Ham's Test for Paroxysmal Nocturnal Hemoglobinuria

(redirected from Acid hemolysis test for PNH)

Ham’s Test for Paroxysmal Nocturnal Hemoglobinuria

Synonym/acronym: Acid hemolysis test for PNH.

Common use

To assist in diagnosing a rare condition called paroxysmal nocturnal hemoglobinuria (PNH), wherein red blood cells undergo lysis during and after sleep with hemoglobin excreted in the urine.


Whole blood (5 mL) collected in lavender-top (EDTA) tube and serum (3 mL) collected in red-top tube.

Normal findings

(Method: Acidified hemolysis) No hemolysis seen.


Paroxysmal nocturnal hemoglobinuria (PNH) is a rare condition in which the patient experiences nocturnal hemoglobinuria, chronic hemolytic anemia, diminished or absent generation of new red blood cells (RBCs), and a tendency to thrombose. It is not considered a primary disease in and of itself but rather a secondary condition caused by an acquired defect in hematopoietic stem cells. Specifically, PNH occurs through acquired mutations in the PIGA gene (Phosphatidyl Inositol Glycan anchor biosynthesis, class A) that eventually result in absence of GPI anchor proteins on cell surfaces of damaged stem cell progeny. Normally the GPI anchor proteins attach a numerous variety of proteins to the cell membrane so they are available when needed. It is believed that PNH is caused by complement mediated cellular lysis that occurs in the absence of GPI anchored complement inhibitors. The gene can have multiple mutations acquired throughout a person's lifetime and can occur in all three cell types (RBCs, WBCs, and platelets) although the type affecting the RBCs is the easiest to identify by the presence of corresponding symptoms. It was originally thought that the nighttime hemolysis was initiated by a state of acidosis that occurred during sleep. This was later disproved and the prevailing logic is that hemolysis takes place continuously. The hematuria is more noticeable when the accumulated contents of the bladder are passed in the morning and the color of the concentrated urine is dramatically different than that seen during the day. The disease affects males and females equally because the mutations take place in somatic or body cells rather than in germ cells which are then inherited. Symptoms of the disease usually manifest between the ages of 20 and 40 years. PNH is frequently associated with aplastic anemia. It has also been associated with myelodysplasia which may point to bone marrow failure syndromes as a condition that favors exposure of GPI anchor protein deficient hematopoietic stem cells.

In patients with PNH, erythrocytes have an increased sensitivity to complement and will lyse when mixed with acidified control serum that contains complement. The patient’s RBCs are also mixed with fresh, normal serum that is ABO compatible with the patient’s cells. Some of the control serum is acidified, and some is heated to inactivate the complement. The result is positive if 10% to 50% cell lysis occurs in the samples mixed with patient and control acidified serum. No hemolysis should occur in the heated control serum.

The sugar water or sucrose hemolysis test can also be performed to investigate the presence of PNH. Low ionic strength isotonic sucrose will cause serum globulin to fix complement on the RBC surface. When a small amount of type specific serum and sucrose solution is added to a sample of the patient's washed RBCs, PNH RBCs will be lysed compared to type specific serum and sucrose added to washed normal control RBCs. Platelet and granulocyte membranes are affected as well, but RBC hemolysis in a positive test is clear evidence of PNH. Greater than 5 percent hemolysis is considered positive for PNH.

Flow cytometry has replaced the Ham’s test as the definitive test for PNH. Levels of CD55 and CD59, membrane glycoproteins that regulate complement, on WBCs and RBCs surfaces are measured using flow cytometry. CD59 is more prevalent in the membrane than CD55 and the use of both is effective in identifying small and large clones of PNH cells. The findings are described as Type I PNH cells which have normal levels of both proteins, Type II PNH cells which have reduced levels, and Type III PNH cells which demonstrate an absence of CD55 and CD59 proteins. Testing is more accurately accomplished using WBCs, because the life of circulating WBCs is normal in PNH, whereas, the life of PNH RBCs is considerably shortened by chronic hemolysis, especially for PNH type III RBCs. Another advantage of WBC studies over RBC is that PNH RBCs are diluted by transfusion, one of the therapeutic modalities used to treat the anemia of PNH.

The treatment strategies for PNH depend on the type and degree of affect. Treatments are limited and there is a need for improvement in treatment options. While transfusions are effectively used to treat the anemia of PNH, iron overload is a significant consideration in the administration of serial blood cell transfusions.

This procedure is contraindicated for



  • Evaluate hemolytic anemia, especially with hemosiderinuria
  • Evaluate suspected congenital dyserythropoietic anemia, type II (also known as HEMPAS [hereditary erythroblastic multinuclearity with positive acidified serum test])
  • Evaluate suspected PNH

Potential diagnosis

Increased in

  • Congenital dyserythropoietic anemia, type II
  • PNH

Decreased in


Critical findings


Interfering factors

  • False positives may occur in the presence of other disorders, such as aplastic anemia, HEMPAS, hereditary or acquired spherocytosis, leukemia, and myeloproliferative syndromes. False positives may also occur with aged RBCs. The sugar water test is negative in HEMPAS.
  • False-negatives can occur if the patient’s serum sample contains a low level of complement.

Nursing Implications and Procedure


  • Positively identify the patient using at least two unique identifiers before providing care, treatment, or services.
  • Patient Teaching:  Inform the patient this test can assist in testing for causes of anemia.
  • Obtain a history of the patient’s complaints, including a list of known allergens, especially allergies or sensitivities to latex.
  • Obtain a history of the patient’s hematopoietic system, symptoms, and results of previously performed laboratory tests and diagnostic and surgical procedures.
  • Obtain a list of the patient’s current medications, including herbs, nutritional supplements, and neutraceuticals (see Effects of Natural Products on Laboratory Values).
  • Review the procedure with the patient. Inform the patient that specimen collection takes approximately 5 to 10 min. Address concerns about pain and explain that there may be some discomfort during the venipuncture.
  • Sensitivity to social and cultural issues,  as well as concern for modesty, is important in providing psychological support before, during, and after the procedure.
  • Note that there are no food, fluid, or medication restrictions unless by medical direction.


  • Potential complications: N/A
  • Avoid the use of equipment containing latex if the patient has a history of allergic reaction to latex.
  • Instruct the patient to cooperate fully and to follow directions. Direct the patient to breathe normally and to avoid unnecessary movement.
  • Observe standard precautions, and follow the general guidelines in Patient Preparation and Specimen Collection. Positively identify the patient, and label the appropriate specimen container with the corresponding patient demographics, initials of the person collecting the specimen, date, and time of collection. Perform a venipuncture.
  • Remove the needle and apply direct pressure with dry gauze to stop bleeding. Observe/assess venipuncture site for bleeding or hematoma formation and secure gauze with adhesive bandage.
  • Promptly transport the specimen to the laboratory for processing and analysis.


  • Inform the patient that a report of the results will be made available to the requesting health-care provider (HCP), who will discuss the results with the patient.
  • Reinforce information given by the patient’s HCP regarding further testing, treatment, or referral to another HCP. Answer any questions or address any concerns voiced by the patient or family.
  • Depending on the results of this procedure, additional testing may be performed to evaluate or monitor progression of the disease process and determine the need for a change in therapy. Evaluate test results in relation to the patient’s symptoms and other tests performed.

Related Monographs

  • Related tests include biopsy bone marrow, CBC, CBC hematocrit, CBC hemoglobin, Coomb's antiglobulin, G6PD, haptoglobin, hemosiderin, LAP, and osmotic fragility.
  • Refer to the Hematopoietic System table at the end of the book for related tests by body system.
Handbook of Laboratory and Diagnostic Tests, © 2013 Farlex and Partners