Seeing is not Believing (13-Nov-2004)

In: 55th Annual Meeting of the American College of Veterinary Pathologists (ACVP) & 39th Annual Meeting of the American Society of Clinical Pathology (ASVCP), ACVP and ASVCP (Eds.) Publisher: American College of Veterinary Pathologists & American Society for Veterinary Clinical Pathology, Middleton WI, USA Internet Publisher: Publisher: International Veterinary Information Service (www.ivis.org), Ithaca, New York, USA. Seeing is not Believing (13-Nov-2004) L. Shanahan Department of Clinical Pathology, Colorado State University, Fort Collins, CO, USA. There are common as well as uncommon interferences in routine chemistry testing. Sample integrity indicators (hemolysis, lipemia and icterus indices) are important roadmaps to valid test results, and should always be included in a chemistry panel as well as with an individual chemistry request. There are also disease states which can cause spurious chemistry results. This will be a discussion of the chemistry clues which can help in test interpretation and lead to more accurate diagnoses. Case Studies: Feline Cases Test Requested Case 1 Case 2 Case 3 Feline Ref Intervals Glucose 163 118 116 69-136 mg/dl BUN 34 55 50 16-35 mg/dl Creatinine 2.3 4.1 3.5 1.3-2.6 mg/dl Phosphorus 5.4 3.1 4.0 2.2-6.5 mg/dl Calcium 11.6 10.8 13.8 8.7-11.1 mg/dl Magnesium 3.2 2.1 2.4 1.8-3.0 mg/dl Total Protein 20.0 11.0 9.4 6.0-8.7 gm/dl Albumin 3.1 3.1 3.7 2.8-4.2 gm/dl Globulin 16.9 7.9 5.7 2.8-5.3 gm/dl A/G Ratio 0.2 0.4 0.6 0.6-1.4 ratio Cholesterol 77 113 127 70-250 mg/dl T-Bilirubin 0.3 32.3 5.7 0-0.2 mg/dl CK 264 60 543 60-350 IU/L ALP 17 13 13 11-61 IU/L ALT 94 103 98 25-120 IU/L AST 77 39 50 12-46 IU/L GGT 0 0 0 0-4 IU/L Sodium 124 146 145 148-158 meq/l Potassium 3.2 4.1 4.3 3.5-5.2 meq/l Chloride 97 113 115 114-124 meq/l Bicarbonate 16.8 17.2 15.5 13-22 meq/l Anion Gap 13 20 19 18-27 Calc Lipemia Index 5 35 8 0-40 mg/dl Hemolysis Index 23 14 8 0-60 mg/dl Icterus Index 0 0 0 0-0 mg/dl Tech Comments

Case Studies: Feline Cases Corrected Test Requested Case 1 Case 2 Case 3 Feline Ref Intervals Glucose 163 118 116 69-136 mg/dl BUN 34 55 50 16-35 mg/dl Creatinine 2.3 4.1 3.5 1.3-2.6 mg/dl Phosphorus 5.4 3.1 4.0 2.2-6.5 mg/dl Calcium 11.6 10.8 1 3.8 8.7-11.1 mg/dl Magnesium 3.2 2.1 2.4 1.8-3.0 mg/dl Total Protein 20.0 11.0 9.4 6.0-8.7 gm/dl Albumin 3.1 3.1 3. 7 2.8-4.2 gm/dl Globulin 16.9 7.9 5.7 2.8-5.3 gm/dl A/G Ratio 0.2 0. 4 0.6 0.6-1.4 ratio Cholesterol 77 11 3 12 7 70-250 mg/dl T-Bilirubin 0.3 1.8 0. 4 0-0.2 mg/dl CK 264 60 543 60-350 IU/L ALP 17 13 13 11-61 IU/L ALT 94 103 98 25-120 IU/L AST 77 39 50 12-46 IU/L GGT 0 0 0 0-4 IU/L Sodium 159 154 145 148-158 meq/l Potassium 4.3 4.1 4.3 3.5-5.2 meq/l Chloride 98 113 115 114-124 meq/l Bicarbonate 16.8 17.2 15.5 13-22 meq/l Anion Gap 49 28 19 18-27 Calc Lipemia Index 5 35 8 0-40 mg/dl Hemolysis Index 23 14 8 0-60 mg/dl Icterus Index 0 0 0 0-0 mg/dl Tech Comments Electrolyte results from ABL505 & Chloridometer T-Bili result from dilution. Na result from ABL 505 T-Bili result from dilution. Globulin interference Case Studies: Canine Case Studies Test Requested Case 1 Case 2 Case 3 Case 4 K9 Ref Intervals Glucose 103 82 109 99 75-130 mg/dl BUN 41 23 24 17 7-32 mg/dl Creatinine 1.7 0.8 1.3 1.4 0.7-1.8 mg/dl Phosphorus 4.8 4.4 7.3 4.9 2.1-6.0 mg/dl Calcium 10.3 9.0 10.1 10.9 9.2-11.7 mg/dl Magnesium 2.0 1.7 1.8-2.5 mg/dl Total Protein 7.5 11.3 12.3 5.9 5.3-7.2 gm/dl Albumin 2.8 1.4 1.6 3.3 2.5-4.0 gm/dl Globulin 4.7 9.9 10.7 2.6 1.9-3.4 gm/dl A/G Ratio 0.6 0.1 0.1 1.3 0.9-2.1 ratio Cholesterol 227 174 98 196 130-300 mg/dl T-Bilirubin 0.2 0.2 0.1 0.1 0.0-0.3 mg/dl CK 58 85 110 73 50-275 IU/L ALP 481 179 60 53 20-142 IU/L

Case Studies: Canine Case Studies Test Requested Case 1 Case 2 Case 3 Case 4 K9 Ref Intervals ALT 84 24 77 72 10-110 IU/L AST 28 28 39 38 16-50 IU/L GGT 11 2 4 5 0-9 IU/L Sodium 141 145 149 150 142-152 meq/l Potassium 4.6 3.9 4.0 4.6 3.5-5.2 meq/l Chloride 109 111 112 116 108-120 meq/l Bicarbonate 17.3 25.5 18.2 21.4 16-25 meq/l Anion Gap 19 12 23 17 13-22 Calc Lipemia Index 15 29 1 32 0-40 mg/dl Hemolysis Index 30 13 13 11 0-60 mg/dl Icterus Index 0 0 0 0 0-0 mg/dl Bile Acids 70 <6 umol/l Tech Comment Case Studies: Canine Cases Corrected Test Requested Case 1 Case 2 Case 3 Case 4 K9 Ref Intervals Glucose 103 82 109 99 75-130 mg/dl BUN 41 23 24 17 7-32 mg/dl Creatinine 1.7 0.8 1.3 1.4 0.7-1.8 mg/dl Phosphorus 4.8 4.4 7.3 4.9 2.1-6.0 mg/dl Calcium 10.3 9.0 10.1 10.9 9.2-11.7 mg/dl Magnesium 2.0 1.7 1.8-2.5 mg/dl Total Protein 7.5 11.3 12.3 5.9 5.3-7.2 gm/dl Albumin 2.8 1.4 1.6 3.3 2.5-4.0 gm/dl Globulin 4.7 9.9 10.7 2.6 1.9-3.4 gm/dl A/G Ratio 0.6 0.1 0.1 1.3 0.9-2.1 ratio Cholesterol 227 174 98 196 130-300 mg/dl T-Bilirubin 0.2 0.2 0.1 0.1 0.0-0.3 mg/dl CK 58 85 110 73 50-275 IU/L ALP 481 179 60 53 20-142 IU/L ALT 84 24 77 72 10-110 IU/L AST 28 28 39 38 16-50 IU/L GGT 11 2 4 5 0-9 IU/L Sodium 146 150 149 150 142-152 meq/l Potassium 5.0 3.9 4.0 4.6 3.5-5.2 meq/l Chloride 109 111 112 116 108-120 meq/l Bicarbonate 17.3 25.5 18.2 21.4 16-25 meq/l Anion Gap 19 12 23 17 13-22 Calc Lipemia Index 15 29 1 32 0-40 mg/dl Hemolysis Index 30 13 13 11 0-60 mg/dl Icterus Index 0 0 0 0 0-0 mg/dl Bile Acids 70 <6 umol/l Tech Comment Electrolytes from ABL 505 Na result from ABL505 Phos may be falsely elevated due to immunoglobulins No evidence of disease

Common Interferences RBC Contamination - Inadequate centrifugation of serum/plasma or poor technique in removal of serum/plasma from the red blood cells can cause RBC contamination in the sample. If this contamination is significant, it can affect the following tests: AST, ALT, Magnesium, Total Protein, UIBC, Potassium, Iron and Phosphorus [1]. An example follows which shows how important it is to obtain a good sample once the blood has been submitted to the laboratory: Initial Spin Sample Re-Spun Sample H Index 0 H Index 13 L Index 241 L Index 6 I Index 0 I Index 0 ALT 455 ALT 425 AST 47 AST 105 CK 115 CK 86 GGT 41 GGT 33 Note - The serum indices ( particularly the hemolysis and lipemia index) are the clues to the poor sample quality. If there are red cells in the sample, the hemolysis index will be normal even though the sample is blood tinged in appearance, and the lipemia index will be elevated due to the haziness from the red blood cells in the serum. Hemolysis - Hemolysis, either from poor collection technique, age of the sample, or poor handling of the sample once in the laboratory, has a serious effect on many chemistry tests. Tests affected most dramatically are the enzymes ALP and GGT and CK. A new sample should be obtained if there is gross hemolysis. The chart "Interpretation Guide for Serum Indexes on Hitachi Systems" reflects interference by hemolysis and lipemia and icterus on Roche methods analyzed on a Hitachi 917. The same interferences can be seen on many chemistry analyzers, because methodologies are the same [2]. Lipemia and Icterus - The amount of lipemia and/or icterus in a sample does not affect as many chemistry tests as does hemolysis, but their interference can be very significant. Icterus greater than 10mg/dl has a negative impact on creatinine, cholesterol, and triglycerides. Icterus also affects Amylase, Fructosamine, and Uric Acid. Obtaining a new sample will not alter the amount of icterus present, so educating the clinician regarding the interpretation of serum index values is the only alternative. Lipemia greater than 500 mg/dl causes a "volume exclusion effect", which lowers the analyte concentrations in the sample. When serum/plasma contains large concentrations of lipid or paraprotein, these extra components occupy volume and displace water. When this happens the serum/plasma contains less water per unit volume and therefore less analytes per unit volume [3]. This effect causes spurious electrolyte results, particularly sodium. If the electrolytes are measured indirectly, there is a predictable decrease in the electrolyte results. Indirect electrolyte measurements involve dilution of the sample with a diluent (i.e., Hitachi 917 analyzes 15 ul of sample with 450 ul of a diluent buffer). Waugh s equation [4] uses the total serum protein concentration and the serum triglyceride concentration to calculate the correct serum/plasma water content. The greater the lipemia or hyperproteinemia, the greater the decrease in the water content, and the greater the decrease in the electrolyte evaluated. A correct result can be obtained by measuring the electrolytes directly (i.e., using a blood gas analyzer such as a Radiometer America or NOVA). The direct measurement analyzes the sample with no intermediate dilution step. Lipemia can also be significantly removed by ultracentrifugation (>10,000 rpm for 5 minutes). The lipemia rises to the top, and the serum/plasma can be carefully removed and analyzed. Note the examples below. Before Lipid Removal After Lipid Removal No Lipid Removal Indirect ISE measurement Indirect ISE measurement Direct ISE measurement Lipemia Index 995 Lipemia Index 137 Lipemia Index 995 Na 148 Na 145 Na 151 K 3.9 K 3.8 K 4.0 Cl 110 Cl 108 CL 109

Other Tests Affected by this Grossly Lipemic Sample Before Lipid Removal After Lipid Removal AST 4 abs? AST 31 ALT 82 abs? ALT 142 ABS! Data flags for turbidity [1] AL T/IFCC AST/IFCC Bicarbonate BUN Interferences from Disease States Monoclonal Gammopathy: Feline Case Study #1, #2, Canine Case Study #1, #2 Monoclonal immunoglobulins, which occur typically in patients with a plasma cell dyscrasia such as Multiple Myeloma or Waldenstrom s Macroglobulinemia, can cause numerous erroneous results in routine chemistry testing. One such effect is the previously mentioned volume exclusion effect, which primarily affects electrolyte testing in automated chemistry analyzers. The serum/plasma contains large concentrations of the immunoglobulin, which displaces water containing the electrolyte to be measured. Thus, the electrolyte values are falsely decreased. Monoclonal Gammopathy: Canine Case Study #3 Feline Case Study #2, #3 Monoclonal immunoglobulins also cause erroneous results through a variety of other mechanisms. 1. The immunoglobulin can precipitate during analysis, causing turbidity. Note the elevated phosphorus result in K9 case study #3. Falsely elevated results were caused by precipitation of the immunoglobulins in the reagent-sample mixture. Dilution of the sample showed no linearity, and the results were not reliable. The use of an alternative method such as an enzymatic or modified molybdate method is recommended [5-7]. Also note the elevated total bilirubin in feline case study #2 and #3. Roche Biochemicals warns that patients with multiple myeloma may show a positive bias with their bilirubin methodology, but the bias and the severity may vary. This bias might also be due to the immunoglobulin precipitating with the Diazo reagent. The clue to this abnormality was the Icterus serum index of 0. The icterus index and the bilirubin value correlate very closely, so an icterus index of 5 or 6 would have been expected, and the physical appearance of the serum would have been "icteric". 2. The high viscosity of the serum/plasma sample due to the increased immunoglobulin can cause inaccurate pipetting by the analyzer, and thus a "short" sample. 3. Reagents can be inactivated because of binding to the immunoglobulin. These errors are very difficult to detect. Some of the chemistry tests prone to interference by hyperparaproteinemia include: Albumin, Phosphorus, Creatinine, BUN, Total Bilirubin, Uric Acid, Thyroxine, C Reactive Protein, and Sodium [8-11]. When marked hyperproteinemia is observed on any sample, the results should be reviewed and questionable values should be repeated by alternate methods. All laboratory personnel should be alert to these situations. Other Chemistry Observations Small Intestine Bacterial Overgrowth: Canine Case Study #4 Humans and animals, with no evidence of hepatic disease, have been observed to have elevated serum bile acids. It has been noted in some of these cases that there is a small intestinal bacterial overgrowth (SIBO) [12]. In the absence of primary disease, the signs of SIBO may be subclinical or have chronic symptoms of diarrhea and/or weight loss. The excess bacteria increases the rate of deconjugation of bile acids in the small intestine, which may result in increases in the circulating

unconjugated bile acids in the serum. Because these unconjugated bile acids have reduced efficiency they can accumulate and result in an increase in the total bile acids concentration in the serum. These physical and biochemical changes can be improved by antibiotic administration and dietary supplementation. Conclusion Laboratory professionals, pathologists, and clinicians have to be constantly alert in identifying potentially erroneous laboratory test results due to sample integrity issues and various disease states, such as hyperproteinemia. Education and intensive review of results can lead to accurate diagnoses and appropriate treatment. References 1. Interpretation Guide for Serum Indexes on Hitachi Systems. Indianapolis, IN: Boehringer Mannheim Corporation, 1993. 2. Glick M, Ryder K, and Glick S. Interferographs: User s Guide to Interferences in Clinical Chemistry Instruments. Indianapolis, IN: Science Enterprises, Inc, 1991. 3. Scott MG, Heusel JW, LeGrys VA, et al. Chapter 31: Electrolytes and blood gases. In: Burtis CA, Ashwood EA, Tietz NW, Eds. Tietz Textbook of Clinical Chemistry, 3rd ed. Philadelphia: WB Saunders 1999:1061-1063. 4. Waugh WH. Utility of expressing serum sodium per unit of water in assessing hyponatremia. Metabolism 1969; 18:706-712. 5. Bakker AJ, Bosma H, Christen PJ. Influence of monoclonal immunoglobulins in three different methods for inorganic phosphorus. Ann Clin Biochem 1991; 28(Pt 2):196. 6. Zaman Z, Sneyers L, Van Orshoven A et al. Elimination of paraprotein interference in determination of plasma inorganic phosphate by ammonium molybdate method. Clin Chem 1995; 41:609-614. 7. Larner AJ. Pseudohyperphosphatemia. Clin Biochem 1995; 28:391-393. 8. Reed RG. Interference by an IgM paraprotein in the bromcresol green method for determination of serum albumin. Clin Chem 1987; 33:1075-1076. 9. Datta P, Graham GA, Schoen I. Interference by IgG paraproteins in the Jaffe method for creatinine determination. Am J Clin Pathol 1986; 85:463-468. 10. Pierce GF, Garrett NC, Koenig J, et al. Interference by monoclonal proteins in the o-phthalaldehyde method for blood urea nitrogen. Clin Chim Acta 1986; 154:233-236. 11. Yu A, Pira U. False increase in serum C-reactive protein caused by monoclonal IgM-Lambda: A case report. Clin Chem Lab Med 2001; 39:983-987. 12. Williams DA, Ruaux CG, Steiner JM, et al. Small intestinal bacterial overgrowth leading to high fasting serum total and unconjugated bile acids in beagle dogs with no evidence of hepatic disease. In: Proceedings of the 10th Congr Int Soc Anim Clin Chem 2002. All rights reserved. This document is available on-line at www.ivis.org. Document No. P1215.1104. This manuscript is reproduced in the IVIS website with the permission of the ACVP & ASVCP www.acvp.org