Answering your questions

March 1, 2010

Edited By Brad S. Karon, MD, PhD

Must an ESR be completed within two hours?

tube on which one can process an erythrocyte sedimentation rate
(ESR)? Our policy is to perform all ESRs within two hours of
collection. Is there any documentation as to the longest one can
wait after collection to perform an ESR on the sample?

A The most important
consideration initially would be the labeling or instructions provided
by the manufacturer of the test and collection tube. If the test is
FDA-approved or cleared, then the test must be performed according to
manufacturer's instructions for sample stability. Most waived or
moderate-complexity tests have instructions relating to specimen
stability. If you find that the manufacturer's instructions do not
provide enough time to complete testing, then it is possible to validate
a longer processing time for the test. Be aware, however, that this may
change the complexity of the test from waived or moderate-complexity to
high-complexity. This has implications for the personnel eligible to
perform the test as well as the extent of in-house validation required
prior to testing.

If you find that the manufacturer's instructions do not provide enough time to complete testing, then it is possible to validate a longer processing time for the test.

In many cases, the requirements for validation
become similar to the validation of a lab-developed test, which requires
much more documentation than verifying the performance characteristics
of an FDA-approved assay.

If neither the manufacturer of the test or
collection tube dictates that the test be completed within two hours,
but your lab policy states this limitation, then you do have options to
expand your acceptable processing times. Good lab practice would dictate
some type of stability study in this instance, though it would not be
strictly required as long as you are using the test within manufacturer
specifications. If the FDA labeling allows longer transport or storage
times but some staff are not comfortable with that, then a small
internal study would be warranted. In our institution, we are using an
ESR method that allows up to eight hours before testing if samples are
stored correctly; and we have validated internally that the test is
stable over that time.

—Brad S. Karon,

Is it necessary to protect all bilirubin specimens from light?

Q The usual practice
for neonatal bilirubin is to protect the specimen from light, but no such
practice is used for specimens obtained from adults. Since bilirubin is
degraded by light, should we protect all specimens from light? What should
be the practice on jaundiced adults?

A In the clinical laboratory,
it is well recognized that bilirubin concentrations decrease when specimens
are exposed to light. The phenomenon of light decreasing bilirubin
concentrations in blood, identified in the 1950s,1 is the basis
of today's current treatment for hyperbilirubinemia in infants —
phototherapy. While protecting specimens from light is well documented in
hyperbilirubinemic samples,2 it has also been documented in more
recent literature where the photodegradation of bilirubin was examined in
human serum with “normal” bilirubin concentrations.3 Whether the
photodegradation of bilirubin will result in a clinically significant change
in concentration depends on the clinical circumstance in which the testing
was requested.

The mechanism of phototherapy uses light (blue
light works particularly well) to create different isomers of
unconjugated bilirubin. Configurational changes of unconjugated
bilirubin, specifically bilirubin IX alpha, involve the rotation of
carbon-carbon bonds on the bilirubin carbon atoms designated 4 and 15.
These changes result in decreased hydrophobicity of the molecule,
resulting in excretion from the body.

Thus, the measurement of bilirubin is subjected to
photodegradation both in vitro and in vivo. The
clinical laboratory can only affect the in vitro formation of
bilriubin photoisomers by protecting samples from light, from sample
collection to analysis. In addition, samples being collected for direct
bilirubin analysis should be “promptly cooled to 4^0C” and minimally kept at
room temperature to prevent their degradation from artificially inflating
the unconjugated bilirubin concentration.4
Protecting all bilirubin specimens from light degradation can be
impractical, though not impossible. Each clinical laboratory's circumstances
are different; thus, each laboratory's ability to minimize bilirubin samples
to room light may be different.

—Stanley F. Lo, PhD

Children's Hospital of Wisconsin

Milwaukee, WI


  1. Dobbs RH, Cremer RJ. Phototherapy. Arch Dis Child.
  2. Ihara H, Nakamura H, Aoki Y, Aoki T, Yoshida M. In vitro effects of
    light on serum bilirubin subfractions measured by high-performance
    liquid chromatography: Comparison with four routine methods. Clin
  3. Rehak NN, Cecco SA, Hortin GL. Photolysis of bilirubin in serum
    specimens exposed to room lighting. Clin Chim Acta.
  4. McDonagh AF. Photolysis and photoisomerization of bilirubin in serum
    specimens exposed to room lighting. Clin Chim Acta.

Brad S. Karon, MD, PhD, is assistant
professor of laboratory medicine and pathology, and director of the
Hospital Clinical Laboratories, point-of-care testing, and
phlebotomy services at Mayo Clinic in Rochester, MN.