Discontinuation and Replacement of Acid Hemolysins (Ham's Test) and Sucrose Lysis Test

SUBJECT: Discontinuation of the Ham’s acid hemolysin and sucrose lysis tests for Paroxysmal Nocturnal Hemoglobinuria (PNH) and their replacement by flow cytometric testing for glycosylphosphatidyl inositol (GPI)-anchored proteins.

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a stem cell disorder in which the affected cells are deficient in GPI-anchored proteins. GPI-anchored proteins include a number of important molecules on the surfaces of blood cells of all lineages. These include CD55 (decay-accelerating factor, DAF) and CD59 (membrane inhibitor of reactive lysis, MIRL) which protect against accidental activation of the complement system and cell lysis. Other GPI-anchored proteins present on leukocytes include CD16 and CD24 on B lymphocytes, CD14, CD16 and CD24 on monocytes, and CD16 and CD66b on granulocytes.

Ham’s acid hemolysin and sucrose lysis tests have been the standard tests used to diagnose PNH, however these assays are both insensitive and labor-intensive. Since monoclonal antibodies specific for GPI-anchored proteins have become available, many studies have shown that PNH can be sensitively and specifically diagnosed by flow cytometric assay of these proteins on both erythrocytes and various leukocytes.

As of this date, the Ham’s acid hemolysin and sucrose lysis tests will be discontinued in the Hematology Laboratory and replaced by a flow cytometric assay for GPI-anchored proteins performed in Immunopathology Laboratory. The flow cytometric assay has already been available from IP Lab for approximately one year. It is performed on whole blood. Both erythrocytes and granulocytes are analyzed for CD55 and CD59 expression. Granulocytes are the most sensitive population in which to detect GPI-anchored protein deficiency, while the erythrocytes are best for categorizing PNH type I, II, and III cells.

Results will be issued as a Bone Marrow (H-6) report interpreted by a pathologist. The number of GPI-anchored protein deficient cells can vary widely from case to case. Those patients with the highest relative numbers of GPI-anchored protein deficient cells are most likely to have classical PNH symptoms, while those with small relative numbers are more likely to present with aplastic anemia or myelodysplastic syndrome. About 20-25% of patients with aplastic anemia and MDS have been found to demonstrate small clones of PNH cells, so studies for PNH may also be indicated in patients with these diagnoses.


1)Richards, S et al. Application of Flow Cytometry to the Diagnosis of Paroxysmal Noctural Hemoglobinuria. Cytometry 2000; 42:223-233.

2)Dunn, D, et al. Paroxysmal Nocturnal Hemoglobinuria in Patients with Bone Marrow Failure Syndromes. Ann Int Med 1999; 131:401-408.

REQUISITION: Please order this test on the IMMUNOPATHOLOGY O-8 (green and white) form under the FLOW CYTOMETRY section at the bottom of the page.

SPECIMEN REQUIREMENTS: The specimen required for flow cytometric analysis for PNH is peripheral blood, 7 ml, in either yellow top or lavender tube. Yellow top (citrate anticoagulant) is preferred because it improves leukocyte viability compared to lavender top (EDTA anticoagulant).


QUESTIONS or COMMENTS: contact Dr. Jim Goeken (6-1966) or Dr. Nancy Rosenthal (4-8751).