VII-A.

Preparing C3b/C4b-Coated RBCs

Purpose To provide instructions for coating RBCs with C3b and C4b for use in
the following:
• Quality control of negative test results with anti-C3 reagents.

Background Under low-ionic conditions, C3b and C4b are bound to RBCs without prior
Information attachment of antibody molecules (ie, via the alternative pathway).

Operational When performing comparative evaluations of different lots of AHG

Policy reagents, vary the ratio of fresh normal serum to RBCs (eg, 5:1, 4:1, 3:1,
2:1, 1:1).
Note: Some IgG may also be bound by this method.

Limitations Unwanted Positive Reactions:

• Positive DAT on donor RBCs.
• Improper storage of reagents or coated RBCs.
• Use of incorrect technique.
• Use of wrong reagent.
Unwanted Negative Reactions:
• Improper storage of reagents or coated RBCs.
• Use of incorrect technique.
• Use of wrong reagent.

Sample Group O whole blood collected into ACD, CPD, or CPD-A1.

Requirements
Note: Before coating, RBCs should have a negative DAT using
polyspecific AHG.
Equipment/ 10% sucrose.
Materials
Anti-C3 reagent known to detect intact C3b.
Note: Some monoclonal anti-C3d will not detect intact C3b.
Anti-IgG.
Alsever’s solution.
Pipettors with disposable tips to deliver 1 mL and 10 mL (eg, VWR,
Batavia, IL).

Quality Test the coated RBCs vs anti-IgG and anti-C3.

Control

Procedure Use the following steps to perform the procedure:

Step Action
1. Mix 1 mL of whole blood with 10 mL of sucrose.
2. Incubate at 37 C for 15 minutes.
3. Wash the RBCs three times with saline. (The RBCs are now
coated with C3b and C4b.)
4. Resuspend the coated RBCs to a 3%-5% suspension with
Alsever’s solution.
5. Test the coated RBCs with anti-IgG and anti-C3 as specified by
the manufacturer.
6. Interpret the reactions as follows:
If test results And/or test Then…
with anti-C3 results with
are… anti-IgG are…
>2+ negative RBCs may be used:
• Store refrigerated.
≤ 2+ positive or do not use RBCs:
negative • Repeat C3b/C4b
coating procedure.
• Consider inactive
anti-C3 reagent.
 Or, when validating negative antiglobulin test results:
If C3-coated RBCs…
give a mixed-field reaction
are nonreactive
Then…
negative antiglobulin test result is
valid.
negative antiglobulin test result is
invalid:
• Repeat antiglobulin test.
• Consider inadequate washing
before addition of AHG or
inactive AHG.

Reference Garratty G, Petz LD. Quality control of antiglobulin serum (letter).

Transfusion 1975;15:397.
Source: Copied from
Judd’s Methods in Immunohematology 3rd edition
VII-B. Preparing IgM/C3b-Coated RBCs

Purpose To provide instructions for coating RBCs with IgM and C3b for use in the
following:
• Evaluation/standardization of AHG reagents.
• Quality control of negative test results with anti-C3 reagents.

Background Le (Lewis) antibodies are used to coat RBCs with C3b via the classical
Information pathway.

Operational The proportions of RBCs and serum may need to be changed depending
Policy on the strength of the Le antibody. However, maintain the ratio of 0.25 mL
of K2EDTA to 2 mL of serum.
To assess the anti-C3 activity in AHG reagents, it will be necessary to
subtract the results of tests with the IgM-coated RBCs from those
obtained with the C3b-coated RBCs.
Anti-Le prepared in goats has been shown to be unsuitable for use in this
procedure. Use human or rabbit anti-Le sera.
C3b-coated RBCs may be converted to C3d-coated RBCs, either by
extended incubation (2 hours) in 20 volumes of freshly collected normal
human serum or by treatment of the RBCs with crude trypsin, as
described in Section III.

Limitations Unwanted Positive Reactions:

• Positive DAT on donor RBCs.
• Improper storage of reagents or coated RBCs.
• Use of incorrect technique.
• Use of wrong reagent.
Unwanted Negative Reactions:
• Improper storage of reagents or coated RBCs.
• Use of incorrect technique.
• Use of wrong reagent.
Sample Anti-C3 reagent under evaluation.
Requirements

Equipment/ Alsever’s solution.

Materials
Antibody: anti-Lea or anti-Lea+b; 2 mL.
Anti-C3 reagent known to detect C3b.
Note: Some monoclonal anti-C3d will not detect intact C3b.
6% BSA.
4.45% wt/vol K2EDTA.2H2O.
Human complement: freshly collected, normal human serum known to
lack unexpected antibodies; 16 mL.
RBCs: packed group O, Le(a+b–) RBCs, washed three times with saline;
2 mL.
Pipettors with disposable tips to deliver 200-500 µ L and 2 mL (eg, VWR,
Batavia, IL).

Quality Test with reactive anti-C3 and 6% BSA.

Control

Procedure Use the following steps to perform the procedure:

Step Action
1. Mix 250 µ L of EDTA with 2 mL of antibody-containing serum.
Incubate at RT for 15 minutes.
2. Add 2 mL of saline to the serum and prepare serial twofold
dilutions of the serum with saline. The dilution range should be
from 1 in 2 to 1 in 256 (8 tubes), and 2-mL volumes should be
prepared.
3. Add 0.2 mL of washed Le(a+b–) RBCs to each dilution.
4. Mix and incubate at 37 C for 60-90 minutes.
5. Wash the RBCs four times with saline. (The RBCs are now
coated with IgM.)
6. Dilute an aliquot to a 3%-5% suspension with Alsever’s solution
and save for later testing.
7. To the washed antibody-coated RBCs, add 2 mL of complement.
 8. Mix and incubate at 37 C for 15-20 minutes.
9. Wash the RBCs four times with saline and dilute to a 3%-5%
suspension with Alsever’s solution. (The RBCs are now coated
with C3.) Store refrigerated when not in use.
10. Test the IgM- and C3-coated RBCs with AHG reagents, as
specified by the manufacturer, and by the same technique with
6% BSA.
11. Interpret the reactions of the C3-coated RBCs as follows:
If test results And/or test Then…
with anti-C3 results with
are… 6% BSA are…
positive negative highest dilution giving 1+
reaction = titer of anti-C3.
negative with negative do not use RBCs:
RBCs coated at • Repeat C3b coating
all dilutions procedure.
• Consider inactive anti-
C3 reagent.
positive or positive RBCs are unsuitable for
negative use as C3-coated RBCs:
• Higher serum dilution
required.
Interpret the reactions of the IgM-coated RBCs as follows:
If test results with 6% Then…
BSA are…
positive highest dilution giving 1+ reaction =
titer of IgM anti-Le.

Reference 1. Issitt PD, Smith TR. Evaluation of antiglobulin reagents. In:

Myhre BA, ed. A seminar on performance evaluation.
Washington, DC: AABB, 1976:25-73.
Source: Copied from
2. Judd’s Methods in Immunohematology 3rd edition