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Refractometric total protein concentrations in icteric serum from dogs Aradhana Gupta, DVM, MVSc, and Steven L. Stockham, DVM, MS Objective—To determine whether high serum bilirubin concentrations interfere with the measurement of serum total protein concentration by refractometry and to assess potential biases among refractometer measurements. Design—Evaluation study. Sample—Sera from 2 healthy Greyhounds. Procedures—Bilirubin was dissolved in 0.1M NaOH, and the resulting solution was mixed with sera from 2 dogs from which food had been withheld to achieve various bilirubin concentrations up to 40 mg/dL. Refractometric total protein concentrations were estimated with 3 clinical refractometers. A biochemical analyzer was used to measure biuret assay– based total protein and bilirubin concentrations with spectrophotometric assays. Results—No interference with refractometric measurement of total protein concentrations was detected with bilirubin concentrations up to 41.5 mg/dL. Biases in refractometric total protein concentrations were detected and were related to the conversion of refractive index values to total protein concentrations. Conclusions and Clinical Relevance—Hyperbilirubinemia did not interfere with the refractometric estimation of serum total protein concentration. The agreement among total protein concentrations estimated by 3 refractometers was dependent on the method of conversion of refractive index to total protein concentration and was independent of hyperbilirubinemia. (J Am Vet Med Assoc 2014;244:63–67)
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tatements in veterinary textbooks regarding the effects of bilirubin on the refractometric total protein concentration are conflicting; some textbooks state that hyperbilirubinemia (icterus) has no effect,1–7 whereas others list hyperbilirubinemia as a cause of a false increase in refractometric total protein concentrations.8–11 Human clinical biochemistry textbooks also state that hyperbilirubinemia will cause a false increase.12–16 Published data indicate that hyperbilirubinemia has little or no effect, but the bilirubin concentrations in those studies17–20 are either not provided or represent only minor increases from physiologic concentrations (ie, < 1.0 mg/dL). Establishing whether bilirubin affects refractometry will allow veterinarians to properly interpret total protein concentrations when there are differences between refractometric total protein and biuret assayderived total protein concentrations in icteric sera. Recently, pseudohypoproteinemia was reported in a hyperbilirubinemic dog.21 The refractometer measurement was in the reference interval, whereas the biuret assay total protein concentration was decreased. The lower biuret assay total protein concentration was atFrom the Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506. Presented in abstract form at the Annual Meeting of the American Society for Veterinary Clinical Pathology, Seattle, November 2012. The authors thank Janice Muller, Jill Newland, Jessica Penner, and Stephanie Ochoa for technical assistance. Address correspondence to Dr. Stockham ([email protected]. edu). JAVMA, Vol 244, No. 1, January 1, 2014
ABBREVIATIONS CV TS%
Coefficient of variation Percentage of total soluble solids
tributed to interference caused by bilirubin and biliverdin, but it was not reported whether the refractometric method was affected by the hyperbilirubinemia. Refractometers measure the refractive index (n), or the bending of light when it passes through a liquid. Refraction (r), also known as refractive index difference, is the difference between the refractive index of an aqueous solution and water (ie, r = n – 1.333)22; the refractive index of water is 1.333. A refractive index is dependent on the soluble solids in the liquid,22 and both the TS% and the type of soluble particles in a solution alter the refractive index. Suspended particles do not alter the refractive index.23 When the liquid is plasma or serum, the refractive index is proportional to the TS% (g of solute/100 g of solution X 100%).22 The refractometric estimation of total protein concentration includes the assumption that changes in the refractive index are caused by changes in total protein concentration, and the concentrations of other soluble solids have not changed. However, higher concentrations of soluble solids such as glucose, urea, electrolytes, and lipoproteins will result in a higher TS% and thus a higher refractive index and, after conversion, an erroneously increased total protein concentration.22,24 Considering the contribution of bilirubin to the TS%, even the amount of bilirubin in extremely icteric sera should not interfere with the refractometric total protein concentration. Scientific Reports
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Handheld refractometers are common in many veterinary laboratories and clinics and provide quick and inexpensive estimations of total protein concentrations in serum, plasma, or other body fluids.24 The total protein scale of clinical refractometers is typically calibrated for the normal soluble constituents of human plasma. Various equations or relationships have been used to convert refractive indices of human serum or plasma to total protein concentations.22 It is also reported that calibration factors for converting refractive indices to total protein concentrations are different among species.17 On the basis of available information, different total protein concentrations may be found when different methods are used to convert a refractive index to a total protein concentration. However, observed differences among refractometers should not be influenced by the presence of increased bilirubin concentrations. The purpose of the study reported here was to confirm or refute the supposition that hyperbilirubinemia causes falsely increased refractometric serum total protein concentrations. Because a variety of refractometers are available for a clinical laboratory, 3 refractometers were used and the refractometric data were compared with data from a biuret assay–based automated spectrophotometric method. Materials and Methods Blood collection—Venous blood samples were collected with syringes from 2 healthy Greyhounds from which food had been withheld overnight and then transferred to evacuated tubes. After approximately 30 minutes, tubes were centrifuged and nonlipemic and nonhemolyzed sera were harvested. Bilirubin stock solution and preparation of icteric sera—Bilirubin powdera (40 mg) was dissolved in 10 mL of 0.1M NaOH25,26 to prepare a stock bilirubin solution of nearly 400 mg/dL. Diluted stock solutions were mixed with a constant volume of sera (1:9 bilirubin solution mixed with serum) to create bilirubin concentrations near 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, and 40 mg/ dL. The 0.1M NaOH solution was mixed with serum in the same ratio. Refractometric total protein concentration measurements—Three temperature-compensated refractometers were used: a refractometerb (refractometer 1) with total protein concentration (g/dL) and refraction scales, a refractometerc (refractometer 2) with a TS% scale, and a refractometerd (refractometer 3) with a total protein concentration scale (g/dL). Each refractometer was calibrated with deionized water to 1.000 on its urine specific gravity scale prior to analyzing samples. Because a conversion table for refractometer 2c was not found in the literature, its TS% values were first converted to refraction values (r) with the aid of a conversion table reported by Wolf 22 and then to total protein concentrations in 4 ways: the conversion table,22 a conversion table27 for refractometer 1, a canine conversion formula,17 and a multispecies conversion formula.17 To assess the analytic precision of the refractometric methods, 2 investigators (AG and a medical technologist) determined refractometric values in the unspiked 64
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serum samples and the mean of those values was calculated. This process was performed 8 times for unspiked sera of 2 dogs. For the assessment of bias, the refractometric values of each unspiked and spiked sample were determined by the same 2 investigators with each refractometer. The mean values of the 2 readings were recorded for data analysis. Clinical biochemical assays for serum total protein, total bilirubin, and direct bilirubin concentrations—Clinical biochemical assays were completed on the day of blood collection with an automated analyzere and the manufacturer’s reagents. The total protein assay (2-point-end, bichromatic biuret assay) was used to measure total protein concentrations in the unspiked serum samples. During the previous 5 months and including the weeks of this study, the CV for the biuret assay was 1.5% for commercial level 1 control serum (mean ± SD concentration, 6.0 ± 0.1 g/dL) and 1.5% for commercial level 2 control serum (mean concentration, 4.4 ± 0.1 g/dL). Unspiked sera of both dogs were assayed 8 times, and the mean concentration for each dog was calculated. The total bilirubin assay was a modification of the Malloy-Evelyn method,f and the direct bilirubin assay was based on the Jendrassik-Grof procedure; automatic dilutions were completed for samples with bilirubin concentrations > 35 mg/dL. Statistical analysis—The CV (%) was calculated for the repeated refractometric and spectrophotometric measurements of total protein concentrations in sera from both dogs. Difference plots were created to assess agreement among the refractometric total protein concentrations. Results Analytic precision of refractometric and spectrophotometric total protein assay methods—For the unspiked canine sera of the 2 dogs, the mean refractometric total protein concentrations were 6.5 g/dL (CV, 0.0%) and 6.2 g/dL (CV, 0.0%) for refractometer 1, 6.2 g/dL Table 1—Refractometric total protein concentrations in unspiked sera from 2 healthy Greyhounds, derived from TS% values obtained with a refractometer with a TS% scale (refractometer 2), which was chosen as the comparative method for refractometer difference plots because total protein concentrations obtained with a conversion table22 agreed with spectrophotometric assay total protein concentrations (provided for comparison). Variable
Dog 1
Dog 2
TS% (g/100 g X 100%) from refractometer 2 Refraction (conversion table from Wolf22) Total protein concentration Conversion table from Wolf22 (g/dL) Conversion table27 for refractometer 1 with a total protein concentration (g/dL) and a refraction scale (g/dL) Canine formula* (g/dL) Multispecies formula† (g/dL) Biuret assay total protein concentration (g/dL)
7.6 0.0139
7.3 0.0133
6.2 6.3
5.8 6.0
5.8 6.3 6.2
5.5 6.0 5.8
*Formula for canine sera17: total protein concentration = (430 X r) – 0.20, where r is refraction. †Formula derived for the combination of bovine, canine, and equine sera17: total protein concentration = (482 X r) – 0.41. JAVMA, Vol 244, No. 1, January 1, 2014
The mean spectrophotometric total protein concentrations in the unspiked sera were 6.2 g/dL (CV, 1.4%) and 5.8 g/dL (CV, 1.4%) for the 2 dogs, respectively. The CV percentages were consistent with the laboratory’s daily quality control data for level 1 and level 2 control sera. Agreement among refractometric total protein concentrations—For refractometer 2, different total protein concentrations were derived by different methods (Table 1). Because the refractometer 2 total protein concentrations determined with the conversion table reported by Wolf22 agreed with the spectrophotometric assay total protein concentrations, refractometer 2 was used as the comparative method for the refractometric difference plots. The biases of refractometric methods for total protein concentrations of serum samples from only 1 dog are reported (Figure 1); similar biases were found in sera from the other dog. Effects of increased bilirubin concentrations on refractometric total protein concentrations—The measured total bilirubin concentration in the NaOHspiked serum sample was 0.1 mg/dL. The measured total bilirubin concentrations in bilirubin-spiked samples 1 through 12 were 0.9, 1.7, 2.9, 4.6, 5.6, 6.8, 10.7, 15.1, 21.8, 27.7, 32.7, and 41.5 mg/dL, respectively (Figure 2). Direct bilirubin concentrations in bilirubin-spiked samples 1 through 12 were 0.3, 0.5, 0.4, 0.5, 0.5, 0.5, 0.6, 0.5, 0.6, 0.6, 0.7, and 0.8 mg/dL, respectively. Because the same biases and analytic precision values were consistently found in samples from both dogs, the bilirubin effects on the refractometric total protein concentrations for serum samples from only 1 dog are reported. For that dog, when samples were spiked with only NaOH (1:9 NaOH and serum), thus diluting the unspiked protein concentration, the spiked sample’s total protein concentrations were 5.7 g/dL for refractometer 1, 5.4 g/ dL for refractometer 2, and 5.5 g/dL for refractometer 3. With increasing bilirubin concentrations (up to 41.5 mg/ dL), the total protein concentrations varied from 5.3 to 5.5 g/dL for refractometer 1, 5.1 to 5.3 g/dL for refractometer 2, and 5.4 to 5.6 g/dL for refractometer 3 (Figure 2).
Figure 1—Difference plots of the refractometric total protein concentrations of unspiked and spiked serum samples (n = 14) of 1 of 2 healthy Greyhounds in a study to determine whether increased serum bilirubin concentrations interfere with the measurement of serum total protein concentration by refractometry and to assess potential biases among measurements obtained with 3 temperature-compensated refractometers: a refractometerb (refractometer 1) with total protein concentration (g/dL) and refraction scales, a refractometerc (refractometer 2) with a TS% scale, and a refractometerd (refractometer 3) with a total protein concentration scale (g/dL). Refractometric total protein concentrations determined with refractometer 2 were considered the comparative values (x-axis). For the x-axis, the highest protein concentration (5.8 g/dL) in each graph represents the concentration in the unspiked sample; the lower concentrations (5.1 to 5.4 g/dL) represent samples after they were spiked and thus had diluted protein concentrations. The numbers within circles represent number of values represented by a plotted point. Concentrations for both refractometer 3 (A) and refractometer 1 (B) had positive biases (approx 0.2 g/dL; dashed lines), compared with the refractometer 2 concentrations. JAVMA, Vol 244, No. 1, January 1, 2014
Figure 2—Line graph of bilirubin concentrations (diamonds) and refractometer 1 (triangles), refractometer 2 (squares), and refractometer 3 (crosses) total protein concentrations in sera of the same dog as in Figure 1 spiked with NaOH only or with increasing amounts of bilirubin up to a total bilirubin concentration of 41.5 mg/dL. The differences in the refractometric total protein concentrations among the refractometers reflect their analytic biases; the variations in concentrations from samples 1 to 12 reflect the methods’ analytic precisions. Scientific Reports
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(CV, 1.2%) and 5.8 g/dL (CV, 0.9%) for refractometer 2, and 6.5 g/dL (CV, 0.6%) and 6.2 g/dL (CV, 0.0%) for refractometer 3. On the basis of these findings and the refractometric scales being marked at 0.1 g/dL (refractometer 1 and refractometer 3) or 0.1 g/100 g (refractometer 2) intervals, any change to total protein concentration in the bilirubin-spiked samples > 0.2 g/dL was accepted as representing a true change.
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Table 2—Contributions of major solutes (in various units) to serum TS% (g/100 g X 100%) when there are physiologic concentrations of the solutes.* Solute Total protein Glucose Urea Na+ Cl− Na+ + Cl− Bilirubin H2O Sum of solutes TS%
Common units
g/dL of serum
g/dL of serum H2O†
7.0 g/dL 100 mg/dL 60 mg/dL§ 150 mmol/L 110 mmol/L — 1 mg/dL — — —
7.0 0.100 0.060 0.345|| 0.391|| 0.736 0.001 93.8¶ — —
7.5 0.107 0.064 — — 0.784 0.001 — 8.4 —
g/100 g‡ 7.3 0.1 0.1 — — 0.8 < 0.1 — — 8.2
*Original solute concentrations and the calculated TS% are reported with appropriate significant figures. Excess figures are present in some calculated values so that minor changes are seen. †Value in column derived by dividing value in previous column by 0.938. ‡Converting solute weight per 102.2 g of solution (solute weight + H2O weight) to solute weight per 100 g of solution. §Equals a urea nitrogen concentration of 28 mg/dL or 10 mmol/L. ||Converted by use of relative molecular mass of Na+ (23 g/mol) and Cl− (35.5 g/ mol). ¶When serum total protein concentration is 7.0 g/dL, the serum water concentration is 93.8 g/dL.22 — = Not applicable. Table 3—Contributions of solutes (in various units) to serum TS% (g/100 g X 100%) when there are increased concentrations of glucose, urea, or bilirubin.* Solute
Common units
g/dL of serum
g/dL of serum H2O†
Total protein Glucose Urea Bilirubin
7.0 g/dL§ 600 mg/dL 600 mg/dL¶ 40 mg/dL
7.0 0.600 0.600 0.040
7.5 0.640 0.640 0.043
g/100 g‡ 7.2/100 0.6/100|| 0.6/100|| < 0.1/100
*Original solute concentrations and the calculated soluble-solids concentrations are reported with appropriate significant figures; additional significant figures were left in some calculated values so that minor changes are seen. †See Table 2 for the conversion of concentrations; each calculation assumed a total protein concentration of 7.0 g/dL and thus a serum water concentration of 93.8 g/dL. ‡Converting solute weight per 101.9 g of solution to solute weight per 100 g of solution, assuming there is an increase in glucose concentration or urea concentration and not both. §A typical serum protein concentration measured with a biuret assay. ¶Equals a urea nitrogen concentration of 280 mg/dL or 100 mmol/L. ||A serum glucose concentration of 700 mg/dL and a urea nitrogen concentration of 300 mg/dL (urea concentration, 643 mg/dL) have been reported to increase the refractometric total protein concentration by 0.6 g/dL.29
Discussion This study found that total bilirubin concentrations up to 41.5 mg/dL did not affect refractometric estimations of serum total protein concentrations, in that there were no detected differences in samples 1 to 12. The minor variations in concentrations obtained with each refractometer reflected the analytic precision of the methods. On the basis of the measured concentrations, nearly all the bilirubin in the spiked samples was considered unconjugated bilirubin. The apparent presence of direct bilirubin (ie, conjugated or delta bilirubin) may have reflected unconjugated bilirubin reacting in the direct diazo reaction.28 The refraction of light by serum is affected by the concentrations of some solutes (eg, proteins, glucose, urea, electrolytes, and lipoproteins),22,24 but marked hyperbilirubinemia does not cause a detectable change in 66
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the serum refractive index. The reason for the apparent higher total protein concentrations in the NaOH-only samples (approx 0.2 g/dL), compared with samples 1 to 12, was not determined. The refractive index of a liquid is proportional to TS%. This key concept is based on a property of solutes called refractivity (R) that is calculated with the following formula: R = r/TS%
As stated by Wolf,22 “This relationship is meaningful because the refractivity is relatively constant over wide ranges of concentrations of any solute.” The clinical refractometer measures a solution’s refractive index, which is used to determine the solution’s refraction, and thus it is useful to rephrase the equation in that context: r = R X TS%
Different solutes have different refractivity values, but the major solutes of serum have similar values (human serum protein, R = 19; urea, R = 15; glucose, R = 14; and sodium chloride, R = 17).22 A refractivity value for bilirubin was not found in the literature. Accordingly, change in serum refraction is mostly attributable to change in the TS%. In serum or plasma, the largest portion (by weight) of the total soluble solids is the protein content. Thus, proteins are the major contributors to the refraction of serum or plasma. To understand the relative contribution of solutes to TS%, it is necessary to express concentrations of common serum solutes in terms of their weights and not other common units of concentration. The sum of the solutes’ ratio (wt/wt) expression X 100% represents a TS% (eg, 8.2 g/100 g X 100% = 8.2%). If there are physiologic concentrations of all serum solutes and the total protein concentration is 7.0 g/dL, the TS% is expected to be 8.4%.22 Physiologic concentrations of sodium and chloride contribute almost 0.8% (0.8 g of solute/100 g of solution) to the TS%, whereas physiologic concentrations of glucose and urea contribute approximately 0.1% each. Most other nonprotein serum solutes contribute negligible amounts to TS%. Major solutes that contribute to serum TS% and the conversion of the common clinical concentrations to g/100 g were tabulated (Table 2). Marked hyperglycemia or marked azotemia can increase the TS% and thus falsely increase the refractive total protein concentration (Table 3).29 However, even with an extreme increase in serum bilirubin concentration, the calculated increase in TS% is too small (ie, < 0.1%) to affect the serum’s refractive index and thus would not falsely increase the refractive total protein concentration. This calculated prediction was confirmed by the measurements of this study. The bilirubin powder used to spike sera contained unconjugated bilirubin, as determined by evaluation of the total and direct bilirubin concentrations, yet none of the various forms of bilirubin should affect the TS%. When it is stated that hyperbilirubinemia causes a falsely increased refractometric total protein concentration, that statement is in error, and the reason for the JAVMA, Vol 244, No. 1, January 1, 2014
a. b. c. d. e. f.
Sigma-Aldrich, St Louis, Mo. TS Meter Refractometer, model 10400A, Leica Microsystems Inc, Buffalo, NY. TS Meter Refractometer, model 10400B, Leica Microsystems Inc, Buffalo, NY. Veterinary Total Solids Refractometer, model 10436, Leica Microsystems Inc, Buffalo, NY. Cobas c501, Roche Diagnostics, Indianapolis, Ind. Wahlefeld AW, Herz G, Bernt E. Modification of the MalloyEvelyn method for a simple, reliable determination of total bilirubin in serum (abstr). Scand J Clin Lab Invest 1972;29(suppl 126):11.12.
References 1. 2. 3. 4.
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Meyer DJ, Harvey JW. Evaluation of plasma proteins. In: Meyer DJ, Harvey JW, eds. Veterinary laboratory medicine: interpretation and diagnosis. 3rd ed. St Louis: Saunders, 2004;156–168. Jain NC. Hematologic techniques. In: Jain NC, ed. Schalm’s veterinary hematology. 4th ed. Philadelphia: Lea & Febiger, 1986;20–86. Benjamin MM. Protein. In: Benjamin MM, ed. Outline of veterinary clinical pathology. 3rd ed. Ames, Iowa: Iowa State University Press, 1978;108–120. Evans EW, Duncan JR. Proteins, lipids, and carbohydrates. In: Latimer KS, Mahaffey EA, Prasse KW, eds. Duncan and Prasse’s veterinary laboratory medicine: clinical pathology. 5th ed. Ames, Iowa: Iowa State Press, 2011;173–209. Kaneko JJ. Serum proteins and the dysproteinemias. In: Kaneko JJ, ed. Clinical biochemistry of domestic animals. 4th ed. San Diego: Academic Press, 1989;142–165. Kaneko JJ. Serum proteins and the dysproteinemias. In: Kaneko JJ, Harvey JW, Bruss ML, eds. Clinical biochemistry of domestic animals. 5th ed. San Diego: Academic Press, 1997;117–138. Allison RW. Laboratory evaluation of plasma and serum proteins. In: Thrall MA, Weiser G, Allison RW, et al, eds. Veterinary hematology and clinical chemistry. 2nd ed. Ames, Iowa: WileyBlackwell, 2012;460–475. McGrotty Y, Tennant K. Disorders of plasma proteins. In: Villiers E, Blackwood L, eds. BSAVA manual of canine and feline clinical pathology. 2nd ed. Gloucester, England: British Small Animal Veterinary Association, 2005;99–112. Lassen ED. Laboratory evaluation of plasma and serum pro-
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21. 22. 23. 24. 25.
26. 27. 28. 29. 30.
teins. In: Thrall MA, Baker DC, Campbell TW, et al, eds. Veterinary hematology and clinical chemistry. Philadelphia: Lippincott Williams & Wilkins, 2004;401–415. Werner LL, Turnwald GH, Willard MD. Immunologic and plasma protein disorders. In: Willard MD, Tvedten HW, eds. Small animal clinical diagnosis by laboratory methods. 4th ed. St Louis: Saunders, 2004;290–305. Thomas JS. Overview of plasma proteins. In: Feldman BF, Zinkl JG, Jain NC, eds. Schalm’s veterinary hematology. 5th ed. Philadelphia: Lippincott Williams & Wilkins, 2000;891–898. Johnson AM, Rohlfs EM, Silverman LM. Proteins. In: Burtis CA, Ashwood ER, eds. Tietz textbook of clinical chemistry. 3rd ed. Philadelphia: WB Saunders Co, 1999;477–540. McPherson RA. Specific proteins. In: Henry JB, ed. Clinical diagnosis and management by laboratory methods. 20th ed. Philadelphia: WB Saunders Co, 2001;249–263. Lindsey BJ. Amino acids and proteins. In: Bishop ML, DubenEngelkirk JL, Fody EP, eds. Clinical chemistry: principles, procedures, correlations. 4th ed. Philadelphia: Lippincott Williams & Wilkins, 2000;151–184. Wu AHB. Tietz clinical guide to laboratory tests. 4th ed. St Louis: Saunders, 2006. Lee-Lewandrowski E, Lewandrowski K. The plasma proteins. In: McClatchey KD, ed. Clinical laboratory medicine. Baltimore: Williams & Wilkins, 1994;239–258. Sutton RH. The refractometric determination of the total protein concentration in some animal plasmas. N Z Vet J 1976;24:141– 148. Briend-Marchal A, Medaille C, Braun JP. Comparison of total protein measurement by biuret method and refractometry in canine and feline plasma. Rev Med Vet (Toulouse) 2005;156:615–619. Schalm OW. The Goldberg refractometer or TS meter. Calif Vet 1965;19:28–30. Barry KG, McLaurin AW, Parnell BL. A practical temperaturecompensated hand refractometer (the TS-meter). Its clinical use and application in estimation of total serum proteins. J Lab Clin Med 1960;55:803–808. Garner BC, Priest H, Smith J. Pseudo-hypoproteinemia in a hyperbilirubinemic dog with immune-mediated hemolytic anemia. Vet Clin Pathol 2013;43:in press. Wolf AV. Aqueous solutions and body fluids. Their concentrative properties and conversion tables. New York: Hoeber Medical Division, Harper & Row, 1966. Goldberg HE. Principles of refractometry. Buffalo, NY: Leica Inc, 1997. George JW. The usefulness and limitations of hand-held refractometers in veterinary laboratory medicine: an historical and technical review. Vet Clin Pathol 2001;30:201–210. Martínez-Subiela S, Tecles F, Montes A, et al. Effects of haemolysis, lipaemia, bilirubinaemia and fibrinogen on protein electropherogram of canine samples analysed by capillary zone electrophoresis. Vet J 2002;164:261–268. Weber JA, van Zanten AP. Interferences in current methods for measurements of creatinine. Clin Chem 1991;37:695–700. American Optical Scientific Instrument Division. Instructions for use and care of the AO TS meter (a Goldberg refractometer). Buffalo, NY: American Optical, 1976. Tisdale WA, Klatskin G, Kinsella ED. The significance of the direct-reacting fraction of serum bilirubin in hemolytic jaundice. Am J Med 1959;26:214–227. Silverman LM, Christenson RH. Amino acids and proteins. In: Burtis CA, Ashwood ER, eds. Tietz textbook of clinical chemistry. 2nd ed. Philadelphia: WB Saunders Co, 1994;625–734. With TK. Spectral absorption of bilirubin. Measurements in pure aqueous solutions and in solutions containing human serum. Acta Physiol Scand 1945;10:172–180.
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error is usually unclear but might be based on concepts of photometry. Bilirubin is a pigmented compound that will absorb light wavelengths near 455 nm.30 However, for clinical refractometry, a broad range of visible light wavelengths (approx 380 to 740 nm) enter the refractometer and only a narrow band of wavelengths are absorbed by the bilirubin. Thus, most of the visible light is not affected by the pigment and it does not affect the measured refraction. Total protein concentrations derived for refractometer 2 (with the conversion table reported by Wolf22) agreed poorly with the concentrations derived for refractometer 1 and refractometer 3 and with other methods of converting refractive index values. However, the increasing bilirubin concentrations did not affect the biases. This study’s data provide evidence that the refractometric total protein concentration is dependent on the method of converting a refractive index value to a total protein concentration.
Refractometric total protein concentrations in icteric serum from dogs Aradhana Gupta, DVM, MVSc, and Steven L. Stockham, DVM, MS Objective—To determine whether high serum bilirubin concentrations interfere with the measurement of serum total protein concentration by refractometry and to assess potential biases among refractometer measurements. Design—Evaluation study. Sample—Sera from 2 healthy Greyhounds. Procedures—Bilirubin was dissolved in 0.1M NaOH, and the resulting solution was mixed with sera from 2 dogs from which food had been withheld to achieve various bilirubin concentrations up to 40 mg/dL. Refractometric total protein concentrations were estimated with 3 clinical refractometers. A biochemical analyzer was used to measure biuret assay– based total protein and bilirubin concentrations with spectrophotometric assays. Results—No interference with refractometric measurement of total protein concentrations was detected with bilirubin concentrations up to 41.5 mg/dL. Biases in refractometric total protein concentrations were detected and were related to the conversion of refractive index values to total protein concentrations. Conclusions and Clinical Relevance—Hyperbilirubinemia did not interfere with the refractometric estimation of serum total protein concentration. The agreement among total protein concentrations estimated by 3 refractometers was dependent on the method of conversion of refractive index to total protein concentration and was independent of hyperbilirubinemia. (J Am Vet Med Assoc 2014;244:63–67)
S
tatements in veterinary textbooks regarding the effects of bilirubin on the refractometric total protein concentration are conflicting; some textbooks state that hyperbilirubinemia (icterus) has no effect,1–7 whereas others list hyperbilirubinemia as a cause of a false increase in refractometric total protein concentrations.8–11 Human clinical biochemistry textbooks also state that hyperbilirubinemia will cause a false increase.12–16 Published data indicate that hyperbilirubinemia has little or no effect, but the bilirubin concentrations in those studies17–20 are either not provided or represent only minor increases from physiologic concentrations (ie, < 1.0 mg/dL). Establishing whether bilirubin affects refractometry will allow veterinarians to properly interpret total protein concentrations when there are differences between refractometric total protein and biuret assayderived total protein concentrations in icteric sera. Recently, pseudohypoproteinemia was reported in a hyperbilirubinemic dog.21 The refractometer measurement was in the reference interval, whereas the biuret assay total protein concentration was decreased. The lower biuret assay total protein concentration was atFrom the Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506. Presented in abstract form at the Annual Meeting of the American Society for Veterinary Clinical Pathology, Seattle, November 2012. The authors thank Janice Muller, Jill Newland, Jessica Penner, and Stephanie Ochoa for technical assistance. Address correspondence to Dr. Stockham ([email protected]. edu). JAVMA, Vol 244, No. 1, January 1, 2014
ABBREVIATIONS CV TS%
Coefficient of variation Percentage of total soluble solids
tributed to interference caused by bilirubin and biliverdin, but it was not reported whether the refractometric method was affected by the hyperbilirubinemia. Refractometers measure the refractive index (n), or the bending of light when it passes through a liquid. Refraction (r), also known as refractive index difference, is the difference between the refractive index of an aqueous solution and water (ie, r = n – 1.333)22; the refractive index of water is 1.333. A refractive index is dependent on the soluble solids in the liquid,22 and both the TS% and the type of soluble particles in a solution alter the refractive index. Suspended particles do not alter the refractive index.23 When the liquid is plasma or serum, the refractive index is proportional to the TS% (g of solute/100 g of solution X 100%).22 The refractometric estimation of total protein concentration includes the assumption that changes in the refractive index are caused by changes in total protein concentration, and the concentrations of other soluble solids have not changed. However, higher concentrations of soluble solids such as glucose, urea, electrolytes, and lipoproteins will result in a higher TS% and thus a higher refractive index and, after conversion, an erroneously increased total protein concentration.22,24 Considering the contribution of bilirubin to the TS%, even the amount of bilirubin in extremely icteric sera should not interfere with the refractometric total protein concentration. Scientific Reports
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Handheld refractometers are common in many veterinary laboratories and clinics and provide quick and inexpensive estimations of total protein concentrations in serum, plasma, or other body fluids.24 The total protein scale of clinical refractometers is typically calibrated for the normal soluble constituents of human plasma. Various equations or relationships have been used to convert refractive indices of human serum or plasma to total protein concentations.22 It is also reported that calibration factors for converting refractive indices to total protein concentrations are different among species.17 On the basis of available information, different total protein concentrations may be found when different methods are used to convert a refractive index to a total protein concentration. However, observed differences among refractometers should not be influenced by the presence of increased bilirubin concentrations. The purpose of the study reported here was to confirm or refute the supposition that hyperbilirubinemia causes falsely increased refractometric serum total protein concentrations. Because a variety of refractometers are available for a clinical laboratory, 3 refractometers were used and the refractometric data were compared with data from a biuret assay–based automated spectrophotometric method. Materials and Methods Blood collection—Venous blood samples were collected with syringes from 2 healthy Greyhounds from which food had been withheld overnight and then transferred to evacuated tubes. After approximately 30 minutes, tubes were centrifuged and nonlipemic and nonhemolyzed sera were harvested. Bilirubin stock solution and preparation of icteric sera—Bilirubin powdera (40 mg) was dissolved in 10 mL of 0.1M NaOH25,26 to prepare a stock bilirubin solution of nearly 400 mg/dL. Diluted stock solutions were mixed with a constant volume of sera (1:9 bilirubin solution mixed with serum) to create bilirubin concentrations near 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, and 40 mg/ dL. The 0.1M NaOH solution was mixed with serum in the same ratio. Refractometric total protein concentration measurements—Three temperature-compensated refractometers were used: a refractometerb (refractometer 1) with total protein concentration (g/dL) and refraction scales, a refractometerc (refractometer 2) with a TS% scale, and a refractometerd (refractometer 3) with a total protein concentration scale (g/dL). Each refractometer was calibrated with deionized water to 1.000 on its urine specific gravity scale prior to analyzing samples. Because a conversion table for refractometer 2c was not found in the literature, its TS% values were first converted to refraction values (r) with the aid of a conversion table reported by Wolf 22 and then to total protein concentrations in 4 ways: the conversion table,22 a conversion table27 for refractometer 1, a canine conversion formula,17 and a multispecies conversion formula.17 To assess the analytic precision of the refractometric methods, 2 investigators (AG and a medical technologist) determined refractometric values in the unspiked 64
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serum samples and the mean of those values was calculated. This process was performed 8 times for unspiked sera of 2 dogs. For the assessment of bias, the refractometric values of each unspiked and spiked sample were determined by the same 2 investigators with each refractometer. The mean values of the 2 readings were recorded for data analysis. Clinical biochemical assays for serum total protein, total bilirubin, and direct bilirubin concentrations—Clinical biochemical assays were completed on the day of blood collection with an automated analyzere and the manufacturer’s reagents. The total protein assay (2-point-end, bichromatic biuret assay) was used to measure total protein concentrations in the unspiked serum samples. During the previous 5 months and including the weeks of this study, the CV for the biuret assay was 1.5% for commercial level 1 control serum (mean ± SD concentration, 6.0 ± 0.1 g/dL) and 1.5% for commercial level 2 control serum (mean concentration, 4.4 ± 0.1 g/dL). Unspiked sera of both dogs were assayed 8 times, and the mean concentration for each dog was calculated. The total bilirubin assay was a modification of the Malloy-Evelyn method,f and the direct bilirubin assay was based on the Jendrassik-Grof procedure; automatic dilutions were completed for samples with bilirubin concentrations > 35 mg/dL. Statistical analysis—The CV (%) was calculated for the repeated refractometric and spectrophotometric measurements of total protein concentrations in sera from both dogs. Difference plots were created to assess agreement among the refractometric total protein concentrations. Results Analytic precision of refractometric and spectrophotometric total protein assay methods—For the unspiked canine sera of the 2 dogs, the mean refractometric total protein concentrations were 6.5 g/dL (CV, 0.0%) and 6.2 g/dL (CV, 0.0%) for refractometer 1, 6.2 g/dL Table 1—Refractometric total protein concentrations in unspiked sera from 2 healthy Greyhounds, derived from TS% values obtained with a refractometer with a TS% scale (refractometer 2), which was chosen as the comparative method for refractometer difference plots because total protein concentrations obtained with a conversion table22 agreed with spectrophotometric assay total protein concentrations (provided for comparison). Variable
Dog 1
Dog 2
TS% (g/100 g X 100%) from refractometer 2 Refraction (conversion table from Wolf22) Total protein concentration Conversion table from Wolf22 (g/dL) Conversion table27 for refractometer 1 with a total protein concentration (g/dL) and a refraction scale (g/dL) Canine formula* (g/dL) Multispecies formula† (g/dL) Biuret assay total protein concentration (g/dL)
7.6 0.0139
7.3 0.0133
6.2 6.3
5.8 6.0
5.8 6.3 6.2
5.5 6.0 5.8
*Formula for canine sera17: total protein concentration = (430 X r) – 0.20, where r is refraction. †Formula derived for the combination of bovine, canine, and equine sera17: total protein concentration = (482 X r) – 0.41. JAVMA, Vol 244, No. 1, January 1, 2014
The mean spectrophotometric total protein concentrations in the unspiked sera were 6.2 g/dL (CV, 1.4%) and 5.8 g/dL (CV, 1.4%) for the 2 dogs, respectively. The CV percentages were consistent with the laboratory’s daily quality control data for level 1 and level 2 control sera. Agreement among refractometric total protein concentrations—For refractometer 2, different total protein concentrations were derived by different methods (Table 1). Because the refractometer 2 total protein concentrations determined with the conversion table reported by Wolf22 agreed with the spectrophotometric assay total protein concentrations, refractometer 2 was used as the comparative method for the refractometric difference plots. The biases of refractometric methods for total protein concentrations of serum samples from only 1 dog are reported (Figure 1); similar biases were found in sera from the other dog. Effects of increased bilirubin concentrations on refractometric total protein concentrations—The measured total bilirubin concentration in the NaOHspiked serum sample was 0.1 mg/dL. The measured total bilirubin concentrations in bilirubin-spiked samples 1 through 12 were 0.9, 1.7, 2.9, 4.6, 5.6, 6.8, 10.7, 15.1, 21.8, 27.7, 32.7, and 41.5 mg/dL, respectively (Figure 2). Direct bilirubin concentrations in bilirubin-spiked samples 1 through 12 were 0.3, 0.5, 0.4, 0.5, 0.5, 0.5, 0.6, 0.5, 0.6, 0.6, 0.7, and 0.8 mg/dL, respectively. Because the same biases and analytic precision values were consistently found in samples from both dogs, the bilirubin effects on the refractometric total protein concentrations for serum samples from only 1 dog are reported. For that dog, when samples were spiked with only NaOH (1:9 NaOH and serum), thus diluting the unspiked protein concentration, the spiked sample’s total protein concentrations were 5.7 g/dL for refractometer 1, 5.4 g/ dL for refractometer 2, and 5.5 g/dL for refractometer 3. With increasing bilirubin concentrations (up to 41.5 mg/ dL), the total protein concentrations varied from 5.3 to 5.5 g/dL for refractometer 1, 5.1 to 5.3 g/dL for refractometer 2, and 5.4 to 5.6 g/dL for refractometer 3 (Figure 2).
Figure 1—Difference plots of the refractometric total protein concentrations of unspiked and spiked serum samples (n = 14) of 1 of 2 healthy Greyhounds in a study to determine whether increased serum bilirubin concentrations interfere with the measurement of serum total protein concentration by refractometry and to assess potential biases among measurements obtained with 3 temperature-compensated refractometers: a refractometerb (refractometer 1) with total protein concentration (g/dL) and refraction scales, a refractometerc (refractometer 2) with a TS% scale, and a refractometerd (refractometer 3) with a total protein concentration scale (g/dL). Refractometric total protein concentrations determined with refractometer 2 were considered the comparative values (x-axis). For the x-axis, the highest protein concentration (5.8 g/dL) in each graph represents the concentration in the unspiked sample; the lower concentrations (5.1 to 5.4 g/dL) represent samples after they were spiked and thus had diluted protein concentrations. The numbers within circles represent number of values represented by a plotted point. Concentrations for both refractometer 3 (A) and refractometer 1 (B) had positive biases (approx 0.2 g/dL; dashed lines), compared with the refractometer 2 concentrations. JAVMA, Vol 244, No. 1, January 1, 2014
Figure 2—Line graph of bilirubin concentrations (diamonds) and refractometer 1 (triangles), refractometer 2 (squares), and refractometer 3 (crosses) total protein concentrations in sera of the same dog as in Figure 1 spiked with NaOH only or with increasing amounts of bilirubin up to a total bilirubin concentration of 41.5 mg/dL. The differences in the refractometric total protein concentrations among the refractometers reflect their analytic biases; the variations in concentrations from samples 1 to 12 reflect the methods’ analytic precisions. Scientific Reports
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(CV, 1.2%) and 5.8 g/dL (CV, 0.9%) for refractometer 2, and 6.5 g/dL (CV, 0.6%) and 6.2 g/dL (CV, 0.0%) for refractometer 3. On the basis of these findings and the refractometric scales being marked at 0.1 g/dL (refractometer 1 and refractometer 3) or 0.1 g/100 g (refractometer 2) intervals, any change to total protein concentration in the bilirubin-spiked samples > 0.2 g/dL was accepted as representing a true change.
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Table 2—Contributions of major solutes (in various units) to serum TS% (g/100 g X 100%) when there are physiologic concentrations of the solutes.* Solute Total protein Glucose Urea Na+ Cl− Na+ + Cl− Bilirubin H2O Sum of solutes TS%
Common units
g/dL of serum
g/dL of serum H2O†
7.0 g/dL 100 mg/dL 60 mg/dL§ 150 mmol/L 110 mmol/L — 1 mg/dL — — —
7.0 0.100 0.060 0.345|| 0.391|| 0.736 0.001 93.8¶ — —
7.5 0.107 0.064 — — 0.784 0.001 — 8.4 —
g/100 g‡ 7.3 0.1 0.1 — — 0.8 < 0.1 — — 8.2
*Original solute concentrations and the calculated TS% are reported with appropriate significant figures. Excess figures are present in some calculated values so that minor changes are seen. †Value in column derived by dividing value in previous column by 0.938. ‡Converting solute weight per 102.2 g of solution (solute weight + H2O weight) to solute weight per 100 g of solution. §Equals a urea nitrogen concentration of 28 mg/dL or 10 mmol/L. ||Converted by use of relative molecular mass of Na+ (23 g/mol) and Cl− (35.5 g/ mol). ¶When serum total protein concentration is 7.0 g/dL, the serum water concentration is 93.8 g/dL.22 — = Not applicable. Table 3—Contributions of solutes (in various units) to serum TS% (g/100 g X 100%) when there are increased concentrations of glucose, urea, or bilirubin.* Solute
Common units
g/dL of serum
g/dL of serum H2O†
Total protein Glucose Urea Bilirubin
7.0 g/dL§ 600 mg/dL 600 mg/dL¶ 40 mg/dL
7.0 0.600 0.600 0.040
7.5 0.640 0.640 0.043
g/100 g‡ 7.2/100 0.6/100|| 0.6/100|| < 0.1/100
*Original solute concentrations and the calculated soluble-solids concentrations are reported with appropriate significant figures; additional significant figures were left in some calculated values so that minor changes are seen. †See Table 2 for the conversion of concentrations; each calculation assumed a total protein concentration of 7.0 g/dL and thus a serum water concentration of 93.8 g/dL. ‡Converting solute weight per 101.9 g of solution to solute weight per 100 g of solution, assuming there is an increase in glucose concentration or urea concentration and not both. §A typical serum protein concentration measured with a biuret assay. ¶Equals a urea nitrogen concentration of 280 mg/dL or 100 mmol/L. ||A serum glucose concentration of 700 mg/dL and a urea nitrogen concentration of 300 mg/dL (urea concentration, 643 mg/dL) have been reported to increase the refractometric total protein concentration by 0.6 g/dL.29
Discussion This study found that total bilirubin concentrations up to 41.5 mg/dL did not affect refractometric estimations of serum total protein concentrations, in that there were no detected differences in samples 1 to 12. The minor variations in concentrations obtained with each refractometer reflected the analytic precision of the methods. On the basis of the measured concentrations, nearly all the bilirubin in the spiked samples was considered unconjugated bilirubin. The apparent presence of direct bilirubin (ie, conjugated or delta bilirubin) may have reflected unconjugated bilirubin reacting in the direct diazo reaction.28 The refraction of light by serum is affected by the concentrations of some solutes (eg, proteins, glucose, urea, electrolytes, and lipoproteins),22,24 but marked hyperbilirubinemia does not cause a detectable change in 66
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the serum refractive index. The reason for the apparent higher total protein concentrations in the NaOH-only samples (approx 0.2 g/dL), compared with samples 1 to 12, was not determined. The refractive index of a liquid is proportional to TS%. This key concept is based on a property of solutes called refractivity (R) that is calculated with the following formula: R = r/TS%
As stated by Wolf,22 “This relationship is meaningful because the refractivity is relatively constant over wide ranges of concentrations of any solute.” The clinical refractometer measures a solution’s refractive index, which is used to determine the solution’s refraction, and thus it is useful to rephrase the equation in that context: r = R X TS%
Different solutes have different refractivity values, but the major solutes of serum have similar values (human serum protein, R = 19; urea, R = 15; glucose, R = 14; and sodium chloride, R = 17).22 A refractivity value for bilirubin was not found in the literature. Accordingly, change in serum refraction is mostly attributable to change in the TS%. In serum or plasma, the largest portion (by weight) of the total soluble solids is the protein content. Thus, proteins are the major contributors to the refraction of serum or plasma. To understand the relative contribution of solutes to TS%, it is necessary to express concentrations of common serum solutes in terms of their weights and not other common units of concentration. The sum of the solutes’ ratio (wt/wt) expression X 100% represents a TS% (eg, 8.2 g/100 g X 100% = 8.2%). If there are physiologic concentrations of all serum solutes and the total protein concentration is 7.0 g/dL, the TS% is expected to be 8.4%.22 Physiologic concentrations of sodium and chloride contribute almost 0.8% (0.8 g of solute/100 g of solution) to the TS%, whereas physiologic concentrations of glucose and urea contribute approximately 0.1% each. Most other nonprotein serum solutes contribute negligible amounts to TS%. Major solutes that contribute to serum TS% and the conversion of the common clinical concentrations to g/100 g were tabulated (Table 2). Marked hyperglycemia or marked azotemia can increase the TS% and thus falsely increase the refractive total protein concentration (Table 3).29 However, even with an extreme increase in serum bilirubin concentration, the calculated increase in TS% is too small (ie, < 0.1%) to affect the serum’s refractive index and thus would not falsely increase the refractive total protein concentration. This calculated prediction was confirmed by the measurements of this study. The bilirubin powder used to spike sera contained unconjugated bilirubin, as determined by evaluation of the total and direct bilirubin concentrations, yet none of the various forms of bilirubin should affect the TS%. When it is stated that hyperbilirubinemia causes a falsely increased refractometric total protein concentration, that statement is in error, and the reason for the JAVMA, Vol 244, No. 1, January 1, 2014
a. b. c. d. e. f.
Sigma-Aldrich, St Louis, Mo. TS Meter Refractometer, model 10400A, Leica Microsystems Inc, Buffalo, NY. TS Meter Refractometer, model 10400B, Leica Microsystems Inc, Buffalo, NY. Veterinary Total Solids Refractometer, model 10436, Leica Microsystems Inc, Buffalo, NY. Cobas c501, Roche Diagnostics, Indianapolis, Ind. Wahlefeld AW, Herz G, Bernt E. Modification of the MalloyEvelyn method for a simple, reliable determination of total bilirubin in serum (abstr). Scand J Clin Lab Invest 1972;29(suppl 126):11.12.
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Meyer DJ, Harvey JW. Evaluation of plasma proteins. In: Meyer DJ, Harvey JW, eds. Veterinary laboratory medicine: interpretation and diagnosis. 3rd ed. St Louis: Saunders, 2004;156–168. Jain NC. Hematologic techniques. In: Jain NC, ed. Schalm’s veterinary hematology. 4th ed. Philadelphia: Lea & Febiger, 1986;20–86. Benjamin MM. Protein. In: Benjamin MM, ed. Outline of veterinary clinical pathology. 3rd ed. Ames, Iowa: Iowa State University Press, 1978;108–120. Evans EW, Duncan JR. Proteins, lipids, and carbohydrates. In: Latimer KS, Mahaffey EA, Prasse KW, eds. Duncan and Prasse’s veterinary laboratory medicine: clinical pathology. 5th ed. Ames, Iowa: Iowa State Press, 2011;173–209. Kaneko JJ. Serum proteins and the dysproteinemias. In: Kaneko JJ, ed. Clinical biochemistry of domestic animals. 4th ed. San Diego: Academic Press, 1989;142–165. Kaneko JJ. Serum proteins and the dysproteinemias. In: Kaneko JJ, Harvey JW, Bruss ML, eds. Clinical biochemistry of domestic animals. 5th ed. San Diego: Academic Press, 1997;117–138. Allison RW. Laboratory evaluation of plasma and serum proteins. In: Thrall MA, Weiser G, Allison RW, et al, eds. Veterinary hematology and clinical chemistry. 2nd ed. Ames, Iowa: WileyBlackwell, 2012;460–475. McGrotty Y, Tennant K. Disorders of plasma proteins. In: Villiers E, Blackwood L, eds. BSAVA manual of canine and feline clinical pathology. 2nd ed. Gloucester, England: British Small Animal Veterinary Association, 2005;99–112. Lassen ED. Laboratory evaluation of plasma and serum pro-
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teins. In: Thrall MA, Baker DC, Campbell TW, et al, eds. Veterinary hematology and clinical chemistry. Philadelphia: Lippincott Williams & Wilkins, 2004;401–415. Werner LL, Turnwald GH, Willard MD. Immunologic and plasma protein disorders. In: Willard MD, Tvedten HW, eds. Small animal clinical diagnosis by laboratory methods. 4th ed. St Louis: Saunders, 2004;290–305. Thomas JS. Overview of plasma proteins. In: Feldman BF, Zinkl JG, Jain NC, eds. Schalm’s veterinary hematology. 5th ed. Philadelphia: Lippincott Williams & Wilkins, 2000;891–898. Johnson AM, Rohlfs EM, Silverman LM. Proteins. In: Burtis CA, Ashwood ER, eds. Tietz textbook of clinical chemistry. 3rd ed. Philadelphia: WB Saunders Co, 1999;477–540. McPherson RA. Specific proteins. In: Henry JB, ed. Clinical diagnosis and management by laboratory methods. 20th ed. Philadelphia: WB Saunders Co, 2001;249–263. Lindsey BJ. Amino acids and proteins. In: Bishop ML, DubenEngelkirk JL, Fody EP, eds. Clinical chemistry: principles, procedures, correlations. 4th ed. Philadelphia: Lippincott Williams & Wilkins, 2000;151–184. Wu AHB. Tietz clinical guide to laboratory tests. 4th ed. St Louis: Saunders, 2006. Lee-Lewandrowski E, Lewandrowski K. The plasma proteins. In: McClatchey KD, ed. Clinical laboratory medicine. Baltimore: Williams & Wilkins, 1994;239–258. Sutton RH. The refractometric determination of the total protein concentration in some animal plasmas. N Z Vet J 1976;24:141– 148. Briend-Marchal A, Medaille C, Braun JP. Comparison of total protein measurement by biuret method and refractometry in canine and feline plasma. Rev Med Vet (Toulouse) 2005;156:615–619. Schalm OW. The Goldberg refractometer or TS meter. Calif Vet 1965;19:28–30. Barry KG, McLaurin AW, Parnell BL. A practical temperaturecompensated hand refractometer (the TS-meter). Its clinical use and application in estimation of total serum proteins. J Lab Clin Med 1960;55:803–808. Garner BC, Priest H, Smith J. Pseudo-hypoproteinemia in a hyperbilirubinemic dog with immune-mediated hemolytic anemia. Vet Clin Pathol 2013;43:in press. Wolf AV. Aqueous solutions and body fluids. Their concentrative properties and conversion tables. New York: Hoeber Medical Division, Harper & Row, 1966. Goldberg HE. Principles of refractometry. Buffalo, NY: Leica Inc, 1997. George JW. The usefulness and limitations of hand-held refractometers in veterinary laboratory medicine: an historical and technical review. Vet Clin Pathol 2001;30:201–210. Martínez-Subiela S, Tecles F, Montes A, et al. Effects of haemolysis, lipaemia, bilirubinaemia and fibrinogen on protein electropherogram of canine samples analysed by capillary zone electrophoresis. Vet J 2002;164:261–268. Weber JA, van Zanten AP. Interferences in current methods for measurements of creatinine. Clin Chem 1991;37:695–700. American Optical Scientific Instrument Division. Instructions for use and care of the AO TS meter (a Goldberg refractometer). Buffalo, NY: American Optical, 1976. Tisdale WA, Klatskin G, Kinsella ED. The significance of the direct-reacting fraction of serum bilirubin in hemolytic jaundice. Am J Med 1959;26:214–227. Silverman LM, Christenson RH. Amino acids and proteins. In: Burtis CA, Ashwood ER, eds. Tietz textbook of clinical chemistry. 2nd ed. Philadelphia: WB Saunders Co, 1994;625–734. With TK. Spectral absorption of bilirubin. Measurements in pure aqueous solutions and in solutions containing human serum. Acta Physiol Scand 1945;10:172–180.
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error is usually unclear but might be based on concepts of photometry. Bilirubin is a pigmented compound that will absorb light wavelengths near 455 nm.30 However, for clinical refractometry, a broad range of visible light wavelengths (approx 380 to 740 nm) enter the refractometer and only a narrow band of wavelengths are absorbed by the bilirubin. Thus, most of the visible light is not affected by the pigment and it does not affect the measured refraction. Total protein concentrations derived for refractometer 2 (with the conversion table reported by Wolf22) agreed poorly with the concentrations derived for refractometer 1 and refractometer 3 and with other methods of converting refractive index values. However, the increasing bilirubin concentrations did not affect the biases. This study’s data provide evidence that the refractometric total protein concentration is dependent on the method of converting a refractive index value to a total protein concentration.
