Sucrose lysis test

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Sucrose lysis test
Sucrose lysis test.jpg
Synonyms Sucrose hemolysis test
Purposescreening and diagnosis of paroxysmal nocturnal hemoglobinuria

The sucrose lysis test is a diagnostic laboratory test used for diagnosing paroxysmal nocturnal hemoglobinuria (PNH), as well as for hypoplastic anemias and any hemolytic anemia with an unclear cause. [1] The test works by using sucrose, which creates a low ionic strength environment that allows complement to bind to red blood cells. [1] In individuals with PNH, some red blood cells are especially vulnerable to lysis caused by complement. [1] The test may also produce suspicious results in other hematologic conditions, including megaloblastic anemia and autoimmune hemolytic anemia. [1] False-negative results can occur when complement activity is absent in the serum. [1] A simpler alternative called the sugar water test also involves mixing blood with sugar and observing for hemolysis, using the same principle as the sucrose lysis test. [1]

Contents

History

The sucrose hemolysis test was developed in the 1960s. [2] Hartmann and Jenkins first described the test in 1966. [3] [4] The test was devised as a superior screen for PNH compared to the Ham's acid hemolysis test (HT) that was developed in the 1930s. [2] For decades, these two tests were the primary methods of diagnosing PNH. [2] The test is now obsolete being replaced by more advanced methods such as flow cytometry with monoclonal antibodies CD55/CD59 that target glycosylphosphatidylinositol-anchored proteins (GPI-AP) with the addition of inactivated fluorescently labeled bacterial toxins, such as fluorescently labeled aerolysin (FLAER). [2] [5] Flow cytometry is the most sensitive and useful assay currently available to screen and diagnosis PNH. [5]

Description

The sucrose lysis test uses an isotonic sucrose solution to lower the ionic strength of serum, which triggers the activation of the classic complement pathway, leading to the lysis of cells that are sensitive to complement. [6]

In the sucrose lysis test, patient RBCs are washed and resuspended in a buffered sucrose solution. [7] The solution is then incubated at a set temperature for a specified period, during which complement-mediated lysis occurs. [7] After incubation, the solution is centrifuged, and the amount of hemoglobin released is measured spectrophotometrically. [7] The degree of lysis is compared to that of control samples. [7]

Although more sensitive than the Ham test, it is not as specific since some RBCs hemolyze to a minor degree in autoimmune hemolytic anemias, leukemia, and aplastic anemia. [6] Although affordable and straightforward to perform, the test is more labor-intensive because PNH RBCs have a short half-life in circulation. [6]

Diagnosis

After one hour of incubation at 25 °C, greater than 5% of red blood cells demonstrating lysis is positive for PNH. [8]

Related Research Articles

<span class="mw-page-title-main">Hemolysis</span> Rupturing of red blood cells and release of their contents

Hemolysis or haemolysis, also known by several other names, is the rupturing (lysis) of red blood cells (erythrocytes) and the release of their contents (cytoplasm) into surrounding fluid. Hemolysis may occur in vivo or in vitro.

<span class="mw-page-title-main">Spherocytosis</span> Medical condition

Spherocytosis is the presence of spherocytes in the blood, i.e. erythrocytes that are sphere-shaped rather than bi-concave disk shaped as normal. Spherocytes are found in all hemolytic anemias to some degree. Hereditary spherocytosis and autoimmune hemolytic anemia are characterized by having only spherocytes.

Microangiopathic hemolytic anemia (MAHA) is a microangiopathic subgroup of hemolytic anemia caused by factors in the small blood vessels. It is identified by the finding of anemia and schistocytes on microscopy of the blood film.

<span class="mw-page-title-main">Hereditary spherocytosis</span> Medical condition

Hereditary spherocytosis (HS) is a congenital hemolytic disorder, wherein a genetic mutation coding for a structural membrane protein phenotype leads to a spherical shaping of erythrocytic cellular morphology. As erythrocytes are sphere-shaped (spherocytosis), rather than the normal biconcave disk-shaped, their morphology interferes with these cells' abilities to be flexible during circulation throughout the entirety of the body - arteries, arterioles, capillaries, venules, veins, and organs. This difference in shape also makes the red blood cells more prone to rupture under osmotic and/or mechanical stress. Cells with these dysfunctional proteins are degraded in the spleen, which leads to a shortage of erythrocytes resulting in hemolytic anemia.

<span class="mw-page-title-main">Paroxysmal nocturnal hemoglobinuria</span> Medical condition

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired, life-threatening disease of the blood characterized by destruction of red blood cells by the complement system, a part of the body's innate immune system. This destructive process occurs due to deficiency of the red blood cell surface protein DAF, which normally inhibits such immune reactions. Since the complement cascade attacks the red blood cells within the blood vessels of the circulatory system, the red blood cell destruction (hemolysis) is considered an intravascular hemolytic anemia. There is ongoing research into other key features of the disease, such as the high incidence of venous blood clot formation. Research suggests that PNH thrombosis is caused by both the absence of GPI-anchored complement regulatory proteins on PNH platelets and the excessive consumption of nitric oxide (NO).

<span class="mw-page-title-main">Hemolytic anemia</span> Medical condition

Hemolytic anemia or haemolytic anaemia is a form of anemia due to hemolysis, the abnormal breakdown of red blood cells (RBCs), either in the blood vessels or elsewhere in the human body (extravascular). This most commonly occurs within the spleen, but also can occur in the reticuloendothelial system or mechanically. Hemolytic anemia accounts for 5% of all existing anemias. It has numerous possible consequences, ranging from general symptoms to life-threatening systemic effects. The general classification of hemolytic anemia is either intrinsic or extrinsic. Treatment depends on the type and cause of the hemolytic anemia.

<span class="mw-page-title-main">Hemoglobinuria</span> Abnormally increased hemoglobin in urine

Hemoglobinuria is a condition in which the oxygen transport protein hemoglobin is found in abnormally high concentrations in the urine. The condition is caused by excessive intravascular hemolysis, in which large numbers of red blood cells (RBCs) are destroyed, thereby releasing free hemoglobin into the plasma. Excess hemoglobin is filtered by the kidneys, which excrete it into the urine, giving urine a purple color. Hemoglobinuria can lead to acute tubular necrosis which is an uncommon cause of a death of uni-traumatic patients recovering in the ICU.

The direct and indirect Coombs tests, also known as antiglobulin test (AGT), are blood tests used in immunohematology. The direct Coombs test detects antibodies that are stuck to the surface of the red blood cells. Since these antibodies sometimes destroy red blood cells they can cause anemia; this test can help clarify the condition. The indirect Coombs test detects antibodies that are floating freely in the blood. These antibodies could act against certain red blood cells; the test can be carried out to diagnose reactions to a blood transfusion.

Autoimmune hemolytic anemia (AIHA) occurs when antibodies directed against the person's own red blood cells (RBCs) cause them to burst (lyse), leading to an insufficient number of oxygen-carrying red blood cells in the circulation. The lifetime of the RBCs is reduced from the normal 100–120 days to just a few days in serious cases. The intracellular components of the RBCs are released into the circulating blood and into tissues, leading to some of the characteristic symptoms of this condition. The antibodies are usually directed against high-incidence antigens, therefore they also commonly act on allogenic RBCs. AIHA is a relatively rare condition, with an incidence of 5–10 cases per 1 million persons per year in the warm-antibody type and 0.45 to 1.9 cases per 1 million persons per year in the cold antibody type. Autoimmune hemolysis might be a precursor of later onset systemic lupus erythematosus.

Paroxysmal cold hemoglobinuria (PCH) or Donath–Landsteiner hemolytic anemia (DLHA) is an autoimmune hemolytic anemia featured by complement-mediated intravascular hemolysis after cold exposure. It can present as an acute non-recurrent postinfectious event in children, or chronic relapsing episodes in adults with hematological malignancies or tertiary syphilis. Described by Julius Donath (1870–1950) and Karl Landsteiner (1868–1943) in 1904, PCH is one of the first clinical entities recognized as an autoimmune disorder.

Cold agglutinin disease (CAD) is a rare autoimmune disease characterized by the presence of high concentrations of circulating cold sensitive antibodies, usually IgM and autoantibodies that are also active at temperatures below 30 °C (86 °F), directed against red blood cells, causing them to agglutinate and undergo lysis. It is a form of autoimmune hemolytic anemia, specifically one in which antibodies bind red blood cells only at low body temperatures, typically 28–31 °C.

Acquired hemolytic anemia can be divided into immune and non-immune mediated forms of hemolytic anemia.

Erythrocyte fragility refers to the propensity of erythrocytes to hemolyse (rupture) under stress. It can be thought of as the degree or proportion of hemolysis that occurs when a sample of red blood cells are subjected to stress. Depending on the application as well as the kind of fragility involved, the amount of stress applied and/or the significance of the resultant hemolysis may vary.

Fluorescein-labeled proaerolysin (FLAER) is used in a flow cytometric assay to diagnose paroxysmal nocturnal hemoglobinuria (PNH). The assay takes advantage of the action of proaerolysin, a prototoxin of aerolysin, a virulence factor of the bacterium Aeromonas hydrophila. Proaerolysin binds to the glycophosphatidylinositol(GPI) anchor in the plasma membrane of cells. Cells affected by PNH lack GPI anchoring proteins, and thus are not bound by proaerolysin. Of note, the FLAER-based assay is not suitable for evaluation of erythrocytes and platelets in PNH but flow cytometry assays based on CD55, CD59 and others are suitable.

The Ham test is a blood test used in the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). Patient red blood cells (RBCs) are placed in mild acid; a positive result indicates PNH or congenital dyserythropoietic anemia. This is now an obsolete test for diagnosing PNH due to its low sensitivity and specificity.

Intravascular hemolysis describes hemolysis that happens mainly inside the vasculature. As a result, the contents of the red blood cell are released into the general circulation, leading to hemoglobinemia and increasing the risk of ensuing hyperbilirubinemia.

Cold autoimmune hemolytic anemia caused by cold-reacting antibodies. Autoantibodies that bind to the erythrocyte membrane leading to premature erythrocyte destruction (hemolysis) characterize autoimmune hemolytic anemia.

Ravulizumab, sold under the brand name Ultomiris, is a humanized monoclonal antibody complement inhibitor medication designed for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome. It is designed to bind to and prevent the activation of Complement component 5 (C5).

Cold sensitive antibodies (CSA) are antibodies sensitive to cold temperature. Some cold sensitive antibodies are pathological and can lead to blood disorder. These pathological cold sensitive antibodies include cold agglutinins, Donath–Landsteiner antibodies, and cryoglobulins which are the culprits of cold agglutinin disease, paroxysmal cold hemoglobinuria in the process of Donath–Landsteiner hemolytic anemia, and vasculitis, respectively.

Pegcetacoplan, sold under the brand name Empaveli, among others, is a medication used to treat paroxysmal nocturnal hemoglobinuria and geographic atrophy of the retina. Pegcetacoplan is a complement inhibitor.

References

  1. 1 2 3 4 5 6 McPherson, Richard A.; Pincus, Matthew R.; Henry, John Bernard (2007). Henry's clinical diagnosis and management by laboratory methods. Philadelphia: Saunders Elsevier. ISBN   978-1-4160-0287-1. OCLC   70327781.
  2. 1 2 3 4 Krauss, Jonathan S. (2012). "The Laboratory Diagnosis of Paroxysmal Nocturnal Hemoglobinuria (PNH): Update 2010". Laboratory Medicine. Oxford University Press (OUP). 43 (1): 20–24. doi:10.1309/lmr59zn0mfzmgqrb. ISSN   0007-5027.
  3. ↑ Hartmann, Robert C.; Jenkins, David E. (1966-07-21). "The Sugar-Water Test for Paroxysmal Nocturnal Hemoglobinuria". New England Journal of Medicine. Massachusetts Medical Society. 275 (3): 155–157. doi:10.1056/nejm196607212750308. ISSN   0028-4793. PMID   5938862.
  4. ↑ Hsu, T. C. S.; Steinberg, J.; Doux, R.; Sawitsky, A. (1979). "The Low Ionic Strength Reaction of Human Blood: Relationship between the Binding of Serum Immunoglobulin and Complement to Red Blood Cells and Surface Charge of the Cells". British Journal of Haematology. Wiley. 42 (3): 403–415. doi:10.1111/j.1365-2141.1979.tb01149.x. ISSN   0007-1048. PMID   38828. S2CID   26343347.
  5. 1 2 Young, Neal S.; Meyers, Gabrielle; Schrezenmeier, Hubert; Hillmen, Peter; Hill, Anita (2009). "The Management of Paroxysmal Nocturnal Hemoglobinuria: Recent Advances in Diagnosis and Treatment and New Hope for Patients". Seminars in Hematology. Elsevier BV. 46 (1 Suppl 1): S1–S16. doi:10.1053/j.seminhematol.2008.11.004. ISSN   0037-1963. PMC   3402209 . PMID   19171207.
  6. 1 2 3 Besa, Emmanuel C (2022-05-12). "Paroxysmal Nocturnal Hemoglobinuria Workup: Approach Considerations, Laboratory Studies, Imaging Studies". Medscape Reference. Retrieved 2023-03-16.
  7. 1 2 3 4 KANN, HERBERT E.; MENGEL, CHARLES E.; MERIWETHER, WILHELM D.; EBBERT, LARRY (1968-07-01). "Production of In Vitro Lytic Characteristics of Paroxysmal Nocturnal Hemoglobinuria Erythrocytes in Normal Erythrocytes". Blood. American Society of Hematology. 32 (1): 49–58. doi: 10.1182/blood.v32.1.49.49 . ISSN   0006-4971. PMID   5658390.
  8. ↑ Krauss, Jonathan S (2003-10-01). "Laboratory Diagnosis of Paroxysmal Nocturnal Hemoglobinuria". Annals of Clinical & Laboratory Science. 33 (4): 401–406. ISSN   0091-7370. PMID   14584753 . Retrieved 2023-03-16.
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