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6TH ANNUAL WORKSHOP

HEMATOLOGY AND BLOOD TRANSFUSION

05 – 09 MUHARRAM 1424 (08-12 MARCH 2003)

BLOOD BANK PROCEDURES


TABLE OF CONTENTS

Specimen Receipt 1-5


Grading of Agglutination Reactions 6-8
Reagent Quality Control 9-12
ABO Grouping Discrepancies – Resolution 13-16
Antibody Screen 17-22
Crossmatch 23-29
Antibody Identification – Basic Panel 30-38
Antibody Identification – Ficin Panel 39-44
Antigen Typing 45-49
Direct Antiglobulin Testing 50-53
Elusion Acid (Elu-kit) 54-57
Elution – Lui Freeze 58-61
Emergency Transfusion 62-67
Transfusion Reaction Investigations 68-79
W.A.R.M 80-82
Adsorption Technique 83-85
Antibody Titration 86-95
King Faisal Specialist Hospital and Research Center
Blood Bank Internal Policy / Procedure

TITLE / DESCRIPTION: INDEX NUMBER(S):

SPECIMEN RECEIPT 610


EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION:

FEB92 REV11 / AUG02 DP XM

PRINCIPLE

Proper collection and labeling of Blood Bank specimens is of the utmost importance to ensure
patient safety.

POLICY

All samples received in the Blood Bank will be labeled in accordance with AABB Standards 5.11
Samples not meeting the labeling requirements must be recollected.

Although samples of insufficient quantity should generally not be received, it may be necessary
to process these samples when difficulties in collection were encountered.

All specimens are drawn according to Specimen Collection and Processing Policies SCP-02-05,
SCP-02-06 and SCP-02-07.

Rejection of inadequately collected or labeled samples will be handled in accordance with LAB-
OM-01-69 Handling and Documentation of Sample Integrity Issues.

Specimen Collections and Processing will receive all Blood Bank samples initially. Where
necessary they may reject samples not meeting Blood Bank criteria.

A second sample receipt and acceptance will occur in the Blood Bank. At this time, a
Transfusion Service staff member will confirm that all identifying information on the request is in
agreement with that on the sample label. Only after this confirmation can testing begin.

In certain situations it may be necessary to process an inadequately labeled sample, but only
with approval of the Blood Bank Supervisor or Medical Director.

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Blood Bank Internal Policy / Procedure

SPECIMEN

CROSSMATCH ADULT VOL. (mL) PEDIATRIC VOL. (mL)


TYPE OF TUBE
TEST (MINIMUM VOL.) (MINIMUM VOL.)
Type and Screen* Red Top 10 (7) 5 (3)
Crossmatch* Red Top 10 (7) 5 (3)
Ab Titre Red Top 10 (7) 5 (3)
ABO Iso Red Top 10 (7) 5 (3)
DAT Lavender Top 5 (3) 3 (1)
Red Cell Phenotype Red Top 10 (7) 5 (3)
Group Confirmation Red Top 5 (3) 3 (1)
Transfusion Reaction Red Top 10 (7) 5 (3)
Lavender Top 5 (3) 3 (1)
Cord Blood Lavender Top N/A 5 (3)
Fetal Blood Lavender Top N/A 3 (1)
Blood Group only Lavender Top 3 (3) 3 (3)
RhIG Red Top 10 (7) N/A
Lavender Top 5
* For Type and Screens or Crossmatches on newborns (collected by heelstick), a minimum of
two full bullets is required

DONOR PROCESSING ADULT VOL. (mL) PEDIATRIC VOL. (mL)


TYPE OF TUBE
TEST (MINIMUM VOL.) (MINIMUM VOL.)
HIV1/2 Red Top 7 (3) 7 (3)
HTLVI/II Red Top 7 (3) 7 (3)
HBsAg Red Top 7 (3) 7 (3)
RIBA Red Top 7 (3) 7 (3)
Delta Red Top 7 (3) 7 (3)
HIV Antigen Red Top 7 (3) 7 (3)
Western Blot Red Top 7 (3) 7 (3)
(HIV1 & HTLVI)
Viral Marker Red Top 7 (3) 7 (3)
(Combination Order)
PCR Green Top 7

REAGENTS, SUPPLIES, EQUIPMENT

 Specimen labels generated by computer


 Blood Bank Specimen Labeling poster (Appendix I)
 Specimen Revision / Rejection Form (Appendix II)

SAFETY PRECAUTIONS

All blood and blood products must be treated as potentially infectious. Follow universal
precautions detailed in the Laboratory Safety Manual.

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Blood Bank Internal Policy / Procedure

PROCEDURE

1. Upon receipt of a sample, check to ensure that the following requirements have been met:
 tube is firmly stoppered.
 label is firmly attached (sticky label is preferred).
 label information is legible and indelible.
 label information matches the information on the request.
 label information includes:
Patient full name.
Patient Medical Record Number.
Date / time drawn.
I.D. number of phlebotomist.

When all the above criteria are met, the sample may be processed.

1.1 Log-in sample by Accession Number. This will be the default method for
receiving samples and Product orders. Samples should be bar-coded
into the application to avoid transcription errors.

1.2 Open the LOG-IN application and click on the button next to Accession.

1.3 Click RETRIEVE and press ENTER. Patient demographics will populate
the Demographics field and the specimen information will populate the
row of the spreadsheet. A red check mark will display in the row header
of the specimen.

1.4 Enter the correct Collection date, Collection time and Collector ID.

1.5 If there is more than one order associated with an accession then you will
see an ellipsis next to the order.

1.5.1 Click on the ellipsis button to view a list of the tests


ordered.

1.5.2 Click on the ellipsis button next to Cont/Vol to view the list
of containers collected.

1.5.3 Click on the DETAILS button to see all details if there is


more than a single test associated with the accession.

If there are tests associated with the accession that are not Blood Bank tests then it is
VERY important to only receive the correct sample.

1.5.4 This can be done by only ticking the line associated with
the Blood Bank specimen.

1.5.5 Clicking the first ticked line will deselect the ticks and allow
you to select the desired container.

1.5.6 Click on DETAILS again to close.

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Blood Bank Internal Policy / Procedure

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1.6 Multiple accession numbers may be entered on the spreadsheet. Patient


demographics will change for each entry selected. If all specimens have
been entered select a location from the location drop-down box where
you want to log the specimen.

1.7 Click on LOG IN. The status will be changed to Collected and the
specimen will be In-Lab.

1.8 The sample is ready for processing now.

2. Do not receive any sample which:


 the tube is cracked or broken, compromising the integrity of the sample
 the label is inadequately attached.
 the label information is incomplete.
 has inadequate volume.
 the specimen date is different than date collected. (patient samples only).
 the information on label does not match the information on the request. (patient
samples only).

3. If patient samples must be rejected for one of the above reasons or if hemolyzed,
they must be cancelled in Cerner.

3.1 Click on CANCEL ORDERS to select the patient for whom you want to
cancel the order. Alternatively, you can go to TASK and select the
CANCEL ORDERS option.

3.2 The top of the screen indicates the mode of Department Order Entry that
you are currently in either :

Result Entry (default),


Cancel Orders or
Accession Add On

The mode for canceling orders is CANCEL ORDERS.

3.3 Enter the patient MRN.

3.4 Click on the FILTER button, located just beneath the patient’s name.

3.4.1 This mechanism allows you to choose to search on


orders by Laboratory or Radiology. Furthermore, it allows
you to search for all encounters associated with the
patient or only the encounter you chose in the Encounter
Search screen when initially selecting the patient. The
column headers will display.

 Orderable,
 Order Status,
 Departmental,
 Start/Time and
 Order Details

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Blood Bank Internal Policy / Procedure

3.4.2 These can be arranged into a desired order by clicking on


any of the column orders and the system will sort to your
selected criteria.

3.5 Select the order you want to cancel and the cancel format will display in
the Details section. All fields are highlighted and mandatory.

3.6 The Cancel Reason can be selected from the drop-down box or by typing
the first letter of the reason you wish to choose.

3.6.1 At this point it is also essential to add a Chartable


Cancel Comment. The cancel reason selected from
the drop-down box does not pass to the on-line chart
and so a cancel comment needs to be added by
clicking the COMMENT icon.

3.6.2 This will open a Cancel Reason Dialogue Box, Click EDIT
and add a more detailed cancellation reason. This can
be viewed in the patients on-line notes attached to the
cancelled order and also can be seen in Order Result
Viewer.

3.7 Click ADD ORDER TO SCRATCH PAD and then SUBMIT ORDERS,
which will process the cancellation.

3.8 Complete a Specimen Revision / Rejection Form (Appendix II) with all the
required information. Especially important is the phlebotomist ID number,
note this under “other” on the form.

3.9 Forward to the Quality Assurance Coordinator for follow-up.

4. If the sample does not meet criteria but must be accepted, get Medical Director or
Supervisor to approve acceptance. Write a variance report (refer to IPP904).

5. If samples from donors cannot be tested, due to the above reasons, the unit must be
discarded physically in and Lifeline with comment stating why the sample could not
be tested.

PROCEDURE NOTES

1. The sample should not be drawn from the tubing used for infusion of intravenous fluid or
from the contiguous vein, but from a fresh venipuncture site.

2. Sample must be drawn from an arm without I.V. If site not available, draw below I.V.

3. Do not allow nursing staff or phlebotomy staff to re-label improperly labeled specimens.
Specimens must be recollected.

4. Do not accept samples for crossmatching and / or Type and Screen if the specimen
date is not the same as the date collected.

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Blood Bank Internal Policy / Procedure

Exception: This does not apply to group confirm samples, cord bloods, or samples
ordered and drawn close to midnight. The collection date must be
handwritten if it doesn’t match the specimen date.

5. If computer-generated labels are not available, imprinted labels (e.g. addressograph


labels) may be used provided all required information is included. The following items
must be handwritten, if they are not included on the addressograph label:
 The specimen number.
 Specimen date.
 Collection time.
 ID # of the person drawing the specimen.

6. Store crossmatch samples for 14 days, donor clot tubes for 1 month and donor segments
for 3 months.

7. Store patient samples for viral marker testing at 1-6°C in walk-in refrigerator #10 in bags
labeled with date of testing for at least 14 days after testing. Refer to viral marker
manufacturer inserts for suitability of sample for testing.

8. If the specimen is improperly labeled, phone floor to explain what is wrong. Send
floor a copy of the Blood Bank Specimen Labeling poster (Appendix I).

9. The Blood Bank Quality Assurance Coordinator will forward the Specimen Revision /
Rejection Form to Laboratory and Nursing Quality Improvement Coordinators who
will initiate the corrective action.

REFERENCE

1. Accreditation Requirements Manual, 6th edition, Washington, DC: American Association


of Blood Banks, 1995.

2. Standards for Blood Banks and Transfusion Services, 21st Edition. Bethesda, MD:
American Association of Blood Banks, 2002.
th
3. Technical Manual, 13 Edition, Bethesda, MD: American Association of Blood
Banks, 1999.

4. Cerner HNA Millenium Integrated Clinical Information System PathNet User Training
st
Manual, 1 Edition, King Faisal Specialist Hospital and Research Center, July 2002.

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Blood Bank Internal Policy / Procedure

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Blood Bank Internal Policy / Procedure

TITLE / DESCRIPTION: INDEX NUMBER(S):

GRADING OF AGGLUTINATION REACTIONS 624


EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION:

JAN92 REV8 / OCT01 DP XM

PRINCIPLE

The grading of agglutination reactions will be standardized among the members of the Blood
Bank staff in the interest of uniformity and reproducibility of test results. Scoring of reactions
will only be used for antibody titration.

POLICY

All Blood Bank staff will assign numerical values (grades) to reactions observed. Score values
will be assigned for antibody titrations.

Correct grading will be verified for each staff member during their annual competency
assessments.

REAGENTS, SUPPLIES AND EQUIPMENT

 Agglutination viewer
 Microscope

SAFETY PRECAUTIONS

All blood and blood products must be treated as potentially infectious. Follow universal
precautions detailed in the Laboratory Safety Manual.

PROCEDURE

1. Spin the cell / serum mixtures at a time appropriate to the calibration of the centrifuge
(refer to time posted on the centrifuge being used).

2. Observe for presence of hemolysis.

3. Gently shake the tube and disrupt the cell button in the tube.

4. Observe the cells as they disperse.

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5. If agglutination is present, record the reactivity by comparing the agglutinates to


descriptions in the table under Reporting Results.

6. Record the results of the test immediately after observations are made.

REPORTING RESULTS

SCORE
GRADE VALUE APPEARANCE

H Red supernatant, few or no intact red cells.

PH Pink supernatant, some intact red cells.


A single agglutinate. No free cells detected. Clear
4+ 12 background.
Strong reaction. A number of large agglutinates.
3+ 10 Clear background.
Large agglutinates in a sea of smaller clumps.
2+ 8 Clear to slightly cloudy background.
Numerous small agglutinates. Cloudy red
1+ 5 background.
Very small agglutinates easily dispersed. Cloudy
W red background.
Macroscopically: appears negative.
M Microscopically: A few agglutinates in most fields.

PROCEDURE NOTES

1. Serum surrounding the centrifuged cell button must be observed for hemolysis.
Hemolysis is generally regarded as a positive reaction, but may be the consequence of
bacterial or chemical contamination and should not be interpreted as a positive result
without further investigation. Most often, antibodies capable of producing hemolysis have
specificity in the ABO, P, Lewis, Kidd, or Vel blood group systems.

2. Mixed field reactions (small – medium aggregates in a field of otherwise unagglutinated


cells) should be noted as such, as well as graded e.g. 2;MF.

REFERENCES

1. Technical Manual, 13th Edition. Bethesda, MD: American Association of Blood Banks,
1999.

2. Rudman, Sally V, Textbook of Blood Banking and Transfusion Medicine. Philadelphia,


PA: W. B. Saunders Company, 1995.

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Blood Bank Internal Policy / Procedure

TITLE / DESCRIPTION: INDEX NUMBER(S):

REAGENT QUALITY CONTROL 760


EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION:

SEP91 REV4 / JAN99 XM


(Reviewed OCT01)

PRINCIPLE

Blood Bank reagents are subject to extensive quality control testing by the manufacturer, but
factors such as duration of storage, severe changes in temperature and contamination during
usage may affect the integrity and performance of the product.

Daily Quality Control testing is performed to confirm that reagents are giving expected results
and to provide a means to recognize deteriorating potency or reactivity of reagents.

POLICY

In accordance with AABB Standard 5.1.3 and Code of Federal Regulations Title 21 Part
606.65(c), quality control of reagent red blood cells and antisera will be performed on each day
of use. Records of this quality control testing will be retained for five years in compliance with
AABB Standard 6.0.

REAGENTS, SUPPLIES, EQUIPMENT

1. Gamma RQC Kit, consisting of:


 A1B D-negative cells 3-4% in Alsevers solution
 O D-positive cells 3-4% in Alsevers solution
 Antibody reagent - polyclonal / monoclonal antibodies in 6% bovine albumin

2. Routine blood grouping reagents, consisting of:


 Anti-A, Anti-B
 Anti-D
 A1 and B cells

3. Routine Antibody Detection Reagents, consisting of:


 Antibody screening cells
 Antibody enhancement media
 Antihuman globulin reagent
 Coombs control cells
 6% bovine albumin

4. Equipment:
 12 x 75 mm glass test tubes
 Serofuge and/or automatic cell washer
 Agglutination viewer
 37C heat block or water bath

5. Reagent Quality Control Weekly Record Sheet (Appendix I)

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SAFETY PRECAUTIONS

1. All blood and blood products must be treated as potentially infectious. Follow universal
precautions detailed in the Laboratory Safety Manual.

2. Do not attempt to pick up broken glass with fingers. Use a dustpan or other scooping
implement and dispose of glass fragments in sharps disposal container.

PROCEDURE

1. Locate the Reagent Quality Control Weekly Record Sheet (Appendix I) specific to your
reagent rack number in the “Daily QC” folder. If filling out a new Record Sheet, record
RQC lot number, expiry date and rack number at the top.

2. Under "Reagent Identification," record the manufacturer, lot number, and expiry date of
the reagents in use in your rack.

3. If any reagents are expired, replace them with in-date reagents. If no in-date reagents are
available, refer to Expired Reagents/Products Certification IPP 113.

4. Examine the visual appearance of the reagents in use for hemolysis, turbidity,
discoloration, etc.

4.1 If the visual appearance is satisfactory, under "Reagent Appearance” mark a tick in
the appropriate column.

4.2 If the visual appearance is NOT satisfactory, under "Reagent Appearance” put an
“X” in the appropriate column and replace the reagent. Make a note of the change
in the “Comment” section.

5. Record any changes of lot numbers on the blank lines under “Reagent Identification”.
6. Label ten (10) 12 x 75 mm test tubes from 1 to 10, which will contain the following:

Tube Test Reagent # drops RQC reagent # drops LISS # drops

#1 Anti-A 1 A1B, D negative cells 1 no 0

#2 Anti-B 1 A1B, D negative cells 1 no 0

#3 Anti-D 1 A1B, D negative cells 1 no 0

#4 Anti-D 1 O, D positive cells 1 no 0

#5 A1 cells 1 Antibody reagent 2 no 0

#6 B cells 1 Antibody reagent 2 no 0

#7 Screening Cell 1 1 Antibody reagent 2 yes 2

#8 Screening Cell 2 1 Antibody reagent 2 yes 2

#9 Screening Cell 3 1 Antibody reagent 2 yes 2

#10 6% Albumin 1 A1B, D negative cells 1 no 0

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7. Add one drop of reagent antisera or red cells to be tested to the appropriately labeled
tube.

8. Add one drop of the corresponding RQC cells or 2 drops of the RQC Antibody reagent to
the appropriately labeled tube.

9. Mix all tubes and centrifuge for time indicated on centrifuge (20 seconds). Examine
macroscopically for agglutination and record on the Record Sheet. Grade reactions
according to Grading of Agglutination Reactions IPPD 624.)

10. Add two drops of LISS additive to tubes 7, 8 and 9.

11. Incubate tubes 3, 7, 8, 9 and 10 for 15 minutes at 37C. Centrifuge and examine for
hemolysis and/or agglutination. Record on Record Sheet. Wash 4 times on automatic
cycle in cell washer (CW2), then add 2 drops of anti-IgG AHG reagent. Centrifuge and
observe for agglutination macroscopically. Record results on Record Sheet.

12. Add one drop of Coombs control cells to all negative AHG tubes. Centrifuge and examine
macroscopically for agglutination. Record results on Record Sheet.

REPORTING RESULTS

1. Interpret results using Record Sheet “Expected Rx” as a guide. If any results are not
acceptable, do not use the reagents. Exchange the reagent(s) in question for a new vial.
Repeat the quality control test. If still not acceptable, report to the Senior in Crossmatch.

2. If QC was not done on the previous day or days, write “Not In Use” in the column(s).

3. When a new lot number of reagent red cells is placed in use, use a new Weekly Record
Sheet rather than recording the new numbers on the previous form. On the previous
form, write “See Other Sheet” in the blank spaces. On the new form, write “See Other
Sheet” in the blank spaces.

4. The Senior Technologist will review, date and initial each Record Sheet at the end of each
week.

5. Tests performed with these reagents are not valid unless the quality control results are
acceptable.

6. Record Sheets will be retained for 5 years.

PROCEDURE NOTES

1. Test each reagent rack each day of use. Make a note on Record Sheet indicating a rack
is “NOT IN USE” for each day the rack is not used.

2. Use a new Weekly Record Sheet at the beginning of each week. Blank forms are in
the front of the Daily Reagent QC binder.

3. When there are multiple components within a reagent kit, the components will only
be used with kits of the same lot numbers.
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REFERENCES

1. Accreditation Requirements Manual, 5th edition, Bethesda, MD: American Association of


Blood Banks, 1994, pages 23-24.
th
2. Technical Manual, 13 Edition, Bethesda, MD: American Association of Blood Banks,
1999.

3. Product Insert, RQC Kit (Reagent Quality Control). Gamma Biologicals Inc. January 1996.
th
4. Standards for Blood Banks and Transfusion Services, 20 Edition. Bethesda, MD:
American Association of Blood Banks, 2000.

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TITLE / DESCRIPTION: INDEX NUMBER(S):

ABO GROUPING DISCREPANCIES - RESOLUTION 702


EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION:

DEC91 REV12 / AUG02 XM

PRINCIPLE

Under most circumstances individuals possess ABO antibodies directed against those ABO
antigens absent from their own cells. Discrepancies, however, do occur and may result from
technical errors, from intrinsic properties of the red blood cells or from intrinsic properties of the
serum. Resolution of ABO problems may depend upon information about the patients age,
diagnosis, medication, and history of pregnancy and transfusion.

POLICY

In accordance with AABB Standards 5.12.1 and 5.12.2 blood shall not be released for a patient
until any discrepancy in patient’s ABO grouping is resolved. If blood must be transfused prior to
resolution of the discrepancy, group O red blood cells will be issued.

SPECIMEN

 Freshly drawn clotted whole blood (Preferred)


 Anti coagulated (EDTA) specimen

REAGENTS, SUPPLIES, EQUIPMENT

 Anti-A, anti-B and Anti-AB


 A1 , A2 and B cells
 Anti-Human Globulin
 Screening cells
 ABO compatible cord serum or plasma
 Anti-A1 (lectin)
 Other antisera and neutralizing substances as indicated.
 Refrigerator (4-6C)
 Resolution of ABO Discrepancies Worksheet (Appendix I)

SAFETY PRECAUTIONS

All blood and blood products must be treated as potentially infectious. Follow safety
precautions detailed in the Laboratory Safety Manual.

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QUALITY CONTROL

1. Routine daily quality control of antisera and cells. Refer to Reagent Quality Control
IPP760.

2. All other anti-sera used must be tested against cells positive and negative for the
corresponding antigen the same day of testing. All reagent red cells must be checked
against the corresponding antisera the same day of testing (refer to Antigen Typing
IPP710).

PROCEDURE

1. If the discrepancy exists between a previous blood group and the current group, order a
third sample with a new accession number under DEPARTMENT ORDER ENTRY and
LABORATORY ORDER ENTRY, and repeat the testing.

1.1. If the new sample confirms the original blood group, the second sample was
incorrectly drawn. Complete a variance report with printouts of the testing results.

1.2. If the new sample confirms the discrepancy, refer to the Senior Technologist or
Supervisor for resolution. After making printouts of the original testing results and
medical record data, remove the historical ABO/Rh. Complete a variance report.

2. If the discrepancy exists between the forward group and the reverse group, obtain patient
history including diagnosis, treatment, medications, and transfusions. Then proceed as
follows:

3. UNEXPECTED NEGATIVE REACTIONS:

3.1. IN REVERSE GROUP

3.1.1. Repeat the testing of both serum and cells with the following
modifications to the reverse group:

3.1.2. Add autocontrol.

3.1.3. Use three to four drops of serum per tube.


o
3.1.4. Incubate for 30 min. at RT, then if negative, 15 minutes at 4 C.

3.1.5. Read microscopically, comparing result against autocontrol.

3.2. IN FORWARD GROUP

3.2.1. Repeat the testing with the following modifications:

3.2.2. Wash patient cells 2-3 times.

3.2.3. Test with different antibody source.


o
3.2.4. Incubate for 30 min. at RT, then if negative, 15 minutes at 4 C.

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3.2.5. Read microscopically, comparing result against negative saline


control.

3.2.6. Look for mixed field and evidence that the patient has been
transfused with group O blood.

3.2.7. Absorb/elute with human source reagents may help to establish true
group.

4. UNEXPECTED POSITIVE REACTIONS

4.1. IN REVERSE GROUP


o
4.1.1. Incubate reverse group reactions for 5 minutes at 37 C to dissipate
any cold non-significant reactivity.

4.1.2. Perform antibody screen, including auto control.

4.1.3 If all screening cells are positive including the positive control

4.1.3.1 If AHG phase is negative, suspect rouleaux

4.1.3.1.1 Repeat positive reactions and read using saline replacement technique
(Technical Manual 13th Ed).

4.1.3.2 If AHG phase is positive, an autoantibody may be present.

4.1.3.2.1 If cold autoantibody, prewarm the cells and serum at 37°C prior to testing.

4.1.3.2.2 Autoabsorb serum with autologous cells and repeat reverse group, antibody
screen and crossmatch. [See W.A.R.M. (Warm Autoantibody Removal
Medium) IPP781].

4.1.4 If one or more screening cells are positive

4.1.4.1 Suspect unexpected antibody in serum. Perform antibody identification according to


Antibody Identification – Basic Panel IPP 707.

4.1.4.2 Type A cells and B cells used in the reverse typing for the corresponding antigen.

4.1.4.3 Repeat reverse group with:

4.1.4.3.1 A and B cells negative for corresponding antigen OR

4.1.4.3.2 Serum neutralized with appropriate neutralizing substance.

4.1.5 If screen is negative

4.1.5.1 Suspect presence of Anti-A1

4.1.5.2 Test serum against A2 cells. Incubate for 5 minutes.

4.1.5.3 Centrifuge at calibrated spin time and read macroscopically.

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4.1.5.4 If Anti-A1 is identified type patient with anti-A1 (refer to Antigen


Typing IPP 710).

4.2. IN FORWARD GROUP

4.2.1 Wash patient cells 3 times with warm saline (37°C) and repeat testing.

4.2.2 If Test Results Now Coincide With Reverse Group

4.2.2.1 Results caused by cold agglutinin or rouleaux.

4.2.3 If Test Results Are Still Discrepant

4.2.3.1 Repeat test with different source reagents.

4.2.3.2 Suspect acquired B if the patient is group A.

4.2.3.3 Perform DAT. Suspect protein-induced agglutination if the DAT is positive.


Look for mixed field agglutination due to blood group chimera, recent
transfusion, contamination of sample, recent bone marrow transplant.

4.2.3.4 Retest with fresh ABO compatible human adult and cord serum. If the adult
serum is positive and the cord serum negative suspect polyagglutinable RBC.

REPORTING RESULTS

1. Record the initial reactions observed on the Resolution of ABO Discrepancies worksheet
(Appendix I) under Initial Blood Group Reactions.

2. Record any repeat testing performed, on a new specimen or treated specimen (e.g.
prewarmed or absorbed) or if alternative reagents (e.g. human source vs. monoclonal, or
antigen typed reverse cells) are used, on the worksheet under Repeat Testing after
Sample Change / Modification. The initial results will indicate what repeat testing is
required, if any.

3. Record the results of an antibody screen or anti-A1 identification on the worksheet in the
appropriate places. If a further panel is required, attach the completed panel sheet to the
worksheet and record the antibody identified on the worksheet.

4. Complete and leave the discrepancy worksheet and any panel sheets or antigen typing
forms for Senior review. All work will be reviewed by Supervisor and Blood Bank
physician prior to filing in Immunohematology binders.

5. Enter final results in computer under BLOOD BANK RESULT ENTRY and the accession
number. Enter reactions with Anti-A, Anti-B, A1 Cells, and B Cells and the ABO
interpretation in the computer. Enter free text comments, by selecting the COMMENTS
icon, as necessary to explain the additional procedures used to obtain the final results. If
anti-A1 is identified, enter results under antibody screen interpretation.

6. Enter antigen typing into computer according to Antigen Typing IPP 710.

Page 18 of 95
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PROCEDURE NOTES

1. Eliminate technical errors by repeating the test with strict adherence to proper procedure
and technique. Use quality controlled reagents, appropriate positive and negative
controls, careful observation, recording and interpretation of reactions.

2. Until correct ABO group can be determined, the patient must be transfused with O cells (if
additional sample is required for investigation, have it collected prior to transfusion).

3. In most cases, the correct ABO group of a patient can be determined once the causes of
discrepancies have been recognized and resolved or interpreted correctly. Problems that
cannot be resolved after investigation should be referred to the supervisor.

4. Recorded discrepancies (e.g. following bone marrow transplant, immunosuppressed


children etc.) do not require reinvestigation each time a specimen is received.

5. Enter identification of anti-A1 under antibody screen results.

6. Refer to AABB Technical Manual (latest edition) for help with difficult resolutions.

REFERENCES
th
1. Technical Manual, 13 Edition, Bethesda, MD: American Association of Blood Banks,
1999.

2. Standards for Blood Banks and Transfusion Services, 21st Edition. Bethesda, MD:
American Association of Blood Banks, 2002.

3. Cerner HNA Millenium Integrated Clinical Information System PathNet User Training
st
Manual, 1 Edition, King Faisal Specialist Hospital and Research Center, July 2002.

Page 19 of 95
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Blood Bank Internal Policy / Procedure

TITLE / DESCRIPTION: INDEX NUMBER(S):

ANTIBODY SCREEN 706


EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION:

DEC91 REV10 / AUG02 XM

PRINCIPLE

Immunization to foreign red cell antigens may occur through pregnancy, transfusion or
deliberate injection with immunization materials. Antibody screening is performed on:

1. Obstetric patients: to identify women with allo-antibodies that might cause hemolytic
disease of the newborn.

2. Potential candidates for transfusion: to detect allo-antibodies that might cause a hemolytic
transfusion reaction.

3. Donors: to detect allo-antibodies that might cause passive transfer of antibody.

Antibody screening tests allow serum of the patient or donor to react with selected red blood
cells under conditions that demonstrate antibodies active at 37C. The antiglobulin phase is
required.

POLICY

Antibody screens will be performed on all samples received with a request for transfusion, and
when specifically requested. In accordance with AABB Standards 5.12.3, samples will be
tested by a method that will demonstrate all clinically significant unexpected antibodies.
o
Pretransfusion testing will include 37 C incubation and an antiglobulin phase using cells that
are not pooled. IgG sensitized cells will be added to all antiglobulin tests interpreted as
negative.

If the patient has been transfused in the preceding 3 months with blood or a blood component
containing red blood cells or has been pregnant within the preceding 3 months or if the history
is uncertain or unavailable, the sample must be obtained from the patient within 3 days of the
scheduled transfusion. Antibodies that are known to exist need not be re-identified; it is
necessary to run a selected cell panel to exclude any additional clinically significant unexpected
antibodies.

SPECIMEN

Serum and cells from 10 mL (minimum 7 mL) clotted whole blood.

Page 20 of 95
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Blood Bank Internal Policy / Procedure

REAGENTS, SUPPLIES, EQUIPMENT

 12 x 75 mm glass test tubes


 Saline (0.9%)
 LISS additive
 Anti-human serum globulin reagent
 Antibody Screening cells
 IgG sensitized RBC's
 Serofuge and/or automatic cell washer
 Agglutination viewer
 Microscope
 37C heat block or water bath
 Disposable pipettes

SAFETY PRECAUTIONS

1. All blood and blood products must be treated as potentially infectious. Follow universal
precautions detailed in the Laboratory Safety Manual.

2. Do not attempt to pick up broken glass with fingers. Use a dustpan or other scooping
implement and dispose of glass fragments in sharps disposal container.

QUALITY CONTROL

Reagent Quality Control IPP 760.

PROCEDURE

1. Place two drops of serum to be tested into three properly labeled test tubes (one tube for
each screening cell I, II, III).

2. Add one drop of 2-5% suspension of red cells to the corresponding tube and mix.

3. Add two drops of LISS additive and mix.

4. Incubate at 37C for 15 to 30 minutes in the dri-bath incubator.

5. Centrifuge immediately upon completion of incubation. Examine for hemolysis. Dislodge


the cell button and observe for agglutination. Record results in computer.

6. Wash 4 times with saline. After last wash decant completely.

7. Add 2 drops of anti-human globulin reagent and mix.

8. Centrifuge. Examine macroscopically with agglutination viewer for agglutination. Record


results in computer.

Page 21 of 95
King Faisal Specialist Hospital and Research Center
Blood Bank Internal Policy / Procedure

9. To all negative tests, add 1 drop of IgG sensitized red cells. Centrifuge and examine for
agglutination. If no agglutination is present, test must be repeated. Record results in
computer.

Interpretation of results

1. A negative test indicates that the serum tested has no antibodies that react with the
screening cells.

2. When a positive antibody screening result is obtained, proceed with Antibody Identification
- Basic Panel IPP 707.

3. When there is a discrepancy between the results of the antibody screening and the
crossmatch, (i.e., reagent red cells are positive and the crossmatch is negative, or vice
versa) one of the following antibodies may be responsible:

3.1 Anti-H, in the serum of A1 and A1B individuals, may agglutinate all group "O" reagent
cells and A2 cells. Group A1 and A1B cells have very little H antigen.
bH
3.2 Anti-Le reacts with group "O" Le(b+) cells, but not with A1 or A1B cells that are
Le(b+).

3.3 Anti-A1 in the serum of A2 individuals will test negative with group O cells (antibody
screen), while A1 donor cells will be positive (80% of A’s are A1).

3.4 Antibody reactive with a low frequency antigen. Low incidence antigens present on
screening cells or donor cells cause sporadic positive results.

3.5 Antibody reactive only with cells homozygous for the corresponding antigen.

4. If a negative result is obtained when the presence of an antibody is suspected, one of the
following enhancement methods may be used:

4.1 Enzymes: use Ficin Panel (see Antibody Identification Ficin Panel 708).

4.2 Increased incubation time: Do not incubate more than 30 minutes with LISS.

4.3 Increased ratio of serum to cells: Do not do this with LISS.

REPORTING RESULTS

1. See procedure for Grading of Agglutination Reactions IPP 624.

2. Record all results as soon as they are read.

3. Record results in the computer under BLOOD BANK RESULT ENTRY.

3.1.1 Complete the New Worksheet Dialogue box. Select the accession
number format, optionally name the worksheet and press OK.

3.1.2 Enter the accession number associated with the Antibody Screen
procedure to be resulted.

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3.1.3 The system will retrieve the order information and reference data
for use in the worksheet.

3.1.4 When this data has been displayed, you can enter additional
accession numbers.

3.1.5 Click on, or use the arrow key to move to the first cell to be
resulted, represented by a white cell in the worksheet.

3.1.6 Enter or select a result from the list. To enter a result, type the
first letter or number associated with the result (e.g. type 1 for 1+,
n for Not Performed) or click the down arrow key to display the list
of options.

3.2 Press ENTER to move to the next result cell.

3.2.1 For negative antibody screens, the system will automatically


interpret the AB Screen Interpretation.

3.2.2 For positive antibody screens, or incomplete antibody screen


results, the AB Screen Interpretation will not automatically
interpret and a warning “Pattern match not found” will be
displayed.

3.2.3 The result of the AB Screen Interpretation will have to be entered


manually either by:

 Typing the first letter of the result, N-Negative or P-Positive or


 Using the drop down box and selecting the result

3.2.4 The system will display the Blood Bank Exception dialogue box.

3.2.5 To accept the result, select YES in the Override group box, then
select a reason for the override and Click OK to return to Result
Entry.

3.10 Click PERFORM or VERIFY to save the results.

4. The Coombs Control cell results must be weakly positive (1+ or 2+). Repeat any
test that is negative with Coombs Control cells.

PROCEDURE NOTES

1. Although the minimum incubation time quoted for the Ortho Antibody Enhancement
o
Solution (LISS) is 10 minutes at 37 C, incubate for a minimum of 15 minutes as Dri-baths
are not thought to heat as quickly as waterbaths.

2. The specimen expiration time may be extended for up to 7 days if it is certain the patient
has not been transfused or pregnant in the past 3 months. Do not rely on computer
transfusion history as the patient may have been transfused elsewhere or have been
pregnant. This is an exception that requires the Medical Director’s approval.

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LIMITATIONS OF PROCEDURE

1. Using LISS additive, the ionic strength of the test system is dependent on the amount of
serum used. Therefore it is not acceptable to increase the serum-to-cell ratio as it will
increase the ionic strength and weaken the sensitivity.

REFERENCES

1. AABB Technical Manual, 13th Edition. Bethesda, MD: American Association of


Blood Banks, 1999.

2. Standards for Blood Banks and Transfusion Services, 21st Edition. Bethesda, MD:
American Association of Blood Banks, 2002.

3. Cerner HNA Millenium Integrated Clinical Information System PathNet User Training
st
Manual, 1 Edition, King Faisal Specialist Hospital and Research Center, July 2002.

Page 24 of 95
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Blood Bank Internal Policy / Procedure

Page 25 of 95
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Blood Bank Internal Policy / Procedure

TITLE / DESCRIPTION: INDEX NUMBER(S):

CROSSMATCH 722
EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION:

DEC91 REV12 / AUG02 XM

PRINCIPLE

The crossmatch is an in-vitro procedure to determine serologic compatibility between the


donor's red cells and the recipient's serum. This is accomplished by adding a suspension of
donor cells to the recipient serum and observing for agglutination and/or hemolysis in the
various phases.

Massive transfusion is defined as infusion, within a 24-hour period, of a volume of blood


approaching or exceeding replacement of the recipients total blood volume (approximately 10
units). So little is left of the patient’s original blood that complete crossmatching has little or no
benefit.

POLICY

Prior to issuing blood, the patient history must be reviewed, and the review documented, to
compare current ABO/Rh with historical results and to check for history of antibodies or severe
reactions.

In accordance with AABB Standards 5.13.1.1, an immediate spin crossmatch is acceptable for
patients with no currently detectable or history of previously identified clinically significant
antibodies.

The following antibodies are considered clinically insignificant and require immediate spin
a b a a
crossmatching only: P1, M, N, Le , Le , Sd , Bg , and A1, if they are only reactive at the
immediate spin phase OR if they become negative upon prewarming the 37C and the AHG
o
phases. If these antibodies are still reactive at 37 C or AHG phase using a prewarming
technique, they will be considered clinically significant and therefore antigen negative, full
crossmatch compatible units are required.

Patients with currently detectable or previously identified clinically significant antibodies must
receive full crossmatch compatible antigen negative units. Patients who have received RhIG
will have full crossmatch compatible units while the anti-D is detectable, once the anti-D is no
longer detectable, an immediate spin crossmatch is acceptable.

In accordance with AABB Standards 5.15.4 and internal policies, massively transfused patients
will receive ABO compatible blood which is antigen negative for any existing or pre-existing
antibodies. The unit group will be confirmed prior to issue and normal crossmatch procedure
will be resumed once a 24-hour period has elapsed since the massive transfusion protocol was
initiated.

Page 26 of 95
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SPECIMEN

See IPP’s 610 - Specimen Receipt- and 787 - Pretransfusion Testing - for specimen labeling
and request requirements and acceptance criteria

 Serum and cells from clotted whole blood: obtained from patient within 3 days of
scheduled transfusion.
 Donor cells from segment originally attached to the unit being crossmatched.

REAGENTS, SUPPLIES, EQUIPMENT

 12 mm x 75 mm glass test tubes


 Disposable Pasteur pipettes
 Saline (0.9%)
 Anti-Human Serum
 IgG Sensitized RBC's
 Serofuge and/or Automatic Cell Washer
 Agglutination Viewer
 Microscope
 37C Heat Block or Water Bath
 LISS additive
 Polyethylene Glycol (PEG)

SAFETY PRECAUTIONS

1. All blood and blood products must be treated as potentially infectious. Follow universal
precautions detailed in the Laboratory Safety Manual.

2. Do not attempt to pick up broken glass with fingers. Use a dustpan or other scooping
implement and dispose of glass fragments in sharps disposal container.

QUALITY CONTROL

See Reagent Quality Control IPP760

PROCEDURE

1. Immediate Spin Crossmatch

Note: If the patient has currently detectable or a history of clinically significant


antibodies (see Procedure Note 1) the Immediate Spin crossmatch is NOT
sufficient. Perform a full crossmatch.

Note: The immediate spin crossmatch is intended to detect ABO incompatibility.


Do not use for neonates (less than 4 months of age) or patients with current
reverse type interpretations of less than 2+; instead perform a Group Check
on the unit.

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Blood Bank Internal Policy / Procedure

1.1 Place 2 drops of recipient serum in a properly labeled test tube (1 tube for each unit
to be crossmatched).

1.2 Add 1 drop of 3-5% saline suspension of donor red cells. Mix.

1.3 Centrifuge immediately. Examine for hemolysis. Dislodge the cell button and
observe for agglutination macroscopically. Record the results in the computer.

2. Coombs (AHG) Crossmatch

Note: If the patient has currently detectable or a history of clinically significant


antibodies (see Procedure Note 1) the Immediate Spin crossmatch is NOT
sufficient. Perform a full crossmatch on units lacking the corresponding red
cell antigens. Refer to Antigen Typing IPP 710.

2.1 Make a 3-4% twice-washed donor red cell suspension.

2.1.1 Place 4 drops of donor whole blood from segment into a


test tube.

2.1.2 Fill the test tube with 0.9% normal saline and mix gently
with a pipette.

2.1.3 Centrifuge for 20 – 30 seconds.

2.1.4 Aspirate and discard the saline supernatant, leaving donor


red cells in the test tube.

2.1.5 Repeat steps 2.2.2 to 2.2.4 for the second wash.

2.1.6 Add 0.9% normal saline to the donor red cells making a 3-
4% red cell suspension.

2.2 Place 2 drops of recipient serum in a properly labeled test tube (1 tube
for each unit to be crossmatched).

2.3 Add 1 drop of 3-4% saline suspension of twice-washed donor red cells. Mix.

2.4 Spin and observe for agglutination or hemolysis. Read and record results.

2.5 Add 2 drops of LISS additive.

2.6 Incubate at 37C for 15 to 30 minutes.

2.7 Centrifuge immediately upon completion of incubation. Examine for hemolysis.


Dislodge the cell button and observe for agglutination macroscopically. Record
results in computer.

2.8 Wash 4 times with saline. After last wash, decant completely.

2.9 Add 2 drops of anti-human globulin and mix.

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2.10 Centrifuge. Dislodge the cell button and examine macroscopically for agglutination
or hemolysis using the agglutination viewer. Record results in the computer
immediately after reading.

2.11 To all negative tests, add 1 drop of IgG sensitized red cells. Centrifuge and examine
macroscopically for agglutination. If no agglutination is present, test must be
repeated.

3. Massive Transfusion

3.1 If the patient's antibody screen is negative, and 10 units of red blood cells
have been transfused in a 24 hour period, group checks (typing with anti-
A and Anti-B) may be performed on ABO compatible red cells (IPP 754)
for all subsequent crossmatches requested in association with the indate
specimen.

3.1.2 For ABO compatible units that have been typed as in 3.1,
result in the computer as “ GP CHK”.

3.2 After a lapse of 24 hours from the time the massive transfusion protocol
was initiated, a new Type and Screen must be requested and tested
irregardless of there being an indate Type and Screen specimen.
The first 10 units using this specimen must be tested for serological
compatibility as dictated by the patient’s serologic history.

3.3 If the specimen outdates before the 24 hours have elapsed,


subsequent to the initiation of the massive transfusion protocol, the
first 10 units crossmatched on the new specimen must be
serologically compatible with the patient’s serum in association with
the patient’s serologic history.

3.4 If the patient has a history of clinically significant alloantibodies, full


crossmatch is only required on the first 10 units transfused.
Additional red cells may be group checked, provided all units are ABO
compatible and antigen negative. Exceptions to this must be
evaluated and approved by the Blood Bank Medical Director.

3.5 Dispense the units according to Dispensing Blood Components (IPP746).

REPORTING RESULTS

1. Interpretation: Both hemolysis and agglutination in immediate spin, 37C or AHG


phases of the crossmatch indicate incompatibility. Perform further testing according
to Antibody Identification - Basic Panel (IPP 707).

2. Enter the unit numbers in the computer for the units being crossmatched. Record
reactions in the computer as they are observed. Grade reactions according to Grading of
Agglutination Reactions - IPP 624. Save all computer results in the computer.

3. For each unit found to be compatible for the patient:


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3.1 Print a Crossmatch Transfusion Tag hand write the date and the accession # of the
specimen in the top right hand corner and attach it to blood bag with mini tach-it.
This will be the unit identifier attached to the unit.

3.2 Place labeled units in Revco #1 in the drawer corresponding to the last digit of the
patient’s medical record number.

4. If an ABO retype was performed on the unit instead of an immediate spin crossmatch,
result the Immediate Spin crossmatch as “ GP CHK”.

PROCEDURE NOTES

1. A clinically significant antibody is one whose specificity has been known to cause
hemolytic disease of the newborn, hemolytic transfusion reactions, or unacceptably
shortened survival of transfused red blood cells. The following antibodies are considered
clinically insignificant: P1, M, N, Lea, Leb, Sda, Bga, and A1, unless they are reacting at
37C and/or AHG using a prewarm technique (Prewarm Technique – IPP 759).

2. Reactions at 37C may be due to carry-over after binding of IgM agglutinins at room
temperature. Prewarm crossmatch according to IPP759. If testing is now negative at
37C and AHG, the previous reactions are considered to be due to cold reacting clinically
insignificant antibodies.

3. Give priority to 'STAT' crossmatch orders over routine orders. Blood should be available
under normal circumstances in one hour.

4. Crossmatch all blood products containing significant amounts of red cells (greater
than 2 mL) for compatibility before issue. This includes Leukapheresis products and
plateletpheresis.

5. Give random platelets that are bloody to group specific patients if the inventory
levels cannot justify discarding them.

6. If the inventory levels are good, discard the blood random donor platelets. Consult
the Senior Technologist if there are excessive numbers of bloody random platelets
before discarding.

5. Do not use an immediate spin crossmatch for neonates, or patients who have current
reverse type interpretation of less than 2+, or in cases where immediate spin crossmatch
are positive due to cold agglutinins or clinically insignificant alloantibodies. Since an
immediate spin crossmatch is intended to detect ABO incompatibilities, the ABO must be
rechecked on the unit by performing a retype (Group Check) on the unit and resulting as
“IS” crossmatch as “ GP CHK”.

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Blood Bank Internal Policy / Procedure

6. Acceptable blood groups of red cells for transfusion:

ACCEPTABLE WITH
PT BLOOD TYPE FIRST CHOICE ACCEPTABLE ALTERNATE APPROVAL OF MEDICAL
DIRECTOR

O pos O pos O neg -----

O neg O neg ----- O pos

A pos A pos A neg, O pos, O neg -----

A neg A neg O neg A pos, O pos

B pos B pos B neg, O pos, O neg -----

B neg B neg O neg B pos, O pos


AB neg, A pos, A neg, B pos,
AB pos AB pos -----
B neg, O pos, O neg
AB neg AB neg A neg, B neg, O neg AB pos, A pos, B pos, O pos

7. Rh negative patients may switched to RH positive red cells, only by approval from
the Blood Bank Medical Director, during exceptional circumstances.

7.1 Enter a Blood Bank comment in the patient’s historical records that approval
was granted by the Medical Director, for the number of units approved, the
date and your ID#.

7.2 The ICIS will allow the crossmatch and dispense of Rh positive units to Rh
negative individuals with an override reason.

8. Rh negative patients transfused with Rh positive red cells will be serologically monitored
by:

8.1 Performing all subsequent antibody screens with polyethylene glycol (PEG) and
low ionic strength (LISS) in parallel until further notice.

8.2 Performing a direct antiglobulin test (DAT) using polyspecific antihuman globulin
on all red cell specimens until further notice.

8.3 Performing any additional testing as requested by the Senior and/or Supervisor.

8.4 A comment will be entered into the patient’s history by the Senior Technologist
outlining 8.1.1 to 8.1.3.

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9. In a normal adult, the volume of blood transfused which is considered to be replacement


of the original volume is 10 units of packed red cells. In pediatric cases, the volume will
be less but as calculation of that volume is virtually impossible; 10 units of packed red
cells is considered the safe cut-off.

10. If there is insufficient serum to perform a crossmatch, perform a group check on the units
until a new sample can be obtained. Order the new sample and perform a Type and
Screen on the new sample prior to crossmatching units.

LIMITATIONS OF PROCEDURE

1. The crossmatch has many limitations. A compatible crossmatch will not guarantee normal
survival of transfused cells, prevent immunization of the recipient, or detect all unexpected
red cell antibodies in the recipient’s serum.

2. An immediate spin crossmatch performed on a patient with low titred or absent


isohemagglutinins may or may not detect an ABO incompatibility. Perform group
check on the units.

3. Insufficient washing of donor red cells for the full crossmatch procedure may result
in false negative reactions due to serum protein interference.

REFERENCES
th
1. Technical Manual, 13 Edition, Bethesda, MD: American Association of Blood
Banks, 1999.

2. Standards for Blood Banks and Transfusion Services, 21st Edition. Bethesda, MD:
American Association of Blood Banks, 2002.

3. Cerner HNA Millenium Integrated Clinical Information System PathNet User Training
st
Manual, 1 Edition, King Faisal Specialist Hospital and Research Center, July 2002.

Page 32 of 95
King Faisal Specialist Hospital and Research Center
Blood Bank Internal Policy / Procedure

TITLE / DESCRIPTION: INDEX NUMBER(S):

ANTIBODY IDENTIFICATION - BASIC PANEL 707


EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION:

DEC91 REV9 / JUL01 XM


(Reviewed JUL02)

PRINCIPLE

When an antibody is detected in patient serum by a positive antibody screen or an incompatible


crossmatch, and/or is eluted from red cells, a panel of red cells of known antigenic configuration
is used to identify the antibody.

Determining the specificity of antibodies encountered in pretransfusion testing is important in


assessing the need to select antigen-negative blood for transfusion. In prenatal testing,
knowing the specificity and immunoglobulin class of an antibody helps predict the likelihood of
HDN.

The serum is tested at all test phases at which antibody activity was initially detected.
Additional antibodies may become apparent at different test phases, and the reactivity of some
antibodies may be increased by altering incubation time and / or temperature, serum/cell ratio,
or by using additive solutions.

POLICY

A full reagent red cell panel is performed as the initial investigation for all patients who have a
positive antibody screen, including Rhig, and no history of previously identified antibodies. Test
as many reagent red cells as necessary to confirm or exclude antibody specificities. At least
three positive homozygous cells are required to confirm an antibody on its first presentation.

For patients with previously identified antibodies, including previously identified Rhig, serum is
tested against a selected cell panel of antigen negative cells to exclude any additional clinically
significant antibodies. Previously identified antibodies need not be re-identified.

All clinically significant antibodies on panel sheet are excluded using a cell with homozygous
expression of the corresponding antigen when available, or with two heterozygous cells when
homozygous is not available. An auto control is run with all serum panels.

SPECIMEN

 Serum and cells from 10mL (min. 7mL) clotted whole blood
 Eluate

Page 33 of 95
King Faisal Specialist Hospital and Research Center
Blood Bank Internal Policy / Procedure

REAGENTS, SUPPLIES, EQUIPMENT

 12mm x 75 mm test tubes


 Pasteur pipettes
 0.9% saline
 LISS additive (Ortho Antibody Enhancement solution)
 Anti-human globulin reagent
 IgG Sensitized red cells
 Commercially prepared red cell panel and corresponding panel sheet
 Immunohematology Report form (Appendix I)
 “Reagent Red Cell Panel; Negative Auto Control” Flow Sheet (Appendix II)

SAFETY PRECAUTIONS

1. All blood and blood products must be treated as potentially infectious. Follow universal
precautions detailed in the Laboratory Safety Manual.

2. Do not attempt to pick up broken glass with fingers. Use a dustpan or other scooping
implement and dispose of glass fragments in sharps disposal container.

QUALITY CONTROL

Reagent Quality Control IPP760.

PROCEDURE

1. Select an indate panel from Jewett refrigerator #5. Select a copy of the panel sheet with
the lot number that matches the lot number of the panel cells.

2. Fill in essential information on the panel sheet:


Date of testing,
Tech ID #
Patient name and medical record number
Phases of testing, additive and conditions:
IS = Immediate Spin phase
37 = 37oC incubation phase
AHG = Antihuman globulin phase
CC = Coombs Control (or Check Cells)
Ficin = Ficin
Neat = No additive
LISS = LISS additive
(Others as indicated)

NEW ANTIBODY
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1. Select appropriate cells for testing. This must be an 11- cell panel at the very minimum.

2. Label reagent red cell tubes with patient identification. Label the last tube "AC" (Auto
Control).

3. Place 2 drops of patient's serum in each of the tubes.

4. Gently mix the panel cells well. Add 1 drop of reagent red cells to the labeled tube, Mix
well.

5. Prepare a 2-5% cell suspension of the patients own cells using saline. Add 1 drop of
this cell suspension to the auto control tube.

6. Centrifuge all tubes in a properly calibrated centrifuge (saline spin time is designated on
specific Serofuge or cell washer).

7. Examine for hemolysis and record if present. Gently resuspend the cell button and
examine for agglutination macroscopically. Record results in the immediate spin column
on the panel sheet.

8. Add 2 drops of LISS Additive to each tube and mix well. (Do not add LISS when testing
eluates or if an enzyme panel used).

9. Incubate the tubes for 15 minutes at 37oC. Incubation can be extended for up to 30
minutes for greater sensitivity.

10. Centrifuge as before. Examine for hemolysis and record if present. Gently resuspend
the cell button and examine for agglutination macroscopically. Record results in the 37
o
C column on the panel sheet.

11. Wash all tubes 4 times with saline. After last wash decant completely.

12. Add 2 drops of anti-human globulin reagent and mix.

13. Centrifuge. Gently resuspend the cell button and carefully examine for agglutination
macroscopically. Record results in the AHG column on the panel sheet.

14. To all negative tests, add 1 drop of IgG-sensitized red cells (Coombs control cells).
Centrifuge and examine for agglutination. Record results in the CC column on the panel
sheet. If no agglutination is present, test must be repeated.

INTERPRETATION:

Interpret positive and negative results according to the following table:

Test
Positive Result Negative Result
Result:
Definition: Agglutination or hemolysis in Absence of hemolysis or
immediate spin or incubation phase, agglutination.
or agglutination in antiglobulin phase.
Indication: Presence of one or more antibodies Absence of detectable antibodies
directed at antigens present on the to antigens present on the cells.
particular cells.
Grade reactions and record on panel sheet according to Grading of Agglutination

Page 35 of 95
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Blood Bank Internal Policy / Procedure

Reactions - IPP624.

Refer to Flow Sheet (Appendix II) for interpretation of patterns of reactivity and suggestions
of further testing to perform.

If a pattern of positive and negative reactions is observed, antibodies can be eliminated to the
antigens present on the non-reactive cells. Using the panel sheet and considering one cell
at a time, eliminate all of the antigens that are represented homozygously on the cells giving
negative reactions. Cross out the antigens at the top of the page.

3.1 All Rh Negative individuals with positive antibody screens will have Anti-D, Anti-C
and Anti-E ruled out / differentiated by testing an Ro, r’r’, and r’’r reagent red cell
with PEG.
3.2 Additional alloantibodies may be excluded using these cells if the corresponding
antigen is in the homozygous state of expression.

3.3 LISS additive will be used for additional exclusions using the applicable zygosity rules.

4. If only one antigen is remaining:

4.1 Observe the overall pattern of positive and negative cells to see if the pattern of
serum reactions matches that of the positive cells of that antigen.

4.1.1 If a match is observed and there are a minimum of 3 positive and 3


negative cells, the antibody has been identified.

4.1.2 If there are not 3 positive and 3 negative cells, select additional cells.

4.2 Antigen type the patient for the corresponding antigen according to Antigen Typing
IPP 710.

4.2.1 If an Rh antibody has been identified, perform a full Rh phenotype.

4.2.2 If the antigen has an allele, type for both alleles.

4.2.3 The patient should be negative for the antigen that the antibody is directed
against. If the patient has been transfused in the past 3 months,
phenotyping may not be reliable due to the presence of transfused cells.

5. Reaction phase:

5.1 Reactivity at room temperature indicates the presence of a cold reacting antibody
to M, N, P, or Lewis system antigens, (for example).

5.2 Reactivity at 37oC and AHG indicates the presence of a warm reacting antibody
such as those reacting to S, s, Rh, Kell, Duffy or Kidd system antigens.

6. If more than one antigen is remaining, refer to Extended Workup in this IPP.

7. Auto Control:

7.1 If the serum reacts with both the panel cells and the autologous cells, this
suggests the presence of an autoantibody, but does not exclude an alloantibody.

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Auto absorption may be necessary to rule out the presence of coexisting


alloantibodies. See Adsorption Technique IPP 703.

7.2 If the autocontrol is positive in the AHG phase, perform a DAT according to Direct
Antiglobulin Testing IPP 728. If DAT is positive, perform an eluate according to
Elution Acid (Elukit) IPP 704.

7.3 If the patient has been recently transfused, a mixed-field reaction in the Auto
Control and DAT may be seen, indicating an alloantibody directed at an antigen
present on surviving donor cells.

8. Dosage:

8.1 Some antibodies react stronger or only with cells homozygous for the
corresponding antigen. Anti-M, anti-c, anti-Jka often show dosage.

9. Antibody to high frequency antigen:

9.1 Reactivity with all cells except the patient control cells may indicate reactivity to a
high frequency antigen. High frequency antigens usually react with equal
strength against all cells tested. It may be useful to test the patients serum
against red cells from siblings and other first degree relatives in the search for
compatible blood.

10. Panagglutination:

10.1 Equal strength reactivity with all cells, including the patient control cells, indicates
non-specific panagglutination activity.

10.2 Perform auto adsorption according to Adsorption Technique IPP 703 to rule out
underlying alloantibodies.

10.3 Additive solutions or Ingredients in preservatives in reagent red cells may cause
reactivity with some patients, which may be reduced by eliminating the additive,
using a different additive, or washing the reagent cells prior to use.

11. Phase of reactivity:

11.1 Take into account the known characteristics of antibody reactivity. For example, if
immediate spin reactions seem to correspond to an anti-Fya, it should not be
considered as the first choice, because its optimum phase of reactivity is AHG
phase.

EXTENDED WORKUP

1. Perform a selected cell panel by choosing additional cells to include cells both positive
(homozygous) and negative for each of those antigens not excluded.
1.1 Consider the possibility of antibodies to additional antigens other than the obvious
ones that are not ruled out in its presence. For example, if the reactions
correspond to an anti-c and all E positive cells are also c positive, the presence of
anti-E cannot be ruled out. Select cells from other red cell panels that are c
negative and E positive and repeat the tests. Anti-c frequently occurs with anti-E
and anti-e with anti-C.

1.2 Similarly, an anti-c may conceal the presence of an anti-K or other antibody.
Select cells negative for c antigen and positive for the questionable antigen.

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2. If the observed pattern does not fit any specific pattern, there may be a combination of
antibodies present. Perform the following steps to identify and rule out antibodies.

2.1 Antigen type the patient for antigens corresponding to all antibodies not ruled out.
A patient will not produce allo-antibodies toward an antigen present on their own
red cells. Therefore, if the patient’s red cells type positive for the antigen, they
cannot develop the corresponding antibody and it can be ruled out as an allo-
antibody.

2.2 If it appears that Anti-Fya or anti-Fyb is present, run an enzyme panel (see
Antibody Identification -- Ficin Panel IPP 708) to detect antibodies that are masked
by the anti-Fya or anti-Fyb.
a b a
2.3 If anti-P1 or anti-Le or anti-Le is present, or if anti-Sd is suspected,
neutralization (Neutralization with Group Specific Substances IPP753) will
frequently confirm an antibody or remove one or more antibodies allowing the
identification of others.

2.4 Adsorption (Adsorption Technique IPP703) and / or adsorption-elution studies


(Elution Acid (Elukit) IPP704) can be used to separate specific antibodies.

3. If a specific antibody pattern isn’t demonstrated, it may be due to weak reactivity of the
antibody. Weak reactions may be enhanced in several ways:

3.1 Increase the incubation time. (Do not incubate LISS additive longer than 30
minutes).

3.2 Increase the ratio of serum to cells e.g., 4 or 5 drops of serum to 1 drop of cells.
(DO NOT use LISS Additive).

3.3 Perform Dry Button technique: (DO NOT use LISS Additive).

3.3.1 Add one drop of each panel cell to appropriately labeled tube.

3.3.2 Wash in cell washer (or Serofuge) for one wash cycle, decant to dry
button.

3.3.3 Add two or more drops of serum to dry button of cells.

3.4 Test serum against a FICIN Panel (Antibody Identification -- Ficin Panel IPP708).

3.5 Use different enhancement reagent -- e.g. PEG, NHance. (Refer to package
insert for directions.)

4. An antibody which reacts best at room temperature and decreases in reactivity as the
temperature increases is usually considered to be non-clinically significant. These
antibodies may mask the presence of a clinically significant warm antibody. Perform a
prewarmed panel (Prewarm Technique IPP 759) or autologous adsorption (Adsorption
Technique IPP 703) to help detect and identify other antibodies.

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5. If anti-M,N, or P1is identified that reacts at the AHG phase, perform prewarm testing.

5.1 If the reactivity is eliminated upon prewarming, report the antibody as non-
clinically significant.

5.2 If it is positive in AHG phase after prewarming, report the antibody as being
clinically significant.

6. Check for extended typings of panel cells. Some cells will have been typed for less
common antigens such as Wra, Chido, etc. and a listing is provided by the manufacturer.

PREVIOUSLY IDENTIFIED ANTIBODY

1. From available panels select enough cells which meet the following criteria:

 Cells that are antigen negative for the previously identified antibody(ies), but
positive for all remaining clinically significant antigens.
 Any additional antibodies that are identified by this process must be confirmed
using a total of 3 cells of homozygous expression for the corresponding individual
antigen.

2. Perform an auto control.

3. Previously identified antibodies need not be re-identified.

4. If the panel of selected cells is positive, test additional cells to identify the additional
antibody(ies) detected.

EXCLUSIONS USING POLYETHYLENE GLYCOL (PEG)

1. If you incorporate a more sensitive technique into an antibody workup, all alloantibodies
should be ruled out using that more sensitive technique.

2. Since liss is our primary potentiator for now, a full panel should be performed using liss.
PEG can be used as an alternate, but do not use a combination of liss and peg to
perform exclusions on the same panel. Use only one potentiator all the way through
your work up.

EXCEPTIONS:

3. Rh exclusions in Rh negative (d-) individual most often these people are rr:

3.1 Test two r'r cells with PEG to rule out anti-C, if r'r' cell unavailable

3.2 Test two r"r cells with PEG to rule out anti-E, if r"r" cell unavailable

3.3 Test one R2R2 cell with PEG to rule out anti-D

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3.4 Other alloantibodies may be ruled out on the aforementioned cells if the antigen is
in the homozygous state of expression.

4. If a homozygous cell is unavailable to rule out a certain antibody with LISS, test two
heterozygous cells with PEG. This would occur most often in multiple antibody
situations.

5. If, in the above two situations, an antibody is detected with PEG. Test the same cells
with LISS as a comparison. If the same antibody (ies) is non-reactive with LISS, the
other exclusions must be repeated using PEG.

REPORTING RESULTS

1. Enter Results in Meditech RESULT ENTRY, ENTER RESULTS. At the ABID prompt
there is a list of possible panel choices. Enter the results for the methods used as
either POSITIVE, NEGATIVE or NP.

1.1. If the initial Antibody Screen has not been resulted and only a rule-out
panel is to be performed, order the ABID in Meditech under
REQUISITIONS, ENTER/EDIT&RESULT.

1.1.1. Add the Antibody Identification to the existing Type and


Screen requisition by performing an <F9> “lookup” at the
REQ # prompt and selecting the current TS order.

1.1.2. At the ORDER prompt order an ABID.

1.1.3 If a new sample is ordered to finish the antibody work up,


for the 1st sample ND the antibody
interpretation and add to the comment that new sample
requested from the ward and add the number of the
sample “BX#” and ND the conclusion too.
nd
1.1.4 Use the ABID sample “2 sample” to enter the result
interpretation for the antibody identification.

2. At the ANTIBODY INTERP (or ELUATE) prompt press <shift + right arrow key> to obtain
the Antibody Interpretation Result Entry Screen. At the ANTIBODY prompt perform an
<F9> “lookup” and select the antibody that was identified. Press ENTER to return to the
regular Result Entry screen.

3. Complete Immunohematology Report form (Appendix I) and leave it together with all
screening cell antigrams, panel sheets and antigen typing form for senior review. All work
will be reviewed by supervisor and pathologist prior to filing in Immunohematology
binders.

4. In Meditech, order Antigen Typing(s) and result according to Antigen Typing IPP 710.

5. Print a new file card.

6. For patients with previously identified antibodies: If the panel of selected cells is negative,
result as “No additional antibodies”.

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7. For patients who have received RhIg and whose selected panel is negative, result as
RHIG- “Passive Anti-D”. Under HISTORY / EDIT BBK HISTORY, enter the date the RhIg
was last given.

PROCEDURE NOTES

1. Grade reactions and record on panel sheet according to Grading of Agglutination


Reactions IPP624.

2. If the specificity and identification is in some doubt, calculation of probability can be


performed using Fisher's Exact Method employing a 2 x 2 table (AABB Technical
Manual)

3. It is sometimes impractical to perform exclusions using cells with homozygous


expressions of certain antigens. It is acceptable to exclude Anti-K using two
heterozygous cells and for patient with anti-D, to exclude Anti-C and Anti-E using two
heterozygous cells. Use two heterozygous cells any time a homozygous cell is not
available.

4. It is acceptable not to eliminate antibodies to low frequency antigens such as Cw Kpa, V,


VS, Jsa.

5. A clinically significant red cell antibody is one that shortens the survival of transfused red
cells or has been associated with hemolytic disease of the newborn (HDN). Clinically
significant antibodies include the Rh antibodies, anti-S, -s, -U, -Lub, -K, -k, -Kpa, -Kpb, -
Jsa, -Jsb, -Fya, -Fyb, -Jka, - Jkb, -Dia, -Dib, -Doa, -Coa.

6. Other antibodies may occasionally cause shortened red cell survival, such as anti-M or -
Yta, and therefore must be treated as clinically significant.

7. If a new sample is required to complete an antibody workup, order it as ABID with a new
BX number. Perform an ABO/Rh on the new sample; order ABO/RH on that BX number
to have space to record the results in the computer. Repeat one more positive cell,
screening cell or selected cells to rule out, and ensure that the 2nd cell, repeated cell,
will react as the 1st cell’s agglutination strength. For multiple antibody workups, only
one positive cell needs to be repeated.

8. Use the ABID sample “2nd sample” to enter the result interpretation for the antibody
identification .

9. If a new antibody is detected on a patient of Dr. Rahman or Dr. Kurdi (OBGYN), perform
and result antibody titration without waiting for specific titration order.

REFERENCES

1. AABB Technical Manual, 12th edition. Bethesda, MD: American Association of


Blood Banks, 1996, pages 349-378.

2. Standards for Blood Banks and Transfusion Services, 21st edition. Bethesda, MD:
American Association of Blood Banks, 2002.

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3. Blood Transfusion in Clinical Medicine, P.L. Mollison, 1993.

4. Gamma Ficin-Panel Systems (Panel One), Package Insert. Houston, TX: Gamma
Biologicals Inc., May 1996.

5. Panel Twenty, Package Insert. Houston, TX: Gamma Biologicals Inc., May 1996.

6. Resolve Panel A, Package Insert. Raritan, NJ: Ortho Diagnostic Systems, May, 1989.

7. Resolve Panel B, Package Insert. Raritan, NJ: Ortho Diagnostic Systems, July 1988.

TITLE / DESCRIPTION: INDEX NUMBER(S):

ANTIBODY IDENTIFICATION FICIN PANEL 708


EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION:

DEC91 REV10 / AUG02 XM

PRINCIPLE

The use of ficin pretreated panel cells increases sensitivity in the identification of some blood
group antibodies, most notably those of Rh, Kidd, Lewis, & Vel blood group systems as well as
cold agglutinins of anti-I specificity. Ficin treated cells will not react with most samples of anti-
a b a
Fy , -Fy , -M, -N,. Xg , and –Pr. Anti-S and anti-s may show loss of specificity when tested with
enzyme-treated cells. For this reason the ficin panel is never used alone in identification tests.

POLICY

The Ficin panel may be used as an aid in the identification of antibodies, either by enhancing or
inhibiting antibody reactivity, in the following circumstances:

1. When a pattern of weak reactions fails to indicate specificity.

2. When the presence of an antibody is suspected but cannot be demonstrated.

3. When the depression of a reaction may aid in the identification of antibody mixtures or
may confirm the identity of an antibody.

Enzyme treated panels must always be used in parallel with the corresponding untreated panel.

SPECIMEN

10mL (min 7mL) serum sample in a red top tube

REAGENTS, SUPPLIES, EQUIPMENT

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 12 mm x 75 mm test tubes
 Pasteur pipettes
 0.9% saline

O
37 C Dri bath
 Waterproof/permanent marker
 Anti-human globulin reagent
 IgG Sensitized red cells
 Ficin treated panel
Ficin treated panel cells
Ficin solution
Enzyme control reagent

SAFETY PRECAUTIONS

1. All blood and blood products must be treated as potentially infectious. Follow universal
precautions detailed in the Laboratory Safety Manual.

2. Do not attempt to pick up broken glass with fingers. Use a dustpan or other scooping
implement and dispose of glass fragments in sharps disposal container.

QUALITY CONTROL

1. Routine daily quality control of antisera and cells.

2. Enzyme Control Reagent.

PROCEDURE

1. Select an indate ficin panel from Jewett refrigerator #5 with the same lot number as the
untreated cell panel used. Select a copy of the panel sheet with the lot number that
matches the lot number of the panel cells.

2. Complete the essential information on the panel sheet:

Date of testing
Tech ID #
Patient name and medical record number
Phases of testing, additive and conditions:
IS = Immediate Spin phase
37 = 37oC incubation phase
AHG = Antihuman globulin phase
CC = Coombs Control (or Check Cells)

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Ficin = Ficin
Neat = No additive
LISS = LISS additive
(Others as indicated)

3. Prepare controls:

3.1 Prepare a 3% cell suspension of washed patient cells.

3.2 Place 2 drops of washed cell suspension into an appropriately labeled tube.

3.3 Add 2 drops of ficin solution to the tube.

3.4 Mix well and incubate for 10 minutes at 37C.

3.5 Immediately wash the treated cells at least 2 times in saline and decant the last
wash.

3.6 Resuspend the dry cell button in 2 drops of Modified Alsever’s Solution to provide a
3-4% cell suspension.

3.7 Confirm the adequacy of ficin treatment by testing as follows:

3.7.1 Place 1 drop of Gamma enzyme control reagent to each of two labeled
tubes, e.g. “AC” for Auto Control and “FP” for random Ficin-Panel cell.

3.7.2 Add 1 drop of the treated auto control cells to “AC” tube and 1 drop of a
random Ficin-Panel cell to the “FP” tube.

3.7.3 Centrifuge for designated saline spin time. Resuspend the cell button and
examine for agglutination.

3.8 Adequate ficin treatment of the auto control is indicated by agglutination of 2-3+ in
both tubes.

3.9 If the ficin treatment is satisfactory, the remaining drop of ficin treated cells may be
used to the auto control tube on the panel.

4. Label 12 tubes with patient identification. Label the first eleven tubes with numbers 1-11
for panel cells and the twelfth tube with AC for the patient auto control.

5. Place 2 drops of patient's serum in each of the 12 tubes.

6. Gently mix the panel cells well. Add 1 drop of cell #1 to tube #1, 1 drop of cell #2 to tube
#2 and so forth through cell #11. Mix well.

7. Add 1 drop of the ficin treated patient cell suspension to the tube labeled AC, mix well.

8. Incubate the tubes for 15 minutes at 37C. Incubation may be extended to 60 minutes.

9. Centrifuge all tubes in a properly calibrated centrifuge (saline spin time is designated on
specific Serofuge or cell washer).

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10. Examine for hemolysis and record if present. Gently resuspend the cell button and
examine for agglutination macroscopically. Record results in the 37C column on
the panel sheet.

11. Wash 4 times with saline. After last wash decant completely.

12. Add 2 drops of anti-human globulin reagent and mix.

13. Centrifuge as in #9 above. Gently resuspend the cell button and carefully examine for
agglutination macroscopically. Do not read microscopically. Immediately record results
on the panel sheet.

14. To all negative tests, add 1 drop of IgG sensitized red cells, mix. Centrifuge as above.
Examine for agglutination. Immediately record results in CC column on panel sheet. If no
agglutination is present, test(s) must be repeated.

Interpretation:

1. Interpret positive and negative results according to the following table:

Test
Positive Result Negative Result
Result:
Definition: Agglutination or hemolysis in Absence of hemolysis or
immediate spin or incubation phase, agglutination.
or agglutination in antiglobulin phase.
Indication: Presence of one or more antibodies Absence of detectable antibodies to
directed at antigens present on the antigens present on the cells.
particular cells.
Grade reactions and record on panel sheet according to Grading of Agglutination
Reactions - IPP624.

a
2. Antibodies that show increased reactions with ficin treated cells are anti-Rh, -Jk and -
Jkb, -Lea and -Leb. Anti-I will also show increased reactions. If a serum sample gives
positive (or negative) results on a specific cell on the untreated panel and stronger (or
positive) results on the same cell on the ficin treated panel it may indicate the presence of
one of these antibodies.

3. Antibodies that show negative or diminished reactions with ficin treated cells are anti-M, -
N, -Fya and -Fyb. Anti-S and -s may show diminished reactions or loss of specificity with
ficin treated cells. If a serum sample gives positive results on a specific cell on the
untreated panel but negative results on the same cell on the ficin treated panel, it may
indicate the presence of one of these antibodies.

4. Enter results under Antibody ID in BLOOD BANK RESULT ENTRY.

4.1 Under the FICIN column, enter or select a result from the list of options.

REPORTING RESULTS

1. Report results according to Antibody Identification - Basic Panel (707). On the panel
sheet(s) label ficin results clearly to differentiate from untreated cell results.

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PROCEDURE NOTES

1. Do not read results microscopically because the use of enzyme treated cells enhances
non-specific reactivity.

2. A positive result in auto control tube indicates the presence of a warm or cold
autoantibody and should be referred to Senior Technologist for evaluation.

LIMITATIONS OF PROCEDURE

1. Warm or cold auto agglutination may be enhanced in tests performed with ficin treated
cells.

2. Excess ficin or over treatment of cells may cause nonspecific agglutination.

3. The reactivity of the ficin treated red cells may diminish over the dating period.

REFERENCES

1. AABB Technical Manual, 13th Edition. Bethesda, MD: American Association of Blood
Banks, 1999.

2. Package Insert, Gamma Biologicals, Reagent Red Blood Cells (Ficin Treated), May
1996.

3. Cerner HNA Millenium Integrated Clinical Information System PathNet User Training
st
Manual, 1 Edition, King Faisal Specialist Hospital and Research Center, July 2002.

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ANTIGEN TYPINGS
Technologist: ______________ Date: _____________
MR # and Date of Last Transfusion:_____________________

Computer
DETAILS Rosette Sickle Anti-C Anti-E Anti-c Anti-e Anti- Anti- Anti- Anti- Anti Entry
Test Test - MT LL
Lot Number
Expiry Date
POS - Lot No.
- Expiry
- Cell No.
- Result
NEG - Lot No.
- Expiry
-Cell No.
- Result

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PAGE No. ______________

REVIEWED BY: _____________________ DATE: ____________________

NOTE: [! ] in same column, indicates negative AHG result confirmed with coombs control
cells and found to give acceptable 2+ reaction

ICIS DOWNTIME: Entered into ICIS By:________________________ Date___________________

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TITLE/DESCRIPTION: INDEX NUMBER(S):

ANTIGEN TYPING 710


EFFECTIVE DATE: REVISION DATE/NUMBER: DISSEMINATION:

DEC91 REV10 / AUG02 XM

PRINCIPLE

Antigen typing is performed to provide red cell products without the corresponding antigen for
patients with clinically significant red blood cell antibodies.

Once a clinically significant alloantibody has been identified in a patient’s serum it is advisable
to test the patient red cells for the corresponding antigen as a confirmatory test.

POLICY

In accordance with AABB Standards 5.14.3, when clinically significant unexpected red cell
antibodies are found or the recipient has a history of such antibodies, whole blood or red blood
cell components shall be prepared for transfusion that do not contain the corresponding
antigen, and are crossmatch compatible. Exceptions may be made, only when approved by the
Medical Director, when clinical circumstances warrant deviation.
a b a a
The following antibodies are considered clinically insignificant: P1, M, N, Le , Le , Sd , Bg ,
o
and A1, and antigen typed units are not required if the antibodies are not reactive at 37 C or
AHG. All units must be crossmatch compatible by the method deemed appropriate for the
antibody and the phase at which it is reactive.

Patients who have one or more Rh antibodies will have their full Rh phenotype determined.
Patients with other clinically significant antibodies will have both alleles of the corresponding
antigen phenotyped. Patient phenotyping may be performed to aid in identifying and/or
excluding antibodies, but should not be done on patients with a recent transfusion history. Red
cell phenotypes may be ordered by physicians after consultation with the Blood Bank Director.

SPECIMEN

10mL (min. 7mL) clotted whole blood - test as soon as possible, store at 2-8C if testing is
delayed.

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REAGENTS, SUPPLIES, EQUIPMENT

 Commercial blood grouping antisera


 Antisera directions for use (Package inserts)
 Isotonic saline
 Anti-Human globulin reagent
 Coombs control cells
 12 x 75 mm test tubes
 37C Dri bath
 Centrifuge
 Disposable pipettes
 Waterproof/Permanent Marker
 Cell Washer
 Antigen Typing form (Appendix I)

SAFETY PRECAUTIONS

All blood and blood products must be treated as potentially infectious. Follow universal
precautions detailed in the Laboratory Safety Manual.

QUALITY CONTROL

1. Test each antisera each day of use with the following:

1.1 Positive Control: a cell with heterozygous (if possible) expression of the antigen it is
directed against.

1.2 Negative Control: a cell negative for the antigen.

If appropriate controls cannot be found among the indate screening cells, use
outdated panel cells.

PROCEDURE

Refer to package inserts for instructions.

REPORTING RESULTS

1. Record all tests performed, including controls, on the Antigen Typing form (Appendix I)
as each test is read. Record the lot number and cell number of all control cells. For
patients, record the MR # and Date of the last transfusion. Grade reactions according to
Grading of Agglutination Reactions - IPP624.

2. Control all negative tests read in the antiglobulin phase with Coombs Control Cells.
Record a tick () in the same column to indicate validation of these results.

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3. Interpret antigen type only when positive and negative control cells react with antisera
as expected. The positive control should give a minimum reaction reading of 2+ or
greater, as defined in IPP 624 Grading of Agglutination Reactions.

4. If the positive control gives a grading of < 2+, it must be repeated with either fresh
control cells or another vial or lot number of antisera. Refer the discrepancy to the
Senior Technologist, if it can not be resolved.

5. Antisera controls must be performed each day of use. If controls have already been
done and recorded on a different antigen typing sheet, record the lot number and expiry
of the reagent on the current antigen typing sheet. In place of the control results, record
that controls were already performed.

6. Order Antigen Typing(s) in DEPARTMENT ORDER ENTRY under the sample


accession number. BMT PHENO’s will be ordered by the wards.

7. To result antigen typings select BLOOD BANK RESULT ENTRY.

8. Complete the New Worksheet Dialogue box, select the accession number format for a
patient or product number and press OK.

9. Enter the accession number associated with the Antigen Type procedure to be resulted
or enter the product number. The Insert Phases/Cells/Interp window will be displayed.

10. Select AG TYPE from the Phase list by using the drop-box and PATIENT or PRODUCT
depending on which you are performing, from the Cell list.

11. For BMT Phenotype (Antigen Types), select BMT PHENO from the phase list and also
BMT PHENO from the cells list.

For BMT Pheno, if all the cells are not going to be required they must be
deleted before result verification.

12. The system retrieves order information and reference data for use in the worksheet.
When this data has been displayed, you can enter additional accession numbers.

13. Click on, or use the arrow key to move to the first cell to be resulted, represented by
a white cell in the worksheet.

13.1.1 Enter the antigen that you have tested in the Ag Tested column
using free text.

13.2 Enter or select a result from the drop down list for the R.T, AHG and AHG
CC columns.

13.3 To enter a result, type the first letter or number associated with the result
(e.g. 1 for 1+, NP for Not Performed) or click the down-arrow key to
display the list of options.

13.4 Press ENTER to move to the next result cell.

14. Enter or select from the list the Antigen Type Interpretation, taking care to select the
correct interpretation.

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The Result Entry spreadsheet does not show all the fields to be resulted on the
screen in a single view. When you get to the Ag Interp field you will not be able to
see the Ag tested box which makes it difficult to follow. To view the Ag tested
once you have got to the Ag interp field use the left/right arrow keys to move
between fields.

15. Click PERFORM or VERIFY to save the results.

16. The system will then prompt “Do you wish to complete the Ag Type”

16.1 Click YES to complete the antigen type entry.

17. Enter donor antigen results in Lifeline computer according to Antigen/Sickledex Result
Entry In Lifeline IPP 723.

18. Write antigen results on red cell donor unit on upper left hand corner of the label.

PROCEDURE NOTES

1. Read and follow the manufacturer’s instructions with each lot number of antisera, as the
procedure can vary from manufacturer to manufacturer and from lot to lot.

2. Every time a new vial of antisera is opened, sign and date the vial. Label the package
insert with the reagent lot number and expiration date and place it in the package insert
binder. Remove the previous package insert.

3. A binder of current package inserts is available and is updated monthly.

4. When screening donor units, take antigenic frequency into account. If the antigen is
relatively low frequency, negative units will be easily found, e.g. if a patient has anti-K,
only a few units will need to be typed as 91% of donors are K negative. If a patient has
multiple antibodies, type for the higher frequency antigen first, e.g. if patient has anti-c
and anti-E, type for c antigen first since 80% will be unsuitable, then type c negative units
for E.

5. Frequency tables for each blood group can be found in the AABB Technical Manual. This
should be used only as a guide, as it is not based on Arabian populations.

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LIMITATIONS OF PROCEDURE

1. If a patient is to be antigen typed, samples should be tested prior to transfusion therapy.


Patients who have been transfused may have surviving donor cells that can cause mixed
field agglutination or false positive or negative reactions.

1.1 Do not antigen type patients who have been transfused within the previous 30 days.

1.2 If patient has been transfused within the previous 3 months, interpret the antigen
type with caution.

1.3 Do not do BMT Phenotype if patient has been transfused in the previous 3 months.
Cancel BMT Phenotype under DEPARTMENT ORDER ENTRY with cancellation
comment of “BMT PHENOTYPE NOT DONE; TRANSFUSED WITHIN 90 DAYS”

2. If a patient has a positive direct antiglobulin test, their cells will give a false positive result
when the indirect antiglobulin test is required for antigen typing.

3. Take into account the characteristics of the antigens when antigen typing patients or
donor units.

4. The strength of P1 antigen reactivity differs markedly among different cell samples and
reactivity declines during storage, thus use of fresh control cells is desirable.

5. Some antigens are not fully expressed on the red cells of infants and young children,
these include P1, Lewis and Lutheran antigens.

REFERENCES

1. Manufacturer’s inserts for each antisera.

2. Issitt, PD. Applied Blood Group Serology, 3rd Edition. Miami, Florida: Montgomery
Scientific Publications, 1989.

3. Technical Manual, 13th ed., Bethesda, MD: American Association of Blood Banks,
1999.

4. Standards for Blood Banks and Transfusion Services, 21st Edition. Bethesda, MD:
American Association of Blood Banks, 2002.

5. Cerner HNA Millenium Integrated Clinical Information System PathNet User Training
st
Manual, 1 Edition, King Faisal Specialist Hospital and Research Center, July 2002.

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BLOOD BANK INTERNAL PROCEDURE / POLICY


TITLE / DESCRIPTION: INDEX NUMBER(S):

DIRECT ANTIGLOBULIN TEST (DAT) 728


EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION:

JAN92 REV9 / AUG02 XM

PRINCIPLE

The direct antiglobulin test (DAT) demonstrates the presence of antibody bound to red cells in vivo. It is
of value in detecting cases of HDN (hemolytic disease of the newborn), auto-immune hemolytic anemia,
transfusion reaction and other clinical conditions.

POLICY

A direct antiglobulin test (DAT) will be performed on all cord samples and will be provided as a
diagnostic tool at the physicians’ request. All post transfusion samples submitted for transfusion reaction
investigation will have a direct antiglobulin test performed on them in accordance with AABB Standards
7.3.3.1.4. A direct antiglobulin test will be performed if the autocontrol performed with an antibody
investigation panel is positive.

SPECIMEN

Whole blood from patient in lavender top (EDTA) tube, 5 mL

REAGENTS, SUPPLIES AND EQUIPMENT

 12 mm x 75 mm disposable glass tubes


 Normal saline
 Disposable pipettes
 Polyspecific Anti-human globulin reagent
 Monospecific (IgG and C3d) Anti-Human globulin reagents
 Automatic cell washer or Serofuge
 IgG sensitized cells (Coombs Control Cells)
 Microscope
 Complement Coombs Control Cells
 Antigen Typings form (Appendix I)

SAFETY PRECAUTIONS

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1. All blood and blood products must be treated as potentially infectious. Follow universal
precautions detailed in the Laboratory Safety Manual.

2. Do not attempt to pick up broken glass with fingers. Use a dustpan or other scooping implement
and dispose of glass fragments in sharps disposal container.

QUALITY CONTROL

1. Test and record anti-IgG results according to Reagent Quality Control IPP 760.

2. Record polyspecific control results of antihuman globulin and anti-C3d monospecific antihuman
globulin on Antigen Typings form (Appendix I) each day of use.

PROCEDURE

1. Make a 3-5% suspension of patient cells in saline.

2. Add one drop of cell suspension to each of three tubes labeled for polyspecific AHG, IgG and C3d.

3. Wash the cells four times using the automatic cell washer.

4. Polyspecific Direct Antiglobulin Testing

4.1 To the dry cell button, add 2 drops of polyspecific antihuman globulin reagent to the
appropriately labeled tube.

4.2 Mix well and centrifuge at calibrated AHG spin time.

4.3 Immediately resuspend the red blood cells by gentle agitation and examine macroscopically
for agglutination.

4.4 Examine any apparent negative tests microscopically. Record results.

4.5 If negative, incubate tube at room temperature for 5-10 minutes after the initial reading.
Centrifuge and read microscopically, record results.

4.6 To each negative test, add one drop of Coombs Control Cells and centrifuge for calibrated
spin time, then examine microscopically immediately for agglutination. Repeat any negative
tests.

4.7 If a positive result is found with polyspecific AHG, perform monospecific direct antiglobulin
testing to determine whether the positive test is due to IgG or complement components bound
to the cell.

5. Monospecific Direct Antiglobulin Testing

5.1 Add two drops of anti-IgG monospecific anti-human globulin reagent into the appropriately
labeled tube. Add 2 drops of anti-C3d monospecific anti-human globulin reagent into the
appropriately labeled tube.

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5.2 Mix well and centrifuge at AHG calibrated spin time.

5.3 Resuspend the cell button gently and examine macroscopically for agglutination.

5.4 Examine any apparent negative tests microscopically.

5.5 If the DAT is positive with anti-IgG, anti-c3d and polyspecific antihuman
globulin, a 6% albumin control must be performed and recorded to rule out the
presence of spontaneous agglutination.

5.6 If anti-C3d is negative, incubate 5-10 minutes at room temperature, spin, read
microscopically, record results.

5.7 To any negative IgG tests, add 1 drop of Coombs Control Cells, centrifuge at calibrated AHG
spin time and examine macroscopically for agglutination. Repeat any negative tests.

5.8 To any negative C3d tests add 1 drop of Complement Control Cells, incubate at
room temperature for 5 minutes, spin and examine macroscopically for
agglutination. Repeat any negative tests.

5.9 Enter DAT results under BLOOD BANK RESULT ENTRY.

REPORTING RESULTS

1. Presence of agglutination, either macroscopically or microscopically, is interpreted as a positive


result. Absence of agglutination is interpreted as a negative result. Refer to procedure on Grading
of Agglutination Reactions - IPP624.

2. A patient who develops a positive DAT subsequent to recent transfusion may be undergoing a
delayed hemolytic transfusion reaction. This possibility must always be considered and if
indicated a full transfusion reaction work-up should be performed. Consult the Senior, Supervisor
(weekdays) and / or Blood Bank Director (evenings, nights, weekends). In this case, the DAT
result is a critical value and the recipient physician should be notified via the Medical Director of
the Blood Bank.

3. Refer to Cord Blood Testing - IPP724 for the reporting format for neonate samples.

PROCEDURAL NOTES

1. If anti-IgG gives a positive result, perform an eluate on the cells. (See #4 below for guidelines.

2. If IgG is negative and only C3d and polyspecific AHG are positive, there is no need to perform an
eluate.

3. Polyspecific anti-human globulin testing requires both an immediate spin reading and a room
temperature incubation for 5-10 minutes and reading again. A negative reaction will sometimes
become positive after this time. This is particularly useful in detecting C3d or IgA sensitization.
Positive reactions due to IgG sensitization will often become weaker following this procedure so
that reading after 5-10 minutes incubation in antiglobulin serum must never be substituted for the
immediate reading.

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4. If a DAT is positive with anti-IgG, anti-C3d, and polyspecific AHG, a 6% albumin control must be
performed and recorded in order to rule out the presence of spontaneous agglutination due to
immunoglobulin coating.

5. Some patients with autoimmune disease process have routine DAT’s requested in order to monitor
treatment. If the DAT is positive perform an eluate on first presentation. Following the initial
elution, perform an eluate only if:

5.1 The DAT or elution has not been done for 6 months.

5.2 The DAT strength increases significantly (2 or greater agglutination gradings).

5.3 The patient has been transfused within the past month.

6. If a DAT is ordered and there is no history of ABO/Rh results in the patient’s medical record data
in computer, order the test “ABO/RH” on the same accession number, perform ABO/Rh, and enter
the results under the same accession number in the BLOOD BANK RESULT ENTRY
application.

LIMITATIONS OF PROCEDURE

Cells from clotted specimens stored at 4 - 6C temperature may absorb complement which will be
detected by the anti-C3d component of polyspecific AHG and by monospecific Anti-C3d. Any positive
DAT performed on a clotted specimen must be confirmed on an EDTA specimen.

REFERENCES

1. Technical Manual, 13th Edition, Bethesda, MD: American Association of Blood Banks, 1999.

2. Standards for Blood Banks and Transfusion Services, 21st Edition. Bethesda, MD: American
Association of Blood Banks, 2002.

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BLOOD BANK INTERNAL PROCEDURE / POLICY

TITLE / DESCRIPTION: INDEX NUMBER(S):

ELUTION - ACID (Elu-kit) 704

EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION:

DEC91 REV8 / AUG02 XM

PRINCIPLE

Elution using the commercial Elu-kit is a procedure for the recovery of antibody from coated red blood cells.

1. Red blood cells coated with antibody are first thoroughly washed in a buffered wash solution to
remove all traces of unbound protein.

2. The washed red cells, with antibody still attached, are then suspended in a low pH glycine buffer.

3. After centrifugation, the supernatant containing any dissociated antibody is separated from the red
cells. Restoration to near normal ionic strength and pH is achieved by adding a concentrated tris
buffer.

POLICY

An acid elution will be performed on patient cell samples with positive DAT due to IgG if six months have passed
since last elution. Patients with a diagnosis of autoimmune disease and a positive DAT must have an eluate
performed on first presentation then every six months thereafter. As well, an eluate must be performed if the DAT
strength increases significantly (equal to or greater than 2 agglutination readings), or if the patient has been
transfused in the past three months.

SPECIMEN

5mL (minimum 3mL) whole blood in a Lavender Top tube

REAGENTS, SUPPLIES, EQUIPMENT

 centrifuge
 normal saline
 distilled water
 12 x 75 mm disposable glass test tubes
 Pasteur pipettes
 timer
 Elu-kit
 Concentrated Wash Solution: Prepare working wash solution by adding one volume of
concentrated wash solution to nine volumes of distilled water. Mix thoroughly before
use. Store at 20 - 80C in a stoppered glass or plastic container; good until turbid.
 Eluting Solution: Low pH glycine buffer

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 Buffering Solution: Tris-aminomethane solution to neutralize acidity of eluting


solution.

SAFETY PRECAUTIONS

1. All blood and blood products must be treated as potentially infectious. Follow universal
precautions detailed in the Laboratory Safety Manual.

2. Do not attempt to pick up broken glass with fingers. Use a dustpan or other scooping implement
and dispose of glass fragments in sharps disposal container.

QUALITY CONTROL
The purpose of this control test is to assure that antibody present in the eluate has been
derived from the bound state on the original cells and is not unbound antibody remaining as the
result of inadequate washing.

1. Retain a small sample of working wash solution from the last wash of the coated red cells to test
for antibody activity in parallel with eluate.

2. Test the last wash against cells homozygous for antigens of clinical significance. Routine reagent
screening cells are adequate for testing last wash.

3. If antibody activity is present in the last wash, repeat the elution procedure after more thorough
washing of the cells.

PROCEDURE
Preparing the Eluate

1.1. Centrifuge the specimen and remove all excess plasma or serum.

1.2. Wash the coated red cells once with saline. Wash the cells an additional four
times with working wash solution. For each wash, mix well after each addition
of working wash solution, centrifuge for one minute, remove supernatant
carefully without losing any red cells.

1.3. Reserve a small aliquot of solution from the last wash to serve as a control (see
Quality Control).

1.4. Place 1 mL (20 drops) of washed red cells in a clean 12 x 75 mm test tube. Add
1 mL (20 drops) of eluting solution and mix gently by inversion four times.
Immediately Serofuge for 45-60 seconds.
Note: If fewer than 20 drops available, add an equal number of drops of eluting solution.
Add the same number of drops of buffering solution.

1.5. Immediately transfer the supernatant eluate to a clean test tube and discard red
cells. Add 1 mL (20 drops) of buffering solution to supernatant and mix well.
The eluate should turn light blue. Continue adding additional buffer, one drop at
a time with mixing, until it turns blue.

1.6. Mix well. Centrifuge for one minute to remove any precipitate of cellular debris,
then transfer the eluate to a clean and properly labeled test tube.

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1.7. The eluate is now ready for testing to detect or identify antibodies.

2. Testing the Eluate

2.1. Place 1 drop of a 3-5% suspension of each cell to be tested in a clean, labeled
test tube. Add a small volume of physiologic saline (5-10 drops). Centrifuge for
30 seconds at 3,400 rpm (RCF 900 to 1,000), decant the saline and blot the tubes
dry.

2.2. Test A and B cells with the eluate if the patient has received ABO incompatible
plasma products or if the patient received an ABO incompatible bone marrow
transplant.

2.3. Add 2 drops of eluate to the dry cell button. Do not add potentiators such as
LlSS or albumin.

2.4. Mix gently to resuspend the cells and incubate for 15 minutes in a 37 C Dribath.

2.5. After appropriate incubation, add 5-10 drops of working wash solution to the
tube and mix.

2.6. Centrifuge for 30 seconds at 3,400 rpm (RCF 900-1,000).

2.7. Decant the working wash solution and blot the tubes dry.

2.8. Without further washing add 2 drops of anti-human globulin and shake to mix.

2.9. Immediately centrifuge in Serofuge for 15 seconds.

2.10. Resuspend the cells by gentle shaking, read for agglutination and record test
results.

2.11. To all tubes with negative reactions, add one drop IgG sensitized cells.
Centrifuge and read. Record results. If negative, repeat testing starting from
step 2.1.

3. Interpretation

3.1. An agglutination reaction, occurring when the eluate is tested against red blood
cells of appropriate phenotypes, indicates that an antibody has been recovered
from the original cells, subject to satisfactory control tests.

3.2. The absence of agglutination indicates that no antibody has been recovered from
the cells.

3.3. If the antibody activity is present in the wash saline, or if the wash saline
hemolyzes test cells, repeat the elution procedure after more thorough washing
of the cells.

REPORTING RESULTS
1. Record graded reactions on panel sheet. Record that it is an eluate being tested
and type of eluate. Record specific phase of test reactions, e.g. “IgG”.

2. If serum panels are included on the same sheet, clearly label results as serum.

3. Record results of control (last wash) on panel sheet.

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4. Order an ELUATE, ACID and enter results under BLOOD BANK RESULT ENTRY by
entering the sample accession number and selecting PATIENT under CELL in the dialogue box
that appears.
5. Click on, or use the arrow key to move to the first cell to be resulted, represented by a white cell in
the worksheet.
6. Enter or select a result from the list. To enter a result, type the first letter associated with the result
(e.g. N for negative, NP for Not Performed) or click the down arrow key to display the list of
options.
7. Press ENTER to move to the next result cell.
8. Enter or select the overall interpretation of the Eluate panel. All eluate interpretations begin with
Eluate eg. Eluate-Fyb.
9. Click PERFORM or VERIFY to save the results.

10. Submit panel sheet(s) to the senior technologist for review.

PROCEDURE NOTES
1. Prolonged immersion of red cells in the elution solution causes hemolysis. The
consequent release of hemoglobin into the eluate alters the pH and may affect
the performance of the eluate in serological testing.

2. Do not add potentiators such as bovine albumin or a low ionic strength additive solution, as the
eluate is already a low ionic substrate. Polyethylene glycol (PEG) may be used to increase to
sensitivity of the eluate as it will not affect the ionic strength.

3. For greater test sensitivity if the direct antiglobulin test on the original red cell was weak (w to 1+),
the sensitivity of the antibody detection test may be enhanced by increasing the number of drops of
eluate per test and/or by extending the incubation time (e.g. 3-4 drops of eluate may be used for the
test instead of 2 drops, and/or incubation at 37 C may be extended to 20 minutes).

4. Not washing the panel cells with saline before adding anti-human globulin improves the sensitivity
of the test by minimizing dissociation of antibody bound to the test cells during incubation.

LIMITATIONS OF PROCEDURE

The yield of antibody obtained upon elution from coated red blood cells is dependent on the
following variable factors:

1. The amount of antibody bound to the cells. Where elution is being attempted from red blood cells
which have been incubated with serum in vitro, this is related to the serum: cell ratio in the
incubating mixture, to the mean binding constant of the particular antibody, to the number, type
and accessibility of the red cell receptor sites and whether or not the reaction has been allowed to
reach equilibrium. These factors are partially under the control of the serologist, who is able to
vary proportions of serum to cells and incubation conditions accordingly.

2. The degree of dissociation of antibody that occurs during the washing in cold 4 C working wash
solution. More dissociation of antibody will occur if the working wash solution is warm, giving
false negative results.

3. The degree of recombination of antibody that occurs before the eluate is separated from the cells.
This is minimal with the acid elution procedure, as separation of the eluate from the cells occurs at
pH that is unfavorable for antibody association.

4. The degree to which immunoglobulin is denatured by the low pH during dissociation. This is
minimal if the procedure is carried out as recommended.

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5. Other factors to be considered are:

5.1. Red blood cells which have a positive direct antiglobulin test due to bound
complement alone will normally yield an eluate showing no antibody activity.

5.2. Incorrect restoration of pH in the eluate may cause hemolysis of red blood cells
in subsequent testing.

5.3. Cells from blood samples stored for longer than 72 hours may yield less potent
eluates than those from freshly drawn samples. An alternative elution procedure
may be preferable for the preparation of eluates from stored cells.

5.4. A false negative test may occur if the test red cells suspension is not washed
sufficiently free of human protein before incubation with eluate, or if the test
system becomes contaminated in any way with human protein other than
antibody dissociated during the elution phase. A negative Coombs control test
indicates this source of error.

REFERENCES

1. Elukit, Package Insert. Houston, TX: Gamma Biologicals, August 1995.

2. Technical Manual, 13th edition. Bethesda, MD: American Association of Blood Banks, 1999.

3. Cerner HNA Millenium Integrated Clinical Information System PathNet User Training
Manual, 1st Edition, King Faisal Specialist Hospital and Research Center, July 2002.

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BLOOD BANK INTERNAL PROCEDURE / POLICY

TITLE/DESCRIPTION: INDEX NUMBER(S):

ELUTION - LUI FREEZE 748


EFFECTIVE DATE: REVISION DATE/NUMBER: DISSEMINATION:

FEB92 REV9 / AUG02 XM

PRINCIPLE

Elution is the removal of antibody that has been adsorbed onto the surfaces of red cells. If an ABO antibody is
suspected, as commonly occurs in neonates whose blood type is ABO incompatible with maternal blood, the method
of choice is Lui freeze elution. After the eluate is prepared, it should be tested with A and B cells, as well as with a
3-cell panel of O red cells of known antigenic make up (antibody screening cells).

POLICY

A Lui freeze eluate will be prepared from cord samples of suspected ABO hemolytic disease of the newborn cases.
The eluate will be tested against A1, B and O cells using antiglobulin technique.

SPECIMEN

Antibody coated red cells from group A, B or A,B cord blood sample which has tested DAT positive.

REAGENTS, SUPPLIES AND EQUIPMENT

 Saline
 Stoppered Test Tube
 -20C to -70C Freezer
 Running Tap Water
 Centrifuge (Calibrated)

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 Reverse grouping cells


 Antibody screening cells
 Cellwasher
 IgG Sensitized Cells
 Anti-human globulin reagent
 12 x 75 mm test tubes
 Marking pen
 Disposable pipettes
 37C Dri Bath

SAFETY PRECAUTIONS

All blood and blood products must be treated as potentially infectious. Follow safety precautions detailed in the
Laboratory Safety Manual.

QUALITY CONTROL

The purpose of this control test is to assure that antibody present in the eluate has been derived from the bound state
on the original cells, and is not unbound antibody remaining as the result of inadequate washing.

1. Retain a sample of saline from the last wash of the coated red cells to test for antibody activity
against A and B cells.

2. If antibody activity is present in the last wash, repeat the elution procedure after more thorough
washing of the cells.

PROCEDURE

1. Wash cells to be eluted with large volumes of saline at least 6 times. Mix well after each addition
of saline and centrifuge for one minute. Remove supernatant carefully without losing any red cells.

2. Retain the last wash. Test as specified in Quality Control (above).

3. Add 3 drops of 0.9% saline to 10 drops of the well washed packed red blood cells.

4. Stopper the tube and mix well.

5. Coat the sides of the tube with red cells by rotating the tube.

6. Rapidly freeze the red cell layer by placing the tube horizontally in a -20C to -70C freezer for 10
minutes.

7. Thaw rapidly under warm running tap water.

8. Centrifuge the hemolysed cells at high speed for 2 minutes. Remove the hemolysate to an
appropriately labeled test tube.

9. Test the hemolysate (2 drops) against A and B cells (1 drop) and screening cells I, II and III (1
drop) by antiglobulin technique. Test last wash against A and B cells.

9.1. Do not add LISS.

9.2. Incubate for 15 minutes at 37C.

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9.3. Wash 4 times in the cell washer.

9.4. Add 2 drops of AHG. Centrifuge and read.

9.5. To all negative tubes, add 1 drop IgG sensitized cells. Centrifuge and read.
Record results.

REPORTING RESULTS

1. Record serological results on panel sheets.

2. Order Eluate ,Lui under the DEPARTMENT ORDER ENTRY application.

3. Enter results in the BLOOD BANK RESULT ENTRY application.

3.2 Agglutination of A cells with a negative antibody screen demonstrates presence of


anti-A.

3.3 Agglutination of B cells with a negative antibody screen demonstrates presence of anti-B.

3.4 Agglutination of A and B cells with a negative antibody screen demonstrates presence of
anti-A,B.

5. If agglutination is present in the screening cells, perform the Elukit elution. See IPP 704 - Elution - Acid
(Elukit).

6. Append comment under mother’s medical record number, e.g., “Cord # (baby MRN) B POS, DAT POS;
ANTI-B ELUTED, date and accession #, using the appropriate comment template.

7. Print a hard copy of LUI Eluate results and attach to panel sheets. Submit to senior technologist for review.

PROCEDURE NOTES

1. This elution is only used for confirming ABO HDN. Do not utilize to identify other antibodies. If positive
DAT is suspected to be other than ABO, utilize Elukit elution. See IPP 704.

LIMITATIONS OF PROCEDURE

1. This is a poor elution technique for antibodies other than ABO.

2. The markedly hemoglobin stained eluate causes difficulty in reading tests prior to antiglobulin stage.

REFERENCES

1. Basic Blood Grouping Techniques and Procedures, 2nd Edition. Victorian Immuno-haematology Discussion
Group. 1992.

2. Technical Manual, 13th edition. Bethesda, MD: American Association of Blood Banks, 1999.

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BLOOD BANK INTERNAL PROCEDURE / POLICY


TITLE / DESCRIPTION: INDEX NUMBER(S):

EMERGENCY TRANSFUSION 730


EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION:

FEB92 REV8 / SEP02 XM

PRINCIPLE

Emergency issue of blood for transfusion, i.e. issue of blood prior to completion of pre-
transfusion testing, must be requested by the patient's physician. That physician must take
responsibility for the transfusion of uncrossmatched blood and its attendant risks.

POLICY

When a delay in transfusion could be detrimental to the patient, blood products will be released
prior to completion of pre-transfusion testing in accordance with AABB Standard 5.17.4 Urgent
Requirement for Blood. These products will be prepared and dispensed in accordance with
AABB Standards 5.11 to 5.17

REAGENTS, SUPPLIES, EQUIPMENT

 Emergency Transfusion Form (Appendix I)


 "NOT CROSSMATCHED" unit tags (Appendix II)

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SAFETY PRECAUTIONS

Follow safety precautions listed in referenced IPP’s.

PROCEDURE

Candidates for emergency dispense of blood for transfusion fall into two categories depending
on the availability of a specimen for pre-transfusion testing, but in both scenarios, the units will
be dispensed in the same manner:

1. Patients with no type and screen in the past 72 hours - specimen accompanies
request for emergency transfusion.

1.1 Locate previous patient records, if available, either in computer or card file.

1.2 Perform ABO/Rh on the blood specimen according to ABO/Rh and Weak D Typing
IPP 701.

1.2.1 If blood group matches previous records, select type specific units from
available blood inventory.

1.2.2 If blood group does not match previous records, select O negative units and
request that a repeat specimen be drawn for pre-transfusion testing and
patient's ID be rechecked. Proceed with procedure step 2 as if no specimen
were available.

1.3 Save segments in labeled tubes from each unit to be issued. If time allows, perform
immediate spin crossmatches on units to be emergency issued.

1.4 Attach "NOT CROSSMATCHED" tags (Appendix II) to units to be issued.

1.5 Go to the DISPENSE AND ASSIGN application.

1.6 Select the DISPENSE mode under TASK.

1.7 Enter the patient’s medical record number.

1.8 Barcode the unit into the Worksheet.

1.9 If the unit group does not match the patient’s group, an exception box will
open. Ensure the unit group is compatible with the recipient’s group, then
select YES to override.

1.10 Select EMERGENCY as the reason.

1.11 Click the Save icon. A Save box will open.

1.12 Enter the ordering physician’s name in the Physician box.

1.13 Select EMERGENCY in the Reason box.

1.14 List OK in the Visual Inspection box.

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1.15 Enter the messenger’s ID number in the courier box.

1.16 Enter the ward in the Location box.

1.17 Press OK at the bottom of the box.

1.18 The print box will open. Select the appropriate printer to print the
Emergency Transfusion Form (Appendix I).

1.19 Attach the Emergency Transfusion Form (Appendix I) to the unit.

1.20 Go the GENERATE TAGS AND LABELS application.

1.21 Click on Option Reprint.

1.22 Click on Emergency Tag under Tag/Label box.

1.23 Select 2 copies.

1.24 Enter the patient’s MRN number and the product number in the
appropriate fields.

1.25 Click the printer icon.

1.26 Place one Emergency Transfusion Request form (Appendix I) with the
unit and give to the messenger.

Note: Ideally a signed request form should accompany the specimen but the
form can be sent with blood products and be signed and returned to
the Blood Bank at a later time.

1.27 Place the other on the Senior’s desk for follow up.

1.28 Complete the type and screen according to ABO/Rh and Weak D Typing
IPP 701 and Antibody Screen IPP 706. Perform the crossmatch
according to Crossmatch IPP 722.

1.28.1 If the antibody screen is negative, perform immediate spin


crossmatches using segments saved from units already
issued.

1.28.2 Before entering the crossmatch results in the computer,


the unit has to be given a transfused status.

1.28.3 Go to the FINAL DISPOSITION application.

1.28.4 Select the Transfuse mode under Task.

1.28.5 Enter the product number and click on the SAVE icon.

1.28.6 A crossmatch may have to be ordered under Accession


Add On in the DEPARTMENT ORDER ENTRY

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application, if not entered by the floor with the Type and


Screen.

1.28.7 Go to the RESULT ENTRY application to enter the


crossmatch results under the accession number.

1.28.8 A message box will appear stating that the unit is not in a
valid state for crossmatch because it has bee dispensed
and transfused already.

1.28.9 Click on YES after the “Do you wish to continue?”


question.

1.28.10 Enter the crossmatch results and interpretation and


click on PERFORM or VERIFY.

1.28.11 A message box will appear stating that the product is


transfused, the crossmatch procedure results will be
saved, but product will not be set to crossmatched. Click
OK.

1.28.12 Another message box will appear asking if you want to


complete the crossmatch for that accession number. Click
on YES and the crossmatch is completed.
.
1.8.2 If antibody screen is positive, inform patient’s physician and the Blood Bank
Supervisor immediately. Complete full AHG crossmatch to determine if units
issued are compatible. Identify the antibody and antigen type the units
according to Antibody Identification - Basic Panel IPP 707 and Antigen
Typing IPP 710.

2. Patient with no type and screen in past 72 hours - no specimen available.

2.1 Notify the requesting location that a sample for crossmatch must be collected Stat.

2.2 Select O negative units from available blood inventory.

2.3 Save segment in labeled tubes from each unit to be issued. Use segments for
crossmatching when the specimen is received. If time allows, group check the units
prior to issue.

2.4 Attach "NOT CROSSMATCHED" tags (Appendix II) to units to be issued.

2.5 Dispense the units according to steps 1.5 – 1.27.

Note: Ideally a signed request form should accompany the specimen but the
form can be sent with blood products and be signed and returned to
the Blood Bank at a later time.

2.6 When specimen is drawn and received, perform the type and screen.

2.7 If antibody screen is negative, perform immediate spin crossmatches


using segments saved from units already issued.

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2.8 Before entering the crossmatch results in the computer, the unit has to be
given a transfused status.

2.8.1 Go to the FINAL DISPOSITION application.

2.8.2 Select the Transfuse mode under Task.

2.8.3 Enter the product number and click on the SAVE icon.

2.8.4 A crossmatch may have to be ordered under Accession


Add On in the DEPARTMENT ORDER ENTRY
application, if not entered by the floor with the Type and
Screen.

2.8.5 Go to the RESULT ENTRY application to enter the


crossmatch results under the accession number.

2.8.6 A message box will appear stating that the unit is not in a
valid state for crossmatch because it has bee dispensed
and transfused already.

2.8.7 Click on YES after the “Do you wish to continue?”


question.

2.8.8 Enter the crossmatch results and interpretation and click


on PERFORM or VERIFY.

2.8.9 A message box will appear stating that the product is


transfused, the crossmatch procedure results will be
saved, but product will not be set to crossmatched. Click
OK.

2.8.10 Another message box will appear asking if you want to


complete the crossmatch for that accession number. Click
on YES and the crossmatch is completed.

2.8.2 If antibody screen is positive, inform patient’s physician and the Blood Bank
Supervisor immediately. Complete full AHG crossmatch, antibody
identification and antigen typing to determine if units issued are compatible.

REPORTING RESULTS

1. Complete the ABO/Rh, antibody screen and crossmatch STAT and record in the
computer.

2. If incompatible crossmatches are found, notify the physician to stop transfusion


immediately and have all issued blood returned to Blood Bank. Notify the Blood Bank
Medical Director immediately.

3. A copy of the Emergency Transfusion Form must be returned to the Laboratory within 24
hours, complete with the signature of the charge nurse and attending physician. The

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original should go into the patient’s chart. Give form to senior for review. Senior will file
forms in the Emergency Transfusion file in the issue bench area.

PROCEDURE NOTES

1. Should any patient with a request for emergency transfusion prove to have a
previously identified clinically significant antibody, units negative for that antigen(s)
must be given and blood should not be issued by emergency issue. Units should be
typed STAT for the antigens in question and a specimen can be obtained in the
meantime. Contact the on-call blood bank pathologist if this is not acceptable with
the requesting physician.

2. Each unit dispensed should have a NOT CROSSMATCHED tag attached to it with a
tach-it.

3. An Emergency Transfusion Form is unique for each unit dispensed. There should
be one attached to the unit itself and one that accompanies the unit.

3. A sample for crossmatch must be collected at the earliest opportunity in the transfusion
sequence.

LIMITATIONS OF PROCEDURE

If the patient has alloantibody and compatible units are not available, refer to the Medical
Director of Blood Bank. The decision may be made to issue incompatible units.

REFERENCES
th
1. Technical Manual, 13 Edition, Bethesda, MD: American Association of Blood Banks,
1999.

2. Standards for Blood Banks and Transfusion Services, 21st Edition. Bethesda, MD:
American Association of Blood Banks, 2002.

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BLOOD BANK INTERNAL PROCEDURE / POLICY


TITLE / DESCRIPTION: INDEX NUMBER(S):

TRANSFUSION REACTION INVESTIGATIONS 778


EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION:

DEC91 REV7 / AUG02 XM

PRINCIPLE

Any transfused blood product can cause an adverse effect which may be due to incompatibility
of donor red cells and recipient plasma, bacterial contamination of blood product, transfusion-
transmitted infection, allergy, white cell antibodies or other miscellaneous causes. The most
common signs and symptoms of an adverse reaction due to transfusion of blood or blood
products are:


o o
fever (increase of equal to or greater than 1 C or 2 F), with or without chills
 chills (rigors), with or without fever
 skin symptoms - hives (urticaria) or itching

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 chest pain and/or dyspnea


 acute hypotension or acute hypertension
 nausea, vomiting
 flushing
 bleeding
 hematuria
 hemoglobinuria
 anaphylaxis

POLICY

Inaccordance with AABB Standards 7.3 The blood bank or transfusion service shall have a
process for the detection, reporting, and evaluation of suspected complications of transfusion.
An adverse event experienced by a patient in association with a transfusion shall be regarded
as a suspected transfusion complication, and followed up accordingly.

SPECIMEN

 10mL (min. 7mL) red top tube


 5mL (min. 3mL) lavender top tube
 Bag from transfused product (if available)

REAGENT, SUPPLIES, EQUIPMENT

 Anti-A
 Anti-B
 Anti-D
 Anti-human globulin
 Coombs control cells
 12 x 75 mL disposable tubes
 Automatic cell washer or Serofuge
 Disposable pipettes
 Transfusion Reaction Form (Appendix I)
 Transfusion Reaction Worksheet (Appendix II) – used only during ICIS downtime
 0.9% Saline

SAFETY PRECAUTIONS

1. All blood and blood products must be treated as potentially infectious. Follow universal
precautions detailed in the Laboratory Safety Manual.

2. Do not attempt to pick up broken glass with fingers. Use a dustpan or other scooping
implement and dispose of glass fragments in sharps disposal container.

PROCEDURE

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Nursing Procedure (Read this section to nurses if they don’t know what to do)

1. Stop the infusion immediately; keep the line open with a slow infusion of saline.

2. Notify the physician immediately.

3. Notify the Blood Bank (extension 38270, 38220, 35841, or 35842) immediately. Notify the
Blood Bank of the unit number(s) of the blood products implicated in the reaction.

4. Reconfirm the identification of the patient and the identification of the blood infused.

5. Complete a Transfusion Reaction Form with the required information. One copy of
the form must accompany the samples to the Blood Bank because there is no
mechanism to note the clinical information in the ICIS.

6. Retain a copy of the Transfusion Reaction Form in the patient’s chart.

7. Order a Transfusion Reaction Careset in the computer which includes a DAT for
Blood Bank, Urinalysis for Microbiology.

6. Labels will print for the specimens. (One lavender and one Red top) labels will also print
for a Urinalysis.

7. Ensure samples are drawn STAT for the workup. Samples must be drawn carefully to
minimize mechanical hemolysis and must be properly labeled.

8. Send blood samples, Crossmatch / Component Transfusion Form, the Transfusion


Reaction Form and the blood bag with infusion set and tag attached to the Blood Bank,
STAT.

9. Send a properly labeled urine sample (when available) to Microbiology for urinalysis.

10. Do not transfuse the patient until the reaction investigation is completed. If transfusion is
indicated prior to completion of the investigation, the physician must consult with the
Blood Bank Director.

Blood Bank Procedure

NOTE: If a severe reaction such as hemolytic, anaphylactic, bacterial contamination or


any other reaction that may be life threatening is suspected at any stage in the
investigation, immediately notify the Blood Bank Medical Director or Consultant
and the Supervisor.

Red Cell Products


Immediate Workup:

11. Transfusion reaction investigations are STAT procedures and must be completed as soon
as possible. If possible, a different technologist other than the one who crossmatched the
blood must perform the investigation.

12. Login the samples when they are received.

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13. Check that all the required clinical information is completed on the Transfusion
Reaction Form.

14. Enter transfusion reaction work up results under RESULT ENTRY.

15. Perform clerical check:

15.1 Check the patient's name, medical record number, blood type, donor unit number
and donor blood type on all patient specimens, components involved, paper and
computer records.

15.2 Record findings in the computer in the Clerical Check cell, either clerical check OK,
or clerical error noted.

15.3 If any discrepancy is noted in clerical check, proceed as follows:

15.3.1 If there is evidence of a transfusion error, notify the Blood


Bank consultant, patient's physician and supervisor
immediately.

15.3.2 If there is indication of a mix-up of patient samples, misdraw of patient, or


mix-up of donor units, check other patient records over the same time period
as there may be another patient at risk.

16. Perform visual inspection of sample:

16.1 Compare pre and post transfusion specimens. Check for hemolysis and jaundice
(yellow or brownish coloring). Record findings in the computer in the PRE and
POST Hemolysis and PRE and POST Icteric cells, either YES or NO.

16.2 If hemolysis or jaundice is observed in post transfusion sample but not in


the pretransfusion sample, refer to Extended Workup below.

17. Perform direct antiglobulin test (DAT):

17.1 Perform a DAT on post transfusion specimen. Record results at the DAT cells.

17.2 If the DAT is positive on the post transfusion sample, Add on a DAT to the
pretransfusion TS. If a DAT was already performed on the pre-transfusion sample
enter the repeated result as a comment.

17.2.1. If the DAT on the pre transfusion sample is the same strength as the post
reaction sample, this indicates the DAT is not caused by the transfusion.

17.2.1.1. If this is the first time that a DAT was performed on the pre
transfusion sample, perform an eluate on the pre-transfusion (or
post-transfusion) sample.

17.2.2. If the DAT on the pre transfusion sample is negative, or is weaker than the
post reaction sample, refer to Extended Workup.

18. Check for temperature increase:

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18.1 If there has been a rise in temperature of at least 1C enter a result of Y
in the TEMPERATURE INC cell otherwise enter N.

18.2 Send blood bag for culture if the temperature increase is 1C or greater.

18.3 Register the unit so tests can be ordered. Go to the DEPARTMENT


ORDER ENTRY application.

18.4 Click in the medical record number box and press ENTER.

18.5 An Encounter Search box will appear, click on ADD PERSON.

18.6 Select KFSHRC Riyadh as the Client and Facility name and the
Registration window will open.

18.7 In the FIRST NAME box, enter Crossmatch.

18.8 In the MIDDLE NAME box, enter Transfusion Reaction .

18.9 In the LAST NAME box, enter the product number i.e GM12345.

18.10 Enter the birth date as the order date for ease of tracking.

18.11 Enter Dr. Roberts for the admitting and the attending physican.

18.12 In the MEDICAL SERVICE box, select LAB- BLOOD BANK.

18.13 In the BUILDING box, select MAIN BUILDING.

18.14 In the AMBULATORY box, select LAB-MAIN-R.

18.15 At the bottom of the box, select OK..

18.16 A new medical record number will be assigned.

18.17 In the ORDERABLE box, select Blood Culture and the collection
detail box will open.

18.18 Ensure the print label box is checked off and that the information is
correct and click ADD under ORDERS.

18.19 Enter Blood Bank – PRBC under the Body Site.

18.20 Enter none under Antibiotics.

18.21 Click on the SUBMIT ORDERS icon and note the accession numbers
that are assigned.

18.22 Retrieve labels from the designated printer and affix to the unit.

18.23 The culture specimen must be submitted to Microbiology as soon as


possible. A delay in receipt of the specimen can disrupt Microbiology’s
blood culture numbering sequence and cause possible further delays.

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18.24 Enter either YES or NO under Bag Sent for Culture.

19. If there is no immediate indication of an acute hemolytic transfusion reaction, enter a


result of N at the HEMOLYTIC cell.

20. After completing result entry, click Perform / Verfy.

21. Go to the ORDER RESULT VIEWER application and print the Transfusion Workup
Results.

22. Look up and record blood culture and urinalysis accession numbers on the print out,
if applicable. Give Report to Senior for review who will then forward it to the Blood
Bank consultant for evaluation and interpretation.

21. Save the blood product bag(s) in Jewett refrigerator for one week. If the bag is sent to
Microbiology, they will retain it for one week.

Extended Workup:

NOTE: If Clerical Check, Visual Inspection or DAT give positive results, further workup must
be done immediately to confirm or rule out an acute hemolytic transfusion reaction.

22. Add on a TS and Crossmatch to the transfusion reaction accession number. Perform
ABO and Rh testing on the patient’s post-transfusion sample and enter the results in
Cerner.

23. Perform an antibody screen with the post-transfusion sample and enter the results in
Cerner. If a previously undetected antibody is detected, perform antibody identification to
identify the antibody. Test the segment from the transfused donor unit for the
corresponding antigen. Order and enter the antigen types on the unit according to IPP710
Antigen Typing.

24. Perform a crossmatch with the post- transfusion sample using an attached segment or on
blood remaining in the blood bag. Perform full AHG crossmatch even if the pretransfusion
crossmatch was only immediate spin.

25. Perform ABO and Rh testing on the patient’s pretransfusion sample and on blood from the
unit or an attached segment.

25.1 Enter the specimen number of the pre-transfusion TS or enter the product number
and the number of the unit that is being retyped.

25.2 Enter repeat results performed on the pre-transfusion sample or unit segment in the
form of a result comment after the initial verified results. For example: 4+ “Pre-
transfusion sample repeat.” or “Unit retype repeat.”

26. Perform an antibody screen on the pre-transfusion sample. Enter results as outlined
above. If a previously undetected antibody is detected, perform antibody identification to
identify the antibody. Test the segment from the transfused donor unit for the
corresponding antigen. Order and enter the antigen types on the unit according to IPP710
Antigen Typing.

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27. Perform a crossmatch with the pre-transfusion sample using an attached segment or on
blood remaining in the blood bag. Enter the results in Cerner as outlined above. Perform a
full AHG crossmatch even if the pretransfusion crossmatch was only immediate spin.

28. If DAT is positive, add on an eluate. Perform an eluate on the post transfusion sample.

29. Consult with the Blood Bank Medical Director or consultant to determine whether further
testing is needed, such as baseline and 6 hours post transfusion levels of:
 Haptoglobin
 Plasma hemoglobin
 Total Bilirubin
 Serum creatinine
 Urea

30. Refer to Technical Manual for additional serological techniques which may be useful.

Delayed Hemolytic Transfusion Reaction Workup:

31. Suspected Delayed Hemolytic Transfusion:

31.1 If DHTR is suspected by floor, request them to order a Transfusion Reaction and
send it to Crossmatch. Perform a Transfusion Reaction investigation as outlined
above in Immediate Workup.

31.2 If DHTR is suspected in Crossmatch due to a positive DAT with antibody eluted,
order a Transfusion Reaction or, if appropriate, add on a Transfusion Reaction to the
existing TS requisition. Enter a comment “Transfusion reaction on unit xxxxx.”

31.3 Result the transfusion reaction as outlined above in Immediate Workup.

32. In the RESULT ENTRY application, enter the results of ABO/Rh, Antibody Screen, DAT
and ELUATE. Attach panel and elution results.

33. In computer, print out:

33.1 Patient’s transfusion history in the PATIENT PRODUCT INQUIRY


application.

33.2 Patient’s laboratory tests in the ORDER RESULT VIEWER


 CBC’s since last transfusion (to track Hct)
 Bilirubins pretransfusion and since last transfusion

34. Attach all results printouts. Look up and record blood culture accession number on
Transfusion Reaction results, if applicable. Give to Senior for review who will then forward
it to the Blood Bank consultant for evaluation and interpretation.

35. The Blood Bank consultants will enter interpretions in the ICIS.

Non-Red Cell Products

Acute Hemolytic Transfusion Reactions are not likely to occur with non-red cell products.
Therefore a limited workup is adequate (as follows):

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36. Login the samples when they are received.

37. Check that all the required clinical information is completed on the Transfusion
Reaction Form.

38. Enter transfusion reaction work up results under RESULT ENTRY.

39. Perform clerical check:

39.1 Check the patient's name, medical record number, blood type, donor unit number
and donor blood type on all patient specimens, components involved and paper and
computer records.

39.2 Record findings in the computer in the Clerical Check cell, either clerical check OK,
or clerical error noted.

40. If any discrepancy is noted in clerical check, proceed as follows:

40.1 If there is evidence of a transfusion error, notify the Blood Bank consultant, patient's
physician and supervisor immediately.

40.2 If there is indication of a mix-up of patient samples, misdraw of patient, or mix-up of


donor units, check other patients’ records over the same time period as there may
be another patient at risk.

41. Check patient MR comments for previous transfusion reactions and record the date and
classification of each on the Transfusion Reaction Form

42. Perform visual inspection of sample:

42.1 Compare pre and post transfusion specimens. Check for hemolysis and jaundice
(yellow or brownish coloring). Record findings in the computer in the PRE and
POST Hemolysis and PRE and POST Icteric cells, either YES or NO.

42.2 If hemolysis or jaundice is observed in post transfusion sample but not in the
pretransfusion sample, refer to Extended Workup.

43. Check for temperature increase:

43.1 A rise in temperature of at least 1C enter a result of Y in the


TEMPERATURE INC cell otherwise enter N.

43.2 Send component bag for culture if the temperature increase is 1C or
greater.

43.3 Register the unit so tests can be ordered. Go to the DEPARTMENT


ORDER ENTRY application.

43.4 Click in the medical record number box and press ENTER.

43.5 An Encounter Search box will appear, click on ADD PERSON.

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43.6 Select KFSHRC Riyadh as the Client and Facility name and the
Registration window will open.

43.7 In the FIRST NAME box, enter Crossmatch.

43.8 In the MIDDLE NAME box, enter Transfusion Reaction .

43.9 In the LAST NAME box, enter the product number i.e GM12345.

43.10 Enter the birth date as the order date for ease of tracking.

43.11 Enter Dr. Roberts for the admitting and the attending physican.

43.12 In the MEDICAL SERVICE box, select LAB- BLOOD BANK.

43.13 In the BUILDING box, select MAIN BUILDING.

43.14 In the AMBULATORY box, select LAB-MAIN-R.

43.15 At the bottom of the box, select OK.

43.16 A new medical record number will be assigned.

43.17 In the ORDERABLE box, select Blood Culture and the collection
detail box will open.

43.18 Ensure the print label box is checked off and that the information is
correct and click ADD under ORDERS.

43.19 Enter Blood Bank – Platelets, FPP, or Cryo under the Body Site.

43.20 Enter none under Antibiotics.

43.21 Click on the SUBMIT ORDERS icon and note the accession
numbers that are assigned.

43.22 Retrieve labels from the designated printer and affix to the unit.

43.23 The culture specimen must be submitted to Microbiology as soon as


possible. A delay in receipt of the specimen can disrupt
Microbiology’s blood culture numbering sequence and cause
possible further delays.

43.24 Enter either YES or NO under Bag Sent for Culture.

44. If there is no immediate indication of an acute hemolytic transfusion reaction, enter a


result of N at the HEMOLYTIC cell

45. Enter NP at the DAT result prompts.

46. After completing result entry, click Perform / Verfy.

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47. Go to the ORDER RESULT VIEWER application and print the Transfusion Workup
Results.

48. Look up and record blood culture and urinalysis accession numbers on the print out,
if applicable. Give Report to Senior for review who will then forward it to the Blood
Bank consultant for evaluation and interpretation.

49. Save the blood product bag(s) in Jewett refrigerator for one week. If the bag is sent
to Microbiology, they will retain it for one week.

REPORTING RESULTS

1. If Microbiology reports a positive blood culture, notify the Blood Bank Director or
Consultant immediately. If bacterial contamination is suspected, the pathologist will notify
the patient’s physician immediately.

2. If any results are indicative of an acute hemolytic transfusion reaction, notify the Blood
Bank Director or Consultant and the Supervisor immediately. If an acute hemolytic
reaction is suspected, the pathologist will notify the patient’s physician immediately.

4. If there is indication there may be further workup needed to rule out a reaction, leave the
HEMOLYTIC cell result pending, to be resulted when the workup is completed.

5. Attach the Transfusion Reactin Form received from the Ward to the completed transfusion
reaction result print out and submit to the Senior Technologist for review and forwarding to
the Blood Bank Director or Consultant. The pathologist will review these results as soon
as possible for interpretation and may recommend further testing. The Blood Bank
Director or consultant will return the completed report to the Crossmatch Senior
Technologist.

6. The Senior Technologist will enter the relevant information into the patient’s Medical
Record Data for future reference and then file the Transfusion Reaction Forms in the
Transfusion Reaction binder.

7. If there is any real or potential adverse reaction caused by blood or components received
from another facility (King Fahad National Guard Hospital or Aramco Hospital in Dhahran),
the Blood Bank Supervisor will notify the facility. Complete a Variance Report with the
details of the problem and document date, time, and contact person at the other facility
who received the information.

PROCEDURE NOTES

1. Delayed hemolytic transfusion reactions may not be apparent for several days following
transfusion. The most common signs are fever, unexpected fall or less than expected rise
in hemoglobin, and jaundice. The DAT may be positive.

2. Patients who experience repeated febrile reactions which are possibly due to white blood
cell antibodies may be transfused with blood that is leukocyte reduced by filtration.
Approval by the Medical Director is required before placing a patient on this protocol
permanently.

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3. All reactions should be handled initially as possible hemolytic reactions. The most
dangerous reactions which can occur are acute hemolytic, septic shock, and anaphylactic
shock. Most acute hemolytic transfusion reaction occur as a result of misidentification or
clerical errors in the procedures for blood drawing, requesting blood, pretransfusion
testing, issuing of blood and administration of blood. Therefore, procedures must be
adhered to strictly.

4. Most acute hemolytic transfusion reactions, transfusion of bacterial contaminated blood


products and anaphylactic reactions will be apparent after the first few mL. of blood has
been infused. Therefore, the infusion should be started slowly and the patient observed
closely for symptoms during the first 15 minutes of infusion.

5. If there is any report or mention of possible disease transmission in response to


transfusion, record the patient’s name, medical record number, physician name, then
report this information to the Medical Director who will decide if there is indication for
followup.

6. In the event of a computer downtime, the transfusion reaction work up may be


ordered on the Blood Bank Downtime Orders Form that will be accompanied with the
Transfusion Reaction Form. The workup and investigation will be documented on
the Transfusion Reaction Worksheet. The urinalysis and blood culture will have to
be ordered on a separate form, Laboratory Miscellaneous Orders Form.

REFERENCES
th
1. Technical Manual, 13 Edition, Bethesda, MD: American Association of Blood Banks,
1999, Pages 543-562.

2. Standards for Blood Banks and Transfusion Services, 21st Edition. Bethesda, MD:
American Association of Blood Banks, 2002.

TRANSFUSION REACTION WORKSHEET – COMPUTER DOWNTIME

MRN_________________________ Accession Number________/______/____


Name__________________________ Unit Number________________________

CLERICAL CHECK
Re-check of identity of patient, Donor unit(s) records and find no discrepancy
Donor OK Tech: Recipient OK Tech:
Yes / No Yes / No

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VISUAL INSPECTION OF PATIENTS SERUM

Haemolysis Icteric Direct Antiglobulin Test

Pre Transfusion AHG CC INTERP.


Yes / No Yes / No
Sample number ____/_____/_____
Post Transfusion AHG. CC INTERP.
Yes / No Yes / No
Sample number ____/_____/_____

Bag sent for culture Results of culture Blood in first voided urine

Yes / No Yes / No
Accession number if yes ___/___/____

CONFIRMATION OF ABO AND RH TYPE

Serum Cells INTERP.


A B D CON A B
Pre Transfusion
Recipient
Post Transfusion

Donor Segment
Donor Segment AHG CC INTERP.
DAT

RECONFIRMATION OF CROSSMATCH

Recipient Serum IS 37 C AHG CC Compatible

Pre Transfusion Donor RBC Segment

Post Transfusion Donor RBC Segment

Technologist : Date:
Conclusions:

Pathologist: Date:

Entered in ICIS By:_______________ Date:___________ Reviewed By: ____________ Date: _______

King Faisal Specialist Hospital and Research Centre


Pathology and Laboratory Medicine - Blood Bank
Riyadh, Saudi Arabia

TRANSFUSION REACTION FORM

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Ordering Doctor________________ Pager Number ________________

Date Requested ____/____/____ Time Requested ______________

Patient Location_____________ Ward Phone Ext. _____________

Type of Product: Unit Number(s):

Time and Date Time and Date Time apparent reaction Amount remaining in
Transfusion Began: Transfusion Stopped: began: bag

ML

Symptoms

Time and Date reported to Blood Clerical check performed by 2 SN Med’s Given for adverse
Bank: I’s or one SN I and one SN II reaction:

OK Y / N

ID ___________/____________

PLEASE NOTIFY BLOOD BANK IMMEDIATELY IF CLERICAL CHECK DOES NOT MATCH

Before transfusion At time of suspected transfusion reaction

Temperature

Pulse
Blood Pressure

Staff Nurse Signature ______________________________________________________

Refer to Nursing Policy Blood Transfusion : Adverse Reactions


Send yellow copy with blood bag, tubing, samples and all transfusion documentation AFTER
completing Transfusion Reaction order in the computer. During ICIS downtime send this form with
Blood Bank Orders Form.
Retain white copy in patient’s chart.
If blueplate not available then the following information is required:
MRN, Full Name, DOB, Sex__________________
BLOOD BANK INTERNAL PROCEDURE / POLICY

TITLE / DESCRIPTION: INDEX NUMBER(S):

W.A.R.M. (Warm Autoantibody Removal Medium) 781

EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION:

OCT93 REV4 / SEP99 XM


(Reviewed MAR02)

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Blood Bank Internal Policy / Procedure

PRINCIPLE

The red cells from a patient with a positive DAT and free serum autoantibody are treated with W.A.R.M. This
treatment removes IgG autoantibody, thereby reducing the strength of the positive DAT and freeing antigen sites for
adsorption. Concomitantly, the enzyme premodifies the red cells. The red cells, thus prepared, are combined with
autologous serum and the mixture is incubated at 37oC. The warm-reactive autoantibody is attached to the antigen
present on the patient's red cells. This antigen-antibody interaction is enhanced by the enzyme treatment of the cells.
Following incubation, the mixture is centrifuged and the adsorbed serum is harvested and tested. By comparing the
results of W.A.R.M.-adsorbed serum with those obtained with the unadsorbed serum, it is usually possible to confirm
the presence of the warm-reactive autoantibody, and detect or identify any additional alloantibody(ies) that may be
present.

POLICY

W.A.R.M. procedure will be performed prior to any other autoadsorption technique to remove a warm reactive auto
antibody from patient serum.

Red cells from recently transfused patients (within past 3 months) should not be used for autoadsorption because
transfused red cells in the circulation are likely to adsorb alloantibodies.

SPECIMEN

5mL (min. mL) whole blood in a Lavender Top tube.


10mL (min. 7mL) whole blood in a Red Top tube.

REAGENTS, SUPPLIES AND EQUIPMENT

 W.A.R.M. (Reconstitute with deionized water prior to use; see label for volume and directions. Store
reconstituted W.A.R.M. at 2-8oC for no more than 5 days after reconstitution. Do not freeze.)
 Isotonic saline
 Test tubes and test tube racks
 Transfer pipettes
 Serological centrifuge
 37C dry bath or waterbath

SAFETY PRECAUTIONS

1. All blood and blood products must be treated as potentially infectious. Follow universal precautions detailed
in the Laboratory Safety Manual.

2. Do not attempt to pick up broken glass with fingers. Use a dustpan or other scooping implement and dispose
of glass fragments in sharps disposal container.

PROCEDURE

1. Appropriately label a test tube.

2. Add 1 ml of packed patient red cells that have been washed with 0.9% saline.

3. Add 2 volumes of reconstituted W.A.R.M. to the tube (1 mL RBC: 2 mL W.A.R.M.).

4. Mix well and incubate at 37C for 30 minutes.

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5. Wash the W.A.R.M.-treated cells with saline a minimum of three (3) times, being careful to remove as much
supernatant as possible after each wash. Hard pack the W.A.R.M. treated red cells after the last saline wash.

6. Add an equal volume of patient serum to the W.A.R.M.-treated, packed red cells.

7. Mix well and incubate for 30-60 minutes at 37C.

8. Centrifuge for two (2) minutes or until red cells are well-packed.

9. Harvest the adsorbed serum (plasma) using a transfer pipette.

10. Test an aliquot of the adsorbed specimen at 37C and the antiglobulin phase to determine if warm-reactive
autoantibody has been removed. (See Interpretation). If results indicate that adsorption was insufficient,
repeat steps 1-9 using a new aliquot of patient red cells. If adsorption is complete, the adsorbed specimen is
ready for use in antibody detection and identification.

Interpretation:

1. Complete Adsorption

Complete adsorption of warm-reactive autoantibody has been achieved when the adsorbed serum no longer
reacts where previously detected with:

1.1 The patient's own W.A.R.M. treated red cells;

1.2 Those reagent red cells that lack antigens toward which additional alloantibodies are directed.

2. Incomplete Adsorption

If warm-reactive autoantibody has not been sufficiently removed, or the autoantibody cannot be adsorbed by
W.A.R.M.-treated cells, agglutination will be detected when the serum is tested as in "1.1" or "1.2" above.

NOTE: Complete adsorption of warm-reactive autoantibodies may not always be possible. However, in
some instances, only partial adsorption of the autoantibody may be sufficient to reveal underlying
alloantibody(ies) that might also be present.

REPORTING RESULTS

Record reactions on panel sheet in parallel with unadsorbed serum results. Label columns clearly to designate which
column is unadsorbed serum results and which is adsorbed serum results. Record the number of autoadsorptions
performed.

LIMITATIONS OF THE PROCEDURE

1. Not all red cells will become DAT negative upon W.A.R.M. treatment. Some examples may only show a
reduction in strength. It is possible that certain red cells will not be demonstrably affected. This does not
obviate their use in an autoadsorption procedure.

2. W.A.R.M. treatment and adsorption cannot be evaluated quantitatively. The number of adsorptions necessary
as well as their effectiveness is dependent on a variety of factors and may differ from patient to patient.

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3. Some autoantibodies have specificity within the Kell blood group system or other systems whose antigens are
enzyme sensitive (Xga). Since W.A.R.M. treatment would destroy these antigens, autoadsorption using
W.A.R.M.-treated cells would not be expected to be effective.

4. The use of W.A.R.M.-treated red cells for antigen typing is not recommended since commercial antisera may
or may not be specific when tested in this manner.

5. On occasion, it is possible that a warm-reactive autoantibody will not be completely adsorbed by W.A.R.M.-
treated red cells. As a result, it may not be possible to assign a definitive specificity to those reactions
obtained following adsorption. Further, complete adsorption of warm-reactive autoantibodies does not assure
that those antibodies remaining in the adsorbed serum can be readily identified.

6. Crossmatches performed on a warm autoadsorbed serum cannot be considered "compatible", since the
antibodies removed by warm autoadsorption procedures are active at 37C. The warm autoadsorption
procedure serves only as an aid in the detection and identification of underlying alloantibodies potentially
masked by a warm-reactive autoantibody.

7. Because the potential exists for adsorption of significant alloantibodies onto transfused donor red cells,
autoadsorption procedures should not be performed on samples from recently transfused patients.

8. Due to the fragility of certain red cells, some hemolysis may be observed upon treatment.

9. The treatment of the red cells with W.A.R.M., should be no more than, but not less than thirty (30) minutes.

REFERENCE

1. Warm Autoantibody Removal Medium, Package Insert. Durham, NC: Organon Teknika, 1992.

BLOOD BANK INTERNAL PROCEDURE / POLICY


TITLE / DESCRIPTION: INDEX NUMBER(S):

ADSORPTION TECHNIQUE 703


EFFECTIVE DATE: REVISION NUMBER / DATE: DISSEMINATION:

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DEC91 REV4 / AUG97 XM


(Reviewed MAR02)

PRINCIPLE

Antibody can be removed from the serum with red cells of the appropriate phenotype. The
antibody forms a complex with the membrane-bound antigen; when the serum and cells are
separated, the antibody remains attached to the cells.

Adsorption techniques may be useful in the following situations:

1. Removing autoantibody activity to permit detection of co-existing alloantibodies.

2. Removing unwanted antibody from a serum that contains an antibody suitable for reagent
use.

3. Separating multiple antibodies present in serum or eluate to aid in antibody identification.

4. Confirming the presence of an antigen on red cells through their ability to remove specific
antibody from serum.

5. Confirming antibody specificity.

POLICY

Autoadsorption for removal of autoantibodies should be used in untransfused patients


whenever an autoantibody is present which may mask the presence of underlying clinically
significant alloantibodies.

Do not perform autoadsorption on a patient who has been transfused in the past three months,
because transfused RBC’s may adsorb alloantibody from the patient’s serum.

SPECIMEN

 10mL (min. 7mL) red top tube


 5mL Lavender top tube

REAGENTS, SUPPLIES, EQUIPMENT

 0.9% saline
 10mL test tube

o
ice or 37 C waterbath
 centrifuge

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SAFETY PRECAUTIONS

All blood and blood products must be treated as potentially infectious. Follow safety
precautions detailed in the Laboratory Safety Manual.

QUALITY CONTROL

1. Test the adsorbed serum against screening cells to determine if the procedure has
removed the antibody.

1.1 Use the temperature and phase previously demonstrated to be optimal for antibody
activity.

1.2 A negative reaction indicates that adsorption is sufficient.

1.3 A positive reaction indicates that further adsorption is necessary.

PROCEDURE

1. Wash the selected cells at least three times with saline. After the last wash centrifuge the
cells for five minutes to pack them. Remove all the supernatant.

2. Mix appropriate volumes of serum and cells, and incubate at the desired temperature for
30-60 minutes.

2.1 The serum to cell ratio used depends upon the strength of the antibody that is to be
removed. The usual ratio is one volume of washed red cells to one volume of
serum.

2.2 The incubation temperature used should be that optimal for the activity of the
antibody:

2.2.1 For cold reacting antibodies place the tube inside a plastic bag filled with
crushed ice, seal, and mix on mechanical specimen rotator.
o
2.2.2 For warm reacting antibodies place the tube in the 37 C waterbath and mix
frequently.

2.3 Use a large tube to increase area of contact between serum and cells, and mix tube
every 5-10 minutes during incubation.

3. Centrifuge and remove serum promptly to avoid antigen-antibody dissociation. Label


serum as “adsorbed serum”.

4. Determine if all the antibody has been removed from the serum (see Quality Control).

5. If eluate is to be performed, save cells.

LIMITATIONS OF PROCEDURE

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Autoadsorption should not be performed on red cells from recently transfused patients because
the circulating transfused red cells may adsorb the alloantibodies that are being sought.

PROCEDURE NOTES

1. In separating mixtures of antibodies, selecting the proper cells for adsorption is extremely
important. The cells must be of an appropriate phenotype. The specificity of at least one
of the antibodies must be known (or suspected) in order to choose cells positive for one
antigen and negative for another.

2. Pretreating red cells with a proteolytic enzyme may enhance antibody uptake, reducing
the adsorptions required for complete removal of the antibody. Since some antigens are
destroyed by proteases, antibodies directed against these antigens may not be removed
by enzyme-treated cells.

3. In the presence of warm reacting auto antibodies circulating autologous red cells are
already coated with autoantibody which must be removed prior to performing an
adsorption. Refer to W.A.R.M. (Warm Autoantibody Removal Medium) IPP781.

REFERENCES
th
1. Technical Manual, 13 Edition, Bethesda, MD: American Association of Blood
Banks, 1999.

2. W.A.R.M. Package Insert, West Chester, PA: BCA Division of Biopool International, 1999.

BLOOD BANK INTERNAL PROCEDURE / POLICY


TITLE/DESCRIPTION: INDEX NUMBER(S):

ANTIBODY TITRATION 709


EFFECTIVE DATE: REVISION DATE/NUMBER: DISSEMINATION:

JAN92 REV9 / AUG02 XM

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PRINCIPLE

Titration is a semi-quantitative technique for measuring the amount of antibody. It is most


frequently performed in the following situations:

1. Prenatal Studies - the mother's serum is tested at one-month or longer intervals during
pregnancy; each clinically significant antibody is titered.

2. ABO mismatched Bone Marrow Transplant – may be requested prior to transplant and
again approximately one year post transplant. Perform saline phase titration of anti-A
and/or anti-B.

3. Antibody identification - mixtures of antibodies may be identified by titration. DTT


treatment can also be used to abolish IgM antibody activity to permit detection of coexisting
IgG antibodies. DTT (Dithiothreitol, or Clelands reagent) treatment of the patient’s serum
will split IgM antibodies, eliminating their agglutination ability, thus differentiating between
IgG and IgM antibodies.

4. Immunodeficiency workup - perform saline phase titration of anti-A and / or anti-B.

POLICY

Antibody titers will be performed when requested by the physician, when a new antibody is
detected on a patient of the high risk OB/GYN physicians or at the discretion of the Blood Bank
Director. Prenatal titers will not be performed once invasive monitoring has begun unless
request is approved by Blood Bank Medical Director. Only clinically significant antibodies will
be titered. Cells with homozygous expression of the corresponding antigen will be used for
testing whenever practical.

IgG titers using DTT are performed to aid in antibody identification.

SPECIMEN

 10ml (min.7ml) clotted whole blood


 Frozen aliquot of previous serum sample containing the antibody to be titered. (stored in
Revco Freezer #3)

REAGENTS, SUPPLIES, EQUIPMENT

 12 mm x 75 mm test tubes
 Calibrated automatic pipette 100 L
 Disposable pipette tips
 Serological centrifuge and/or cell washer
 Saline 0.9%
 6% Albumin
 Anti-human globulin
 Coombs Control cells
 Red blood cells appropriate for titration (see Procedure)
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 Antibody Titration worksheet (Appendix I)


 IgG Titration Using DTT worksheet (Appendix II)

For DTT titration only:

 0.01M DTT in saline. Prepare in a chemical fume hood.

1. Place 0.077g DTT in flask (a Mettler scale is located in Flow Cytometry)


and mix to a volume of 50 mL with isotonic saline.
2. Adjust pH to 8.0 by adding .5M NaOH (obtain in Chemistry). One-two
small drops may be adequate. (pH meters are found in Immunology,
Chemistry and Hematology)
3. Solution is stable for 3 months at 4C.

SAFETY PRECAUTIONS

1. All blood and blood products must be treated as potentially infectious. Follow
universal precautions detailed in the Laboratory Safety Manual.

2. Handle DTT and NaOH in a chemical fume hood, wearing gloves. DTT is
harmful if inhaled and is irritating to eyes. NaOH causes eye and skin burns and
causes digestive and respiratory tract burns.

QUALITY CONTROL

1. The previous sample will be tested in parallel and the titer obtained from it will be
compared to the previous reported titer prior to reporting results.

2. The current sample is frozen for titrating in parallel with subsequent sample.

PROCEDURE

1. Prenatal titration:

1.1 Locate previous sample, if available, in Revco #3. Record previous results on
Antibody Titration Worksheet (Appendix I).

1.2 Perform ABO/Rh on current sample according to IPP701 ABO/Rh and


Weak D Typing.

1.2.1 Perform an antibody screen (IPP706 Antibody


Screen) if one hasn’t been done previously.

1.2.2 Perform antibody identification (IPP707 Antibody


Identification) if antibody has not been previously
identified.

1.2.3 If an antibody has been previously identified,


ensure no additional antibodies have been formed.
Run at least one cell positive for the antigen
corresponding to each previously identified

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antibody in order to get an indication of the


expected titration and to ensure the antibody is still
reactive.

1.3 Titre each clinically significant antibody identified.

1.4 Label 2 sets of test tubes, one set for current sample; one set for the
previous sample. The first tube is 1:1 (undiluted), then 1:2, 1:4, etc.
through 1:1024.

1.5 To both sets of tubes, add 100 L of 6% albumin to all tubes except the
first.

1.6 Add 100 L of serum to tube #1 and tube #2.

1.7 Using a clean pipette tip, mix the contents of tube #2 several times.
Transfer 100 uL to the next tube.

1.8 Continue with the same technique through all dilutions. From the last
tube, remove 100 L of the diluted serum and save for use in further
dilutions ineeded.

1.9 Prepare the red cell suspension. See Procedure Notes for selection of
red cells. Wash the red cells and decant the saline completely.
Resuspend to approximately 2-5% concentration with 6% albumin.

1.10 Add 100 L of the appropriate red cell suspension to each tube.

1.11 Mix well and incubate at 37C for 30 minutes.

1.12 After incubation, wash cells four (4) times and proceed with the antiglobulin
phase.

1.13 Beginning with final dilution, gently re-suspend the cell button and
observe each tube macroscopically for agglutination until undiluted
sample is read.

1.14 Record the results on the Antibody Titration Worksheet (Appendix I).
Grade reactions according to IPP624 Grading of Agglutination
Reactions.

1.15 Report the results. Store the remainder of the serum in freezer #3
according to Procedure Note 3.

2. ABO Iso Titration:

2.1. Locate previous sample, if available, in Revco #3. Record


previous results on Antibody Titration Worksheet (Appendix I).

2.2. Perform ABO/Rh on current sample according to IPP701 ABO/Rh


and Weak D Typing.

2.3. Titer against A1 and/or B reagent red cells, as appropriate.

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2.4. Label 2 sets of test tubes, one set for current sample; one set for
the previous sample. The first tube is 1:1 (undiluted), then 1:2,
1:4, etc. through 1:1024.

2.5. To both sets of tubes add 100L of saline to all tubes except the
first tube.

2.6. Add 100 L of serum to tube #1 and to tube #2.

2.7. Using a clean pipette tip, mix the contents of tube #2 several
times. Transfer 100 L to the next tube.

2.8. Continue with the same technique through all dilutions. From the
last tube, remove 100 L of the diluted serum and save for use in
further dilutions if needed.

2.9. Prepare the red cell suspension. Wash the red cells and decant
the saline completely. Resuspend to approximately 2-5%
concentration with saline.

2.10. Add 100 L of the appropriate red cell suspension to each tube.

2.11. Mix well and incubate at room temperature for 30 minutes.

2.12. Centrifuge for time indicated on centrifuge.

2.13. Beginning with final dilution, gently resuspend the cell button and
observe each tube macroscopically for agglutination until
undiluted sample is read.

2.14. Record the results on the Antibody Titration Worksheet (Appendix


I). Grade reactions according to IPP624 Grading of Agglutination
Reactions.

2.15. Report the results. Store the remainder of the serum in freezer #3
according to Procedure Note 3.

3. DTT Titration:

3.1. Treatment of Serum:

3.1.1. Mix 0.3 mL of serum and 0.3 mL of 0.01M DTT in a


labeled tube.

3.1.2. In another tube (control), mix 0.3 mL of serum and


0.3 mL of saline.

3.1.3. Incubate at 37C for 15 minutes.

3.2. Titration:

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3.2.1. Label 2 sets of tubes, one set for the DTT treated
serum and one set for the saline / serum control.
The first tube is a 1:2 dilution, then 1:4, 1:8, 1:16,
etc. through 1:512.

3.2.2. To both sets of tubes add 100 L of 6% albumin to


all tubes except the first.

3.2.3. To the appropriate set of tubes, add 0.1 mL of the


DTT or saline treated serum to the first and the
second tubes of each titration.

3.2.4. Using a clean pipette tip, mix the contents of tube


#2 several times. Transfer 100 L to the next tube.

3.2.5. Continue the same technique through all dilutions.


From the last tube remove 100 L of the diluted
serum and save for use if further dilutions are
needed.

3.2.6. Prepare the red cell suspension. See Procedure


Notes for selection of red cells. Wash the red cells
and decant the saline completely. Resuspend to
approximately 2-5% concentration with 6%
albumin.

3.2.7. Add 100 L of the appropriate red cell suspension


to each tube.

3.2.8. Mix well and incubate at 37C for 30 minutes.

3.2.9. After incubation, wash cells four (4) times and


proceed with the antiglobulin phase.

3.2.10. Beginning with final dilution, gently resuspend the


cell button and observe each tube macroscopically
for agglutination until undiluted sample is read.

3.2.11. Record the results on the IgG Titers Using DTT


worksheet (Appendix II). Grade reactions
according to IPP624 Grading of Agglutination
Reactions.

3.2.12. Report the results. Store the remainder of the


serum in freezer #3 according to Procedure Note 3.

CALCULATIONS

Assign each agglutination a score and total all score values for each sample.

REPORTING RESULTS

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Report the highest dilution that produces 1+ macroscopic agglutination. Do not report weak or
microscopic reactions.

Report results as the reciprocal of the highest dilution that shows 1+ macroscopic agglutination.
(Report titer as 32; not 1:32). If 1:1024 dilution is positive report as “Greater than 1024.”

Reporting Antibody Titre Results in Cerner

Antibody titre results will be entered into Cerner using textual result entry under BLOOD BANK
RESULT ENTRY.

3.1 Complete the New Worksheet Dialogue box, select the accession number
format and press OK.

3.2 Enter the accession number associated with the antibody titration to be resulted.

3.3 The system retrieves order information and reference data for use in the
worksheet.

3.4 Click on, or use the arrow key to move to the cell to be resulted,
represented by a white cell in the worksheet.

3.5 Press the SPACE BAR to open text editor window and press F2 to open the
Select Template Dialogue box.

3.6 In the Activity Type box, select BLOOD BANK.

3.6.1 If the name of the template is known, type it in the Name box and
press FIND. If not, a search can be performed by:

 Entering the first letter of the template and clicking FIND or ENTER.
 Pressing the SPACE BAR and clicking FIND or ENTER.

This will bring up all Blood bank templates.

3.7 Select the Antibody Titre template and press OK.

3.8 Press F3 to move from one underscore (_) to the next.

3.9 Click OK to close the text editor window and Click PERFORM or VERIFY to save
the results.

3.10 Add a comment, “DTT used for titration.” if applicable.

4. A two tube change in titer or a score difference of 10 is considered significant.

5. If previous titer is greater than 1024, do not repeat. Enter a comment as above "Not
Indicated; previous titer on (date) was greater than 1024”.

Reporting ABO ISO Titer Results:

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6.1. Report current results of previous sample at the Previous Titer prompt. This
can be done using the same titer and score templates as described in AB
Titer result entry above. (Do not report the result that was previously
reported). If these results are significantly different (greater than 1 tube)
from the previous reported results discuss with senior technologist prior to
reporting results.

6.3 Add result comment, “DTT used for titration” if applicable.

7 DTT Interpretation:

7.1 Identical titers and strength of agglutination in both the test (DTT) and
control is (saline) tubes denotes that neutralization has not taken place
and that the antibody is IgG.

7.2 Inhibition of agglutination in the test (DTT) sample but not the control
(saline) sample denotes that neutralization has occurred, and that the
antibody is IgM.

7.3 Mixtures of IgG and IgM will show partial neutralization.

PROCEDURE NOTES

1. Select cells which will react strongest with the antibody being titrated; i.e., cells
which are homozygous for the corresponding antigen.

If a single antibody is present, select cell according to the following chart:

Antibody Cell Phenotype


anti-D, anti-c, anti-E R2R2
anti-C, anti-e R1R1
anti-K Kk
anti-A A1
anti-B B

If multiple antibodies are present, select cells positive for antigens of the antibody to be
titrated and negative for antigens of the other antibodies present.

Select a cell with heterozygous expression when titrating anti-K, since it is usually not
possible to select a cell with homozygous expression of the Kell antigen. This will ensure
consistency from one anti-Kell titration to the next.

2. If an IgM antibody, other than Anti-A or Anti-B, is to be titered; 0.9% Saline is the
preferable diluent. Incubate at room temperature for 30 minutes.

3. Specimen storage:

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3.1 Place frozen sample in freezer #3 in Antibody Titer Box #2; place sample in row
corresponding to last digit of medical record number.

3.2 Write sample information in Frozen Specimen Log. Cross out specimen when
removing from box for subsequent testing or for discard.

3.3 Retain frozen samples for one year.

LIMITATIONS OF PROCEDURES

Technical factors will affect the results. To reduce these variables to a minimum:

1. Meticulous pipetting is essential. Change tips between tubes.

2. Optimum incubation time, temperature and dilution medium should be determined


and used consistently.

3. The age, phenotype and concentration of the test RBC’s may affect the results.
When the titers of several different examples of an antibody are to be compared,
all sera should be tested against a pool of reagent RBC’s from donors of the same
phenotype

4. Completely reproducible results are virtually impossible to achieve. Comparisons are


only valid when specimens are tested concurrently. In prenatal testing, when sequential
serum samples are tested for changing antibody titer, samples should be frozen for
comparison with subsequent specimens. Each new sample should be tested in parallel
with the immediate preceding sample.

REFERENCES

1. AABB Technical Manual, 13th edition. Bethesda, MD: American Association of Blood
Banks, 1999.

2. Cerner HNA Millenium Integrated Clinical Information System PathNet User Training
st
Manual, 1 Edition, King Faisal Specialist Hospital and Research Center, July 2002.

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ANTIBODY TITRATION
PATIENT NAME PATIENT MR#

ANTIBODY (IES) IDENTIFIED:

CURRENT TITRATION PREVIOUS TITRATION


DILUENT: RED CELL USED: DATE DONE:

INCUBATION INCUBATION RESULTS:


TEMPERATURE: TIME:

USE A NEW SHEET FOR EACH ANTIBODY TITERED

CURRENT SPECIMEN PREVIOUS SPECIMEN


SPECIMEN DATE: SPECIMEN DATE:
ANTIBODY BEING TITERED: ANTIBODY BEING TITERED:
DILUTION GRADE CCC SCORE GRADE CCC SCORE

1:1

1:2

1:4

1:8

1:16

1:32

1:64

1:128

1:256

1:512

1:1024

TITER: TOTAL SCORE TITER: TOTAL SCORE

AGGLUTINATION SCORE: 4+ = 12 3+ =10 2+ = 8 1+ = 5

ALIQUOT FROZEN? YES / NO

Performed By____________________________ Date_______________

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IgG TITRATION USING DTT

PATIENT NAME PATIENT MR#

ANTIBODY:

CURRENT TITRATION PREVIOUS TITRATION


DILUENT: RED CELL USED: DATE DONE:

INCUBATION INCUBATION RESULTS:


TEMPERATURE: TIME:

USE A NEW SHEET FOR EACH ANTIBODY TITERED

DTT TREATED SERUM SALINE TREATED SERUM


(CONTROL)
CURRENT PREVIOUS CURRENT PREVIOUS
SAMPLE SAMPLE SAMPLE SAMPLE

DILUTION GRADE CCC GRADE CCC GRADE CCC GRADE CCC

1:1 ND ND ND ND ND ND ND ND

1:2

1:4

1:8

1:16

1:32

1:64

1:128

1:256

1:512

TITRE: TITRE: TITRE: TITRE:

ALIQUOT FROZEN? Y / N

Interpretation (IgG and/or IgM) : _______________________________________

Performed By____________________________ Date_______________

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