College of American Pathologists
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  Q & A





cap today

March 2003

Q. Is 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?

A.  Examining 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.

Pathologists 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 practices.

Regardless of 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.

Patrick L. Fitzgibbons, MD
Department of Pathology
St. Jude Medical Center
Fullerton, Calif.

Advisor, CAP Surgical
Pathology Committee

Q.  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?

A.  Contradictory 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.

Branda and 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 5,8-dihydromethotrexate.7

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.

Clinical specimens 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.

This conflicting 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 (4°C).12

In conclusion, B12 and folate specimens should be protected from light until a study using current methods demonstrates that light does not significantly alter results.

1.  Branda RF, Eaton JW. Skin color and nutrient photolysis: an evolutionary hypothesis.
. 1978;201:625-626.
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. 1999;52: 581-582.
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 (Bogota). 2002;22:46-50.
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. 1999;69: 77-85.
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.

Frederick L. Kiechle MD, PhD
Chair, Department
of Clinical Pathology

J. Douglas Ferry, PhD
Clinical Chemist
Department of Clinical Pathology
William Beaumont Hospital
Royal Oak, Mich.

Dr. Kiechle is a member
of the CAP Publications Committee.