
suspensions. If anti-A1 or anti-H is identified, blood for transfusion should be selected on the
basis of A subgroup.
Rh Type:
Recheck recipient and donor, determination of the Rh phenotype may be helpful in some
cases.
Auto Control:
Test Patients serum with his own cells to determine if the problems are due to blood group
isoantibody, autoantibody, or nonspecific reaction. This control should be run concurrently
with crossmatch or antibody identification.
5.

Stage of incompatibility:
Procedure will be determined to a large extent by the stage at which
the incompatibility is most pronounced, as suggested by the following
table.
Stage of apparent incompatibility
Saline or serum at RT
Saline, Serum or High protein at 37o
C
Antiglobulin or Enzyme

Possible Cause
- ABO Error.
- Cold autoagglutinin
or irregular
agglutinin
- Irregular Antibody
- Autoagglutinin
- Rouleaux
- Other serum direct
Antiglobulin test
- Irregular Antibody
- Autoantibody
- Positive Direct
Antiglobulin test
If some donors are incompatible in an early stage of the crossmatch but
other donors are not incompatible until a later stage, this might indicate
two or more antibodies.
6.
Percentage of incompatible donors:
The approximate percentage of incompatible donors may help in elucidate
the problem, for example, with an antibody reaction n the Antiglobulin
phase: six or seven bloods positive out of 10 bloods tested suggests antiFya; one blood positive out of 10 tested suggests anti-K.

7.
Grading donor reactions:
Are the reactions of incompatible bloods all of the same strength? If not

There may be two or more antibodies of varying strength.
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The antibody may be exhibiting a dosage phenomenon.
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8.
9.
What was the patient diagnosis?
Is the direct Antiglobulin test of either recipient or donor positive?
a)
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If recipient has a positive direct Antiglobulin tests:
Serum may or may not contain autoantibody. If an autoantibody is present, the
serum may react with all donor samples tested. The technique by which the
incompatibility is detected depends upon the type of antibody (cold or warm).
All minor crossmatches will be incompatible.
If the recipient has been recently transfused, the positive Antiglobulin test may
indicate incompatibility of infused donor red cells, specially if the appearance is that
of a mixed filed reaction
b)
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10.
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If donor has a positive direct Antiglobulin test.
Major Crossmatch will be incompatible.
Minor crossmatch may or may not be incompatible.
Other donor units will crossmatch satisfactorily.
Abnormal proteins, autoagglutinin and cold agglutinins. Factors relating to
disease or medication may cause agglutination or pseudo agglutination.
If Rouleaux occurs:
 Check patient's diagnosis and serum protein level.
 Autologous red cells and serum at 22o C and 37o C should give the
same reactions as in the compatibility test.
 Compatibility testing with strong Rouleaux, the saline anti-globulin
crossmatch may be the only reliable test since the Antiglobulin
reactions is not affected by properties of serum that cause Rouleaux.
High protein techniques are affected.

Cold agglutinins are the most common cause of difficulty in compatibility
testing. Although they react best at 4o C, they may cause agglutination in
the room temperature phase of the crossmatch and on immediate
centrifugation of the high protein test. They also may cause a positive
Antiglobulin test, especially in autoimmune disease.


Strong cold autoagglutinin, especially those with wide thermal amplitude, must
be absorbed from the patient's serum since they may mask the presence of
specific blood group antibodies. If the autoantibody is active at 22o C or
lower, it can usually be removed from the serum by placing a fresh recipient
blood specimen in ice and allowing it to clot in the refrigerator.
After the cold active antibody is adsorbed onto autologous red cell, the
absorbed serum is used for antibody detection and compatibility testing. A
suspension of the red cells (For Control) should be prepared from blood that
has not been refrigerated.
11)
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12)
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Does the serum contain irregular antibodies?
Test with reagent blood cells (DiaCell), if this has not been done as part
of the compatibility test, identify any antibodies present.
If the crossmatch is incompatible only with one donor, and antibody
detection tests are negative, the recipient's serum should be tested for
antibodies directed against low-incidence antigens.
Technical Causes of apparent incompatibility (False Positive):
Dirty Glassware
Bacterial Contamination.
Chemical or other contamination or reagents, including saline.
Fibrin clots.
Over-centrifugation.
What to Do With Crossmatch Clues
Crossmatch for Newborns and infants:

In case of Erythroblastosis:
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The corssmatch should include a crossmatch with the mother's serum. If
mother's serum is not available crossmatch with baby's serum.
In ABO incompatibility: choose blood compatible with mother's or group O
cells suspended in-group specific plasma. Perform major and minor
crossmatch.
In Rh incompatibility: Mother and infant are of the same blood group,
transfer with compatible group specific Rh Negative
In Rh incompatibility: Mother and infant are of different blood group, choose
O Rh Negative cells suspended in-group specific fresh plasma.
In Erythroblastosis due to (c): use blood which is c/c also Rho(D).
In case transfusion is to be repeated, use the same group and method as for
the first transfusion
In Case of No Erythroblastosis:


When Infant's RBC is compatible with mother's serum; do crossmatch with
mother's serum.
When infant's RBC's are incompatible with mother's serum use infant's
serum for crossmatch.
Selection of Blood for Exchange transfusion