Reference Range

The reference range of hemolysis on sucrose hemolysis testing (screening and confirmatory tests) is listed below.^[1]

Sucrose hemolysis test (screening)

See the list below:

No hemolysis visible - Negative result
Hemolysis present - Positive result

Sucrose hemolysis test (confirmatory)

Hemolysis findings in supernate are as follows:

< 5% - Inconsequential or negative
6-10% - Questionable or borderline
>10% - Positive

The reference range does not vary with age, gender, race, or ethnicity. Note that the percentage of hemolysis on the sucrose hemolysis test can vary based on the temperature at which the test is performed, the type of blood used (defibrinated or whole blood), and recent multiple blood transfusions that can dilute the percentage of paroxysmal nocturnal hemoglobinuria (PNH) cells. Therefore, the test must be run at an appropriate temperature, with the appropriate type of blood and an adequate history of recent blood transfusions to avoid false-positive and false-negative results, respectively.^[1]

Sucrose hemolysis test (whole blood screening test)

See the list below:

Positive result - Hemolysis present, need to run further confirmatory test
Negative result - No hemolysis

Sucrose hemolysis test (confirmatory)

See the list below:

< 5% hemolysis - Inconsequential or negative
6-10% hemolysis - Borderline or questionable; found in some leukemia, aplastic anemia, autoimmune hemolytic anemia, megaloblastic anemia, and myelofibrosis
>10% hemolysis - Positive, diagnostic for paroxysmal nocturnal hemoglobinuria (results should be correlated with acid lysis/Ham test or flow cytometry); also found in congenital dyserythropoietic anemia type II.

All results are subjected to vary with temperature changes, level of complement in serum, type of blood used, and recent multiple blood transfusions.

Principle

Washed red blood cells (RBCs) are incubated in an isotonic sucrose solution containing normal ABO-compatible serum. At low ionic concentrations, RBCs absorb complement components from serum. Because PNH RBCs are much more sensitive than normal red cells, they hemolyze under these conditions. Normal RBCs do not. At the end of the incubation period, the mixture is examined for hemolysis.

Variables that affect results

See the list below:

False-positive results - Aplastic anemia, autoimmune hemolytic anemia, megaloblastic anemia, heparin use, ethylenediaminetetraacetic acid (EDTA) use, higher temperature of incubation, use of defibrinated blood
False negative - Low complement levels, recent blood transfusions

To minimize variables, the following should be noted:

Temperature: 23 º C is an appropriate temperature because non-PNH blood cells are more stable at this temperature. More hemolysis is seen if the cells are incubated at 37 ºC in non-PNH cells, whereas negligible change in percentage of hemolysis is appreciated in PNH cells.
Hematocrit: With dilution of the blood, a higher percentage of hemolysis is seen, with negligible effect on the visible hemolysis.
Type of blood: Defibrinated blood should not be used because false-positive results can occur in non-PNH blood disorders.
Complement levels: Low levels in the serum can be interpreted as a false negative.
Calcium and magnesium levels: These ions are used in complement-mediated hemolysis; thus, restoring the ions in blood helps decrease the chance of false-negative results.
Blood transfusion: This can dilute the percentage of PNH cells, resulting in a false negative.
Percent of PNH cells: As the number of PNH cells varies from 10-80%, the percent of hemolysis also varies with the number of cells available in the blood.

Sucrose hemolysis (screening test)/sugar water test

See the list below:

Pretest preparation - No preparations before the test; heparin, EDTA, defibrinated blood, blood without anticoagulant are avoided
Specimen - Blood (whole blood, citrated)
Ideal amount - 2.7 mL whole blood, 5 mL of blue top
Minimum required blood - 2.4 mL of whole blood
Container - Blue top, keep at room temperature, 23ºC
Method - 1 part citrated whole blood, 9 parts of sugar water/sucrose solution incubated for 30 minutes; centrifuge and observe supernate for hemolysis

Sucrose hemolysis (confirmatory test)

See the list below:

Pretest preparation - No preparations before the test; heparin, EDTA, defibrinated blood, blood without anticoagulant are avoided
Specimen - Blood (whole blood, citrated)
Ideal amount - 2.7 mL whole blood, 5 mL of blue top
Minimum required blood - 2.4 mL of whole blood
Container - Blue top, keep at room temperature, 23 º C
Method
- To 1 mL of patient blood, add normal saline (sodium chloride 0.85%). Mix and centrifuge at high speed for 5 minutes.
- From supernatant, prepare washed cell 50% solution.
- Add 3 drops of cells from washed cell tube and 3 drops of normal saline.
- Add reagent and incubate as shown below.
- Transfer mixtures to a cuvet and record optical density (OD) at wavelength 540 nm and calculate the percentage of hemolysis using the following formula: % Hemolysis = OD test/OD total X 100

The incubation information using Drabkin reagent is as follows:^[2]

After complete 30 minutes incubation, remix blood-serum tube (250 mcL, mix incubation 10 min).
After complete 30 minutes incubation, remix blank tube (250 mcL, mix incubation 10 min).
After complete 5 minutes, centrifuge of remaining blood-sucrose tube from the supernatant (250 mcL, mix incubation 10 min).

Background

Paroxysmal nocturnal hemoglobinuria is a rare clonal hematopoietic stem-cell disorder (approximately 1-2 cases per 1 million people in the US) with protean clinical manifestations.^[3]The disease manifests with complement-mediated intravascular hemolysis, smooth muscle dystonia, and thrombosis.

Virtually all clinical manifestations of PNH result from the absence of a class of cell membrane proteins known as glycophosphatidylinositol (GPI)-anchored proteins. At least 2 of these GPI-anchored proteins (CD55 and CD59) normally function as complement regulatory proteins; their absence from PNH erythrocytes explains the complement-mediated hemolysis that is characteristic of PNH. PNH erythrocytes are vulnerable to complement activation through any of these pathways; however, the alternative pathway is in a state of continuous activation, which explains why hemolysis can occur at any time in patients with PNH.

Clinical indications to test for PNH include the following^[4]:

Intravascular hemolysis - With or without anemia
Bone marrow failure syndromes - Such as aplastic anemia and myelodysplastic syndromes (particularly refractory cytopenia with unilineage dysplasia)
Thrombosis with unusual features and/or manifesting at unusual sites - Such as the hepatic veins (as in Budd-Chiari syndrome), other intra-abdominal veins, the cerebral sinuses, and the dermal veins

Historically, in order to diagnose PNH, the first test done is the sugar water test/sucrose hemolysis test. In this test, the RBCs are exposed to low isotonic solution. Erythrocytes of PNH, adsorb complement on their surfaces when exposed to such solution in the presence of serum in the reaction mixture. and that results in hemolysis of the erythrocytes. The visible hemolysis(no OD measured, sugar water test) or the percentage of hemolysis (OD measurement, sucrose hemolysis test) defines the results of the test.

In order to decrease the manual error, the sugar water test (which was earlier used as screening test) is not done. Currently, the sucrose hemolysis test is used as the screening test. The results are then confirmed with the Ham test or flow cytometry.

Nocturnal hemoglobinuria, jaundice, and anemia indicate PNH as the most likely diagnosis.

The tests are also used for the following:

Classical PNH, PNH with aplastic anemia or myelodysplastic syndrome, subclinical PNH in patients with aplastic anemia
Hemolytic anemia of obscure origin
Congenital dyserythropoietic anemia type 2

Sucrose hemolysis testing is sensitive but is less specific for PNH because some RBCs hemolyze from autoimmune hemolytic anemias, leukemia, and aplastic anemia to a minor degree.

The sucrose lysis test has been the standard screening test for PNH. More than 5% hemolysis is considered positive for PNH. The sucrose hemolysis test can also be used to determine complement lysis sensitivity.

In a retrospective study of laboratory tests for PNH, the RBCs of several patients gave conflicting results when different testing procedures were used. Reports in the literature described an association of PNH with various lymphoproliferative and myeloproliferative disorders; testing procedures for PNH should be more carefully controlled for a better understanding of the nature of the membrane defect. Certain standards should be maintained, such as incubation at room temperature (23ºC) and the use of freshly prepared sugar solutions, oxalated or citrated blood, human serum, adequate calcium, magnesium, and complement levels in human serum.

For many years, the Ham test has been the standard way to identify the PNH clone among RBCs.^[5]Only one other disease is associated with a positive Ham test result: hereditary erythroid multinuclearity with positive acidified serum (HEMPAS) or congenital dyserythropoietic anemia (CDA) type 2, which can be readily differentiated from PNH by the medical history, bone marrow aspirate morphology, and a negative sucrose hemolysis test result. Hence, the Ham test is highly specific for PNH.

Although these tests are inexpensive and simple to perform, they are more labor intensive and less sensitive due to the short half-life of circulating PNH RBCs and the other variables that affects percentage of hemolysis.

Flow cytometry (FCM) has replaced the Ham test as the definitive test for PNH. Usually, CD55 and CD59 are both measured by FCM; depressed levels of both of these glycoproteins are consistent with PNH. FCM identification of small PNH clones (< 5%) shows greater sensitivity than the Ham test.^{[6, 3]}