Direct Coombs Test Manual Procedure

The Direct Coombs Test, also known as the Direct Antiglobulin Test (DAT), is a laboratory procedure used to detect antibodies or complement proteins attached to the surface of red blood cells. This test is essential for diagnosing autoimmune hemolytic anemia, hemolytic disease of the newborn, and drug-induced hemolytic anemia. Below are key sections for test principles, materials, procedure, interpretation, quality control, and troubleshooting.

Test Principle

The Direct Coombs Test detects immunoglobulin (IgG) or complement (C3d) components bound to red blood cells in vivo. Anti-human globulin (AHG) reagent is added to washed red blood cells, causing agglutination if antibodies or complement are present on the cell surface.

Materials and Reagents

Required materials for performing the Direct Coombs Test:

1. Anti-human globulin reagent (polyspecific and monospecific)
2. Patient red blood cells (EDTA anticoagulated blood)
3. Positive control (IgG-coated red cells)
4. Negative control (normal red cells)
5. Normal saline (0.9% NaCl)
6. Test tubes (10x75 mm or 12x75 mm)
7. Centrifuge
8. Serological pipettes and droppers
9. Light source and viewing mirror
10. Timer

WARNING! Use reagents before expiration date; store according to manufacturer instructions.

Specimen Requirements

Proper specimen collection and handling are critical for accurate results.

1. Specimen type: Whole blood in EDTA (lavender top tube)
2. Volume: 3-5 mL for adults; 1-2 mL for pediatric patients
3. Storage: Process within 48 hours if refrigerated at 2-8°C
4. Rejection criteria: Hemolyzed, clotted, or contaminated specimens
5. Labeling: Patient identification with two unique identifiers

CAUTION! Do not use heparinized specimens as heparin may interfere with complement binding.

Procedure Steps

Step-by-step procedure for performing the Direct Coombs Test:

1. Label three test tubes: Test, Positive Control, Negative Control
2. Add 1 drop of washed patient red cells to Test tube
3. Add 1 drop of positive control cells to Positive Control tube
4. Add 1 drop of negative control cells to Negative Control tube
5. Wash all tubes 3-4 times with normal saline
6. Decant saline completely after final wash
7. Add 2 drops of anti-human globulin reagent to each tube
8. Mix gently and centrifuge at 1000 RPM for 15-20 seconds
9. Resuspend cell button and examine for agglutination macroscopically
10. If negative, incubate at room temperature for 5-10 minutes and re-centrifuge

Tip: Ensure complete decanting after washing to prevent false negatives.

Interpretation of Results

• Positive Result: Visible agglutination indicates presence of antibody or complement on red cells
• Negative Result: Smooth suspension with no agglutination
• Grading: 4+ (one solid agglutinate) to 1+ (many small agglutinates)
• Weak Positive: Microscopic agglutination visible only under microscope
• Mixed Field: Presence of both agglutinated and non-agglutinated cells

Controls must show expected results: Positive control must be positive, negative control must be negative.

Quality Control

Quality control measures to ensure test validity:

Daily QC: Run positive and negative controls with each batch of tests

Reagent QC: Check anti-human globulin reactivity with known weakly positive cells

Procedural QC: Verify washing technique and centrifugation conditions

Documentation: Record all QC results and any deviations from standard procedure

Limitations

Understanding test limitations is crucial for proper interpretation:

1. May not detect very low levels of bound antibody
2. Cannot differentiate between IgG and complement without monospecific reagents
3. False negatives may occur with improper washing or reagent deterioration
4. False positives may occur with polyagglutinable cells or cold agglutinins
5. Does not identify the specific antibody causing sensitization

Note: Additional testing may be required for complete diagnosis.

Safety Precautions

Standard precautions for handling biological specimens:

Wear appropriate PPE: gloves, lab coat, and eye protection. Treat all specimens as potentially infectious. Dispose of contaminated materials in biohazard containers. Decontaminate work surfaces with appropriate disinfectants.

CAUTION! Follow universal precautions and institutional biosafety guidelines.

Troubleshooting

Technical Support: Consult laboratory supervisor for unresolved issues.

References

American Association of Blood Banks Technical Manual. Clinical and Laboratory Standards Institute (CLSI) guidelines. Manufacturer's package inserts for reagents used. Standard hematology and immunohematology textbooks.