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September 2003
Q.
What can we use as a positive control for a Heinz body prep test?
What constitutes adequate proficiency testing? No one can remember
the last time we had a positive result, but our hematologists want
to retain the test as part of their hemolytic anemia workup.
A. Heinz bodies are focal precipitates
of oxidatively denatured hemoglobin within the red blood cell that
are visible only with supravital dyes. Methyl violet, crystal violet,
and brilliant green generally stain only Heinz bodies; reticulocyte
stains, such as new methylene blue, can also be used to demonstrate
Heinz bodies.
Heinz bodies appear as single or multiple irregular
inclusion bodies adjacent to the cell membrane. Some Heinz bodies
are formed in all people, but the spleen rapidly clears them. Increased
numbers of Heinz bodies are seen in patients who are splenectomized;
have decreased levels of red cell enzymes, such as individuals with
glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, especially
if exposed to oxidant drugs; or have certain unstable hemoglobins,
such as hemoglobin Zurich. Heinz bodies also form in anticoagulated
blood incubated at room temperature for several hours and can be
formed in vitro by exposing blood to oxidizers such as phenylhydrazine.
Valid Heinz body evaluations need the following:
- procedural controls—a sample run with the patient samples that will
reliably demonstrate Heinz bodies to confirm that your staining procedure
worked
- appropriate normal reference intervals for the types of patients you will
be evaluating based on your specimen type and staining procedure
- appropriate proficiency testing.
As
a procedural control, two alternatives are potentially viable. The
first is to identify individuals who have been splenectomized or
have Hb Zurich and who are willing to provide a sample using a protocol
approved by your institution’s human protection program. This
alternative is usually limited to very large institutions. The second
is to use anticoagulated blood that exceeds what is needed for testing
and has been retained at room temperature for a known duration.
The latter obviously is an easier alternative, but it still requires
appropriate treatment of the specimen.
An
internally validated reference range is important because different
staining protocols can themselves induce Heinz body formation. The
age of the blood sample to be used is important. These ranges must
also attempt to include all groups you are likely to test—neonates,
for instance, have more Heinz bodies than older children and adults
because they have developmentally lower levels of G-6-PD. Fortunately,
individuals with true Heinz body anemias often demonstrate multiple
Heinz bodies in more than 50 percent of cells, levels that far exceed
those seen in healthy people.
Since no proficiency program offers Heinz body testing materials, laboratories
must develop their own proficiency testing. It would be appropriate to send
split samples to another certified lab that uses a procedure similar to that
used by your lab or to conduct blinded testing of known samples.
Bibliography
- Heinz bodies. In: Howanitz JH, Howanitz PJ, eds. Laboratory Medicine.
New York, NY: Churchill Livingstone; 1991: 479–480.
- Heinz body staining. In: Beutler E, Lichtman
MA, Coller BS, et al, eds. Williams Hematology, 5th ed. New York,
NY: McGraw-Hill; 1995:L26.
Robert
Novak, MD
Department of Pathology
Children’s Hospital
Medical Center of Akron (Ohio)
Chairman, CAP Hematology/Clinical Microscopy Resource Committee
Q. What
are some applications of tandem mass spectrometry, particularly
for use in metabolic screening programs?
A.
The application of mass spectrometry that has gained the most notoriety
during the past 10 years is in the field of newborn screening. By
the end of this year, more than one fourth of all infants born in
the United States will have their filter paper blood specimens analyzed
by mass spectrometry.
Among the metabolites
analyzed are amino acid and acylcarnitine biomarkers indicative
of more than 35 diseases. Other mass spectrometry applications in
screening programs may include the analysis of homocysteine, 17-hydroxyprogesterone,
bile acids, steroids, very long chain fatty acids, thyroxin, and
other small molecules.
Although mass
spectrometry can be used in protein analysis, especially hemoglobin
and the characterization of sickle cell disorders, it has not been
widely used in routine screening. Other mass spectrometers, such
as time of flight, will likely be used in the next five to 10 years
for protein analysis in screening programs. Furthermore, many new
methods will be developed for use in high-risk analysis, including
second-tier confirmatory tests.
The success of mass spectrometry applications in clinical screening
has been a result of a single broad spectrum metabolite analysis
for more than 50 biomarkers. The cost per analysis relative to the
number of disorders detected is incredibly low. In fact, this same
principle has produced a new quantitative method for measuring free
carnitine in the plasma of dialysis patients, an area not specifically
associated with metabolic screening programs.
Detailed reviews and discussions of mass spectrometry in clinical chemistry
can be found in Chemical Reviews (Chace DH. 2001; 101: 445–477) and Annual
Reviews of Genomics and Human Genetics (Chace DH, et al. 2002;3:17–45).
Donald H. Chace, PhD
Section Chief
Division of Bioanalytical
Chemistry and Mass Spectrometry
Pediatrix Screening
Bridgeville, Pa.
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