4.5: Refractometry
- Page ID
- 121672
\( \newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} } \) \( \newcommand{\vecd}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash {#1}}} \)\(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\) \(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\)\(\newcommand{\AA}{\unicode[.8,0]{x212B}}\)
A laboratory is using a refractometer to measure serum protein in the STAT lab area. A technologist measures the total protein in one specimen as 89 g/L. When the technologist enters this value in the laboratory’s information system (LIS), a delta check flag warns that this value is significantly different from the previously reported value of 78 g/L.
QUESTION
- How can this difference be explained?
- The technologist retrieves the previous sample and checks to ensure that the patient name and identification number are the same. Next, the technologist looks at both serum samples and then performs a “renal” profile on the current specimen (glucose, BUN, creatinine and electrolytes). The results of this analysis are glucose = 10,340 mg/L, BUN = 1050 mg/L, creatinine = 51 mg/L, and electrolyte levels within normal range. The technologist checks in the LIS and determines that the initial sample had a protein determination performed by a biuret assay as part of a routine chemistry profile. How do these test results explain the differences between the refractive index values for the two specimens?
Questions to Consider
- What is the principle of measuring total serum protein by refractometry?
- What other method is commonly used to quantitate total serum protein?
- What are the major interferences for each method?
- Why did the technologist look at the specimen, and what do you think was seen?
- Answer
-
- The results of the “renal” profile explain the difference between the total protein results because: Hemolysis and turbidity caused by hyperlipidemia are also causes of false positive results (see p 105). The refractive index difference was probably caused by a change in the concentration of one or more of the non-protein analytes.
- The very large amount of glucose and serum urea nitrogen present in the current sample (reported total protein, 89 g/L) was the cause of the falsely elevated results obtained by refractometry. There is an additional 11.8 g/L of material (glucose + BUN + creatinine) in the sample. Since the initial sample was measured by the more specific biuret procedure, there was no false positive interference.
Answers to Questions to Consider
- The refractometer is not specific for protein but measures the total mass of dissolved solids (page 104). The greater the mass of solids, the greater the angle of refraction of the incident light passing through the solution. The angle of refraction is indicated on a scale as mass/volume. The assumptions are that almost all the mass in serum is protein and that the concentration of the other serum components remains essentially constant from patient to patient (p. 104-105).
- The most commonly used method for measuring serum total protein is the biuret reaction (see Total Protein on CD-ROM). The reaction is based on the color formed when copper complexes with the peptide bond. The refractometer is an infrequently used method for measuring serum total protein.
- The biuret reaction has very few interferences. Since the refractometer is only a non-specific measure of total dissolved solids, any substantial increase of non-protein solutes will give rise to false positive increases in protein determination. Large increases in glucose, BUN, or bilirubin (in order of importance) can affect the refractive index.
- The technologist examined the specimen to determine if the specimen was either lipemic or hemolyzed, both possible causes of false positive results. Since the technologist went on to do a renal profile, one can assume that the specimen did not appear lipemic or hemolyzed.