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
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
- Baker J, Cornbleet PJ. Erythrocyte disorders. In: Howanitz JH, Howanitz
PJ, eds. Laboratory Medicine. Churchill Livingstone: New York, NY;
- Vettore L, Zanella A, Molaro GL, et al. A new test for the laboratory diagnosis
of spherocytosis. Acta
- Judkiewicz L, Szczepanek A, Bugala I, et al. Modified end-point glycerol
hemolysis as a screening test for hereditary spherocytosis that requires no
J Hematol. 1987;1:89-91.
- Rutherford CJ, Postlewaight BF, Hallowes M. An evaluation of the acidified
glycerol lysis test. Brit
J Haematol. 1986;63:119-121.
- 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.
- 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.
Robert Novak, MD
Department of Pathology
Children’s Hospital Medical Center of Akron (Ohio)
Chair, CAP Hematology/Clinical Microscopy Resource Committee