Hemolysis as a Factor in Clinical Chemistry and Hematology of the Dog

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<ul><li><p>Hemolysis as a Factor in Clinical Chemistry and Hematology of the Dog Shamn L. ONeill, M.S. Bernard F. Feldman, D.V.M., Ph.D. Department of Clinical Pathology School of Veterinary Medicine University of California, Davis Davis, California 95616 </p><p>S U M . . Y </p><p>bumin, all of which appemd to increase with increasing hemolysis. The results of testing for the remainder of the biochemical, hemostatic, and hematologic analytes vaned great&amp; among analyzen or methoris, m l y in a predictable manner, indicatingthat each laboratoy should evaluate the effects of hemolysis on each analyzer and for each method used, in order to make informed decisions on the use of hemolyzed, but irreplaceable, samples. </p><p>Pey words: hemolysis, canine, interferographs, serum biochemistry, hemogram, hemostatic analytes </p><p>Introduction Hemolyzed blood is often submitted to the veterinary </p><p>laboratory for testing and is a source of error in testing. Hemolysis is sometimes the result of a disease state but is most often due to sampling difficulty. Some animals, such as pigs, have increased erythrocyte fragility, par- ticularly when young. Other factors may ar t i f i d l y in- duce hemolysis, such as prolonged storage of the blood before separation of the serum, rapidly forcing blood through small needles, excessive agitation when mixing, the use of evacuated tubes which may collapse small veins or cause blood to enter the tube too vigorously, or the physical act of centrifugation and separation of sen1m.3~ </p><p>Hemolysis will not be apparent until the sample is centrifuged at the laboratory. In dogs, hemolysis can easi- ly be seen by eye when hemoglobin content of serum is 0.1 g/dl or greater. The laboratory may receive samples with hemolysis as much as 1.7 @dl, but more often in the range of 0.1 to 0 3 g/dl. Policy often dictates rejection of </p><p>hemolyzed samples without rationale, assuming they will be unsuitable for any analysis. Problems in obtaining another sample arise in the veterinary clinic when animals are too small, too ill, have already left the clinic, or when samples were part of a timed analysis. Arbitrari- ly rejecting hemolyzed samples may not be necessary in all cases. </p><p>Hemolysis causes an increase in analytes contained in erythrocytes. This may result in a serum increase in red cell constituents that are more concentrated in red cells than serum such as lactic dehydrogenase and aspartate aminotransferase. Conversely, if red cell damage is ex- cessive, constituents such as sodium, calcium and cho- lesterol, present in serum in higher concentration than red cells, could be diluted when hemolysis occurs? Hemolysis may interfere in other ways, sometimes directly increasing absorbance in the hemoglobin absor- bance spectral range (540 to 590 nm), acting directly on the chemical reactions of a method used, or slightly </p><p>PAGE 58 0 Vol, 18, No. 3 0 VETEaZNARY CLINICAL PATFIOLOGY </p></li><li><p>TABLE 1 Instrumentation </p><p>Instruments Used for Serum Biochemical Analytes </p><p>SMA-2C Technicon Instruments Corp., Tarrytown NY 10591 SMA 12160 Technicon Instruments Corp., Tarrytown, NY 10591 Model 8000 Bichromatic Analyzer Boehringer Mannheim Diagnostics, Inc., </p><p>Discrete Analyzer with Continuous Optical Scanning </p><p>Coulter Electronics, Inc., Hialeah, FL 33012 Coleman 91 Analyzer - Lipase only Perkin Elmer Corp., Norwalk, CT 06856 400E Spectrophotometer - </p><p>Creatine Phosphokinase, Amylase Gilford Instrument Laboratories, Inc., Oberlin, OH 44074 Colormetric Analyzer - Lipase only Chemtrix, Inc., Hillsboro, OR 97123 Chloridometer - Chlorides Buchler Instruments, Inc. Fort Lee, NJ 07024 Flame Photometer Model 143 - Sodium, Potassium Instrument Laboratories, Inc., Wilmington, MA 02173 Spectronic 700 - Cholinesterase only Bausch &amp; Lomb, Rochester, NY 14625 </p><p>Indianapolis, IN 46250 </p><p>(DACOS) </p><p>Instruments Used to Analyze Hemostatic Factors </p><p>and to Complete the Hemogram </p><p>BBL Fibrosystem - PT, APTT, lT, Fibrinogen, </p><p>Becton, Dickinson &amp; Company, Cockeysville, MD 21030 Spectronic 700 - Antithrombin Ill, Plasminogen Bausch &amp; Lomb, Rochester, NY 14625 Horizontal Electrophoresis Cell Model 1415 - </p><p>von Willebrands Factor Antigen BioRad Laboratories, Richmond, CA 94804 Counter Model ZBI with Hemoglobinometer - </p><p>Coulter Electronics, Inc., Hialeah, FL 33012 Electra 750 Coagulation Timer - PT, APTT, TT Medical Laboratory Automation, Inc., </p><p>Thrombotest, Factors V, VII, VIII, IX, X </p><p>RBC, WBC, Hemoglobin </p><p>Mount Vernon, NY 10550 </p><p>altering the pH in enzymatic reactions. Decisions need to be made as to what analyses are </p><p>most important, whether the presence of hemoglobin will interfere with the testing, and whether valid information can still be obtained from the affected sample. </p><p>Some researchers have assessed the effect of increas- ing hernoglobin on biochemical analytical testing using absolute values compared by anal* of variance,s per- cent error or a fold difference from the starting nonhe- molyzed concentration,7o or a graphical display of the data (termed interferographs) which easily allows an informed decision at a gIauce?qs </p><p>In this study, we have analyzed a set of hemolyzed sam- ples for biochemical, hematologic, and hemostatic analytes employing all the analyzers pertinent to our veterinary samples. Interferographs have been prepared to enable laboratorians to make decisions concerning hemolyzed samples. </p><p>Materials and Methods BIOCHEMISTRY </p><p>A blood sample was taken by syringe from the jugular vein of a healthy, fasted donor dog. Nonautiaqulated blood for chemistry was allowed to clot in siliconized glass tubes for 60 minutes, centrifuged at lo00 x g for 20 minutes and the serum aliquot4 into six smaller tubes. A portion of the whole blood was frozen at -60C for 30 minutes, defrosted 10 minutes at 37C and centrifuged 20 minutes at lo00 x g. Hemolysate was analyzed by Coul- ter hemoglobinometer (Coulter Electronics) for hemoglobin. Appropriate amounts of hemolysate, es- timated by calculation, were added to the serum aliquots to produce a series of samples with increasing hemolysis (0.0 to 2 5 g/dl hemoglobin), (Fig. 1). These hemolyzed samples were divided into four and submitted to analysis by different veterinary laboratories using four different analyzers - SMA-2C (Technicon Instruments), SMA 12/60 (Technicon Instruments) Model 8OOO BCA (Bi- chromatic Analyzer:Boehringer Mannheim Diagnos- tics), DACOS (Discrete Analyzer With Continuous Op- tical Scanning:Coulter Electronics). No correction for dilution was made in order to simulate a hemolytic event in practice. </p><p>Final hemoglobin content was determined on each sample by the cyanmethemoglobin method. Hemoglobin in all the original sera or plasmas was less than 0.1 g/dL The instruments and analyzers utilized in this study are listed in Table 1. Specific information showing which in- struments were used for each analyte can be found in Tables 2-6. </p><p>VETERINARY CLINICAL PATHOLOGY 0 Vol. 18, No. 3 0 PAGE 59 </p></li><li><p>compared percent of original analyte concentration (Final concentrationforiginal con- centration x 100%) with the content of added hemolysate. </p><p>HEMOGRAM Blood for complete blood </p><p>counts was drawn into a syringe containing 0.15% liq- </p><p>quoted into nine tubes. An- ticoagulated hemolysate was prepared as for biochemistry samples above. A series of EDTA containing whole blood samples of increasing hemo- lysis was prepared as above </p><p>(ranges from 0.0 to 3.5 g/dl). Concentration of hemo- globin was determined as described for biochemistry samples above. No correction for dilution was made. </p><p>uid potassium EDTA, and ali- </p><p>Flg. 1. - Visual hemolysis in samples submitted for biochemical analysis. Interferographs were prepared comparing the ability of </p><p>instruments to yield useful results when hemolysis is present for a variety of analytes. Graphical presentation </p><p>~ ~~ </p><p>190 </p><p>sz 180 </p><p>~ </p><p>a. TECHNICON SYA 12/80 b. </p><p>180 r I </p><p>240 230F / COULTERDACOS C. 210 </p><p>0 </p><p>UJ 180 U I 150 </p><p>BOEHRINGER MANNHEIM d. </p><p>/ / MODEL 8000 170 160 </p><p>- - </p><p>CHLORIDE- </p><p>80 70 </p><p>- - </p><p>*LREA NTROOEN 00 'TOTAL BILIRUBIN </p><p>'SODIUM -mTAssIw </p><p>- 50 40 I- </p><p>- </p><p>80 ' I I I 1 I I I I I 0 0.3 0.8 1.6 2.5 0 0.3 0.8 1.6 2.5 </p><p>HEMOLYSATE ADDED (g/dl Hemoglobin} HEMOLYSATE ADrJED (g/dl Hemoglobin) </p><p>Flg. 2 - lnterferographs comparlng the Mect of hemolysis on biochemical analytes by instrument. a. Technicon SMA-2C, b. Technicon SMA 12/60, c. Coulter DACOS, d. BMD Model 8OOO. (Note: CK = creatine kinase, LDH = lactic dehydrogenase, AST = aspartate aminotransferase, ALT = alanine aminotransferase.) </p><p>PAGE 60 0 Vol. 18. No. 3 0 VETERINARY CLTNICAL PATHOLOGY </p></li><li><p>TABLE 2 Serum Biochemical Analytes, Instrumentation, and Methods with Results of Analysis. Enzymes. </p><p>Sample (gldl Hemoglobin) Instrument Wavelength Analvte Dialysis (nml References 0.0 0.3 0.8 1.6 2.5 Units </p><p>Alanine Aminotransferase (ALT) W P T ) </p><p>Alkaline phosphatase </p><p>Amylase </p><p>Aspartate Aminotransferase (AST) (SGOT) </p><p>Creat i ni ne kinase (CK or CPK) y -Glutamyl </p><p>transferase (GGT) Lactate dehydrogenase </p><p>Lipase </p><p>Technicon SMA 2C Technicon SMA 12/60' BMD Model 8000 BCA CoulterlDacos Technicon SMA 2Ca Technicon SMA 12/60' BMD Model 8000 BCA Cou I terlDacos Technicon SMA 2C Gilford 400E BMD Model 8000 BCA Technicon SMA 2C Technicon SMA 12/60' BMD Model 8000 BCA Cou IterlDacos Technicon SMA 2Ca Gilford 400E Technicon SMA 2C </p><p>Technicon SMA 2C Technicon SMA 12/60 Technicon SMA 2C Perkin Elmer </p><p>Chemet rics Coleman 91 Analyzer </p><p>Colorimetric Analyzer </p><p>340 340 340 340 410 410 340 410 340 620 405 340 340 340 340 520 340 410 </p><p>340 340 340 </p><p>&gt;600 </p><p>340 </p><p>34,27 34,27 34,27 </p><p>4 6,48 6,48 </p><p>d 8 52 10 d </p><p>34,27 34,27 34,27 </p><p>5 68 </p><p>57,65 71,50 </p><p>79,49 79,49 </p><p>28 84 </p><p>82 </p><p>58 90 86 84 63 63 73 74 </p><p>107 119 83 82 </p><p>102 104 6b c </p><p>1004 976 197 202 957 879 </p><p>35 54 52 48 33 43 43 52 38 143 32 52 3 C </p><p>238 716 279 452 160 163 0.5 0.6 </p><p>311 431 </p><p>87 76 83 lUlL </p><p>76 93 98 lUlL 72 74 76 lUlL </p><p>106 104 105 lUlL 83 - 79 IUlL 97 62 73 lUlL </p><p>C C c UIL 861 798 779 lUlL 153 180 205 UIL 784 763 721 lUlL 66 59 63 lUlL </p><p>49 64 147 lUlL 64 90 98 lUlL 96 169 244 IUIL 85 158 210 lUlL </p><p>C C c UIL </p><p>83 - 79 lUlL </p><p>62 - 119 IUIL </p><p>C C c lUlL 713 - &gt;800 lUlL 168 774 306 lUlL 0.6 0.8 0.8 lUlL </p><p>361 1263 843 UIL </p><p>a = dialysis; b = different dog serum submitted; c = not abie t o read d u e t o hemolysis; d = BoehringeiMannheim; ( - ) = not tes ted. </p><p>HEMOSTASIS Blood for assay of hemostatic parameters was drawn </p><p>into a syringe containing 3.8% sodium &amp;rate in a ratio of one part citrate plus nine parts whole blood. This was centrifuged immediately for 20 minutes at lo00 x g, the plasma removed and divided into six tubes, and adjusted with citrate hemolysate to produce samples ranging from 0.0 to 4.4 gldl of hemoglobin. These were divided for analysis by two different instruments when possible (BBL Fibrometer:Becton Dickinson; MLA Electra 750Medi- cal Laboratory Automation). Hemoglobin content was determined as described for biochemistry samples above. No correction was made for dilution in order to simulate a hemolytic event in practice. </p><p>Results BIOCHEMISTRY </p><p>Theinstrume nt used, referenced method of anahlsis, and results of 22 biochemical assays are summaLlzed in Tables 24. Hemoly&amp; interfered Consistently with the determina- tion of creatine phosphokinase, lactic dehydrogenase, aspartate aminotransferase, lipase, and albumin, causing these analytes to increase. Hemow did not affect the analysis of total bilirubin, urea nitrogen, creatinine, potas- sium, sodium and cholinesterase, even though some final assay products were read spectrophotometrically at 505 or 520 nm. The results of testing for the remainder of anal* varied greatly among analyzrs or methods employed, rarely in a predictable manner. </p><p>VElElUNARY CLINICAL PATHOLOGY 0 Vol. 18, No. 3 0 PAGE 61 </p></li><li><p>TABLE 3 Miscellaneous Serum Biochemical Analytes, Instrumentation, and Methods with Results of Analysis </p><p>Sample (gldl Hemoglobin) Instrument Wavelength </p><p>Analyte Dialysis (nm) References 0.0 0.3 0.8 1.6 2.5 Units Albumin </p><p>Bit iru bin, Direct </p><p>Bilirubin, Total </p><p>BUN, Urea Nitrogen </p><p>Cholesterol </p><p>Creatinine </p><p>Glucose </p><p>Protein, Total </p><p>Technicon SMA 2C Technicon SMA 12/60 BMD Model 8000 BCA Col uterlDacos Technicon SMA 2C BMD Model 8000 BCA CoulterlDacos Technicon SMA 2C Technicon SMA 12/50 BMD Model 8000 BCA CoulterlDacos Technicon SMA 2C Technicon SMA 12/60 BMD Model 8000 BCA Cou I terlDacos Technicon SMA 2C Technicon SMA 12/60 BMD Model 8000 BCA Coulter Dacos Technicon SMA 2C BMD Model 8000 BCA Coul terlDacos Technicon SMA 2Ca Technicon SMA 12/6Oa BMD Model 8000 BCA CoulterlDacos Technicon SMA 2C Technicon SMA 12/60 BMD Model 8000 BCA CoulterlDacos </p><p>630 630 630 630 600 </p><p>6001675 550 600 600 </p><p>6001675 550 520 520 405 340 525 51 0 520 520 505 505 520 340 520 340 340 550 550 540 550 </p><p>64,16 64,16 64,16 </p><p>17 32,21 32,21 </p><p>80 32,21 32,21 32,21 80 46 46 72 72 </p><p>73, 1 73,l </p><p>1 1 </p><p>30 , l l 30 45 </p><p>42, d 77 </p><p>67,59 78 </p><p>81,69 81,69 </p><p>81 25 </p><p>2.9 2.8 2.3 2.7 0.0 0.0 0.1 0.1 0.1 0.1 0.1 10 11 12 12 </p><p>242 243 231 242 1.1 1.1 1 .o 84 90 92 </p><p>62b 6.9 7.5 6.8 6.6 </p><p>3.4 3.0 2.5 2.8 0.1 0.0 0.2 0.2 0.0 0.1 0.0 </p><p>9 11 12 12 </p><p>273 249 232 235 1 .o 1.1 1.1 88 89 88 61 7.7 7.6 8.2 6.4 </p><p>3.4 3.4 2.6 3.1 0.0 0.0 0.5 0.1 0.0 0.1 </p><p>C </p><p>9 12 12 13 </p><p>243 260 224 227 1.1 1.1 1 .o 88 91 89 60 7.8 8.1 9.1 6.1 </p><p>3.6 </p><p>3.1 3.7 0.1 0.0 1.1 0.1 </p><p>0.1 </p><p>9 </p><p>12 13 </p><p>262 </p><p>207 206 1.1 1.1 1 .o 84 </p><p>85 55 8.8 </p><p>12.0 5.4 </p><p>- </p><p>- </p><p>C </p><p>- </p><p>- </p><p>- </p><p>- </p><p>c gldl 4.5 gldl 3.5 gldl 3.9 gldl 0.2 mgldl 0.0 mgldl 1.4 mgldl 0.3 rngldl 0.0 rngldl 0.1 mgldl 0.0 mgldl </p><p>9 rngldl 11 mgldl 13 rngldl 13 mgldl </p><p>281 mgldl 283 mgldl 205 rngldl 204 rngldl 1.1 mgldl 0.8 mgldl 1.0 mgldl 77 mgldl 88 mgldl 84 rngldl 54 mgldl c gldl </p><p>9.1 gldl 14.0 gldl 5.4 gldl </p><p>a = dialysis, b = different dog serum submitted, c = not able lo read due lo hemolysis, d = Federal Register (39), 1974, ( - ) = not tested </p><p>Interferographs are included for four biochemistry analyzers: SEA 12/60, SMA-2C, DACOS, and BMD Model 8ooo (Fig. 2). Laboratory personnel can easily use these to make decisions for analysis of critical analytes in hemolyzed specimens when it is not possible to obtain a nonhemolyred sample. Interferographs for two selected analytes, total protein and aspartate aminotransferase, are included as Figure 3. These exemplify the manner in which graphs might be used to make decisions on method, or analyzer to be purchased by laboratories. </p><p>These also show how variable the effect of hemolysis can be between methods and among instruments. </p><p>HEMOGRAM Table 5 summarizes the hematologic analytes, instru- </p><p>ment or method used, and results of testing. Analytes not affected by increasing hemolysis were: leukocyte count, erythrocyte count, hemoglobin, mean corpuscular hemoglobin (MCH), reticulocyte and platelet counts, and the differential, including red cell and platelet morphol- </p><p>PAGE 62 0 Vol. 18, No. 3 0 VEIEIUNARY CLINICAL PATHOLOGY </p></li><li><p>a 80 z LL 70 Y </p><p>190 </p><p>170 X - 160 2 150 </p><p>140 K 130 </p><p>d 180 </p><p>2 120 p 100 5 110 </p><p>\ - - </p><p>I I I I </p><p>b. 1 AST / L / </p><p>"" 0 0.3 0.8 1.6 2.5 </p><p>HEMOLYSATE ADDED (gldl Hemoglobin) </p><p>Fig. 3. - Examples of interferographs comparing the effect of hemolysis among instruments for each...</p></li></ul>