VII-F.

Preparing IgG-Coated RBCs

To provide instructions for coating RBCs with IgG for use in the following:
Purpose
• Evaluating AHG reagents.
• Validating negative test results with anti-IgG.

When incubated together, IgG antibody coats antigen-positive RBCs with

Background
IgG. The coated RBCs can be used in the evaluation of AHG reagents
Information
and in the quality control of AHG tests.

Operational When evaluating AHG reagents, vary the proportions of RBCs and serum
Policy to prepare weakly (1+), moderately (2+), or strongly (4+) reactive RBCs
for evaluating AHG reagents.
For quality control of negative antiglobulin test results, the IgG-coated
RBCs should, at a minimum, give a 2+ (score 8) reaction in tests with
reagents containing anti-IgG.

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
Antiglobulin reagent under evaluation or negative antiglobulin tests with
Requirements
anti-IgG.
Equipment/
Alsever’s solution.
Materials
Antibody: weakly reactive IgG anti-D.
Note: Use a pool of 10 anti-D sera for preparing IgG-coated RBCs for
use as control RBCs in AHG tests. Alternatively, modified-tube
anti-D may be used; dilute 1 in 1500 or 1 in 3000 in 6% BSA
before use.
Anti-IgG.
Anti-C3d.
K2EDTA: 4.45% wt/vol.
RBCs: group O, R1r RBCs, washed three times with saline.
Pipettors with disposable tips to deliver 200-500 L and 2 mL (eg, VWR,
Batavia, IL).

Quality
Test the coated RBCs vs anti-IgG and anti-C3d.
Control

Procedure
Use the following steps to perform the procedure:

Step Action
1. Mix 250 L of EDTA with 2 mL of anti-D.
2. Incubate at RT for 15 minutes.
3. Add 0.2 mL of RBCs.
4. Mix and incubate at 37 C for 15-60 minutes.
5. Wash the RBCs four times with saline and dilute to a 3%-5%
suspension with Alsever’s solution. Store at 4 C.
6. Test vs AHG reagents as described by the manufacturer.
Alternatively, add to negative tests with anti-IgG reagents.
 7. Interpret the reactions as follows:
If test results And test Then…
with anti-IgG results with
are… anti-C3d
are…
>2+ negative RBCs may be used:
• Store at 4 C.
2+ positive or do not use RBCs:
negative • Repeat IgG coating
procedure.
• Consider inactive AHG
reagents or
agglutinating anti-D.
Or, when validating negative antiglobulin test results:
If IgG-coated RBCs… Then…
give a mixed-field reaction negative antiglobulin test result is
valid.
are nonreactive negative antiglobulin test result is
invalid:
• Repeat antiglobulin test.
• Consider inadequate washing
before addition of AHG or
inactive AHG.

Reference
Issitt PD, Smith TR. Evaluation of antiglobulin reagents. In: Myhre BA, ed.
A seminar on performance evaluation. Washington, DC: AABB, 1976:25-
73.