it appropriate for a surgeon to open and inspect the large intestine
in the operating room during surgery? Shouldn't this procedure be
limited to the surgical pathology room in the surgical suite? Doesn't
this practice contaminate the operating room?
specimens during surgery and discussing the findings with the surgeon
can reveal important information that directly affects treatment.
Such intraoperative consultations are a valuable medical service
provided by pathologists and must be available when needed.
usually prefer to examine fresh surgical specimens in a room that
has sufficient space, proper equipment, and hygienic disposal facilities
for blood and fecal material. Specimens, however, can also be examined
in the operating room without contaminating the operative field,
and this may provide the surgeon with valuable visual information.
Whether this is appropriate depends on such factors as the size
and character of the specimen, the equipment available in the operating
room, and the proximity to a more suitable examination room. The
pathologist should inform the surgeon if specimen examination in
the operating room is inappropriate, but the hospital must determine
whether such an examination violates institutional infection control
where the specimen is examined, the pathologist's primary responsibility
is to the patient. Handling specimens in an appropriate location
is an important practice, but concern about location should not
override a needed intraoperative consultation.
L. Fitzgibbons, MD
Department of Pathology
St. Jude Medical Center
Advisor, CAP Surgical
Is it no longer necessary to protect B12 and
folate from light? We use Quest Diagnostics to perform our B12
and folate tests, and the company does not require us to wrap the
specimen containers in aluminum foil or a similar material. Will
this significantly affect the results?
studies have been published describing the stability of vitamin
B12 and folate in serum (in vitro) or patients (in vivo)
exposed to sunlight.1-6 These reports span a 24-year
period from 1978 to 2002. Before 1993, serum vitamin B12
and folate were analyzed simultaneously using radioimmunoassays.
After that year, assays for each vitamin were developed on chemiluminescent
autoanalyzers. The literature has not addressed how the differences
in these methodologies influence the light-induced stability of
B12 and folate.
Eaton1 reported that exposing human plasma to sunlight
reduced the folate concentration by 30 to 50 percent within 60 minutes.
Ultraviolet light exposure reduces serum folate concentration in
patients with dermatologic disorders.1-3 Furthermore,
light activation of the folate analogue methotrexate in the presence
of nicotinamide adenine dinucleotide phosphate (reduced form) generates
In the presence
of light, cyanocob(III)alamin (vitamin B12) in aqueous
solution is photolysed to hydroxycobalamin.8,9 Cyanocobalamin
is composed of four pyrrole rings. Ultraviolet light treatment of
a substituted allyl pyrrole induces photochemical rearrangement.10
Glycerol (>25 percent) prevents photolysis of pharmaceutical
preparations of B12 by fluorescent lights.11
The photolysis or photodegradation of folate and B12
is well documented in vitro and in vivo.
may be stored at room temperature, refrigerated (4°C), or frozen
(-20°C or -70°C). Plasma folate specimens protected from
light and stored at -70°C remained stable for 120 days.6
Using the Quantaphase combination assay kit (Bio-Rad Laboratories,
Hercules, Calif.), Mastropaolo and Wilson4 reported B12
to be more stable than serum folate in the presence of light when
stored at room temperature (20-25°C). They concluded that it
is acceptable to measure serum folate in specimens stored at room
temperature in light for less than eight hours and to measure B12
after less than 24 hours of light exposure.
Using the chemiluminescence
immunoassay for B12 and folate on the ACS:180 (Bayer
Diagnostics, Tarrytown, NY), Komaromy-Hiller et al5 found
serum folate more stable than B12 in the presence of
light when frozen or refrigerated. They concluded that refrigerated
serum unprotected from light that is to be used for folate analysis
is acceptable if the test is performed within three days of collection;
otherwise the specimen should be frozen. Light protection is required
for B12 assays if the assay is not performed within four
hours. This delay would also necessitate freezing the specimen.
stability data for B12 and light at different storage
temperatures may reflect variable interferences generated by accumulated
photodegradation products when various immunoassays are performed.
Stability studies need to be completed using current immunoassays,
focusing attention on how adding photodegradation products affects
these two vitamins. Alternately, a more stable collection method
may be used-for example, the microbiological assay of folate from
dried-serum spots on ascorbate-treated paper is stable for one week
at room temperature (20°C) and two weeks at refrigerated temperature
B12 and folate specimens should be protected from light
until a study using current methods demonstrates that light does
not significantly alter results.
RF, Eaton JW. Skin color and nutrient photolysis: an evolutionary
2. Roe DA. Photodegradation of carotenoids in
human subjects. Federation Proceedings. 1987;46:1886-1889.
3. Jablonski NG. A possible link between neural
tube defects and ultraviolet light exposure. Med Hypotheses.
4. Mastropaolo W, Wilson MA. Effect of light on
serum B12 and folate stability. Clin Chem. 1993;39:913.
5. Komaromy-Hiller G, Nuttall KL, Ashwood ER.
Effect of storage on serum vitamin B12 and folate stability.
Ann Clin Lab Sci. 1997;27:249-253.
6. Noy V, Baracaldo CM, Forero Y, et al. Stability
and effect of ingestion on the levels of folate in plasma. Biomedica
7. Chen YQ, Gulotta M, Cheung HT, et al. Light
activates reduction of methotrexate by NADPH in the ternary complex
with Escherichia coli dihydrofolate reductase. Photochem Photobiol.
8. Ahmad I, Hussain W, Fareedi AA. Photolysis
of cyanocobalamin in aqueous solution. J Pharm Biomed Anal.
1992; 10: 9-15.
9. Cole AG, Yoder LM, Shiang JJ, et al. Time-resolved
spectroscopic studies of B(12) coenzymes: a comparison of the primary
photolysis mechanism in methyl-, ethyl-, n-proply-, and 5'deoxyadenosylcobalamin.
Journal of American Chemical Society. 2002;124:434-441.
10. Patterson JM, Ferry JD, Boyd MR. The photoisomerization
of (subtituted allyl)-dialkylpyrroles.
Journal of American Chemical Society. 1973;95:4356.
11. Grissom CB, Chagovetz AM, Wang Z. Use of viscosigens
to stabilize B12 solutions against photolysis.
J Pharm Sci. 1993: 82; 641-643.
12. O'Broin S, Gunter E. Dried-serum spot assay
for folate. Clin Chem. 2002;48; 1128-1130.
L. Kiechle MD, PhD
of Clinical Pathology
Department of Clinical Pathology
William Beaumont Hospital
Royal Oak, Mich.
is a member
of the CAP Publications Committee.