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Chapter 162 Resources for the Hematologist e7

Homocysteine
Summary points • Used in differentiating vitamin B 12 from folate deficiency, evaluating hereditary metabolic disorders, and assessing
cardiovascular risk
Methodology Liquid chromatography—tandem mass spectrometry, stable isotope dilution analysis, quantitative enzymatic
Specimen requirements Plain, EDTA, heparin, or serum separator tube (fasting state preferred)
Indications Suspected B 12 or folate deficiency with equivocal initial testing (folate, vitamin B 12 level), disorder of methionine
metabolism, assess cardiovascular or prothrombotic risk
Reference range Table 162.16
Interpretation • Testing for pathway intermediates homocysteine and MMA can help distinguish between folate and vitamin B 12
deficiency in patients with borderline levels of either
• Homocysteine—elevated in both folate and vitamin B 12 deficiency
• MMA—elevated in vitamin B 12 but not folate deficiency
• Levels decrease with vitamin supplementation
• May be elevated due to age, medications, smoking, renal disease, poor nutrition, thyroid disease, MTHFR 677C→T
polymorphism
• Serum/plasma should be separated quickly from red blood cells to prevent false elevations
• Recent consumption of high-protein meal may falsely elevate level
Related tests Folate, vitamin B 12 , MMA, complete blood count, methionine, plasma amino acids, urine organic acids
Reference Refsum H, Smith AD, Ueland PM, et al: Facts and recommendations about total homocysteine determinations: An expert
opinion. Clin Chem 501:3, 2004.
EDTA, Ethylenediaminetetraacetic acid; MMA, methylmalonic acid.

Methylmalonic Acid

Summary points • Used in differentiating vitamin B 12 from folate deficiency and evaluating hereditary metabolic disorders
Methodology Liquid chromatography—tandem mass spectrometry
Specimen requirements Plain, EDTA, heparin, or serum separator tube; urine levels may also be measured
Indications Suspected vitamin B 12 or folate deficiency with equivocal initial testing (folate, vitamin B 12 level), metabolic disorder
(methylmalonic acidemia)
Reference range Table 162.17
Interpretation • Testing for pathway intermediates homocysteine and MMA can help distinguish between folate and vitamin B 12
deficiency in patients with borderline levels of either
• Homocysteine—elevated in both folate and vitamin B 12 deficiency
• MMA—elevated in vitamin B 12 but not folate deficiency
• Elevated in vitamin B 12 deficiency, renal failure, thyroid disease, bacterial overgrowth of small bowel
• Markedly elevated levels in a child suggest hereditary metabolic disorder but must be confirmed
Related tests Folate, vitamin B 12 , homocysteine, complete blood count
Interfering substances Hemolyzed sample, lipemia

EDTA, Ethylenediaminetetraacetic acid; MMA, methylmalonic acid.

Osmotic Fragility
Summary points • Used in differential diagnosis of hemolytic anemia
• Usually increased osmotic fragility with hereditary spherocytosis
• Increased osmotic fragility can be seen in other conditions with increased spherocytes and other hematologic disorders
Methodology Lysis of erythrocytes in gradient of hyposmolar solutions; degree of hemolysis commonly measured by spectrophotometry
Specimen requirements Whole blood: EDTA or heparin
Indications Hemolytic anemia, suspected hereditary spherocytosis
Reference range Normal curves generated by individual laboratories; patient results are plotted against a normal curve
Interpretation • Patients with hereditary spherocytosis will show increased lysis of red blood cells in hyposmolar solutions compared
with normal controls
• Increased osmotic fragility also seen in other causes of spherocytosis (autoimmune hemolytic anemia, posttransfusion),
other hematologic disorders (pyruvate kinase deficiency, G6PD deficiency, hereditary stomatocytosis, hereditary
pyropoikilocytosis), Rh-null phenotype
• Decreased osmotic fragility seen in sickle cell disease, thalassemia, iron deficiency
• Normal test does not exclude hereditary spherocytosis (the most fragile cells may have already lysed) —repeat testing
at time when patient is at baseline state and hematologically stable may be useful as may perform test with incubation
overnight (increases sensitivity)
• Measurements taken either immediately or after incubation for 24 h at 37°C enhance assay sensitivity
• Results may vary depending on patient age, gender, medication history, and presence or absence of active hemolysis
Flow cytometry for band 3 using EMA is more sensitive and specific for hereditary spherocytosis and related hereditary
disorders than osmotic fragility
Related tests Flow cytometry for band 3 using EMA, peripheral smear, direct antiglobulin test, complete blood count
Interfering substances Hemolyzed specimen, specimen age may falsely increase background hemolysis (laboratory may require submitting
concurrent sample from normal nonrelative to control for this)
References King MJ, Zanella A: Hereditary red cell membrane disorders and laboratory diagnostic testing. Int J Lab Hematol 35:237,
2013.
Park SH, Park CJ, Lee, BR, et al: Comparison study of the eosin-5′-maleimide binding test, flow cytometric osmotic
fragility test, and cryohemolysis test in the diagnosis of hereditary spherocytosis. Am J Clin Pathol 142:474, 2014.

EDTA, Ethylenediaminetetraacetic acid; EMA, eosin-5′-maleimide; G6PD, glucose-6-phosphate dehydrogenase.