Tests Used to Diagnose
Hemolytic Transfusion Reactions

(These tests should be compared to pretransfusion sample results whenever possible)

1.   ABO Grouping (Specimen - Red Top Tube): The severity of a transfusion reaction due to incompatibility in the ABO system is greater than that in other blood group systems. Pre-transfusion and post-transfusion specimens of the patient, RBCs from the segment used in the original crossmatch and RBCs from the blood bag (if available) should be tested.

2.  DAT (Direct Antiglobulin Test; Specimen - Lavender Top Tube): At the time of a transfusion reaction, the DAT may be + or -, depending on the type of antibody involved and the degree of cell lysis at the time the specimen is drawn. A positive DAT (+ DAT) usually indicates the presence of_ recipient antibody on the surface of donor RBC’s. NOTE: A + DAT may be due to infusion of ABO incompatible plasma which frequently occurs in platelet transfusion. Although this rarely causes RBC destruction, the + DAT may cause confusion in interpreting other evidence of a hemolytic transfusion reaction.

3.  IAT (Indirect Antiglobulin Test; Specimen - Red Top Tube): A positive IAT indicates the presence of free antibody in the patient’s serum. The antibody screening test will be negative only if all the antibodies are bound to RBCs. This may be the situation immediately following the transfusion; however, the antibody will reappear in the serum within a few hours to days.

4.  Plasma Free Hemoglobin: In intravascular hemolysis, hemoglobin is released directly into the bloodstream. It is bound to haptoglobin, a plasma protein, and transported to the RE system where it is catabolized.  Haptoglobin binding capacity is rapidly saturated, so the level of free plasma hemoglobin rises. An increase in plasma free hemoglobin is characteristic of intravascular hemolysis. It is not necessary to perform a haptoglobin test if free hemoglobin is visible in the plasma.

5.   Haptoglobin (Specimen - Red Top Tube): When haptoglobin becomes saturated with hemoglobin, the hemoglobin-haptoglobin complex is removed by the RE system. Since the removal of this complex exceeds the synthetic capacity of the liver to produce haptoglobin, plasma haptoglobin levels are decreased in intravascular hemolysis.  NOTE: Because there are other reasons for low haptoglobin levels, a low post-transfusion haptoglobin is meaningful only when compared with a pre-transfusion value.

6.   Hemoglobinuria/Hemosiderinuria (Specimen - Urine): Intact RBCs in the urine (Hematuria) is a sign of hemorrhage in the urinary tract and is not caused by hemolytic transfusion reactions. Free hemoglobin in the plasma is filtered by the kidney and excreted in the urine. Hemoglobinuria occurs following intravascular hemolysis. When hemolysis is chronic (example: autoimmune hemolytic anemia) the heme iron is stored as hemosiderin in the renal epithelial cells. Prussian blue staining of urinary sediment is evidence of hemosiderin and indicates chronic RBC destruction.

7.   Bilirubin (Total and Indirect): (Specimen - Red Top Tube): Bilirubin, the breakdown product of the heme portion of hemoglobin, is released into the circulation (unconjugated or indirect bilirubin), binds to albumin and is carried to the liver where is conjugated with glucuronates. In both intravascular and extravascular hemolysis, the plasma level of unconjugated bilirubin is elevated. The presence of bilirubin in the plasma is rarely observed in less than 6 hours. For this reason, plasma bilirubin should be drawn 6-8 hours after a suspected hemolytic reaction.

8.   Coagulation Tests (Specimen - Blue Top Tube): DIC is a significant sequela to massive intravascular hemolysis. For this reason, post-transfusion specimens drawn immediately after the reaction provide diagnostic information. These tests should include PT/PTT fibrinogen Ddimer, and platelet count.

9.   Serum Creatinine (Specimen - Red Top Tube): An immediate post-transfusion creatinine determination provides evidence of baseline renal function. The renal involvement following an acute, hemolytic transfusion reaction is due to hypotension in which blood flow to the kidney is severely restricted. Although free hemoglobin is found in the kidney tubules, it is not the cause of the ischemia. Free hemoglobin itself is not toxic.