Background/Case Studies: The Acid Hemolysis Test (HAM) was developed by Thomas Ham in 1939 to help diagnose Paroxysmal Nocturnal Hemoglobinuria (PNH). It was later found that a modified version of the test could help diagnose hereditary erythroblastic multinuclearity with positive acidified serum lysis test (HEMPAS). HEMPAS or congenital dyserythropoietic anemia type II is a rare congenital anemia caused by a glycosylation deficiency in humans. Flow cytometry has since replaced the HAM’s test. However, as this case shows, it is still an important and pertinent diagnostic tool.
Study
Design/Methods: A review of the patient’s medical chart was performed for clinical diagnosis and laboratory tests performed outside of the immunohematology reference laboratory. Antibody screen was performed by gel column agglutination method. Direct antiglobulin testing (DAT) was performed by tube method with polyspecific anti-human globulin, rabbit and human derived anti-IgG and anti-C3b/C3d. The modified HAM’s test was performed with 9 drops serum and acidified serum (9 drops serum + 1 drop 0.2M HCl) with 1 drop of 50% saline suspended group O indicator cells. Incubations were conducted on ice for 30m and 60m at 37C. Due to the presence of anti-I (I1) at 4C, HAM’s testing was repeated at 37C only for hemolysis detection. The control was prepared from dithiothreitol (DTT) treated group O red cells. Fresh normal serum was heated for 30m at 56C for a complement-inactivated serum source. Serial dilutions of anti-I (I1) and anti-i (I2) were tested with patient cells and control cells (adult I (I1) + and cord I (I2) + cells) to evaluate antigen expression.
Results/Findings: A 78-year-old woman with a history of normocytic anemia, rheumatoid arthritis, atrial fibrillation, valvular heart disease, congestive heart failure, and pulmonary hypertension. She later showed evidence of hemolysis, a C3+ DAT and spherocytosis consistent with autoimmune hemolytic anemia although no autoantibodies were identified. Two separate flow cytometry tests were conducted with negative results for PNH and her cold agglutinin test was within normal limits. The patient’s antibody screen was nonreactive. Her acid elution and Donath-Landsteiner (D-L) tests were negative and her DAT was positive for C3 only. A cold-reactive autoantibody with anti-I (I1) specificity was detected at 4C. Titers of the i (I2) antigen on our patient’s red cells showed an abnormally high i (I2) antigen expression compared to cord cells and slightly higher I (I1) antigen expression. The HAM’s test result showed hemolysis only when the patient’s cells were incubated with normal serum and acid (See Table 1). Conclusions: The HAM’s test was the gold standard for many decades before the invention of flow cytometry. It was a valuable diagnostic tool for various dyserythropoietic diseases. This case demonstrates it is still an important tool in the investigation HEMPAS.
Importance of research: This report highlights a method that could be used to help diagnose dyserythropoietic diseases. The acid hemolysis test has been considered obsolete for many years but this case proves there is still a need for this important diagnostic tool.
