Approach Considerations
The principal laboratory studies used in the diagnosis of hereditary spherocytosis (HS) include the following:
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Complete blood cell count
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Reticulocyte count
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Mean corpuscular hemoglobin concentration (MCHC)
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Peripheral blood smear
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Lactate dehydrogenase (LDH) level
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Haptoglobin
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Fractionated bilirubin
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Combs testing
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Flow cytometry using the eosin-5'-maleimide (EMA) binding test [18]
Osmotic gradient ektacytometry, although recognized as the reference technique for diagnosis of red blood cell (RBC) membrane disorders, has rarely been used in clinical practice because of its limited availability. However, a new generation of ektacytometers has been developed that may result in broader use of this technique. [19] This technique does not differentiate between HS and autoimmune hemolytic anemia (AIHA), but it distinguishes HS from other hereditary membrane disorders. [20]
The classic laboratory features of HS include the following [5, 6] :
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Mild to moderate anemia
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Reticulocytosis
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Increased MCHC
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Spherocytes on the peripheral blood smear
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Hyperbilirubinemia
RBC morphology in HS is distinctive yet not diagnostic. Anisocytosis is prominent, and the smaller cells are spherocytes. Unlike the spherocytes associated with immune hemolytic disease and thermal injury, HS spherocytes are fairly uniform in size and density. Spherocytes are characterized by the following:
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Lack of central pallor
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Decreased mean corpuscular diameter
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Increased density.
Spherocytic RBCs are not specific to HS. Autoimmune hemolytic anemia also may produce spherocytosis, but this disorder usually can be excluded by negative findings on a direct antiglobulin test.
An increased MCHC is a characteristic feature of RBCs cells in HS. MCHC values greater than the upper limit of normal (35-36%) are common. This increased MCHC is a result of mild cellular dehydration. The mean cell volume (MCV) in patients with HS actually is low, presumably because of membrane loss and cell dehydration.
The most sensitive test for HS is incubated osmotic fragility testing (OFT), which is performed after incubating RBCs for 18-24 hours under sterile conditions at 37°C. OFT with RBCs that have not been incubated may demonstrate hemolysis of HS cells in some patients but is not reliable. This is especially true of newborns, as fetal RBCs are generally more resistant to osmotic hemolysis. Incubated OFT results usually are abnormal. Sample stability is critical for OFT and blood specimens should not be refrigerated for more than 2 days prior to testing. [21]
Although OFT has traditionally been used for diagnosis of HS, it is labor intensive and time-consuming to perform. Instead, current guidelines recommend the use of flow cytometry to screen for HS. [22]
Other laboratory tests used to diagnose HS include the autohemolysis test and the glycerol lysis test. These rarely are used and offer no advantage over OFT.
Further characterization of the specific membrane lesion by looking for abnormalities in spectrin, ankyrin, pallidin, or band 3 is possible. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) can detect the defective protein in most cases; this may be useful for distinguishing HS from other hematologic disorders that may mimic it, such as congenital dyserythropoietic anemia type II. [23] However, these studies are not routinely performed and are available only in select research laboratories.
Osmotic gradient ektacytometry is the reference technique for diagnosis of RBC membrane disorders. In this technique, a laser-diffraction viscometer (ektacytometer) measures the deformability of erythrocytes as a continuous function of the osmolality of the medium in which they are suspended. Osmotic gradient ektacytometry distinguishes HS from other hereditary membrane pathologies but does not differentiate HS from autoimmune hemolytic anemia. [24]
Other tests are indicated in patients who have experienced an aplastic crisis. Testing for herpes simplex virus, parvovirus B19, and infectious mononucleosis may help identify an infectious etiology for the aplastic crisis. Vitamin B12 and folate levels should be measured to determine the nutritional stores during recovery from an aplastic crisis.
If the diagnosis is being made late in life, patients must have an evaluation of their iron status, especially if they have received frequent blood transfusions (eg, because of multiple hemolytic episodes or an inaccurate diagnosis) or prolonged oral iron supplementation for anemia. This evaluation includes measurement of iron stores and serum ferritin levels. Liver dysfunction or cardiac problems may be present in patients with severe iron overload.
Cholecystitis and cholelithiasis are common complications of HS. If the patient presents with signs and symptoms of hemolysis in addition to right upper abdominal quadrant pain, fever, and leukocytosis, an ultrasound of the biliary tree should be performed.
If an aplastic crisis is suggested, further evaluation of white blood cells and platelets should be pursued. This may require a bone marrow aspiration and biopsy to rule out aplasia or megaloblastosis. Obtaining bone marrow aspirate for testing rarely is necessary except in cases of aplastic or megaloblastic crisis. Test results help evaluate marrow function and the development of the lineage.
