Sucrose Hemolysis Test - An Overview

Introduction:

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of hematopoiesis (production of acellular components of blood cells) in which the red blood cells are abnormally sensitive to lysis by normal constituents of plasma. It is characterized by hemoglobinuria (high hemoglobin concentration in the urine) during sleep (nocturnal hemoglobinuria), jaundice, and hemosiderinuria. PNH is not a hereditary disease. It is an acquired condition that occurs due to variations in the PIG-A gene, which causes a deficiency of glycosylphosphatidylinositol-anchored proteins (GPI-AP) on the surface of hematopoietic stem cells.

What Is Sucrose Hemolysis Test?

Sugar-water hemolysis is a blood investigation done for the detection of fragile red blood cells. It determines the fragility of cells by testing their extent of swelling when immersed in a low-salt solution. The test may be recommended when there are manifestations of paroxysmal nocturnal hemoglobinuria or hemolytic anemia. Hemolytic anemia occurs due to the lysis of RBCs earlier than their normal lifespan, whereas, in paroxysmal nocturnal hemoglobinuria, RBCs get destroyed by the complement system of the body (which enhances the ability of antibodies). The symptoms include pale skin, tiredness, weakness, and dizziness. If left untreated, it can cause thrombosis (blood clots), chronic kidney disease, pulmonary hypertension, organ damage, and anemia.

What Are the Characteristic Features of Paroxysmal Nocturnal Hemoglobinuria (PNH)?

The characteristic features of cells belonging to the PNH are that they are deficient in several cell-membrane-bound proteins, including red cells. Such proteins are:

• Acetylcholine esterase (an enzyme that catalyzes the breakdown of acetylcholine and other choline esters that function as neurotransmitters).

• Neutrophil alkaline phosphatase (an enzyme present in white blood cells).

• CD55 (decay-accelerating factor or DAF).

• CD59 (prevents reactive lysis of membrane or MIRL).

CD55, CD59, and HRF play a role in the protection of the cell against complement-mediated damage. CD59 prevents the terminal complex formation of complement, and it is found that the deficiency of CD59 causes the complement sensitivity of PNH red cells.

PNH type III red cells result in a complete deficiency of CD59, and PNH type II red cells are only partially deficient, and it is this contrast that accounts for their varying sensitivities to complement attack.

PNH red cells are easily affected by complement lysis. This can be exhibited in vitro by certain tests, such as:

• Sugar water test.

• Sucrose lysis test.

• The acidified-serum test [Ham test].

How Is Sucrose Hemolysis Test Performed?

First and foremost, to obtain a blood sample from the body, the antiseptic is applied to the skin. Then, with the help of a tourniquet in the upper arm, pressure occurs, and the veins swell with blood. A needle is then inserted into the vein to draw some amount of blood by the syringe, which is then stored in a vial. The sample is sent to the laboratory for its examination. Usually, the ratio of old RBCs to new RBCs can signify if any imbalance is present. However, rarely bone marrow biopsy is also advised, which includes getting a sample from bone marrow and analyzing it for abnormalities.

Reagents and types of equipment used for the test:

• Specimen- Whole blood.

• Volume- 1 ml.

• Container- 7 mL ACD or lavender top (EDTA).

How Does the Sucrose Hemolysis Test Work?

Washed erythrocytes are incubated in an isotonic sucrose solution carrying normal ABO- compatible serum. In low ionic solution, red blood cells tend to absorb the components of complement from serum. As PNH erythrocytes have higher sensitivity than normal cells, they get hemolyzed (broken and dissolved blood cells) in this environment. The normal red blood cells do not undergo this change. After the completion of the incubation period, the mixture is looked for hemolysis.

What Are the Uses of the Sucrose Hemolysis Test?

Normal test results show that less than five percent of erythrocytes undergo lysis when tested. This phenomenon is known as hemolysis. Mostly, a normal test is not able to rule out paroxysmal nocturnal hemoglobinuria, and usually, a false normal result is obtained if the serum is deficient in complement or exhibits recent blood transfusions. The abnormal results of the test or positive results suggest that more than ten percent of RBCs go through lysis, thus concluding the presence of paroxysmal nocturnal hemoglobinuria in the patient.

How Is the Sucrose Hemolysis Test Measured?

The hemolysis reference range on sucrose hemolysis testing, including screening and confirmatory tests, is as follows:

Sucrose Hemolysis Test (Screening):

• No Hemolysis Visible - Negative result.

• Hemolysis Present - Positive result.

Sucrose Hemolysis Test (Confirmatory):

Hemolysis findings in the supernatant are as follows:

• Less Than 5 % - Inconsequential or negative.

• 6 to 10 % - Questionable or borderline.

• More Than 10 % - Positive.

The reference range does not differ with parameters like age, gender, race, or ethnicity. However, the percentage of hemolysis on the sucrose hemolysis test can fluctuate depending on the temperature at which the investigation is carried out, the blood type used (defibrinated or whole blood), and the latest blood transfusions that dilute the presence of percentage of paroxysmal nocturnal hemoglobinuria (PNH) cells. So, the test must be performed at an appropriate temperature, with the optimal blood type and adequate history of recent blood transfusions to avoid false-positive and false-negative results, respectively.

What Does the Sucrose Hemolytic Test Result Mean?

Sucrose Hemolysis Test (Whole Blood Screening Test):

• Positive Result - Hemolysis detected; further confirmatory test required.

• Negative Result - No hemolysis.

Sucrose Hemolysis Test (Confirmatory):

• Less Than 5 % Hemolysis - Inconsequential or negative.

• 6 to 10 % Hemolysis - Borderline or questionable; found in some leukemia, aplastic anemia, autoimmune hemolytic anemia, megaloblastic anemia, and myelofibrosis.

• More Than 10 % Hemolysis - Positive, diagnostic for paroxysmal nocturnal hemoglobinuria (results should be correlated with acid lysis or Ham test or flow cytometry); also found in congenital dyserythropoietic anemia type II.

Conclusion:

Hemolysis of five percent or less is considered negative within normal limits. Hemolysis of six to ten percent is thought to be borderline. Positive results will show greater than ten percent hemolysis. Increased hemolysis (less than ten percent) may be found in some patients with leukemia or myelofibrosis, whereas patients with PNH show ten percent to 80 % hemolysis (will only be as low as 370). The results of the sucrose hemolysis test should correlate with the acid serum test.