Hemolysis as a Factor in Clinical Chemistry and Hematology of the Dog Shamn L. O’Neill, 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

S U M . . Y

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& 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.

Pey words: hemolysis, canine, interferographs, serum biochemistry, hemogram, hemostatic analytes

Introduction Hemolyzed blood is often submitted to the veterinary

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~

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

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.

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

PAGE 58 0 Vol, 18, No. 3 0 VETEaZNARY CLINICAL PATFIOLOGY

TABLE 1 Instrumentation

Instruments Used for Serum Biochemical Analytes

SMA-2C Technicon Instruments Corp., Tarrytown NY 10591 SMA 12160 Technicon Instruments Corp., Tarrytown, NY 10591 Model 8000 Bichromatic Analyzer Boehringer Mannheim Diagnostics, Inc.,

Discrete Analyzer with Continuous Optical Scanning

Coulter Electronics, Inc., Hialeah, FL 33012 Coleman 91 Analyzer - Lipase only Perkin Elmer Corp., Norwalk, CT 06856 400E Spectrophotometer -

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 & Lomb, Rochester, NY 14625

Indianapolis, IN 46250

(DACOS)

Instruments Used to Analyze Hemostatic Factors

and to Complete the Hemogram

BBL Fibrosystem - PT, APTT, lT, Fibrinogen,

Becton, Dickinson & Company, Cockeysville, MD 21030 Spectronic 700 - Antithrombin Ill, Plasminogen Bausch & Lomb, Rochester, NY 14625 Horizontal Electrophoresis Cell Model 1415 -

von Willebrand’s Factor Antigen BioRad Laboratories, Richmond, CA 94804 Counter Model ZBI with Hemoglobinometer -

Coulter Electronics, Inc., Hialeah, FL 33012 Electra 750 Coagulation Timer - PT, APTT, TT Medical Laboratory Automation, Inc.,

Thrombotest, Factors V, VII, VIII, IX, X

RBC, WBC, Hemoglobin

Mount Vernon, NY 10550

altering the pH in enzymatic reactions. Decisions need to be made as to what analyses are

most important, whether the presence of hemoglobin will interfere with the testing, and whether valid information can still be obtained from the affected sample.

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’

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.

Materials and Methods BIOCHEMISTRY

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 -60°C for 30 minutes, defrosted 10 minutes at 37°C 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.

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.

VETERINARY CLINICAL PATHOLOGY 0 Vol. 18, No. 3 0 PAGE 59

compared percent of original analyte concentration (Final concentrationforiginal con- centration x 100%) with the content of added hemolysate.

HEMOGRAM Blood for complete blood

counts was drawn into a syringe containing 0.15% liq-

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

(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.

uid potassium EDTA, and ali-

Flg. 1. - Visual hemolysis in samples submitted for biochemical analysis.

Interferographs were prepared comparing the ability of instruments to yield useful results when hemolysis is present for a variety of analytes. Graphical presentation

~ ~~

190

sz 180

~

a. TECHNICON SYA 12/80 b.

180 r I

240 230F / COULTERDACOS C.

210 0

UJ 180 U I 150

BOEHRINGER MANNHEIM d.

/ / MODEL 8000

170 160

- -

CHLORIDE-

80 70

- -

*LREA NTROOEN 00 'TOTAL BILIRUBIN

'SODIUM -mTAssIw

- 50 40 I-

-

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

HEMOLYSATE ADDED (g/dl Hemoglobin} HEMOLYSATE ADrJED (g/dl Hemoglobin)

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.)

PAGE 60 0 Vol. 18. No. 3 0 VETERINARY CLTNICAL PATHOLOGY

TABLE 2 Serum Biochemical Analytes, Instrumentation, and Methods with Results of Analysis. Enzymes.

Sample (gldl Hemoglobin) Instrument Wavelength Analvte Dialysis (nml References 0.0 0.3 0.8 1.6 2.5 Units

Alanine Aminotransferase (ALT) W P T )

Alkaline phosphatase

Amylase

Aspartate Aminotransferase (AST) (SGOT)

Creat i ni ne kinase (CK or CPK) y -Glutamyl

transferase (GGT) Lactate dehydrogenase

Lipase

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

Technicon SMA 2C Technicon SMA 12/60 Technicon SMA 2C Perkin Elmer

Chemet rics Coleman 91 Analyzer

Colorimetric Analyzer

340 340 340 340 410 410 340 410 340 620 405 340 340 340 340 520 340 410

340 340 340

>600

340

34,27 34,27 34,27

4 6,48 6,48

d 8 52 10 d

34,27 34,27 34,27

5 68

57,65 71,50

79,49 79,49

28 84

82

58 90 86 84 63 63 73 74

107 119 83 82

102 104 6b c

1004 976 197 202 957 879

35 54 52 48 33 43 43 52 38 143 32 52 3 C

238 716 279 452 160 163 0.5 0.6

311 431

87 76 83 lUlL

76 93 98 lUlL 72 74 76 lUlL

106 104 105 lUlL 83 - 79 IUlL 97 62 73 lUlL

C C c UIL 861 798 779 lUlL 153 180 205 UIL 784 763 721 lUlL 66 59 63 lUlL

49 64 147 lUlL 64 90 98 lUlL 96 169 244 IUIL 85 158 210 lUlL

C C c UIL

83 - 79 lUlL

62 - 119 IUIL

C C c lUlL 713 - >800 lUlL 168 774 306 lUlL 0.6 0.8 0.8 lUlL

361 1263 843 UIL

a = dialysis; b = different dog serum submitted; c = not abie t o read d u e t o hemolysis; d = BoehringeiMannheim; ( - ) = not tes ted.

HEMOSTASIS Blood for assay of hemostatic parameters was drawn

into a syringe containing 3.8% sodium &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.

Results BIOCHEMISTRY

Theinstrume nt used, referenced method of anahlsis, and results of 22 biochemical assays are summaLlzed in Tables 24. Hemoly& 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.

VElElUNARY CLINICAL PATHOLOGY 0 Vol. 18, No. 3 0 PAGE 61

TABLE 3 Miscellaneous Serum Biochemical Analytes, Instrumentation, and Methods with Results of Analysis

Sample (gldl Hemoglobin) Instrument Wavelength

Analyte Dialysis (nm) References 0.0 0.3 0.8 1.6 2.5 Units Albumin

Bit iru bin, Direct

Bilirubin, Total

BUN, Urea Nitrogen

Cholesterol

Creatinine

Glucose

Protein, Total

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

630 630 630 630 600

6001675 550 600 600

6001675 550 520 520 405 340 525 51 0 520 520 505 505 520 340 520 340 340 550 550 540 550

64,16 64,16 64,16

17 32,21 32,21

80 32,21 32,21 32,21 80 46 46 72 72

73, 1 73,l

1 1

30 , l l 30 45

42, d 77

67,59 78

81,69 81,69

81 25

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

242 243 231 242 1.1 1.1 1 .o 84 90 92

62b 6.9 7.5 6.8 6.6

3.4 3.0 2.5 2.8 0.1 0.0 0.2 0.2 0.0 0.1 0.0

9 11 12 12

273 249 232 235 1 .o 1.1 1.1 88 89 88 61 7.7 7.6 8.2 6.4

3.4 3.4 2.6 3.1 0.0 0.0 0.5 0.1 0.0 0.1

C

9 12 12 13

243 260 224 227 1.1 1.1 1 .o 88 91 89 60 7.8 8.1 9.1 6.1

3.6

3.1 3.7 0.1 0.0 1.1 0.1

0.1

9

12 13

262

207 206 1.1 1.1 1 .o 84

85 55 8.8

12.0 5.4

-

-

C

-

-

-

-

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

9 rngldl 11 mgldl 13 rngldl 13 mgldl

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

9.1 gldl 14.0 gldl 5.4 gldl

a = dialysis, b = different dog serum submitted, c = not able lo read due lo hemolysis, d = Federal Register (39), 1974, ( - ) = not tested

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.

These also show how variable the effect of hemolysis can be between methods and among instruments.

HEMOGRAM Table 5 summarizes the hematologic analytes, instru-

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-

PAGE 62 0 Vol. 18, No. 3 0 VEIEIUNARY CLINICAL PATHOLOGY

a 80 z LL 70 Y

190

170 X - 160 2 150

140 K 130

d 180

2 120

p 100 5 110

\ - -

I I I I

b. 1 AST / L /

"" 0 0.3 0.8 1.6 2.5

HEMOLYSATE ADDED (gldl Hemoglobin)

Fig. 3. - Examples of interferographs comparing the effect of hemolysis among instruments for each analyte. a total prateln,b. AST (aspartate aminatransferase).

ogy. Affected by hemolysis were: packed cell volume (PCV) and mean corpuscular volume (MCV), which decreased, and the mean corpuscular hemoglobin con- centration (MCHC), erythrocyte sedimentation rate (ESR), plasma protein and fibrinogen, all of which a p peared to increase with increasing hemolysate added. Fibrinogen became difficult to estimate by the refrac- tometric method because hemoglobin caused the result- ing line to become indistinct or fuzzy. Icteric index could not be estimated at all when hemolysis was greater than 0.1 gldl hemoglobin.

HEMOSTASIS The results of the addition of hemolysate on hemos-

tatic analytes are included in Table 6. When samples were analyzed by a fibrometer, no sigdicant change was found in the determination of prothrombin time (FT), activated partial thromboplastin time (AP'IT), throm- botest for protein-induced vitamin K antagonists

(PIVKA), and factors V, W, n C , IX and X Affected by hemolysis were: thrombin time ('IT), which decreased, and fibrinogen, which increased. Plasminogen and an- tithrombin III, assayed by a spedrophotometric assay which read p-nitroaniline cleaved from synthetic sub- strate at 405 nm, appeared decreased by increasing hemolysis. Rocket immunoelectrophoresis was severely decreased by the presence of greater than 0.3 gldl hemoglobin, when testing for von Wdebrand's factor an- tigen (vWfAg),

An MLA Electra 750 coagulation timer (Medical Laboratory Automation) was used to determine PT, AP", and 'IT. Hemolysis caused an increase in FT and TT, but had no effect on AP".

Discussion BIOCHEMISTRY

Recent studies have evaluated the effect of hemolysis on human biochemical analyte~?424~ These results, how-

VETERLNARY CLINICAL PATHOLOGY 0 Vol. 18, No. 3 0 PAGE 63

TABLE 4 Serum Biochemical Analytes, Instrumentation, and Methods with Results of Analysis. Electrolytes and Minerals.

Instrument Wavelength Sample (gldl Hemoglobin)

Analyte Dialysis (nm) References 0.0 0.3 0.8 1.6 2.5 Units Calcium Technicon SMA 2C

Technicon SMA 12/60a BMD Model 8000 BCA CoulterlDacos

Buchler-cot love Chloridorneter

BMD Model 8000 BCA Phosphorus, Technicon SMA 2Ca Inorganic Technicon SMA 12/60.

BMD Model 8000 BCA CoulterlDacos

Potassium Technicon SMA 2C IL, Inc., Boston, MA BMD Model 8000 BCA

Sodium Technicon SMA 2C IL, Inc., Boston, MA BMD Model 8000 BCA

Total C02 Technicon SMA 2C Gilford 400E Chemetrics

Chloride Technicon SMA 2Ca

Colorimetric Analyzer

570 570 570 575 480 -

5051600 340 660 340 340 - - - - - -

550 340 340

CoulterlDacos 340

36,22 36 36 12

69,49 13

66 14,2 38 38 14 63 d e

63,19 d e 69

20,53 20

20

10.2 10.1 10.2 9.8 10.5 11.0 10.0 10.0 117 116 116 112

110 112 3.1 3.6 3.4 3.6 3.0 C

3.5 3.4 4.7 4.8 4.7 4.7 4.8 5.0 147 147 151 151 152 154 20 18 21 19 19 18

27b 27

10.4 10.2 10.0 - 11.3 12.6 10.2 10.3 116 114 116 115

116 121 3.4 3.6 3.7 -

3.3 3.5 4.8 4.8

4.9 5.0 148 147 152 151 153 152 20 19 16 11 12 13

C C

4.8 4.8

28 28

9.9 rngldl 9.6 rngldl

13.5 rngldl 10.3 rngldl 112 rnEqll 113 mEqll

122 rnEqll 3.6 mgldl 4.0 mgldl

c rngldl 3.7 rngldl 4.9 rnEqll 4.9 rnEqll 5.0 rnEqll 146 rnEqll 151 mEqll 150 mEqli 17 rnrnolll 9 rnrnolll 8 rnrnolll

27 rnrnolll a = dialysis; b = different dog serum submitted; c = not able to read due to hemolysis; d = IL, Inc.; e = Boehringer-Mannheim; ( - ) = not tested.

ever, can not be applied to analyses of other species' serum analyte~~." due to differences in the concentration of analytes contained in erythrocytes, different methods and reagents used, and the use of a variety of analyzers among veterinary laboratories.

Dorner et ul.'s predicted that the selection of wave- lengths between 505 and 570 nm would enhance the in- terference by hemoglobin in samples whose endpoints were read in this range. Analytes with reaction endpoints read at 505-570 nm in this study were: creatine kinase (included dialysis), chloride (included dialysis), total COZ, urea nitrogen, cholesterol, creatinine, glucose, and total protein. Of this group, creatinine, urea nitrogen, glucose, and chloride results were not affected by hemolyis up to 25 gldl hemoglobin. Total protein was expected to in- crease due to released erythrocyte proteins. Cholesterol found in red cell membranes could be expected to in- crease serum cholesterol slightly as could be seen in the two Technicon systems. However, a decrease, although slight, in cholesterol concentration with increasing hemolysis was reported for BMC Model 8000 and

DACOS analyzers. All four analyzers employed the same method. Instrumental differences, such as in blanking or correcting for hemolysis, may account for the variation in results, rather than the absorbance used to read the endpoint. Total carbon dioxide decreased slightly with the Technicon SMA-2C, an effect that may result from a change in hemolysate pH and loss of COZ due to processing of the hemolysate, rather than to the absor- bance used. Creatine kinase increased sharply, pre- sumably due to adenylate kinase released from ery- throcytes. The effect of reading an endpoint at wave- lengths close to the range where hemoglobin absorbs a p pears to be minimal. Results are more likely to be affected by other factors when using the more sophisti- cated analyzers.

The slight increase in serum phosphate seen in the results of testing by the two Technicon systems would be expected since erythrocytes contain phosphate ester. The BMD Model 8000, however, could not adapt to any hemolysis. The DACOS results were not affected by hemolysis. Methodology was not the only reason for this,

PAGE 64 0 Vol. 18, No. 3 0 VETERMARY CLINICAL PATIIOLQGY

TABLE 5 Hemogram Analytes, Method and Results.

Sample (@dl Hemoglobin) Analyte Method. or Instrument 0.0 0.1 0.2 0.3 0.5 0.8 1.7 2.5 3.5 Units Erythrocyte Wintrobe Tube 0.5 0.5 0.5 0.5 0.5 1.0 1.0 1.0 2.0 mm

Sedimentat ion Rate

Fibrinogen Ref ractometer; 100.0 100.0 100.0 100.0 50.0 150.0 300.0 300.0 b mgldl

Hemoglobin Coulter Hemoglobinometer 15.3 16.7 15.8 15.5 16.1 16.1 15.7 15.4 15.6 GIL lcteric Index Visual Comparison 2.0 2-5 b b b b b b b units

MCV 89.6 79.3 79.6 84.7 79.2 81.2 69.7 72.2 62.1 f l MCHC 32.2 36.3 34.3 33.7 35.8 36.6 38.3 39.5 43.3 gldl MCH 28.9 28.8 27.3 28.5 28.3 29.7 26.7 28.5 26.9 pg

Plasma Protein Refractometer 6.8 7.0 7.2 7.3 7.6 8.2 9.8 10.6 est 12 Gldl Platelet Count Unopettel 260.0 285.0 220.0 280.0 230.0 220.0 275.0 265.0 225.0 x1031ml

Heat precipitation

to standards

Packed Cell Volume Clay Adams Centrifuge 47.5 46.0 46.0 46.0 45.0 44.0 41 .O 39.0 36.0 YO

Hemacytometer count Red Cell Count Coulter Counter ZBI 5.30 5.88 5.78 5.43 5.68 5.42 5.88 5.40 5.80 xI@/pl Reticulocyte Count Blood Smear/ 0.3 0.2 0.2 0.1 0.3 0.2 0.1 0.2 0.3 O/O

New Methylene Blue Stain White Cell Count Coulter Counter ZBI 8.3 8.7 8.3 9.1 9.4 9.5 9.1 8.2 8.3 XlO31pI

Procedures can be found in Schalm’s Vetennarv Hematoloav. 4th ed. N.C Jam led). Lea & Febiger, Philadelphia, 1986.

~

-. Unable to read due to hemolysis.

nor was the inclusion of dialysis in the procedure. Canine erythrocytes contain lactic dehydrogenase and

aspartate aminotransferase. The anticipated increases in these serum enzymes with hemolysis were seen in all laboratories as a severe increase (Fig. 2).

The effect of hemolysis on biochemical analysis is usually not predictable or constant among methods and analyzers. It is unique to each system and should be deter- mined by each Laboratory in order to obtain the maximum information from hemolyzed, irreplaceable samples. Data presented here, which is based on the ady& of samples prepared from one dog‘s blood, should not be applied in a laboratoxy using a similar instnrmen t or method. Instead, the effect of hemolysis on biochemical analysk should be determined by the individual laboratory.

HEMOGRAM The presence of hemdysis did not interfere in the

evaluation of blood smears (differential, red cell and platelet morphology and reticulocyte counts) or in the

counting of red and white cells, and platelets. Hema- tologic analytes which reflected significant changes with increasing hemolyses were PCV, MCV, MCHC, plasma proteins, fibrinogen, icteric index, and ESR.

Icteric index, a measure of the plasma concentration of b h b i q is read by comparison to potassium d i h m a t e standards. Due to the visual interference from the red color of hemoglobin, icteric index could not be estimated when plasma hemoglobin was greater than 0.1 g/dL

The most sipXcant change was seen in the PCV which decreased as hemolysis increased. This would be a predictable response as more and more hemolysate containing lysed red cell material replaced whole cells. Indices such as MCV and MCHC which are calculated from the PCV value were also changed.

Although PCV changed, the Coulter Counter con- tinued to count red cell ‘ghosts’ as whole cells. The erythrocyte count, therefore, did not decrease in parallel to the PCV and the calculation of MCH was not affeded. This is an artifact of the hemolysate preparation which

VEXERINARY CLINICAL PATHOLOGY 8 Vol. 18, No. 3 8 PAGE 65

TABLE 6 Hemostatic Analytes, Instrumentation and Methods, with Results of Analysis. Enzymes.

Parameter WaveLength Dialysis References 0.0 0.8 1.5 2.5 3.5 4.4 Units

Instrument Hemolyzed Samples (gldl Hemoglobin)

Activated Partial

Thromboplastin Time

Ant it hrorn bin I I I

Factors: V VI I Vl l l IX X

Fibrinogen

PIVKA (Thrombotest)

Plasminogen

Prothrombin Time

Thrombin Time

vWf:Agl

Bectin, Dickinson & Co., BBL Fibrosystem MLA Electra 750, Coagulation Tirnera.f Bausch & Lomb Spectronic 7001 Bectin, Dickinson & Co., BBL Fibrosystem

Bectin, Dickinson & Co, BBL Fibrosystem Bectin, Dickinson & Co., BBL Fibrosystern Bausch & Lomb Spectronic 700' Bectin, Dickinson & Co., BBL Fibrosystem MLA Electra 750, Coagulation Ti m e W Bectin, Dickinson & Co., BBL Fibrosystem MLA Electra 750, Coagulation Timeralc BioRad Laboratories, Electrophoresis Cell Model 1415

60

60

7,54

61 37,76 40,18 18 3

51,56

58

47 75 62

62

33,44

33,44

41,83

9.9

10.2

1 500

117 91 100 94 97 200

15.4

1030

5.9

6.1

6.5

5.2

225

-h

10.5

10.1

129

108 97 120 106 100 200

15.1

110

5.9

6.2

6.2

d

192

-

10.4

10.3

114

109 97

1 38 106 100 202

15.6

86 96 6.5

6.7

5.9

5.6

136

10.5

10.0

121

106 97 118 101 96 192

15.9

e 96 6.3

7.2

5.5

6.0

104

10.4

10.1

140h

109 90 113 99 100 180

15.8

e 84 6.5

7.4

5.3

6.3

i

9.9 sec

10.4 sec

150" '/a

1 17 Y o 97 O/O

1 13 O/O

100 O/O

175 mgldl

16.6 sec

a4

e 58 '/o

6.3 sec

7.6 sec

4.9 sec

6.4 sec

i O/O

a = thrombin mode; b = lamp A; c = lamp B d = insufficient sample size; e = off scale; t = 405 nm; g = 10 p I sample - usual sample size for method; h = 5 flI sample - necessary due to hemolysis exceeding absorbance range; I = no migration of protein. No peaks detected; j = von Willebrand's factor antigen.

may not reflect the effect of in vivo hemolysis on red cell count, as debris from lysed cells would be cleared from the blood by the spleen. Centrifugation of the hemolysate before adding hemolysate to the samples did not bring down the red cell debris for this study. P h protein and fibrinogen were estimated by refrac-

tometer. Plasma proteins increased with increasing hemolysiq however, the increases were not in proportion to the amount of hemolysate added. Hemoglobia or the

refradometer. The line of Werentiation became increas- inglyindhinct, which, in turn, made fibrinogen difficult to

p r m of debris interfered directly with the use of the

determine. It is unknown if fibrinogen appears to in- crease (greater than 0.8 g/dl hemoglobin) because of the difficulty in reading the line or the presence of cell debris.

Erythrocyte sedimentation rate (ESR) is signifucantly influenced by red cell numbers. As whole red cell count falls (as evidenced by the decreasing PCV), the ESR should increase. Our corrected ESR when hemoglobin is 3.5 g/dl is -12 (observed +?, anticipated -14). Negative ESR is usually associated, in vivo, with reticulocytosis and hypoproteinemia?' The decrease in PCV with our induced hemolysis did not result in a significant increase.

PAGE 66 0 Vol. 18, No. 3 0 YETEBINAaY CLINICAL PATaoLoGY

in ESR as would be predicted. This could be due to the red cell proteins and debris which remained in the hemolysate and resulted in a normal erythrocyte count.

HEMOSTASIS Hemolysis present in citrated plasma samples with up

to 3.6 g/dl hemoglobin, submitted for coagulation testing, did not significantly increase PT, APTT, PIVKA, or Fac- tors II through X when analyzed by a fibrometer (Becton Dickinson). When samples were tested using the MLA Electra coagulation timer, PT and 'IT were increased slightly, while APTT was not affected. With the fibrometer method, fibrinogen concentration was decreased slightly. However, the TT became shorter which is an estimated measure of increased fibrinogen. The reason for these seemingly opposing results is not known. It is assumed that the sensitivity of the 'IT test to tissue factors contained in red cell debris but the dilu- tion of the samples and these factors for fibrinogen deter- mination created the opposing effects.

Hemoglobin directly interfered with spectropho- tometric determinations of antithrombin Ill ( A m ) and plasminogen. Hemolysis elevated the initial absorbance above the linear range and decreased the change of ab- sorbance in these kinetic methods. Plasminogen con- centrations were more severely decreased than were ATIII. Other factors may interfere, including the prepar- ation of hemolysate, direct interference from hemo- globin, red cell debris inhibiting the conversion of plas- minogen to plasmin by wokinase or the activity of plas- min on chromogenic substrate. The action of freezing and thawing hemolysate may also have destroyed some of the enzyme.

Rocket immunoelectrophoresis for von Willebrand's factor antigen (vWfAg) was most severely affected by the presence of hemoglobin which inhibited the migra- tion of antibody and antigen. The presence of 0.3 g/dl hemoglobin decreased the concentration of antigen 15% in a sample containing 225% vWfAg. Hemoglobin con- centration of 3.6 g/dl completely blocked the formation of a protein rocket. The effect of hemolysis on a sample containing less than 100% vWfAg could be more severe. Samples for electrophoretic studies of vWfAg should not be accepted if the sample is hemolyzed.

Hemolysis, as artificially induced in this study, may un- derestimate the effects of hemolysis due to a traumatic venipuncture which may result in the addition of more tissue thromboplastin. When tissue factors are present clots may form in the sample CoIlSuming factors and in- creasing hemostatic test times, or hemostatic test times could be shortened if factors are prematurely activated.

Extra care must be taken in evaluating results of hemos- tasis testing on hemolyzed samples. If possible, the sample should always be redrawn.

AVOIDING THE EFFECTS OF HEMOLYSIS

The effects of hemolysis on chemical, hematologic and hemostatic analyses should be determined by each laboratory for each species, method and instrument used for analysis, since variable and even opposing effects may be produced. When interference is determined, changes in method or instrumentation might be attempted to eliminate the effect. For instance, one could try the use of dialysis or deproteinization, kinetic assays rather than endpoint, sample bhking, method pH alterations, or measurement at two different wavelengths.p~m ~n some cases a hemolyzed sample might be analyzed manually on another spectrophotometer which is not affected as much by hemolysis.

The use of interferographs in decision making is re- commended as they are specific to one instrument, and one method.

Avoiding hemolysis is always the best solution to in- terference. Processing blood in a timely manner, storing it at proper temperature and avoiding mechanical agita- tion will help to prevent hemolysis. With especially fragile erythrocytes, the use of larger needles and of plasma in- stead of serum, whenever possible, can help.

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