4.21: Sample Analysis I

During a day’s work, using several types of chemical analyzers, the laboratory’s technologists have prepared a number of dilutions on many different analytes, for a variety of reasons.

QUESTION

Which of the following dilutions might be considered acceptable or unacceptable? Why?

1. Alkaline phosphatase exceeded the reportable range (assay linearity) and a ten-fold dilution with saline was prepared.
2. Albumin exceeded the linear range of the assay. A ten-fold dilution was prepared with saline.
3. Lactate dehydrogenase (LD) showed very early substrate exhaustion, and a two-fold dilution was prepared with saline.
4. A sample to be analyzed for creatine kinase was highly lipemic, and a two-fold dilution was prepared for analysis.
5. Chloride in the specimen exceeded the linear range of the ferric thiocyanate method being used for analysis. A two-fold dilution was prepared for this specimen.
6. For a gas chromatography method for barbiturates, the peak for barbiturate went slightly off scale when a 50 microliter injection was used. The technologist prepared a 1:3 dilution of the sample for further analysis.
7. A sample is diluted two-fold for an analyte measured by a radioimmunoassay procedure because the initial result exceeded the highest standard employed.

Questions to Consider

1. What are the two most common reasons for preparing a dilution?
2. What are the major limitations on the extent to which an analyte should be diluted?

Before answering the problems, the student should read the appropriate analyte chapters in the CD-ROM and Chapter 23.

Answer

1. Acceptable. Most enzymatic methods have linear ranges wide enough so that a ten-fold dilution will usually bring the measured activity well into the acceptable range for measurement.
2. Unacceptable. Dilution should be made to bring the concentration of the analyte into the linear range but the concentration must be kept high enough that the analyte has measurable and reproducible activity. A ten-fold dilution of albumin will result in an albumin concentration so low that its measurement will likely be extraordinarily inaccurate.
3. May be unacceptable. LD assays tend to have relatively narrow linear ranges and thus a two-fold dilution might not be great enough to get the LD concentration within range. A five- or ten- fold dilution probably would have been better.
4. Probably unacceptable. Multiple dilutions would be more appropriate. Each result is multiplied by the dilution factor and the final results compared. When the results begin to repeat, one can assume that the lipemia effect has been diluted out and the results and valid. If the enzyme activity is too low for dilutions, the sample may have to be rejected for analysis or the lipid particles removed by ultracentrifugation.
5. Unacceptable. The ferric thiocyanate method is not linear below 70-75 mmol/L. Therefore, dilution of the specimen would most likely have reduced the concentration of Cl below the linear range. The analysis should be performed on another instrument, e.g. a chloridometer.
6. Unacceptable. There is no reason for introducing a possible dilution error for this type of analysis. Instead, the technologist should have re-injected 20-25 uL of sample.
7. May be acceptable. The technologist must look carefully at the dynamic range of the RIA used. Some standard curves extend over a 10-fold (1-log) range of concentrations while others may extend over a 1000-fold (3-log) range. For a 10-fold range standard curve, a single two-fold dilution may be sufficient. However, for a 1000-fold standard curve, a 10-fold dilution may be more appropriate. In either case, multiple dilution steps of the sample should be used to ensure that a result will be obtained, since many RIA’s are not performed on a daily basis. If a single inadequate dilution is analyzed and the result obtained is still not within range, the analysis will have to be repeated a third time, which may result in an unacceptable delay.

Answers to Questions to Consider

1. Most patient samples are diluted: 1) to remove a possible inhibitor to the analysis (such as lipemia) or 2) because the analyte concentration exceeds the reportable range of the assay.
2. The major limitations are the reference (normal) range of the analyte, the analytical reportable (linear range. see p 410) of the method being employed, and the lower limit of detection of the assay. Dilutions an make the final concentration of the analyte fall within the upper portion of the reference interval of the analyte. This will usually be well within the linear range of the assay and well above the limit of detection.