Test Id : RBCME

Osmotic fragility and eosin-5-maleimide binding (Band3) flow cytometry testing will always be performed. A normal shipping control is necessary to exclude false-positive results due to preanalytical artifact. Testing will be canceled if no shipping control is received or if the shipping control is abnormal. A consultative interpretation will be provided.

For more information see Benign Hematology Evaluation Comparison.

Specimens must arrive within 72 hours of collection.

Include recent transfusion information and most recent complete blood cell count results.

Metabolic Hematology Patient Information (T810) is strongly recommended, but not required, to be filled out and sent with the specimen. This information aids in providing a more thorough interpretation of test results. Ordering providers are strongly encouraged to complete the form and send it with the specimen.

A whole blood EDTA specimen, an EDTA shipping control specimen, and 2 well-made peripheral blood smears (Wright stained or fixed in absolute methanol) are required for testing. The shipping control is used to evaluate whether a patient result has been compromised by handling conditions such as temperature, motion, or other transportation interferences. Temperature and handling extremes can adversely impact the integrity of the specimen.

Patient:

Specimen Type: Whole blood

Container/Tube: Lavender top (EDTA)

Specimen Volume: 4 mL

Collection Instructions:

1. Refrigerate specimen immediately after collection.

2. Send whole blood specimen in original tube. Do not aliquot.

3. Rubber band patient specimen and control vial together.

Patient:

Specimen Type: Slides

Container/Tube: Blood smears

Specimen Volume: 2 Well-made peripheral blood smears

Collection Instructions:

1. Prepare 2 peripheral blood smears from the EDTA tube collected from the patient.

2. Either stain the smear with Wright stain or fix the smear with absolute methanol prior to shipping.

Normal Shipping Control:

Specimen Type: Whole blood

Container/Tube: Lavender top (EDTA)

Specimen Volume: 4 mL

Collection Instructions:

1. Collect a shipping control specimen from a normal (healthy), unrelated, nonsmoking person at the same time as the patient.

2. Clearly hand write "normal control" on the outermost label.

3. Refrigerate specimen immediately after collection.

4. Send control specimen in original tube. Do not aliquot.

5. Rubber band patient specimen and control vial together. The control and patient specimens must be handled in the same manner from specimen collection to receipt in the testing laboratory.

• Metabolic Hematology Patient Information
• Benign Hematology Evaluation Comparison

1. Metabolic Hematology Patient Information (T810)

2. If not ordering electronically, complete, print, and send a Benign Hematology Test Request (T755) with the specimen.

Patient whole blood, shipping control: 2 mL; Slides: See Specimen Required

Investigation of suspected red cell membrane disorders, such as hereditary spherocytosis or hereditary pyropoikilocytosis

This test is not useful for hereditary elliptocytosis.

Osmotic fragility and eosin-5-maleimide binding (Band3) flow cytometry testing will always be performed. A normal shipping control is necessary to exclude false-positive results due to preanalytical artifact. Testing will be canceled if no shipping control is received or if the shipping control is abnormal. A consultative interpretation will be provided.

For more information see Benign Hematology Evaluation Comparison.

The functional red cell membrane is composed of a cholesterol and phospholipid bilayer anchored by integral proteins to an elastic cytoskeletal network. These interactions form the shape, deformability, and proper ion balance of the cell. Abnormalities in these moieties result in red cell membrane disorders.

Hereditary spherocytosis (HS) is a common membrane disorder, which can be present in many ethnic groups. Its prevalence has been estimated at approximately 1 in 3000 persons of Northern European ancestry. It is usually associated with visible spherocytes on the peripheral blood smear and can be associated with variable clinical features of hemolysis ranging from completely compensated to mild to severe.

Hereditary elliptocytosis (HE) is another fairly common and clinically variable disorder that can range from normal red blood cell indices in the large majority of cases to rare patients with moderate to severe anemia.

Common hereditary elliptocytosis is characterized by the presence of elliptocytes on the peripheral blood smear. Genetic variants associated with HE have been reported in widely variable ethnicities, with greater prevalence in populations overlapping the malaria belt.

Hereditary pyropoikilocytosis is best classified as a severe form of hereditary elliptocytosis. It is uncommon and presents in early childhood as a severe hemolytic anemia. These disorders are associated with marked poikilocytosis on the peripheral blood smear.(1,2) Red cell membrane disorders can result from abnormalities involving several red cell membrane proteins, such as band 3, alpha and beta spectrin, protein 4.1, protein 4.2, glycophorin C, and ankyrin.

Most often, red cell membrane disorders are diagnosed in childhood, adolescence, or early adult life. The diagnosis of HS is usually made by a combination of patient and family history, laboratory evidence of hemolysis, and review of a peripheral blood smear. The osmotic fragility (OF) test is usually markedly abnormal in HS cases. However, factors such as age, iron status, and medications can affect the OF test. OF is nonspecific and can be increased in acquired disorders, such as autoimmune hemolytic anemia. Coombs testing should be negative prior to ordering this test.

The addition of eosin-5-maleimide (EMA) binding (band 3) flow cytometry to this profile increases specificity if a typical moderately decreased pattern is present. Hereditary pyropoikilocytosis can have normal or only mildly increased OF results and often displays a very dim and sometimes dual peak pattern with EMA-binding testing. Common hereditary elliptocytosis cases are not discriminated from normal patients in either OF and EMA binding testing, and this profile does not add confirmatory information for HE.

> or =12 months:

0.50 g/dL NaCl (unincubated): 3-53% hemolysis

0.60 g/dL NaCl (incubated): 14-74% hemolysis

0.65 g/dL NaCl (incubated): 4-40% hemolysis

0.75 g/dL NaCl (incubated): 1-11% hemolysis 

NaCl = sodium chloride

An interpretive report will be provided.

Reference values have not been established for patients who are younger than 12 months of age.

An interpretive report will be provided.

Interfering factors:

-Recent transfusion

-Oral contraceptives

-H2 blockers

Infrequently, other hemolytic disorders may also be associated with positive osmotic fragility results, as in patients with congenital nonspherocytic hemolytic anemia due to glucose-6-phosphate dehydrogenase or pyruvate kinase deficiency. Eosin-5-maleimide (EMA) binding (band 3) results can be masked by reticulocytosis. Results must be interpreted within the clinical, familial, and peripheral blood smear findings.

Patients with an immunohemolytic anemia or who have recently received a blood transfusion may also have increased red blood cell lysis.

The shipping control specimen is required to adequately interpret these cases, as temperature extremes can increase fragility of the specimen.

Resulting Cautions:

Osmotic fragility results will be reported if the shipping control is normal.

If the shipping control is abnormal and the osmotic fragility results are within normal range, the results will be reported; however, a comment will be added to the report indicating that the shipping control was not entirely satisfactory.

The test will be canceled if the patient specimen and shipping control are both abnormal.

1. King MJ, Garcon L, Hoyer JD, et al: International Council for Standardization in Haematology. ICSH guidelines for the laboratory diagnosis of nonimmune hereditary red cell membrane disorders. Int J Lab Hematol. 2015 Jun;37(3):304-325

2. Lux SE, IV: Anatomy of the red cell membrane skeleton: unanswered questions. Blood. 2016 Jan 14;127(2):187-199 doi: 10.1182/blood-2014-12-512772

3. Gallagher PG: Abnormalities of the erythrocyte membrane. Pediatr Clin North Am. 2013 Dec;60(6):1349-1362

4. Bianchi P, Fermo E, Vercellati C, et al: Diagnostic power of laboratory tests for hereditary spherocytosis: a comparison study in 150 patients grouped according to molecular and clinical characteristics. Haematologica. 2012 Apr;97(4):516-523

Osmotic Fragility:

Specimens for erythrocyte osmotic fragility tests are anticoagulated with EDTA. Osmotic lysis is performed using sodium chloride solution, 0.5 g/dL. An incubated fragility test is performed following 24-hour incubation at 37 degrees C at the following sodium chloride concentrations: 0.60, 0.65, and 0.75 g/dL. Results are reported and interpreted.(Larson CJ, Scheidt R, Fairbanks VF: The osmotic fragility test for hereditary spherocytosis: use of EDTA-anticoagulated blood stored at 4 degrees C for up to 96 hours. Am Soc Clin Pathol Meeting Abstract, 1988; Larson CJ, Scheidt R, Fairbanks VF: The osmotic fragility test for hereditary spherocytosis: objective criteria for test interpretation. Am Soc Clin Pathol Meeting Abstract, 1988; King MJ, Zanella A: Hereditary red cell membrane disorders and laboratory diagnostic testing. Int J Lab Hematol. 2013 Jun;35(3):237-243)

Band 3/Eosin-5-Maleimide Binding Assay:

Eosin-5-maleimide (EMA) is a fluorescent dye that binds to Lys-430 of the extracellular loop of the band 3 protein. Using a 1-color flow cytometry method (number of events plotted against fluorescence), the fluorescent intensity of EMA-stained red blood cells is assessed and compared to normal-value patients.(King MJ, Behrens J, Rogers C, Flynn C, Greenwood D, Chambers K: Rapid flow cytometric test for the diagnosis of membrane cytoskeletal associated hemolytic anemia. Br J Haematol. 2000 Dec;111(3):924-933; King MJ, Zanella A: Hereditary red cell membrane disorders and laboratory diagnostic testing. Int J Lab Hematol. 2013 Jun;35(3):237-243)

Peripheral Blood Smear Review:

A hematopathologist who is an expert in these disorders evaluates the slides and an interpretive report is issued.

Monday through Saturday

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This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

85557-Osmotic fragility

88184-Flow cytometry; first cell surface, cytoplasmic or nuclear marker x 1

85060-Morphology review