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Technical Briefs |
1
Div. of Clin. Biochem., Depts. of Pediatric Lab. Med. and
2
Cardiol., Critical Care Med. and Pediatrics, Univ. of Toronto, The Hosp. for Sick Children, 555 University Ave., Toronto, ON, M5G 1X8, and
3
Dept. of Clin. Biochem., Univ. of Toronto, 100 College St., Toronto ON, M5G 1L5, Canada;
a address
for correspondence: 17 Runnymede Rd., Toronto, ON, M6S 2Y1, Canada;
E-mail graham.ellis{at}utoronto.ca
Much attention has been focused recently on the role of nitric oxide (NO) in physiology. NO has important effects as a bactericidal agent released by macrophages, as a relaxing factor causing vasodilatation, as a neurotransmitter, and as a paracrine substance (local "hormone") in many other physiological processes (1)(2). In humans, NO is rapidly converted to nitrite and nitrate, and these metabolites in blood or urine are often used to investigate its metabolism.
Disposable surgical gloves are needed in areas where specimens are collected, received, separated, and analyzed. Several types of gloves are available, in latex or latex-free formulation, with powder or powder-free. During the development of assays for nitrate and nitrite, we identified disposable gloves and glass Pasteur pipettes as potential sources of contamination.
We measured nitrite and nitrate with an HPLC method essentially
following the method of Wennmalm et al.
(3)(4). Water was 18 M
(MilliQ water
system, Millipore Canada). The solution to be tested was placed in a
1.0-mL disposable WispTM vial (Waters Ltd., cat. no.
WAT025054). Then 5 µL, diluted with water if necessary, was injected
into a Waters HPLC system run isocratically at 1.0 mL/min with mobile
phase of 10 mmol/L phosphate buffer, pH 8.0, a Waters IC-Pak Anion HR
anion-exchange column [4.6 x 75 mm (cat. no. WAT026765)] with a
Guard-Pak precolumn module containing IC-Pak Anion Guard-Pak Insert
(cat. no. WAT010551). Spectrophotometric detection was at 214 nm.
Samples were also tested for nitrite + nitrate (NOx) with the
colorimetric Griess reaction as modified by Verdon et al.
(5), after treatment of the sample with nitrate reductase
from Aspergillus species (Boehringer Mannheim Canada, cat.
no. 981249).
Several samples were also purified through Sep-Pak® Vacc 3cc (500 mg) tC18 cartridges (Waters cat. no. WAT036815) to further confirm the identity of the HPLC peaks (by removing possible organic interferents). The solution to be tested (2.0 mL) was passed through a Sep-Pak column that had been conditioned by 2 mL of methanol followed by 2 mL of water. The first 1.0 mL of effluent was discarded. The next 1.0 mL was collected into a disposable Wisp vial and analyzed by HPLC.
Solutions from four gloves were also treated with nitrate reductase followed by HPLC. The peak eluting at the retention time of nitrate (~7.2 min) was eliminated by nitrate reductase and replaced by a peak eluting at 4.2 min, corresponding to nitrite, with a recovery of 104.6% ± 5.8% (1 SD).
Gloves from different manufacturers (of various sizes, as available)
were obtained from several areas of the hospital, the laboratory, and
local dental offices. Generally, one pair of each type of glove was
tested. Each glove was taken from the box or removed from the packaging
material, and 100 mL of water was poured inside the glove. Knots were
tied at the cuff, and the glove was inverted, squeezed several times,
and left for 60 min before the water was removed. Particulates were
centrifuged at 1300g for 15 min. The supernatant was
pipetted to a 1.0-mL Wisp vial, and 5–20 µL (diluted if necessary)
was analyzed for nitrite and nitrate by HPLC. A 500-µL aliquot was
also assayed for NOx colorimetrically. The values obtained are listed
in Table 1
as inside values. Some gloves were removed from the box or
package and turned inside-out before water was added and analyzed as
described above. The values obtained in that way are listed as outside
values. Samples were also tested for calcium in the Ektachem 700
Analyzer in the urine mode (Johnson and Johnson Clinical Diagnostics).
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Cornstarch is commonly used as powder lubricant in gloves. Domestic
cornstarch (Canada Corn Starch, Best Foods Canada) was obtained from a
local store. One gram was suspended in 100 mL of cold water, mixed, and
left for 60 min at room temperature. The mixture was centrifuged at
1300g for 10 min, and the supernatant was also tested for
nitrite and nitrate by HPLC (Table 1
).
Glass Pasteur pipettes and polypropylene transfer pipettes were tested (in quintuplicate) by placing 1 mL of water into a 1.0-mL Wisp vial, aspirating it into the pipette, and dispensing it back into the vial 10 times. The solution (20 µL) was then assayed by HPLC. Tap water samples (20 µL) from three faucets in our hospital were also tested by HPLC and the NOx reaction.
The results (Table 1
) showed that disposable gloves and glass
Pasteur pipettes are potential sources of nitrite and nitrate
contamination. The wide variation in the nitrate and nitrite content of
the gloves far exceeded potential experimental errors introduced by
having different glove sizes or tying the cuff of the gloves in
slightly different ways. The contamination on the inside of the gloves
was not uniformly greater than that on the outside (even for
powder-containing gloves). Also glove C, a powder-free glove, contained
more nitrate inside than outside the glove. Polyethylene/methyl
acrylate copolymer gloves had the lowest contamination, but they are
possibly the least suitable for laboratory use in preventing infections
because they tear more easily, fit loosely, and have slippery surfaces.
Several other synthetic nonlatex gloves showed little contamination.
The domestic cornstarch solutions that we prepared contained <1
µmol/L nitrate. Absorbable Dusting Powder USP used in gloves is
cornstarch containing 20 g/kg magnesium oxide (6). If its
nitrate content is similar to that of the domestic product, glove
contamination could not be explained by its cornstarch content.
Generally, the NOx concentration as determined by the Griess reaction
agreed well with the HPLC method. With solutions prepared from the
inside of glove H, and also tap water, there appeared to be inhibitors
present that interfered with either color development or the nitrate
reductase step. This was reproducible, but was not studied further. The
concentrations in water from the latex-containing gloves were very much
greater than those of tap water in our hospital (by HPLC), suggesting
that if domestic water supplies are used in the glove manufacturing
process, tap water would be unlikely as a major source of this
contamination. With some gloves, the calcium concentrations in the
solutions obtained from gloves were approximately one-half those of
nitrate, suggesting that calcium nitrate may be the major source of
nitrate contamination. With others, there appeared to be more calcium
than nitrate, suggesting that other or additional calcium sources were
present. Our study was based on testing a limited number of gloves and
should be regarded as a guide to the potential of contamination from
this source. Laboratory workers should test glove samples from the
specific lot numbers that they are currently using.
No literature exists on the average concentrations of potential contaminants in disposable gloves. Manufacturers are required to leak-test and thickness-test their products, but testing for extractables, such as nitrate, is left to the discretion of the manufacturer and may be done quarterly, rather than for every batch. Consequently, products from a given manufacturer could have batch-to-batch differences. Gloves vary in their chemical composition. Usually they are made from polyethylene, latex, or nonlatex synthetic polymers, as summarized by Vandenplas (7). During manufacture, different lubricants are used to prevent the gloves from sticking to the porcelain formers on which they are made. The formers are also coated with coagulants to cause the latex to set on the former. Lubricant powders such as starch are used to facilitate easy insertion and removal of the hand. Lubricants may become contaminated with latex proteins, antioxidants added to prevent the gloves from hardening, plasticizers (left over from the polymerization process), or other additives. The composition of these additives and coatings varies with different manufacturers and is proprietary. However, calcium nitrate is often used in latex glove manufacture as a coagulant (7), and this would seem to be a potential source of some of the contamination that we have observed.
Glove powders may inadvertently contaminate patients during surgical procedures and have caused granulomata in some patients (6). Latex proteins and other chemicals from gloves and glove powder have caused allergies (7)(8). Glove powder has contaminated ELISA tests for HIV (9) and PCR reactions in DNA diagnostic laboratories (10). Moncada and colleagues (11) briefly mentioned contamination of nitrate and nitrite assays from laboratory ware and reagents.
We urge caution in the use of disposable gloves and glass Pasteur pipettes in areas where nitrite and nitrate specimens are collected, prepared, or analyzed. Glass Pasteur pipettes should be thoroughly washed or (preferably) plastic pipettes should be used. Specimen containers or test tubes should be kept free of glove powder. Low nitrite/nitrate content gloves should be used, and gloves should be washed after they have been put on. However, even thorough washing does not completely remove all traces of glove powder (12), which should be kept in mind when ultramicro quantities of nitrates and nitrites are being measured.
Acknowledgments
This work was supported by grants from The Physician Services Incorporated Foundation.
Footnotes
References
The following articles in journals at HighWire Press have cited this article:
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  B. Childress, J. K. Stechmiller, and G. S. Schultz Arginine Metabolites in Wound Fluids From Pressure Ulcers: A Pilot Study Biol Res Nurs, October 1, 2008; 10(2): 87 - 92. [Abstract] [PDF]
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