Contamination

There are many sources of contamination, in the clinical laboratory. Some of these may affect a random specimen and be overlooked or not detected, even with inclusion of standards and controls. Thorough cleaning of glassware is essential. Detergents frequently contain large quantities of sodium and inorganic phosphate, and these will interfere if not completely removed. A simple check to detect residual detergent consists of filling pipets or rinsing glassware with a dilute solution of phenolphthalein. No pink color should appear.

Other sources of contamination are more subtle. If cuvets are not rinsed adequately, phosphomolybdic acid from glucose determinations may remain to interfere with subsequent measurements of inorganic phosphate. Nessler's reagent can leave a film of mercury salts on glass that could inhibit subsequent assays of enzyme. Similarly, traces of iodine or mercury salts interfere in the determination of protein-bound iodine.

Calcium from tap water forms a film on glass that is difficult to remove by simple rinsing with distilled water. Subsequent use of such glassware for flame photometric calcium determinations involving the use of a sequestering agent (such as Sterox SE) can liberate calcium into the solution to produce falsely elevated results.38 In other calcium procedures, serum protein alone tends to remove this film.

Evans blue dye also binds to -glass surfaces. This could lead to errors in standardization for blood volume determinations unless the standard is further diluted immediately in plasma.40

Traces of cupric ion in distilled water interfere with the determination of oxyhemoglobin, presumably by conversion of oxyhemoglobin to methemoglobin.63 Free chlorine may also be an unsuspected contaminant in distilled water. As such, it behaves as a strong oxidant and results in appreciable losses in the measurement of serum bilirubin and uric acid.36

Ordinary filter paper contains calcium, readily leached out by acid solution; hence, acid-washed filter paper should be used in determinations of calcium. On the other

May 1962

hand, acid-washed filter paper is not suitable for determinations of nitrogen, inasmuch as such papers readily absorb ammonia from the air.157 Special low-fat filter papers, e.g., Whatman No. 43, are available for use in estimations of lipid.

Some lots of reagent-grade absolute methanol contain impurities capable of reacting with Ehrlich's diazo reagent to produce a pink color. This results in falsely elevated bilirubin values by the MalloyEvelyn technic in which the diazo reagent is omitted from the control. Methanol may be purified by shaking it with activated carbon and filtering.36

In determinations of serum amylase, great care should be taken in order to avoid contamination of mixtures with saliva. Results obtained by an amyloclastic procedure reveal that salivary amylase activity is nearly 700 times that of serum.120 It is important, therefore, to deliver serum into the substrate from a pipet calibrated between 2 marks and to avoid use of pipets .calibrated to contain or to "blow-out."

Strips of paper impregnated with glucose oxidase, peroxidase, and a chromogen are widely used for the detection of glucose in urine (Clinistix, Uristix, Combistix, the Ames Co.; Tes-Tape, Eli Lilly & Co.). The test for glucose depends on the following reactions :

Glucose + O2

glucose oxidase > gluconic add + Hi02

H2O2 + chromogen peroxidase > cojor

Numerous reports have emphasized the specificity of this preparation for glucose;16' 43, 66'68,113'115,161 however, a number of strong false-positive reactions were encountered in our laboratories on urine specimens that were negative for glucose by copper reduction methods. Admitting the high specificity of glucose oxidase, it seemed probable that contamination had occurred either with hydrogen peroxide or with some strong oxidizing agent such as hypochlorite.

With Combistix test paper, a strong positive test for "glucose" was obtained with 0.003 per cent hydrogen peroxide or 0.005

per cent sodium hypochlorite. When hydrogen peroxide was added to random specimens of urine, a final concentration of 0.006 per cent usually resulted in a strong positive test. With sodium hypochlorite, a final concentration of 0.2 per cent was required to produce a strong positive test. Apparently, reducing substances normally present in urine destroy lesser quantities of hypochlorite. All such treated urines were negative for reducing sugar by copper reduction tests.

Several commercial bleaches contain approximately 5.5 per cent sodium hypochlorite, and various detergents contain larger quantities. If such detergents are used to clean urinals or specimen bottles, the containers must be rinsed thoroughly prior to collection of urine specimens for laboratory examinations. Our false-positives were traced in part to contamination with hydrogen peroxide. In 1 instance, ail catheters during the cleaning process had been soaked routinely in 3 per cent hydrogen peroxide. In another instance, sterile bottles, used as containers for 3 per cent hydrogen peroxide for irrigating wounds, were subsequently used to collect urine specimens.

Addition of 2 drops of 3 per cent hydrogen peroxide to 50 ml. of urine is sufficient to produce a strong positive reaction for glucose by means of the glucose oxidase strip technic. It has also been observed that very dilute solutions of hydrogen peroxide (0.01 per cent) are not destroyed by autoclaving. A routine procedure at present is to eliminate glucose-negative urines with the strip test; those manifesting positive results are then graded by means of using a copper reduction tablet (Clinitest, Ames Co.). The presence of reducing sugars other than glucose may be detected by means of applying the copper reduction test to glucose-negative urines.

Therapeutic agents may change the physiologic level of the substance measured. Many such changes are predictable or known from experience. Typical examples include: increased serum amylase activity from administration of morphine24 and codeine;77 increased serum glutamic-oxalacetic trans-

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