Ham's Test (Acidified Serum Test)

Paroxysmal nocturnal hemoglobinemia (PNH) is an acquired hematopoietic stem cell disorder in which somatic mutation of the X-linked PIG-A gene results in partial or absolute deficiency of all proteins normally linked to the cell membrane by a glycopphosphatidylinositol (GPI) anchor. The abnormal gene occurs in somatic cells, especially hematopoietic stem cells, but not in germ cells, making PNH an acquired disorder. So far, PIG-A gene mutations have not been detected in somatic cells outside of the hematopoietic system. All hematopoietic cell lineages are affected including erythrocytes, platelets, granulocytes and monocytes. Approximately 174 somatic mutations in the PIG-A gene have been identified. The initial mutagenic event remains unknown.

PNH is rare; the estimated incidence is 1.3 new cases per one million individuals per year. Development of clinical disease requires the combination of a hypoplastic or dysplastic bone marrow, somatic mutation in the PIG-A gene, and clonal expansion of PNH stem cells. Clinical features vary greatly from patient to patient during the course of the disease but there are three main pathophysiologic features:

• Intravascular hemolysis  
• Venous thrombosis 
• Bone marrow aplasia or dysplasia

Hemolysis is a consequence of abnormal erythrocyte sensitivity to complement-mediated lysis. PNH erythrocytes are abnormally sensitive to complement because they are deficient in two complement regulatory proteins, decay accelerating factor (DAF, CD55) and membrane inhibitor of reactive lysis (MIRL, CD59).  DAF regulates the early portion of complement activation including C3-C5 while MIRL inhibits the incorporation of C9 into the membrane attack complex. The absence of CD59 is primarily responsible for red cell hemolysis and may contribute to thrombosis.

Ham’s test was also known as the Acidified Serum Test. Red blood cells from patients with PNH hemolyze when incubated with acidified normal and acidified patient's serum, whereas normal RBC's do not.  A positive result is not sufficient to diagnose PNH.

Patients with marked spherocytosis or with the rare condition, congenital dyserthropoietic anemia, type II, (HEMPAS) may have a positive test. In HEMPAS, the patient's red cells will lyse only in acidified normal serum, and not in the patient's own acidified serum.  In marked spherocytosis, lysis takes place with heat inactivated serum, whereas in PNH this is not the case. 

Ham’s test is not as sensitive or specific in detecting PNH as flow cytometry. Ham’s test is now considered an outdated laboratory test. 

See Paroxysmal Nocturnal Hemoglobinuria by Flow Cytometry.  

References

Rosse WF, Dr Ham’s Test Revisited, Blood, 1991;78:547-550.

Krauss JS, Laboratory Diagnosis of Paroxysmal Nocturnal Hemoglobinuria, Ann Clin Lab Science, 2003;33(4):401-406.