Q & A

August 2005

Richard A. Savage, MD, Editor

Q.  Is it acceptable to use immunological stains for human papillomavirus?

A.  Though HPV antibodies are available, they are not sensitive enough for diagnostic purposes. Routine histology is a better indicator of HPV infection than immunohistochemistry; however, even more sensitive than routine histology or immunohistochemistry is DNA or RNA in situ hybridization or polymerase chain reaction.

Kumarasen Cooper, MBChB, PhD
Department of Pathology
Fletcher Allen Health Care
Burlington, Vt.

Vice Chair, CAP Cell Markers Committee

Q.  Are the osmotic fragility test and the pink test screen useful for diagnosing spherocytosis?

A.   Hereditary spherocytosis is an autosomal dominant syndrome that occurs when red cells have a defect in the structural elements spectrin (reduced synthesis or unstable or dysfunctional spectrin), ankyrin (reduced synthesis or unstable or dysfunctional ankyrin), or band 3 protein (reduced band 3 incorporation into membrane or loss of band 3 and its associated lipids from the membrane). The defect leads to membrane instability with loss of lipid microvesicles, resulting in a decrease in red cell membrane surface area, which reduces cellular deformability and causes red cell stagnation and destruction in the spleen.

The standard for diagnosis of hereditary spherocytosis is increased osmotic fragility of red cells in hypotonic saline solutions. Some cases are harder to detect than others because hereditary spherocytosis is a syndrome with variable severity of the structural protein defect. Osmotic fragility testing on fresh cells may fail to show increased fragility in 20 percent of cases of hereditary spherocytosis.

Most laboratories consider the incubated osmotic fragility test (incubation of red cells for 24 hours at 37°C prior to testing) to be the most sensitive test,¹ however, several alternative tests have been proposed. The autohemolysis test, acidified glycerol test, and pink test (a modification of the acidified glycerol test that uses a Bis-Tris buffer) have been suggested as alternatives to the osmotic fragility test.²

The autohemolysis test involves incubating sterile blood in plasma for 48 hours with and without additional glucose and measuring the amount of hemoglobin spontaneously released into the plasma. Hereditary spherocytosis is associated with increased autohemolysis, with some decrease in the degree of hemolysis in the glucose-added tube. The autohemolysis test is also abnormal in patients with red cell enzyme deficiencies and has been found to offer no advantage over osmotic fragility. The acidified glycerol lysis test involves adding acidified hypotonic glycerol solution to red cells and determining the lysis rate of the red cells (increased in hereditary spherocytosis). The pink test is a modification of the acidified glycerol lysis test in which red cells obtained by heel- or finger-stick are added to a solution of acidic hypotonic glycerol with a somewhat lower pH and evaluated for hemolysis.

The literature is inconsistent regarding the utility of the acidified glycerol test and pink test. Some authors find these tests to be highly specific and reliable,³ whereas others believe they lack specificity and are inferior to osmotic fragility.^4,5

All the aforementioned tests are limited by the fact that they detect all types of spherocytes, not just those of hereditary spherocytosis, and require additional correlative findings (smear review and antiglobulin testing) to be factored into the diagnostic equation. To specifically diagnose the protein abnormalities of hereditary spherocytosis, one must analyze red cell membrane proteins in gels, which separate proteins based on size and change. This is a technically demanding procedure that is not widely available. Recently, there has been interest in flow cytometric diagnosis of membrane protein abnormalities using either dyes, which specifically bind band 3, or conjugated antibodies to proteins of interest, in hopes of developing a rapid and specific test.⁶

References

1. Baker J, Cornbleet PJ. Erythrocyte disorders. In: Howanitz JH, Howanitz PJ, eds. Laboratory Medicine. Churchill Livingstone: New York, NY; 1991:480-483.
2. Vettore L, Zanella A, Molaro GL, et al. A new test for the laboratory diagnosis of spherocytosis. Acta Haematol. 1984;4:258-263.
3. Judkiewicz L, Szczepanek A, Bugala I, et al. Modified end-point glycerol hemolysis as a screening test for hereditary spherocytosis that requires no venipuncture. Am J Hematol. 1987;1:89-91.
4. Rutherford CJ, Postlewaight BF, Hallowes M. An evaluation of the acidified glycerol lysis test. Brit J Haematol. 1986;63:119-121.
5. Buck MJ, Breed WP, Hoffman JJ. Comparison of acidified glycerol lysis test, pink test, and osmotic fragility test in hereditary spherocytosis. European J Haematol. 1988;3:227-231.
6. King MJ, Behrens J, Chris R, et al. Rapid flow cytometric test for the diagnosis of cytoskeletal-associated haemolytic anaemia. Brit J Haematol. 2000;111:924-933.