Background

The sucrose hemolysis test or sugar water test is a diagnostic test which is used to detect the paroxysmal nocturnal hemoglobinuria (PNH). It is a rare acquired hematopoietic stem cell disease. This occurred with other diseases like thrombosis, smooth muscle dystonia, and complement mediated intravascular hemolysis.

PNH is caused because of the absence of cell membrane protein GPI (glycophosphatidylinositol) anchored protein. CD55 and CD59 are the 2 GPI anchored proteins. These are the complement regulatory protein. If they are not present in the PNH erythrocytes, hemolysis occurs. PNH can be detected by the breakdown of RBCs because of increased reactivity of RBCs to complement. Patient who has PNH, and RBCs are more at risk for complement mediated lysis because of lack of protective proteins on their surfaces.

Sucrose hemolysis test is the 1^st test which is used to diagnose PNH. The RBCs are places into a low isotonic solutions, Erythrocytes of the PNH absorbs the complement on the surface in presence of serum. This can lead to the hemolysis of erythrocytes. The result is determined by the visible hemolysis with no OD measurements or % of hemolysis with OD measurement.

The sugar water test is avoided to reduce the error. The sucrose hemolysis test is performed as a screening test. Flow cytometry or Ham test confirms the results. This test is sensitive and less specific to diagnose PNH due to the hemolysis of RBCs from leukemia, autoimmune hemolytic amenia, or aplastic anemia. The sucrose lysis test is standard screening method for the PNH. About 5% of the hemolysis is positive for PNH. It is also used to detect complement lysis sensitivity.

In a study of laboratory test for PNH, RBCs of many patient had conflict with the results because of the various methods were used. There is some link of PNH to different myeloproliferative and lymphoproliferative disease. The test to diagnose PNH must be choose carefully to get the good results. Some specific standards must be followed like incubation at 23 ºC and use fresh sugar solution, citrated or oxalate blood, proper Ca, Mg, and complement level in human serum and human serum.

Ham test is used to detect the PNH for many years. A positive Ham test result is observed in only one other condition hereditary erythroid multinuclearity with positive acidified serum also known as congenital dyserythropoietic anemia type 2. This condition is easily differentiated from PNH on the basis of medical history of patient, the morphology of a bone marrow aspirate, and a negative sucrose hemolysis test. The Ham test is specific to PNH.

Flow cytometry is replaced to Ham test to diagnose PNH. CD55 and CD59 are measured by flow cytometry. Reduced level of these glycoproteins are indication of PNH. Flow cytometry detection of tiny PNH clones (less than 5%) is more sensitive than Ham test.

Indications/Applications

The indication to test for the diagnosis of PNH are:

Intramuscular hemolysis without or with anemia

Disease caused because of the bone marrow failure like myelodysplastic syndrome and aplastic anemia

Thrombosis at areas like hepatic veins, cerebral sinus, intra-abdominal vein, and dermal vein

Jaundice

Anemia

Nocturnal hemoglobinuria

The sucrose hemolysis test is performed in the conditions like:

Classic PNH

PNH with myelodysplastic syndrome or aplastic anemia

Congenital dyserythropoietic anemia type 2

Hemolytic anemia

Reference Range

The normal result of sucrose hemolysis test:

Screening result of sucrose hemolysis test:

Negative result: No visible hemolysis

Positive result: Presence of hemolysis

Confirmatory results of sucrose hemolysis test:

Hemolysis result in supernate:

Less than 5 %: Negative or insignificant

6 to 10%: Borderline

More than 10%: Positive

The normal results do not change with race, gender, age, or ethnicity. The % of hemolysis may differ on the basis of temperature, type of blood which is used, and multiple transfusions which can dilute the & of PNH cells.

Interpretation

Positive Result

PNH: A sucrose hemolysis test yielding a positive outcome indicates the presence of PNH as the erythrocytes in this condition exhibit increased sensitivity to complement activation due to the lack of protective surface glycoproteins (CD55 and CD59).

A positive results generally needs further diagnostic tests through flow cytometry to assess the expression levels of CD55 and CD59 on erythrocytes which facilitates a more definitive diagnosis of PNH.

Negative Result

Normal Hemolytic Response: A negative outcome indicates that the erythrocytes possess standard complement resistance, rendering PNH an improbable diagnosis.

Alternative Diagnoses: In instances where clinical suspicion for PNH persists despite a negative sucrose hemolysis result, supplementary diagnostic assessments (flow cytometry) may still be used.

Limitations

False Positives/Negatives: The sucrose hemolysis assay is relatively limited in sensitivity and has largely been supplanted by flow cytometry, which offers enhanced accuracy. The occurrence of false positives or negatives is possible, particularly in cases of suboptimal sample handling.

To reduce the variables, the below conditions must be followed:

Temperature: The proper temperature of this test is 23 ºC because non PNH cells are more stable. If cells are incubated at temperature of 37 ºC, more hemolysis will occur.

Hematocrit: A greater percentage of hemolysis is observed with blood dilution, whereas the amount of clear hemolysis is relatively unchanged.

Blood type: Defibrinated blood must not be used due to false positive result may take place in non-PNH blood disease.

Complement level: Reduced level in serum is considered as false negative.

Magnesium and calcium level: These are used in complement mediated hemolysis. Restoration of these can be helpful to reduce the false negative results.

Blood transfusion: This may cause a false negative by decreasing the proportion of PNH cells.

% of PNH cell: The percentage of hemolysis fluctuates with the amount of cells present in the blood, much as the quantity of PNH cells fluctuates between 10 and 80%.

Collection And Panels

Screening test:

Sample type: Whole blood (citrated)

Sample volume: 2.7 ml whole blood

Minimum sample volume: 2.4 ml

Sample container: 5 ml blue top tube

Sample storage: Keep at 23 ºC

Test preparation: There is no need of preparation before test.

Sample rejection: EDTA, heparin, blood without anticoagulants, and defibrinated blood is avoided.

Method: 1 portion of citrated whole blood, 9 portions of sucrose solution which is incubated for 30 min. Centrifuge the mixture and observe for any hemolysis in the supernate.

Confirmatory test:

Sample type: Whole blood (citrated)

Sample volume: 2.7 ml whole blood

Minimum sample volume: 2.4 ml

Sample container: 5 ml blue top tube

Sample storage: Keep at 23 ºC

Test preparation: There is no need of preparation before test.

Sample rejection: EDTA, heparin, blood without anticoagulants, and defibrinated blood is avoided.

Method: 1 ml of blood, add saline NaCl 0.85%. Mix it well and centrifuge it for 5 min at high speed. Prepare the washed cell of 50% solution from supernatant. Add 3 drops of the cells from the washed cell tube and normal saline. Add agents and incubate.

% of hemolysis = OD test result/Total OD X 100

Below is the Drabkin reagent incubation details:

Mix the blood-serum tube (250 mcL, mix incubation for 10 min) after the whole 30 minutes of incubation.

Mix the blank tube (250 mcL, mix incubation for 10 min) after the whole 30 minutes of incubation.

Centrifuge the remainder of the blood-sucrose tube from the supernatant (250 mcL, mix incubation or f10 min) after the whole 5 minutes.

Normal ABO-compatible serum combined with isotonic sucrose solution is used to incubate washed RBCs. RBCs absorb components of complement from serum at low ionic levels. PNH RBCs hemolyze in these situations because they are far more susceptible than healthy red blood cells. Normal red blood cells don’t. The mixture is checked for hemolysis at the final stage of the incubation time.

Panels:

Flow Cytometry: Assesses the expression levels of CD55 and CD59 on erythrocytes, thereby substantiating the diagnosis of PNH.

Ham Test: Traditionally used in conjunction with the sucrose hemolysis test for the identification of PNH.

Complete Blood Count (CBC): Determines the presence of anemia and thrombocytopenia, which may be linked with PNH.

Lactate Dehydrogenase (LDH): Typically elevated in conditions of hemolysis and frequently observed at increased levels in patients diagnosed with PNH.

Modifying factors:

False positive result – autoimmune hemolytic anemia, aplastic anemia, heparin, megaloblastic anemia, EDTA, high temperature, defibrinated blood

False negative result – Reduced complement level, blood transfusion

References

https://www.semanticscholar.org/paper/The-sucrose-haemolysis-test-in-paroxysmal-nocturnal-Hansen/a86643640fe9ae02faf5802721e6ff06f7f9b211