Analyte Information and Courses from MediaLab, Inc.

Several manufacturers offer semi-automated instruments (dipstick readers) for reading reagent strips. Use of an instrument removes the subjectivity of visually interpreting color changes on reagent strips, and assures that tests will be read at the correct time. Transcription errors will also be avoided if the instrument is interfaced with the laboratory information system. The technology employed is based on the principle of reflectance, with the amount of light reflected being inversely related to the concentration of substances present. An example of reflectance is the light which is scattered after light strikes an unpolished surface. Since each component on the dipstick produces a different color reaction, the light source for each test must be at the appropriate wavelength. This is accomplished either by using filters or monochromatic light sources. The percent reflectance is determined by dividing the test reflectance by the calibration reflectance and multiplying by 100. Algorithms are used to change the results obtained into a linear relationship with concentration of analyte.

Urine reagent strips are normally adequate for urine screening, but occasionally, it may be necessary to perform a secondary procedure to ensure the accuracy of the test result. Confirmatory or secondary procedures are usually performed for one or more of these reasons: To confirm a result that has been obtained on the reagent strip. To obtain a result from a highly pigmented urine that masks the result on the reagent strip. To test for a specific analyte (or analytes) that are not included in the specificity of the reagent strip test. For example, the glucose reagent strip test is specific for glucose, but you want to test for other reducing substances.

External quality control is performed to ensure the reliability of test results between different laboratories. It is also required by CLIA for laboratory accreditation. External quality control is generally accomplished through proficiency testing (PT). In proficiency testing, simulated patient samples are sent out to laboratories for testing. The CLIA standards for handling proficiency testing specimens are as follows: PT samples must be tested with the laboratory's regular patient load. PT samples must be tested the same number of times that patients' samples are tested routinely. Laboratories participating in PT programs must not engage in interlaboratory comparison of PT sample results. Laboratories may not send PT samples to another laboratory for analysis. Laboratories must document all steps of processing for PT samples. PT is required for only the primary method used for testing of analytes in patients' samples during the period covered by the PT event.In return for their participation, the laboratory will receive the following information: results for each analyte sample mean result for each analyte standard deviation of results by the comparative method number of laboratories using the same method standard deviation index (SDI) lower and upper limits of acceptability of resultsPT results that are between the lower and upper limits of acceptability are considered satisfactory. For chemistry, 80% of samples must test within the acceptable range for the PT to be considered successful. External quality control serves several purposes, including: providing a check on internal quality control detecting errors in a lab's methods providing a comparison of testing methods, which is useful in selecting new methods

Fluoresence polarization immunoassay (FPIA) is also a homogenous competitive immunoassay. In this system, fluorescein-labeled drug competes with unlabeled drug from the patient's serum sample for binding sites on an antibody reagent. The patient's sample, presumably containing the therapeutic drug that is being monitored, and the fluorescein-labeled drug are added to a chamber containing antibody for that drug. The labeled and unlabeled drug will compete for binding sites on the antibody. The greater the amount of drug in the sample, the fewer the number of binding sites that are available for the labeled analyte, leaving a greater number of small, free fluorescein-labeled molecules in the solution.When the chamber is excited with plane polarized light, fluorescein will absorb the light and emit it at a higher wavelength as fluorescent light. A small, free fluorescein-labeled drug rotates randomly and faster than it would if it were bound to antibody, interrupting the light and leading to less emission of light. The larger antibody-drug-fluorescein complexes rotate slower and emit more light in the measured plane. A lower level of drug in the patient's sample results in greater emission of polarized light because there are more antibody-drug-fluorescein complexes present to produce light in the measured plane. A higher level of drug in the patient's sample results in a lower emission of polarized light. This inverse relationship between the concentration of the drug and the polarization units (signal) is illustrated in the image below.

Assayed controls have been analyzed by the manufacturer so that the range of values for the analytes they contain is known. Unassayed controls are unknowns. The laboratory purchasing the controls must determine the concentration of each analyte.

Looking at the example to the right, we can see that the instrument on which we are doing a calibration curve is linear up to 1000 mg/100ml of blood for a particular analyte. Accordingly, we can be fairly certain that any results obtain up to 1000 milligrams are accurate. Above 1000 milligrams our curve begins to bend. This means that any results greater than 1000mg may not reflect a true measurement of the analyte being tested. The specimen must be diluted down to the linear range.

Reference ranges can help show when a test result is drastically out-of-line with expectations by providing a range of most likely values for any given analyte. Reference ranges should reflect the mean value in the population and a certain level of variation (usually 2 standard deviations). 95% of all normal patients will fall inside the reference range of an analyte. Values outside the reference ranges could indicate not only an abnormality in the patient, but also a problem with the test results. For example, should 20% of results suddenly begin to exceed a given reference range, there is most likely an testing error.