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  Q & A

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cap today

October 2001

Q.  What is the value of doing the Ictotest (tablet method for bilirubin detection) on urine specimens that have already tested positive for bilirubin by dipstick?

A.  The urine bilirubin dipstick test and urine bilirubin tablet test (Ictotest) both detect the presence of conjugated bilirubin. (Unconjugated bilirubin is not excreted into the urine.) Dipsticks use the binding of conjugated bilirubin molecules to diazotized salts fixed in the test pad in a strong acidic environment to form a colored compound that is various shades of tan or magenta. Dipsticks have a sensitivity of 0.5 to 1.0 mg of bilirubin/dL. Bilirubin tablet tests use a similar reaction but a different environment. In the tablet test, urine is placed on an absorbent test mat that captures substances within the urine. The reagent tablet is then placed on the mat over the absorbed urine, and water is added to the tablet such that the reactive substances, a solid diazonium salt and acid, run onto the mat. The reaction of conjugated bilirubin with the diazonium salt in the acidic environment results in the formation of a blue ring around the dissolving tablet. The sensitivity of the bilirubin tablet test is 0.05 to 0.1 mg of bilirubin/dL.

The tablet test is approximately tenfold more sensitive than the dipstick test, so it seems counterintuitive that a tablet test should be used to confirm a less sensitive dipstick test. This is done to allow for detection of false-positive results. In some circumstances, there are yellow, orange, or brown pigments (blood breakdown products or products related to drugs such as phenazopyridium or rifampin) in the urine that can "stain" the dipstick pad and cause a false-positive interpretation. Since the urine is placed on the mat first in the tablet test, this staining is evident before the reaction is performed and can be accounted for. Also, because the reaction product is blue rather than tan or magenta, there are fewer interpretation problems.

Other nonbilirubin molecules usually not present in the urine can bind to diazotized salts in an acidic environment. These molecules include etodolac, mefenamic acid, and flufenamic acid (nonsteroidal anti-inflammatory agents). Since the diazotized salts are fixed to the test pads in dipsticks, the presence of these molecules can cause an apparently positive test. These molecules, however, do not bind as readily to the test mat used in the tablet test and are often washed-through, yielding a weaker or negative result.

To further evaluate one of these unexpectedly weaker reactions in a tablet test, a procedure using a water wash-through method is used where water is added to the urine spot before it is tested. This does not usually effect a result due to bilirubin but results in a loss of reactivity when the reaction is caused by other molecules. The reaction of dipstick pads with these other nonbilirubin molecules is often exploited for making liquid urine dipstick control and testing materials, since conjugated bilirubin is light-sensitive and unstable and cannot readily be incorporated into such materials. Such controls and testing materials can be used only for dipstick evaluation and not tablet test evaluation.

Bibliography
Henry JB. Clinical Diagnosis and Management by Laboratory Methods. 18th ed. Philadelphia, Pa.: WB Saunders; 1991:413-440.

Robert Novak, MD
Department of Pathology
Children's Hospital Medical Center of Akron
Akron, Ohio

CAP Hematology/Clinical
Microscopy Resource Committee

Q.  Does the BD Directigen line of group A streptococcus, respiratory syncytial virus, and influenza A meet the CAP requirement of having built-in controls? The manufacturer's package insert says: "ColorPAK devices contain two built-in controls. The appearance of a colored dot (pink or purple—depending on the test) provides an internal positive antigen control that validates the immunologic reactivity of the device, proper detection of reagent function, and assures that the correct detection procedure was followed. The membrane area surrounding the triangle is the internal negative control for the device. The lack of any significant color development in this background area indicates that the test has been performed correctly." Each inspector tends to interpret differently whether the BD Directigen or other manufacturers' products have built-in controls.

A.  In principle, the CAP Laboratory Accreditation Program does not rule on the applicability of a checklist item for specific instrument-reagent systems. The checklists set general—and sometimes specific—guidelines as a way for inspectors and those being inspected to have a common level of understanding. Beyond that, the uniqueness of each laboratory's setting can be properly evaluated only during the on-site inspection. In the case of rapid bacterial antigen tests, two updated items are included in the new Microbiology Checklist.

MIC.21760: For direct bacterial antigen tests on patient specimens that DO include internal positive and negative controls and are NOT classified as "high complexity" under CLIA '88, is a positive and negative external control tested with each new kit lot number or separate shipments of a given lot number?

NOTE: Manufacturers' recommendations must be followed. For panels or batteries, controls must be employed for each bacterial antigen sought. For tests classified as "high complexity" under CLIA '88 that include an extraction phase, the system must be checked each day of use with a positive organism.

MIC.21770: For direct bacterial antigen tests on patient specimens that do NOT include internal positive and negative controls, is a positive and negative control tested with each run of patient specimens?

NOTE: An analytical run is the interval within which the accuracy and precision of the measuring system is expected to be stable, based upon manufacturers' recommendations, and shall not exceed 24 hours. For panels or batteries, controls must be employed for each bacterial antigen sought in patient specimens. For tests classified as "high complexity" under CLIA '88 that include an extraction phase, the system must be checked each day of use with a positive organism.

The core of these paired questions relates frequency of external controls to the presence or absence of built-in controls. From the description given, the built-in positive control apparently contains true antigen, but the negative control is really a measure of process, not reactivity. The requirement for "reactivity" has been eliminated as a requirement of accreditation. Thus, this system would appear to meet the working definition of built-in controls. (Review of the Food and Drug Administration Web site at www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfPMN/pmn.cfm shows that these particular BD kits are 510(k) cleared. This particular Web site is a convenient place for laboratories to search for FDA-cleared products.)

Albert Rabinovitch, MD, PhD
Chief medical officer/vice president
Specialty Laboratories
Santa Monica, Calif.

Checklist commissioner
CAP Commission
on Laboratory Accreditation