THE DIRECT ANTIGLOBULIN TEST (DAT) and Elution/Eluate Testing When performed as part of an antibody identification Jean Purcelli MT(ASCP)SBB May, 2011 Version 2, May 2012

Detects in vivo coating of red cells with IgG or complement components or both. Blood collected in EDTA is the best source of red cells to use for a DAT. Red cells from clotted samples may give false positive results when using polyspecific AHG or monospecific anti-C3d AHG.

The DAT may be ordered by physicians to investigate: Autoimmune hemolytic anemia Drug induced hemolylsis Hemolytic disease of the fetus or (HDFN) Alloimmune reactions due to recently transfused rbcs. DAT and IAT, ordered by physicians is usually tested at the hospital. If positive, the hospital lab would ask for a more complete workup if the patient requires a transfusion.

The DAT is also included in a basic antibody investigation: The DAT has similar significance as the auto control, but they cannot be substituted for one another. A negative DAT implies that complete or partial phenotyping can be performed easily, without red cell modification

The DAT may be positive in: An immediate or delayed transfusion reaction. Circulating donor rbcs become coated with antibodies in the patient’s plasma. Auto immune diseases : cold and warm autoimmune hemolytic anemia. Unexpected findings may help physicians diagnose or treat their patients. A drop in hemoglobin may not mean the patient is bleeding. Please keep in mind that some patients are hemolysing their own red cells but IgG cannot be detected by the DAT.

DAT positive Hemolytic disease of the fetus or newborn (HDFN) The cord cells and circulating fetal rbcs are coated with maternal antibodies. The fetus’ rbcs may be coated with antibodies from any clinically significant maternal antibody including the ABO system.

ABO antibodies are usually IgM and do not cross the placenta ABO antibodies are usually IgM and do not cross the placenta. Group O individuals often have an IgG component of anti-A and –B, as well. HDN due to ABO system antibodies are usually not severe and high levels of bilirubin are controlled with bilirubin lights. The most common ABO incompatibility is when the mother is group O and the fetus is group A, but also group B babies and less often a mother of A or B and the baby of B or A.

DAT positive Collagen-vascular diseases: SLE, RA Drug induced hemolytic anemia (alpha methyldopa, phenacetin, penicillin, quinine, any cephalosporins, procainamide. Paroxysmal Cold Hemolysis (PCH)

DAT positive Treatment with IVIG: may contain ABO or anti-D antibodies or other specificities. Bone marrow or organ transplant: passenger lymphocytes of donor origin may produce ABO or other antibodies against the recipient cells.

DAT positive Treatment of transplant patients with antilymphocyte globulin (ALG) or antithymocyte globulin (ATG). Treatment with IV RHIG. Nonspecific adsorption of proteins. The cause of non-specific adsorption is unknown. “Normal” blood donors have + DAT’s.

In the laboratory, the DAT is included as part of an antibody investigation. Washed rbcs are directly tested with AHG reagents. Polyspecific and/or monospecific reagents are used. In addition to aiding physicians, for the laboratory, you know right away if you can type the patients cells for Duffy, Kell, Kidd, Ss

Polyspecific AHG contains both anti-IgG and anti-complement components Polyspecific AHG contains both anti-IgG and anti-complement components. Monospecific anti-IgG contains only anti-IgG. Monospecific anti-complement contains anti-C3d and usually anti-C3b. Polyspecific reagents may contain some anti-IgM and IgA; therefore poly=+, IgG=0, and C’=0. Requires an explanation.

Polyspecific AHG is used as a screening test Polyspecific AHG is used as a screening test. If the test is negative the DAT is reported as negative. If the test is positive, repeat the test with monospecific reagents. The result of the test is reported as “ positive, the patient’s cells were coated with IgG” or “the patients red cells were coated with complement components”, or “the patient’s rbcs were coated with IgG and complement components”

A saline control tube is included in all DAT tests A saline control tube is included in all DAT tests. Microscopic examination should be included. “mixed field” agglutination may be observed in the recently transfused patient. M-f agglutination indicates surviving coated donor cells in a transfusion reaction

After examination at the IS (immediate spin) phase: The results are recorded. If the tube with anti-IgG is nonreactive, Checkcell is added. If non-reactive, the polyspecific tube and the complement tube are placed at RT for 5-10 minutes, centrifuged and examined micro again, recorded and Check Cell added. Tubes to which anti-IgG has been added must be centrifuged and read immediately

An anti-complement “check cell” should be used. Checkcells are IgG coated red cells and will not react with tubes containing anti-C3d,-C3b. An anti-complement “check cell” should be used. An anti-complement control can be made-- in-house and instructions will be provided for Phase III.

ELUTION and ELUATE TESTING An elution should be performed as part of the antibody identification ONLY if the patient was recently transfused. Each reference lab must establish their own guidelines for defining “recently”. In the USA, 4 weeks to 3 months is usually used.

Elution/Eluate An eluate is prepared and tested to detect a newly formed antibody that is not yet detectable in the serum. Antibody production peaks at about 10-18 days after exposure to a foreign antigen.

Elution/Eluate The eluate is tested by the IAT and usually the same panel cells that are chosen to test the serum are also used for the eluate. Enhancement media is commonly used. A1 and B cells should also be tested if there is ABO incompatibility between a mother and baby. Also in adults if there is a history of recent transfusion of ABO incompatible platelets or plasma. Show example panel sheets If the mother’s antibody screening is negative, an elution with cord cells and subsequent testing of eluate with A and B cells is not necessary.

Elution/Eluate Elutions are not performed on rbcs coated only with complement. The last wash is always saved and tested for unusual dissociation of antibody from the red cells. The last wash test, is an antibody screen with enhancement media by IAT.

Elution/Eluate The last wash should be negative or additional washes of the red cells must be performed. A positive “last wash” would make the results of the eluate testing invalid. It is recommended that LISS or normal saline, kept at 4 C is used for washing the red cells. It is unusual for the last wash to be reactive

The final report may contain comments about eluate, for example: Anti-Jka was identified in the eluate and may indicate the patient is having a delayed transfusion reaction. The eluate was non-reactive. The eluate was reactive with all rbcs without apparent specificity.

If the DAT is positive with anti-IgG, but the eluate is nonreactive it may indicate: The DAT was induced by drugs The DAT was caused by non-specific uptake of proteins.

Pease refer to “Selected Procedures for Fast Referral”. In the lab we will use Elukit IITM reagents and procedure.

Preparing red cells for elution The goal is to wash a large volume of red cells so no trace of contaminating unattached plasma proteins remain. Large 13 mm x 100 mm or 16 mm x 100 mm test tubes are preferred. A centrifuge to accommodate large tubes must be available

LISS or normal saline stored a 4oC is recommended for washing the red cells to prepare them for elution. It is recommended to change to clean test tubes 2x during the washing procedure.

If 13 mm or 16 mm x 100 are used, five washes are adequate, then washed a final time in a 12 mm x 75 mm tube. An aliquot of the last wash is saved and tested before the elution is performed. If 12 x 75 mm are used, then 10 washes would be adequate with 3 tube changes.

“Last Wash” The last wash is tested using the three antibody screening cells. Add two drops of last wash to each tube. Add two drops of LISS or PEG. Place at 370 for 15’ to 30’, then wash 3-4 times and convert to the antiglobulin test.

The last wash test should be non-reactive before proceeding with the elution. If the last wash is reactive, the red cells should be washed 4 more times and the “last wash” saved and tested again.

Elution To 1 ml washed red cells, add 1 ml of Eluting Solution Cover with parafilm and gently mix 4X. Centrifuge for 90” and quickly harvest the eluate. The red cells may be discarded.

Elution 4. Centrifuge the eluate and transfer to a clean tube. 5. Add drops of Buffering Reagent until the color turns to blue. 6. Centrifuge again and transfer to a clean tube.

Eluate Testing Test the eluate with the same cells you tested with the serum. LISS or PEG may be used for enhancement. In general, if the DAT is 2-4+, use LISS for the last wash and the eluate. If the DAT is 1+ or micro +, use PEG for testing.