Me t h o d s S e c t i o n 3

Antibody Detection, Identification,

and Compatibility Testing
⽧

ME T H O D 3 - 1 . U SI N G EDTA saline because high-titer anti-A or

IM M ED I A T E - SPIN anti-B can initiate complement coating,
CO M PAT IB IL IT Y TE ST ING TO which can cause steric hindrance of
agglutination.2 The use of a patient’s sam- ⽧M3
DEMONSTRATE ABO
ple collected in EDTA is an alternative
IN C O M P ATIB IL IT Y
approach to prevent this phenomenon.
Principle 2. Label a tube for each donor red cell sus-
See Chapter 15 for a discussion of the princi- pension being tested with the patient’s
ples of compatibility testing. serum.
3. Add 2 drops of the patient’s serum or
Specimen plasma to each tube.
4. Add 1 drop of the suspension of donor red
Patient serum or plasma may be used. The age cells to the appropriate test tube.
of the specimen must comply with the pre- 5. Mix the contents of the tube(s) and cen-
transfusion specimen requirements in AABB trifuge according to the calibration of the
Standards for Blood Banks and Transfusion centrifuge.
Services.1 6. Examine the tube(s) for hemolysis, gently
resuspend the red cell button(s), and
Reagents examine for agglutination.
7. Read, interpret, and record test results.
1. Normal saline.
2. Donor red cells. Interpretation

Procedure 1. Agglutination or hemolysis constitutes a

positive (incompatible) test result.
1. Prepare a 2% to 5% suspension of donor 2. A smooth suspension of red cells after
red cells in normal saline or EDTA saline. resuspension of the red cell button con-
Some serologists using serum for testing stitutes a negative result and indicates a
prefer to suspend the donor red cells in compatible immediate-spin crossmatch.

897
898 ⽧ AABB TECHNICAL MANUAL

References 6. Group O antibody detection cells. Pooled

group O antibody-detection cells may be
1. Carson TH, ed. Standards for blood banks and
used only for donor testing. Testing of
transfusion services. 27th ed. Bethesda, MD:
AABB, 2011. patient samples must be performed with
2. Judd WJ, Steiner EA, O’Donnell DB, Oberman unpooled cells.
HA. Discrepancies in ABO typing due to pro- 7. A 2% to 5% suspension of donor red cells
zone: How safe is the immediate-spin cross- in saline.
match? Transfusion 1988;28:334-8. 8. IgG-coated red cells.

Note
ME T H O D 3 - 2 . D ET E C T IN G
A N T IBO D IE S T O RED CE L L LISS may be used as an additive (see Method
A N T IG E N S — IN DI REC T 3-2-2) or for the suspension of test red cells
A N T IG L O B U L I N TE S T S (see Method 3-2-3). LISS preparations are also
available commercially.
Principle
Reference
For a discussion of the principles of saline test-
1. Carson TH, ed. Standards for blood banks and
ing, albumin testing, low-ionic-strength saline
transfusion services. 27th ed. Bethesda, MD:
(LISS) testing, and polyethylene glycol (PEG)
AABB, 2011.
indirect antiglobulin testing, see Chapter 15.

Specimen ME T H O D 3- 2 - 1 . SA LI N E
IN D IRE C T A N T I G L O BU L I N TE S T
Serum or plasma may be used. The age of the
PR OC E D U RE
specimen must comply with pretransfusion
specimen requirements in AABB Standards for Procedure
Blood Banks and Transfusion Services.
1. Add 2 drops of serum or plasma to prop-
Reagents erly labeled tubes.
2. Add 1 drop of 2% to 5% saline-suspended
1. Normal saline. reagent group O red cells or donor red
2. Bovine albumin (22%). cells to each tube and mix.
3. LISS, made as follows: 3. Centrifuge and observe for hemolysis and
a. Add 1.75 g of NaCl and 18 g of glycine agglutination. Grade and record the
to a 1-liter volumetric flask. results.
b. Add 20 mL of phosphate buffer pre- 4. Incubate at 37 C for 30 to 60 minutes.
pared by combining 11.3 mL of 0.15 M 5. Centrifuge and observe for hemolysis and
KH2PO4 and 8.7 mL of 0.15 M Na2HPO4. agglutination. Grade and record the
c. Add distilled water to the 1-liter mark. results.
d. Adjust the pH to 6.7 ± 0.1 with NaOH. 6. Wash the red cells three or four times with
e. Add 0.5 g of sodium azide as a preser- saline, and completely decant the final
vative. wash.
4. PEG, 20% w/v: To 20 g of 3350 MW PEG, 7. Add AHG to the dry red cell button
add phosphate-buffered saline (PBS) pH according to the manufacturer’s direc-
7.3 (see Method 1-7) to 100 mL. PEG is tions. Mix well.
also available commercially. 8. Centrifuge and observe for agglutination.
5. Antihuman globulin (AHG) reagent. Grade and record the results.
Polyspecific or anti-IgG may be used 9. Confirm the validity of negative results by
unless otherwise indicated. adding IgG-coated red cells.
 METHODS SECTION 3 Antibody Detection and Identification ⽧ 899

ME T H O D 3 - 2 - 2 . A L B UM I N O R ME T H O D 3- 2 - 4 . PE G IN D I RE C T
L IS S - AD DI T IVE IN D IRE C T A N T IG L O B U L I N TE S T
A N T IG L O B U L I N TE S T PR OC E D U RE
PRO C E D URE
Procedure
Procedure
1. For each red cell sample to be tested, mix
1. Add 2 drops of serum or plasma to prop- 2 drops of test serum, 4 drops of 20% PEG
erly labeled tubes. in PBS, and 1 drop of a 2% to 5% suspen-
2. Add an equivalent volume of 22% bovine sion of red cells.
albumin or LISS additive (unless the 2. Incubate at 37 C for 15 minutes.
manufacturer’s directions state other- 3. DO NOT CENTRIFUGE.
wise). 4. Wash the red cells four times with saline,
3. Add 1 drop of a 2% to 5% saline-sus- and completely decant the final wash.
pended reagent or donor red cells to each 5. Perform the IAT described in Method 3-2-
tube and mix. 1, steps 7 through 9, using anti-IgG.
4. For albumin, incubate at 37 C for 15 to 30 Note: The manufacturer’s instructions
minutes. For LISS, incubate for 10 to 15 should be followed for the proper use of
minutes or follow the manufacturer’s commercial PEG solutions.
directions.
5. Centrifuge and observe for hemolysis and Interpretation (for Antiglobulin Tests,
agglutination. Grade and record the Methods 3-2-1 through 3-2-4)
results.
6. Perform the indirect antiglobulin test 1. The presence of agglutination/hemolysis
(IAT) described in Method 3-2-1, steps 6 after incubation at 37 C constitutes a pos-
through 9. itive test result.
2. The presence of agglutination after addi-
ME T H O D 3 - 2 - 3 . L IS S IN D IRE C T tion of AHG constitutes a positive test
ANTIGLOBULIN TEST PROCEDURE result.
3. Antiglobulin test results are negative
Procedure when no agglutination is observed after
initial centrifugation followed by aggluti-
1. Wash reagent or donor red cells three nation with the addition of IgG-coated
times in normal saline, and completely red cells and centrifugation. If the IgG-
decant the saline. coated red cells are not agglutinated, the
2. Resuspend the cells to a 2% to 3% suspen- negative result is invalid and the test must
sion in LISS. be repeated.
3. Add 2 drops of serum to a properly
labeled tube. Controls
4. Add 2 drops of LISS-suspended red cells,
mix, and incubate at 37 C for 10 to 15 The procedure used for the detection of unex-
minutes or follow the manufacturer’s pected antibodies in pretransfusion testing
directions. should be checked daily with weak examples
5. Centrifuge and observe for hemolysis and of antibody. Control sera can be prepared from
agglutination by gently resuspending the reagent grade typing sera diluted with 6%
red cell button. Grade and record results. bovine albumin to give 2+ reactions by an IAT.
6. Perform the IAT described in Method 3-2- Human sources of IgG antibodies are also
1, steps 6 through 9. acceptable.
900 ⽧ AABB TECHNICAL MANUAL

Notes ME T H O D 3 - 3 . P RE W A R M I N G
PR OC E D U RE
1. The incubation times and the volume and
Principle
concentration of red cells indicated are
those given in the literature. Individual Prewarming may be useful in the detection
laboratories may choose to standardize and identification of red cell antibodies that
techniques with somewhat different val- bind to antigen only at 37 C. The test is partic-
ues. See Chapter 15 for other limitations ularly useful for testing sera of patients with
when modifying procedures. In all cases, cold-reactive autoantibody activity that may
mask the presence of clinically significant
the manufacturer’s package insert should
antibodies. However, use of the prewarming
be consulted before modifying a proce-
technique for this application has become
dure. controversial.1,2 It has been shown to result in
2. For the saline procedure (Method 3-2-1),
decreased reactivity of some potentially signif-
step 3 may be omitted to avoid the detec- icant antibodies, and weak antibodies can be
tion of antibodies reactive at room tem- missed.3 The technique should be used with
perature. caution and not used to eliminate unidentified
3. For the PEG procedure, omit centrifuga- reactivity.
tion after 37 C incubation because red Strong cold-reactive autoantibodies may
cells will not resuspend readily. Use anti- react in prewarmed tests; other techniques
IgG rather than polyspecific AHG to avoid such as cold allo- or autoadsorption or dithio-
unwanted positive reactions caused by threitol treatment of plasma may be required
C3-binding autoantibodies. Precipitation to detect underlying clinically significant anti-
of serum proteins when PEG is added bodies.
appears to be related to elevated serum
globulin levels. The problem becomes
Specimen
apparent when the IgG-coated red cells Serum or plasma may be used. The age of the
are nonreactive or unexplained weak specimen must comply with pretransfusion
reactions are detected.2 At least four specimen requirements in AABB Standards for
washes of the red cells at AHG phase, with Blood Banks and Transfusion Services.4
agitation, will fully resuspend the red cells
and usually prevent the problem from Reagents
occurring. Alternatively, the test may have
1. Normal saline.
to be repeated using a method that does
2. Anti-IgG.
not use PEG. 3. Commercially available group O antibody
4. Steps 6 through 9 of the IAT (Method 3-2-
detection red cells. Pooled group O anti-
1) should be performed without interrup- body detection red cells may be used only
tion. for donor testing. Testing of patient sam-
ples must be performed with unpooled
References red cell samples.
1. Carson TH, ed. Standards for blood banks and 4. IgG-coated red cells.
transfusion services. 27th ed. Bethesda, MD:
AABB, 2011. Procedure
2. Hoffer J, Koslosky WP, Gloster ES, et al. Precipi-
tation of serum proteins by polyethylene 1. Prewarm a container of saline to 37 C.
glycol (PEG) in pretransfusion testing. Immu- 2. Label one tube for each reagent or donor
nohematology 1999;15:105-7. sample to be tested.
 METHODS SECTION 3 Antibody Detection and Identification ⽧ 901

3. Add 1 drop of 2% to 5% saline-suspended References

red cells to each tube.
1. Judd WJ. Controversies in transfusion medi-
4. Place the tubes containing red cells and a
cine. Prewarmed tests: Con. Transfusion
tube containing a small volume of the 1995;35:271-5.
patient’s serum and a pipette at 37 C; 2. Mallory D. Controversies in transfusion medi-
incubate for 5 to 10 minutes. cine. Prewarmed tests: Pro—why, when, and
5. Using the prewarmed pipette, transfer 2 how—not if. Transfusion 1995;35:268-70.
drops of prewarmed serum to each tube 3. Leger RM, Garratty G. Weakening or loss of
containing prewarmed red cells. Mix antibody reactivity after prewarm technique.
without removing tubes from the incuba- Transfusion 2003;43: 1611-14.
tor. 4. Carson TH, ed. Standards for blood banks and
6. Incubate at 37 C for 30 to 60 minutes. transfusion services. 27th ed. Bethesda, MD:
7. Without removing the tubes from the AABB, 2011.
incubator, fill each tube with prewarmed
(37 C) saline. Centrifuge and wash three ME T H O D 3 - 4 . D E T E C T I N G
or four times with 37 C saline. A N T IB OD I ES IN T H E PRE S E N C E
8. Add anti-IgG according to the manufac-
O F RO U LE A U X — SA L IN E
turer’s directions.
9. Centrifuge and observe for reaction. RE PL A C E M E N T
Grade and record the results. Principle
10. Confirm the validity of negative results by
adding IgG-coated red cells. Patient samples with abnormal concentra-
tions of serum proteins, altered serum-protein
Notes ratios, or high-molecular-weight volume
expanders can aggregate reagent red cells and
1. The prewarming procedure described can mimic agglutination. Rouleaux are red cell
earlier will not detect alloantibodies that aggregates that adhere along their flat sur-
agglutinate at 37 C or lower and are not faces, giving a “stacked coin” appearance
reactive in the antiglobulin phase. If microscopically.
detection of these antibodies is desired,
testing and centrifugation at 37 C are Specimen
required. If time permits, a tube con- Serum or plasma to be evaluated.
taining a prewarmed mixture of serum
and cells can be incubated at 37 C for 60 Reagents
to 120 minutes, and the settled red cells
can be examined for agglutination by 1. Saline.
resuspending the button without centrif- 2. A1, B, and O reagent red cells.
ugation.
2. Cold-reactive antibodies may not be Procedure
detectable when room temperature saline
instead of 37 C saline is used in the wash After routine incubation and resuspension,
step.2 The use of room temperature saline proceed with the following steps if the appear-
may avoid the elution of clinically signifi- ance of the resuspended red cells suggests
cant antibody(ies) from reagent red cells rouleaux formation. The saline replacement
that can occur with the use of 37 C saline. technique is best performed by the test tube
Some strong cold-reactive autoantibod- method.
ies, however, may still react and therefore
require the use of 37 C saline to avoid 1. Recentrifuge the serum (or plasma) or cell
their detection. mixture.
902 ⽧ AABB TECHNICAL MANUAL

2. Remove the serum, leaving the red cell Procedure

button.
3. Replace the serum with an equal volume 1. Place 1 g of powdered ficin in a 100-mL
of saline (2 drops). volumetric flask. Handle the ficin care-
4. Resuspend the red cell button gently, and fully; it is harmful if it gets in the eyes or is
observe for agglutination. Rouleaux will inhaled. It is desirable to wear gloves,
disperse when suspended in saline. True mask, and apron, or to work under a
agglutination is stable in the presence of hood.
saline. 2. Add PBS, pH 7.3, to 100 mL to dissolve the
ficin. Agitate vigorously by inversion,
Notes
rotate for 15 minutes, or mix with a mag-
netic stirrer until mostly dissolved. The
1. In some instances, simple dilution of
powder will not dissolve completely.
serum 1:3 with saline is sufficient to pre-
3. Collect clear fluid, either by filtration or
vent rouleaux and to detect ABO isoagglu-
centrifugation, and prepare small ali-
tinins.
quots. Store the aliquots at –20 C or
2. Review of the patient’s recent medical his-
tory and other laboratory results may be colder. Do not refreeze a thawed solution.
helpful (eg, history of multiple myeloma).
ME T H O D 3- 5 - 2 . PREP A R I N G
Reference PAPAIN ENZYME STOCK, 1% W/V
1. Issitt PD, Anstee DJ. Applied blood group Principle
serology. 4th ed. Durham, NC: Montgomery
Scientific Publications, 1998:1135. The papain preparations used in blood bank-
ing differ from lot to lot. Each time a stock
ME T H O D 3 - 5 . E N Z YM E enzyme solution is prepared, its reactivity
PRO C E D URE S should be tested, and incubation periods
should be standardized for optimal effective-
For a discussion of the principles of enzyme ness (see Method 3-5-3).
testing, see Chapter 16.
Reagents
ME T H O D 3 - 5 - 1 . PREP A R I N G
FI C I N EN Z Y M E ST O C K , 1% W / V 1. L-cysteine hydrochloride (0.5 M), 0.88 g
in 10 mL distilled water.
Principle 2. Dry papain powder, 2 g.
The ficin preparations used in blood banking 3. Phosphate buffer (0.067 M at pH 5.4), pre-
differ from lot to lot. Each time a stock enzyme pared by combining 3.5 mL of Na2HPO4
solution is prepared, its reactivity should be and 96.5 mL of KH2PO4.
tested, and incubation periods should be stan-
dardized for optimal effectiveness (see Procedure
Method 3-5-3).
1. Add 2 g of powdered papain to 100 mL of
Reagents phosphate buffer (pH 5.4). Handle papain
carefully; it is harmful to mucous mem-
1. Dry ficin powder, 1 g. branes. Use appropriate protective equip-
2. Phosphate-buffered saline (PBS), pH 7.3 ment.
(see Method 1-7). 2. Agitate enzyme solution for 15 minutes at
3. Phosphate buffer, pH 5.4. room temperature.
 METHODS SECTION 3 Antibody Detection and Identification ⽧ 903

3. Collect clear fluid by filtration or centrifu- 5. Immediately wash the red cells three
gation. times with large volumes of saline.
4. Add L-cysteine hydrochloride, and incu- 6. Resuspend treated red cells to 2% to 5% in
bate solution at 37 C for 1 hour. saline.
5. Add phosphate buffer (pH 5.4) to final 7. Label four tubes for each serum to be
volume of 200 mL. Store aliquots at –20 C tested: untreated, 5 minutes, 10 minutes,
or colder. Do not refreeze aliquots. and 15 minutes.
8. Add 2 drops of the appropriate serum to
METHOD 3-5-3. STANDARDIZING each of the four tubes.
9. Add 1 drop of the appropriate red cell sus-
EN ZY M E PRO C E D URE S
pension to each of the labeled tubes.
Principle 10. Mix and incubate at 37 C for 15 minutes.
11. Centrifuge and examine for agglutination
For a two-stage enzyme procedure, the opti-
by gently resuspending the red cell but-
mal treatment time must be determined for
ton.
each new lot of stock solution. The following
12. Proceed with the IAT test described in
technique for ficin can be modified for use
Method 3-2-1, steps 6 through 9.
with other enzymes.
Interpretation
Reagents
Table 3-5-3-1 shows possible results with D+,
1. 1% stock solution of ficin in PBS, pH 7.3. Fy(a+b–) cells and the sera indicated. In this
2. Several sera known to lack unexpected case, the optimal incubation time would be 10
antibodies. minutes. Incubation for only 5 minutes does
3. Anti-D that agglutinates only enzyme- not completely abolish Fya activity or maxi-
treated D+ red cells and does not aggluti- mally enhance anti-D reactivity. Incubation
nate untreated D+ red cells. for 15 minutes causes false-positive AHG reac-
4. Anti-Fya of moderate or strong reactivity. tivity with inert serum.
5. D+ and Fy(a+b–) red cell samples. If incubation for 5 minutes overtreats the
6. Antihuman globulin (AHG) reagent. red cells, it is preferable to use a more dilute
Polyspecific or anti-IgG may be used working solution of enzyme than to reduce
unless otherwise indicated. incubation time, because it is difficult to accu-
7. IgG-coated red cells. rately monitor very short incubation times.
Additional tests can evaluate a single dilution
Procedure at different incubation times, or a single incu-
bation time can be used for different enzyme
1. Prepare 0.1% ficin by diluting one volume dilutions.
of stock ficin solution with nine volumes
of PBS, pH 7.3.
2. Label three tubes: 5 minutes, 10 minutes, ME T H O D 3- 5 - 4 . EVA L UA T IN G
and 15 minutes. EN ZY M E - TRE A T ED RE D CE L L S
3. Add equal volumes of washed red cells Principle
and 0.1% ficin to each tube.
4. Mix and incubate at 37 C for the time des- After optimal incubation conditions have been
ignated. Incubation times are easily con- determined for a lot of enzyme solution,
trolled if the 15-minute tube is prepared treated red cells should be evaluated before
first, followed by the 10- and 5-minute use to demonstrate that they are adequately,
tubes at 5-minute intervals. Incubation but not excessively, modified. Satisfactory
will be complete for all three tubes at the enzyme treatment should produce red cells
same time. that are directly agglutinated by an antibody
904 ⽧ AABB TECHNICAL MANUAL

TABLE 3-5-3-1. Hypothetical Results with D+, Fy(a+b–) Red Cells

Cells and Enzyme Inert Serum Anti-D Anti-Fya

Untreated 37 C incubation 0 0 0
Antihuman globulin (AHG) test 0 1+ 3+
5 minutes 37 C incubation 0 1+ 0
AHG test 0 2+ 1+
10 minutes 37 C incubation 0 2+ 0
AHG test 0 2+ 0
15 minutes 37 C incubation 0 2+ 0
AHG test w+ 2+ w+

that reacts only by IAT with untreated cells; 7. Examine macroscopically for the pres-
however, enzyme-treated red cells should not ence of agglutination.
be agglutinated or aggregated by inert serum. 8. Perform the IAT described in Method 3-2-
1, steps 6 through 9, on the tube labeled
Specimen “negative.”
Enzyme-treated red cells.
Interpretation
Reagents There should be agglutination in the “positive”
tube and no agglutination in the “negative”
1. Sera known to contain antibody that will tube. If agglutination occurs in the “negative”
agglutinate enzyme-treated red cells. tube, the red cells have been overtreated; if
2. Sera free of any unexpected antibodies. agglutination does not occur in the “positive”
3. Antihuman globulin (AHG) reagent. tube, treatment has been inadequate.
Polyspecific or anti-IgG may be used
unless otherwise indicated.
4. IgG-coated red cells.
ME T H O D 3- 5 - 5 . ON E- ST A G E
EN ZY M E PR OC E D URE
Procedure Specimen

1. Select an antibody that agglutinates Serum or plasma to be tested.

enzyme-treated red cells positive for the
antigen but gives only AHG reactions with Reagent
unmodified red cells. Many examples of
human-source anti-D behave in this way. 1. Reagent red cells.
2. Add 2 drops of the selected antibody-con- 2. Antihuman globulin (AHG) reagent.
taining serum to a tube labeled “positive.” Polyspecific or anti-IgG may be used
3. Add 2 drops of a serum free of unexpected unless otherwise indicated.
antibodies to a tube labeled “negative.” 3. IgG-coated red cells.
4. Add 1 drop of 2% to 5% suspension of
enzyme-treated red cells to each tube. Procedure
5. Mix and incubate 15 minutes at 37 C.
6. Centrifuge and resuspend the red cells by 1. Add 2 drops of serum to an appropriately
gentle shaking. labeled tube.
 METHODS SECTION 3 Antibody Detection and Identification ⽧ 905

2. Add 2 drops of a 2% to 5% saline suspen- Notes

sion of reagent red cells.
3. Add 2 drops of 0.1% papain solution and 1. An alternative method for steps 4 and 5
mix well. (Method 3-5-5) or steps 7 and 8 (Method
4. Incubate at 37 C for 15 minutes. 3-5-6) is to incubate the serum and
5. Centrifuge; gently resuspend the red cells enzyme-treated cells at 37 C for 60 min-
and observe them for agglutination. utes and to examine the settled cells for
Grade and record the results. agglutination without centrifugation.
6. Proceed with the IAT described in Method This examination can be useful for serum
3-2-1, steps 6 through 9. with strong cold-reactive agglutinins and
can sometimes prevent false-positive
ME T H O D 3 - 5 - 6 . TWO - ST A G E results.
EN ZY M E PRO C E D URE 2. Microscopic examination is not recom-
mended for routine use and is particu-
Specimen larly inappropriate with enzyme-
enhanced tests; false-positive reactions
Serum or plasma to be tested.
will often be detected.
3. Either papain or ficin may be used in a
Reagents
two-stage procedure.
4. Enzyme preparations are available com-
1. Reagent red cells.
mercially. The manufacturer’s directions
2. Antihuman globulin (AHG) reagent.
should be followed for appropriate use
Polyspecific or anti-IgG may be used
and quality control.
unless otherwise indicated.
3. IgG-coated red cells.
References
Procedure 1. Issitt PD, Anstee DJ. Applied blood group
serology. 4th ed. Durham, NC: Montgomery
1. Prepare a diluted enzyme solution Scientific Publications, 1998.
(papain or ficin) by adding 9 mL of PBS, 2. Judd WJ, Johnson S, Storry J. Judd’s methods in
immunohematology. 3rd ed. Bethesda, MD:
pH 7.3, to 1 mL of stock enzyme.
AABB Press, 2008.
2. Add one volume of diluted enzyme to one
volume of packed, washed reagent red
cells. ME T H O D 3- 6 . P E R F O R M IN G A
3. Incubate at 37 C for the time determined DIREC T A N T IGL O B UL I N TE S T
to be optimal for that enzyme solution.
4. Wash treated red cells at least three times Principle
with large volumes of saline, and resus- See Chapter 17 for a discussion of the princi-
pend the red cells to a 2% to 5% concen- ples of direct antiglobulin testing.
tration in saline.
5. Add 2 drops of serum or plasma to be Specimen
tested to an appropriately labeled tube.
6. Add 1 drop of 2% to 5% suspension of Red cells from an EDTA-anticoagulated blood
enzyme-treated red cells. sample.
7. Mix and incubate for 15 minutes at 37 C.
8. Centrifuge; gently resuspend the red cells Reagents
and observe for agglutination. Grade and
record the results. 1. Antihuman globulin (AHG) reagent: poly-
9. Proceed with the IAT described in Method specific antiglobulin reagent, anti-IgG,
3-2-1, steps 6 through 9. anti- complement antisera.
906 ⽧ AABB TECHNICAL MANUAL

2. A control reagent (eg, saline or 6% albu- 2. The DAT is negative when no agglutina-
min) is required when all antisera tested tion is observed at either test phase and
give a positive result. the IgG-coated cells added in step 7 are
3. IgG-coated red cells. agglutinated. If the IgG-coated cells are
4. Complement-coated red cells, if instructed not agglutinated, the negative DAT result
by the manufacturer. is considered invalid, and the test must be
repeated. A negative DAT does not neces-
Procedure sarily mean that the red cells have no
attached globulin molecules. Polyspecific
1. Dispense 1 drop of a 2% to 5% suspension and anti-IgG reagents detect 150 to 500
of red cells into each tube. molecules of IgG per cell, but patients
2. Wash each tube three or four times with may still experience autoimmune
saline. Completely decant the final wash. hemolytic anemia when IgG coating is
3. Immediately add antisera and mix. For below this level.2
the amount of antisera required, refer to 3. No interpretation can be made if the con-
the manufacturer’s directions. trol reagent is reactive. This may indicate
4. Centrifuge according to the manufac- the presence of a strong cold autoaggluti-
turer’s directions. For anticomplement, nin or spontaneous agglutination due to
the manufacturer may indicate a delay warm-reactive IgM or IgG antibodies.
before centrifugation. Warming the red cells to 37 C and/or
5. Examine the cells for agglutination. Grade washing with warm (37 C) saline should
and record the reaction. resolve reactivity due to cold autoaggluti-
6. If using polyspecific AHG or anticomple- nins. Spontaneous agglutination requires
treatment of the red cells with dithiothrei-
ment, incubate nonreactive tests at room
tol or 2-aminoethylisothiouronium bro-
temperature if indicated by the manufac-
mide (see Method 3-10).
turer; then centrifuge and read again.
7. Confirm the validity of negative results as
Notes
indicated by the manufacturer (eg, add
IgG-coated red cells to tests containing
1. Steps 2 through 5 should be performed
anti-IgG).
without interruption.
8. Centrifuge according to the manufac-
2. Initial testing may be performed with
turer’s directions.
polyspecific reagent only. If the DAT is
9. Examine the cells for agglutination and
negative with polyspecific reagent, no
record the reaction.
further testing is necessary. If the DAT is
positive with polyspecific reagent, per-
Interpretation
form the DAT with monospecific
reagents, anti-IgG, and anticomplement,
1. The direct antiglobulin test (DAT) is posi- to determine which globulins are present.
tive when agglutination is observed either 3. Additional washes may be needed when
after immediate centrifugation or after testing cord samples contaminated with
the centrifugation that followed room- Wharton’s jelly.
temperature incubation. IgG-coated
red cells usually give immediate reac- References
tions, whereas complement coating may
1. Klein HG, Anstee DJ. Mollison’s blood transfu-
be more easily demonstrable after incu-
sion in clinical medicine. 11th ed. Oxford,
bation.1,2 Monospecific AHG reagents are England: Blackwell Publishing, 2005.
needed to confirm which globulins are 2. Petz LD, Garratty G. Immune hemolytic ane-
present. mia. Philadelphia: Churchill-Livingstone, 2004.
 METHODS SECTION 3 Antibody Detection and Identification ⽧ 907

ME T H O D 3 - 7 . A N T I BO D Y 2. Deliver one volume of saline to all test

TIT R A T I O N P RO C E DU RE tubes except the first (undiluted, 1 in 1)
tube.
Principle 3. Add an equal volume of serum to each of
Titration is a semiquantitative method used to the first two tubes (undiluted and 1 in 2).
determine the concentration of antibody in a 4. Using a clean pipette, mix the contents of
serum sample or to compare the strength of the 1-in-2 dilution several times, and
antigen expression on different red cell sam- transfer one volume into the next tube
ples. The usual applications of titration studies (the 1-in-4 dilution).
are as follows: 1) estimating antibody activity 5. Continue the same process for all dilu-
tions, using a clean pipette to mix and
in alloimmunized pregnant women to deter-
transfer each dilution. Remove one vol-
mine whether and when to perform more
ume of diluted serum from the final tube,
complex invasive investigation of the fetal
and save it for use if further dilutions are
condition (see Chapter 22); 2) elucidating
required.
autoantibody specificity (see Chapter 17); 3)
6. Label 10 tubes for the appropriate dilu-
characterizing antibodies as having high titer
tions.
and low avidity, traits common in antibodies
7. Using separate pipettes for each dilution,
to antigens of the Knops and Chido/Rodgers transfer 2 drops of each diluted serum
systems, Csa, and JMH (see Chapter 16); and 4) into the appropriately labeled tubes, and
observing the effect of sulfhydryl reagents on add 2 drops of a 2% red cell suspension.
antibody behavior, to determine immunoglo- Alternatively, for convenience, add 1 drop
bulin class (IgG or IgM; see Method 3-8). of a 3% to 4% suspension of red cells as
supplied by the reagent manufacturer,
Specimen although this method is less precise.
Serum or plasma antibody to be titrated. 8. Mix well and test by a serologic technique
appropriate to the antibody (see Chapter
Reagents 16).
9. Examine test results macroscopically;
1. Red cells that express the antigen(s) cor- grade and record the reactions. The pro-
responding to the antibody specific- zone phenomenon (see Chapter 10) may
ity(ies), in a 2% to 5% saline suspension. cause reactions to be weaker in the more
Uniformity of red cell suspensions is concentrated serum preparations than in
very important to ensure comparability of higher dilutions. If one is to avoid misin-
terpretation of results, it may be prefera-
results.
2. Saline. (Note: Dilutions may be made ble to examine first the tube containing
the most dilute serum and then to pro-
with albumin if desired.)
ceed through the more concentrated
Procedure samples to the undiluted specimen.

The master dilution technique for titration Interpretation

studies is as follows:
1. Observe the highest dilution that pro-
1. Label 10 test tubes according to the duces 1+ macroscopic agglutination. The
serum dilution (eg, 1 in 1, 1 in 2, etc). A 1- titer is reported as the reciprocal of the
in-1 dilution means one volume of serum dilution level (eg, 32—not 1 in 32 or 1:32).
undiluted; a 1-in-2 dilution means one (See Table 3-7-1.) If there is agglutination
volume of serum in a final volume of two, in the tube containing the most dilute
or a 50% solution of serum in the diluent. serum, the endpoint has not been
 908
⽧
TABLE 3-7-1. Examples of Antibody Titers, Endpoints, and Scores

AABB TECHNICAL MANUAL

Reciprocal of Serum Dilution

1 2 4 8 16 32 64 128 256 512 Titer* Score

Sample #1 Strength 3+ 3+ 3+ 2+ 2+ 2+ 1+ ± ± 0 64(256)

Score 10 10 10 8 8 8 5 3 2 0 64
Sample #2 Strength 4+ 4+ 4+ 3+ 3+ 2+ 2+ 1+ ± 0 128(256)
Score 12 12 12 10 10 8 8 5 3 0 80
Sample #3 Strength 1+ 1+ 1+ 1+ ± ± ± ± ± 0 8(256)
Score 5 5 5 5 3 3 3 2 2 0 33
*The titer is often determined from the highest dilution of serum that gives a reaction 1+ (score 5). This reaction may differ significantly from the titra-
tion endpoint (shown in parentheses), as with the reactions of an antibody with high-titer, low-avidity characteristics, manifested by Sample #3.
 METHODS SECTION 3 Antibody Detection and Identification ⽧ 909

reached, and additional dilutions should 2. Careful pipetting is essential. Pipettes

be prepared and tested. with disposable tips that can be changed
2. In comparative studies, a significant dif- after each dilution are recommended.
ference in titer is three or more dilutions. 3. The age, phenotype, and concentration of
Variations in technique and inherent bio- the test red cells will influence the results.
logic variability can cause duplicate tests 4. The optimal time and temperature of
to give results that differ by one dilution incubation, and the time and force of
in either direction. Serum containing centrifugation, should be used consis-
antibody at a true titer of 32 may show, on tently.
replicate tests, the endpoint in the 1-in-32 5. When the titers of several antibody-con-
tube, the 1-in-64 tube, or the 1-in-16 tube. taining sera are to be compared, all of
3. Titer values alone can be misleading if the them should be tested against red cells
strength of agglutination is not also evalu- (preferably freshly collected) from the
ated. The observed strength of agglutina- same donor. If this is not possible, the
tests should use a pool of reagent red cells
tion can be assigned a number, and the
from donors of the same phenotype.
sum of these numbers for all tubes in a
Comparisons are valid only when speci-
titration study represents the score,
mens are tested concurrently.
another semiquantitative measurement
6. When a single serum is to be tested
of antibody reactivity. The arbitrarily against different red cell samples, all red
assigned threshold for significance in cell samples should be collected and pre-
comparing scores is a difference of 10 or served in the same manner and diluted to
more between different test samples (see the same concentration before use. Mate-
Table 3-7-1). rial from the master dilution should be
4. Antibodies with high-titer and low-avidity used for all the tests. Comparisons are
characteristics generally have a titer valid only when specimens are tested
greater than 64, with most tubes showing concurrently.
consistently weak reactivity. 7. When performing a titration for anti-D for
5. Table 3-7-1 shows the results obtained hemolytic disease of the fetus and new-
with three sera, each of which shows no born, see Method 5-3.
more agglutination after 1:256 dilution. 8. Other titration methods have been
The differences in score, however, indi- described that may show less variation.1
cate considerable variation in strength of
reactivity. Reference
1. AuBuchon JP, de Wildt-Eggen J, Dumont LJ, et
Notes al. Reducing the variation in performance of
Titration is a semiquantitative technique. antibody titrations. Arch Pathol Lab Med
2008;132:1194-201.
Technical variables greatly affect the results,
and care should be taken to achieve the most
uniform practices possible. METHOD 3-8. USING SULFHYDRYL
RE A G E N T S T O DIS T IN G U IS H
1. Measurements are more accurate with IgM F RO M IgG ANTI BO D IE S
large volumes than with small volumes; a
master dilution technique (see earlier) Principle
gives more reliable results than individual Treating IgM antibodies with sulfhydryl
dilutions for a single set of tests. The vol- reagents abolishes both agglutinating and
ume needed for all planned tests should complement-binding activities. Observations
be calculated, and an adequate quantity of antibody activity before and after sulfhydryl
of each dilution should be prepared. treatment are useful in determining immuno-
910 ⽧ AABB TECHNICAL MANUAL

globulin class. Sulfhydryl treatment can also Interpretation

be used to abolish IgM antibody activity to
permit detection of coexisting IgG antibodies. 1. Reactivity in the dilution control serum
For a discussion of IgM and IgG structures, see and no reactivity in the DTT-treated
Chapter 10. serum indicate an IgM antibody.
2. Reactivity in the dilution control serum
Specimen and in the DTT-treated serum indicates
an IgG antibody or an IgG and IgM mix-
2 mL of serum or plasma to be treated. ture. Titration studies may be neces-
sary to distinguish between them (see
Reagents Table 3-8-1).
3. No reactivity in the dilution control
1. Phosphate-buffered saline (PBS) at pH serum indicates dilution of weak anti-
7.3. body reactivity and an invalid test.
2. 0.01 M dithiothreitol (DTT) prepared by
dissolving 0.154 g of DTT in 100 mL of pH Control
7.3 PBS. Store at –18 C or lower. A serum or plasma sample known to contain
an IgM antibody should be treated and tested
Procedure in parallel.

1. Dispense 1 mL of serum or plasma into Notes

each of two test tubes.
2. To one tube (labeled dilution control), 1. 2-mercaptoethanol can also be used for
add 1 mL of pH 7.3 PBS. this purpose (see Method 2-18 for prepa-
3. To the other tube (labeled test), add 1 mL ration).
of 0.01 M DTT. 2. Sulfhydryl reagents used at low concen-
4. Mix and incubate at 37 C for 30 to 60 min- tration may weaken antigens of the Kell
utes. system. For investigation of antibodies in
5. Use the DTT-treated and dilution control the Kell system, it may be necessary to
samples in standard procedures. use other methods.

TABLE 3-8-1. Effect of Dithiothreitol on Blood Group Antibodies

Reciprocal of Serum Dilution

Test Sample 2 4 8 16 32 Interpretation

Serum + DTT 3+ 2+ 2+ 1+ 0
IgG
Serum + PBS 3+ 2+ 2+ 1+ 0
Serum + DTT 0+ 0+ 0+ 0+ 0
IgM
Serum + PBS 3+ 2+ 2+ 1+ 0
Serum + DTT 2+ 1+ 0+ 0+ 0
IgG + IgM*
Serum + PBS 3+ 2+ 2+ 1+ 0
*May also indicate only partial inactivation of IgM.
Note: DTT = dithiothreitol; IgG = immunoglobulin, gamma class; IgM = immunoglobulin M; PBS = phosphate-buffered saline.
 METHODS SECTION 3 Antibody Detection and Identification ⽧ 911

3. Gelling of a serum or plasma sample may should be from 1 in 2 to 1 in 512, or to one

be observed during treatment with DTT. tube beyond the known titer (Method 3-
This gelling can occur if the DTT has been 7). The volume prepared should be not
prepared incorrectly and has a concentra- less than 0.3 mL for each red cell sample
tion above 0.01 M. Gelling may also occur
to be tested.
if serum and DTT are incubated too long.
2. For each red cell sample to be tested,
An aliquot of the sample undergoing
place 2 drops of each serum dilution into
treatment can be tested after 30 minutes
of incubation; if the activity thought to be each of two sets of appropriately labeled
caused by IgM has disappeared, there is 10 or 12 × 75-mm test tubes.
no need to incubate further. Gelled sam- 3. To one set, add 2 drops of pooled plasma
ples cannot be tested for antibody activity to each tube.
because overtreatment with DTT causes 4. To the other set, add 2 drops of 6% albu-
the denaturation of all serum proteins. min to each tube.
5. Gently agitate the contents of each tube
Reference and incubate the tubes at room tempera-
1. Klein HG, Anstee D. Blood transfusion in clini- ture for at least 30 minutes.
cal medicine. 11th ed. Oxford, England: Black- 6. Add 1 drop of a 2% to 5% suspension of
well Scientific, 2005. red cells to each tube.
7. Gently agitate the contents of each tube
ME T H O D 3 - 9 . U S IN G PL A S M A and incubate the tubes at 37 C for 1 hour.
8. Wash the cells four times in saline, add
IN H I B I T IO N T O D I S T I N G U I S H
anti-IgG, and centrifuge according to the
A N T I- C H A N D - R G FR OM
manufacturer’s directions.
OT H E R A N T I B O D IE S W I T H
9. Resuspend the cell buttons and examine
S IM IL A R C H A RA C T ER IS T I C S
for agglutination; confirm all nonreactive
Principle tests microscopically. Grade and record
the results.
For a discussion of the principles of plasma 10. Confirm the validity of negative results by
inhibition of anti-Ch and -Rg, see Chapters 14 adding IgG-coated red cells.
and 16.
Interpretation
Specimen
Serum or plasma to be tested. 1. Inhibition of antibody activity in the
tubes to which plasma has been added
Reagents suggests anti-Ch or anti-Rg specificity;
this inhibition is often complete.
1. Reactive red cell samples. 2. The presence of partial inhibition sug-
2. A pool of six or more normal plasma sam- gests the possibility of additional alloanti-
ples. bodies. This can be tested by preparing a
3. 6% bovine albumin (see Method 1-5).
large volume of inhibited serum and test-
4. Anti-IgG.
5. IgG-coated red cells. ing it against a reagent red cell panel to
see if the nonneutralizable activity dis-
Procedure plays antigenic specificity.
3. Lack of reactivity in the control (6% albu-
1. Prepare serial twofold dilutions of test min) indicates dilution of weakly reactive
serum in saline. The dilution range antibody and an invalid test.
912 ⽧ AABB TECHNICAL MANUAL

Notes mL volumes, and freeze aliquots at –18 C

or colder.
1. Antibodies to other plasma antigens may 2. PBS at pH 7.3 (see Method 1-7).
also be partially inhibited by plasma.1 3. Prepare 6% AET by dissolving 0.6 g of AET
2. Adsorption with C4-coated red cells is an in 10 mL distilled water, and bring the pH
alternative procedure that may be used to 8 by slowly adding 5 N NaOH.
for identifying anti-Ch or anti-Rg and for 4. Red cells known to be positive for the
detecting underlying alloantibodies.2 antigen in question and, as a control, red
cells known to be positive for K, which is
References consistently disrupted by DTT or AET.
5. Anti-K, either in reagent form or strongly
1. Reid ME, Lomas-Francis C. The blood group reactive in a serum specimen.
antigen factsbook. 2nd ed. San Diego: Aca-
demic Press, 2004. Procedure—DTT
2. Ellisor SS, Shoemaker MM, Reid ME. Adsorp-
tion of anti-Chido from serum using autolo-
gous red blood cells coated with homologous 1. Wash one volume of the test cells and the
C4. Transfusion 1982;22: 243-5. control cells with PBS. After decanting,
add four volumes of 0.2 M DTT, pH 8.0.
2. Incubate at 37 C for 30 to 45 minutes.
ME T H O D 3 - 1 0 . TRE A T IN G RE D 3. Wash four times with PBS. Slight hemoly-
C E L LS U S I N G D T T O R A E T sis may occur; if hemolysis is excessive,
repeat the procedure using fresh red cells
Principle
and a smaller volume of DTT (eg, two or
Dithiothreitol (DTT) and 2-aminoethylisothio- three volumes).
uronium bromide (AET) are efficient reducing 4. Resuspend the cells to a 2% to 5% suspen-
agents that can disrupt the tertiary structure of sion in PBS.
proteins by irreversibly reducing disulfide 5. Test DTT-treated cells with serum con-
bonds to free sulfhydryl groups. Without ter- taining the antibody in question. Test K+
tiary structure, protein-containing antigens red cells with anti-K.
can no longer bind antibodies that are specific
for them. Red cells treated with DTT or AET Procedure—AET
will not react with antibodies in the Kell blood
group system, most antibodies in the Knops 1. Combine four volumes of the prepared
system, or most examples of anti-LWa, -Yta, -Ytb, AET solution with one volume of washed,
-Doa, -Dob, -Gya, -Hy, and -Joa. These inhibi- packed red cells to be treated.
tion techniques may be helpful in identifying 2. Incubate for 20 minutes at 37 C, mixing
some of these antibodies or in determining if a every 5 minutes.
serum contains additional underlying 3. Wash treated cells with PBS five to seven
times or until the supernatant is clear.
alloantibodies.
4. Resuspend the cells to a 2% to 5% suspen-
sion in PBS.
Specimen
5. Test AET-treated cells with serum con-
Red cells to be tested. taining the antibody in question. Test K+
red cells with anti-K.
Reagents
Interpretation
1. Prepare 0.2 M DTT by dissolving 1 g of
DTT powder in 32 mL of phosphate-buff- 1. The control K+ red cells should give nega-
ered saline (PBS), pH 8.0. Divide it into 1- tive reactions when tested with anti-K; if
 METHODS SECTION 3 Antibody Detection and Identification ⽧ 913

not, the DTT or AET treatment has been it for 10 minutes. Cool. Using 10-mm-
inadequate. Other antigens in the Kell interior-diameter cellulose membrane
system can also serve as the control. tubing (12,400 MW cut off), dialyze it
2. If reactivity of the test serum is elimi- against phosphate-buffered saline (PBS),
nated, the suspected antibody specificity pH 7.3, at 4 C for 48 hours. Change PBS
may be confirmed. Enough red cell sam- several times. Centrifuge. Dispense super-
ples should be tested to exclude most natant into aliquots, which can be stored
other clinically significant alloantibodies. at –20 C until thawed for use.
2. PBS, pH 7.3 (see Method 1-7).
Note
Procedure
Treatment of red cells with 0.2 M DTT or 6%
AET will denature or weaken all antigens of the
1. Mix equal volumes of thawed urine and
Kell, Cartwright, LW, Dombrock, and Knops
test serum.
systems. Lower concentrations of DTT may
2. Prepare a dilution control tube con-
selectively denature particular blood group
taining equal volumes of serum and PBS.
antigens (ie, 0.002 M DTT will denature only
3. Prepare a urine control tube by mixing
Jsa and Jsb antigens, while other Kell antigens
equal volumes of thawed urine and PBS.
will not be affected). This property may aid in
4. Incubate all tubes at room temperature
certain antibody investigations.
for 30 minutes.
5. Mix 1 drop of each test red cell sample
References
with 4 drops from each of the tubes: neu-
1. Advani H, Zamor J, Judd WJ, et al. Inactivation tralized serum, serum with PBS, and
of Kell blood group antigens by 2-aminoethyl- urine with PBS. Test each one using stan-
isothiouronium bromide. Br J Haematol 1982; dard procedures.
51:107-15.
2. Branch DR, Muensch HA, Sy Siok Hian S, Petz Interpretation
LD. Disulfide bonds are a requirement for Kell
and Cartwright (Yta) blood group antigen
integrity. Br J Haematol 1983;54: 573-8. 1. Persistent agglutination in the serum
sample incubated with urine means
either that partial or no neutralization
ME T H O D 3 - 1 1 . NE UT RA L IZ IN G was achieved or that underlying
A N T I- S d a W IT H UR IN E antibodies are present. Microscopic
examination may be helpful; agglutina-
Principle tion caused by anti-Sda has a refractile,
For a discussion of anti-Sda neutralization by mixed-field appearance on microscopic
urine, see Chapter 16. examination.
2. No agglutination in the neutralized tube
Specimen with persistent agglutination in the dilu-
tion control tube and absence of hemoly-
Serum or plasma suspected of containing anti- sis and agglutination in the urine control
Sda. tube indicate that the antibody has been
neutralized and is quite probably anti-
Reagents Sda.
3. The absence of agglutination in the dilu-
1. Urine from a known Sd(a+) individual, or tion control tube means that the dilution
from a pool of at least six individuals of in the neutralization step was too great
unknown Sda type, and prepared as fol- for the antibody present, and the results
lows: Collect urine and immediately boil of the test are invalid.
914 ⽧ AABB TECHNICAL MANUAL

4. The urine control tube provides assur- possible, to avoid dissociation of anti-
ance that no substances in the urine are body from the red cell membranes.
agglutinating or damaging the red cells. 6. Transfer the supernatant fluid, which is
the adsorbed serum, to a clean test tube.
Note If an eluate is to be prepared, save the red
cells.
Urine may also contain ABO and Lewis blood
7. Test an aliquot of the adsorbed serum,
group substances, depending upon the
preferably against a reserved unused ali-
ABO, Lewis, and secretor status of the donor.
quot of the red cells used for adsorption,
to see if all antibody has been removed.
Reference
1. Judd WJ, Johnson S, Storry J. Judd’s methods in Interpretation
immunohematology. 3rd ed. Bethesda, MD:
If reactivity remains, the antibody has not
AABB Press, 2008.
been completely removed. No reactivity signi-
fies that antibody has been completely
ME T H O D 3 - 1 2 . AD S O RP T I ON adsorbed.
PRO C E D URE
Notes
Principle
See Chapter 16. 1. Adsorption is more effective if the area of
contact between the red cells and serum
Specimen is large. Use of a large-bore test tube (13
mm or larger) is recommended.
Serum or plasma containing antibody to be
2. Multiple adsorptions may be necessary to
adsorbed.
remove an antibody completely; however,
Reagents each successive adsorption increases the
likelihood that the serum will be diluted
Red cells (eg, autologous or allogeneic) that and unadsorbed antibodies weakened.
carry the antigen corresponding to the anti- 3. Repeat adsorptions should use a fresh ali-
body specificity to be adsorbed. quot of red cells and not the red cells from
the earlier adsorption.
Procedure 4. Enzyme pretreatment of adsorbing red
cells can be performed to increase anti-
1. Wash the selected red cells at least three body uptake for enzyme-resistant anti-
times with saline. gens.
2. After the last wash, centrifuge the red
cells at 800 to 1000 × g for at least 5 min- Reference
utes, and remove as much of the superna-
1. Judd WJ, Johnson S, Storry J. Judd’s methods in
tant saline as possible. Additional saline
immunohematology. 3rd ed. Bethesda, MD:
may be removed by touching the red cell AABB Press, 2008.
mass with a narrow piece of filter paper.
3. Mix appropriate volumes of the packed
red cells and serum, and incubate at the ME TH O D 3-1 3 . US ING THE
desired temperature for 30 to 60 minutes. AMERICAN RARE DONOR PROGRAM
4. Mix the serum or cell mixture periodically
Principle
throughout the incubation phase.
5. Centrifuge the red cells at 800 to 1000 × g The American Rare Donor Program (ARDP)
for 5 minutes to pack cells tightly. Centri- helps to locate blood products for patients
fuge at the incubation temperature, if requiring rare or unusual blood. The ARDP
 METHODS SECTION 3 Antibody Detection and Identification ⽧ 915

maintains a database of rare donors submitted 4. The institution contacting the ARDP
by immunohematology reference laboratories (requesting institution) must confirm the
(IRLs) that are accredited by the AABB or the identity of the antibody(ies) by serologic
American Red Cross (ARC). Donors are consid- investigation or by examining the sero-
ered rare because of the absence of a high- logic work performed by another institu-
prevalence antigen, the absence of multiple tion.
common antigens, or IgA deficiency. 5. ARDP staff search their database for cen-
All requests to the ARDP must originate ters that have identified donors with the
from an AABB- or ARC-accredited IRL to needed phenotype and contact the cen-
ensure that the patient in question has been ters for availability of units. The ARDP
accurately evaluated and reported. All ship-
staff give the name(s) of the shipping cen-
ping and rare-unit fees are established by the
ter(s) to the requesting institution.
shipping institution.
6. The requesting and shipping institutions
should discuss and agree on charges and
Procedure
testing requirements before units are
shipped.
1. A hospital blood bank, transfusion ser-
7. If an initial search does not result in a suf-
vice, or blood center identifies a patient
ficient number of units, the following
who needs rare blood.
mechanisms can be used by the ARDP
2. The institution contacts the nearest
AABB- or ARC-accredited IRL to supply staff to obtain needed units: 1) commu-
the needed blood. nicating with all ARDP participating cen-
3. If the IRL cannot supply the blood, it con- ters, alerting them to search their
tacts the ARDP. All requests to the ARDP inventories and/or recruit donors
must come from an AABB- or ARC- matching the needed phenotype, or 2)
accredited IRL (or another rare donor contacting other rare donor files such as
program). Requests received directly from those administered by the World Health
a nonaccredited facility will be referred to Organization, Japanese Red Cross, or sim-
the nearest accredited institution. ilar organizations.