Toxicology for Industrial and Regulatory Scientists

Cli i l P th lClinical Pathology Principles for Industrial c p es o dust a

and Regulatory ScientistsRobert L. Hall, DVM, PhD, DACVP

Covance LaboratoriesCovance LaboratoriesMadison, WI

A il 28 2015April 28, 2015

Learning ObjectivesLearning Objectives• Role of clinical pathologyRole of clinical pathology• Study design considerations

Principles of data interpretation• Principles of data interpretation• Common test article effects

– patterns, correlations, importance

2

Clinical PathologyClinical Pathology• Hematology and coagulationgy g• Clinical chemistry• Urinalysis and urine chemistry• Urinalysis and urine chemistry• Other (e.g., hormones and some

bi k )biomarkers)– these are serum and/or urine chemistry tests

3

Objectives of Clinical PathologyObjectives of Clinical Pathology• Broad screen of important tissues, organs, p , g ,

and metabolic functions• Screen for efficacy (pharmacodynamic y (p y

effect)• Establish dose-response relationshipsp p• Quantitative in vivo assessment• Support for other findings; corroborativeSupport for other findings; corroborative

data• Meet regulatory and clinical expectationsMeet regulatory and clinical expectations

4

PerspectivePerspective• Subtle or non-adverse effects are common• Rarely the only evidence of important or

adverse effectsadverse effects• Specific mechanisms are often undetermined

St d d i d d i t lt• Study design and procedures impact results and affect interpretation

• Monkeys are not little people, and mice are not little dogs

5

Test Selection, Timing, and FrequencyTest Selection, Timing, and Frequency

• Study objectivesy j• Study duration• Dosing regimen• Dosing regimen• Test article• Species• Regulatory guidelinesg y g• Corporate inertia

6

MOHW – Toxicity Studies of Drugs 1990MOHW Toxicity Studies of Drugs, 1990

• Repeated-dose toxicity studyRepeated dose toxicity study

“ it is desirable to include as many…it is desirable to include as many parameters as possible.”

7

Common Hematology TestsCommon Hematology Tests• Red blood cell count, hemoglobin, and g

hematocrit• MCV, MCH, MCHC, RDWMCV, MCH, MCHC, RDW• White blood cell count• Absolute differential white blood cell count• Absolute differential white blood cell count• Platelet count, MPV• Absolute reticulocyte count• Blood cell morphology

8

Common Coagulation TestsCommon Coagulation Tests• Prothrombin time (PT)( )• Activated partial thromboplastin time

(APTT)(APTT)• Fibrinogen

9

Common Clinical Chemistry TestsCommon Clinical Chemistry Tests• Glucose • Creatine kinase• Urea nitrogen, creatinine• Total protein, albumin,

• Calcium, inorganic phosphorusp

globulin, A/G ratio• Cholesterol, triglycerides

• Sodium, potassium, chlorideg y

• Total bilirubin• ALT ASTALT, AST• ALP, GGT

10

Common Urinalysis TestsCommon Urinalysis Tests• Volume if timed collection

(e.g., 16 or 24 hours)• Appearance (color and turbidity)pp ( y)• Specific gravity or osmolality• Reagent strip tests (pH protein occult blood• Reagent strip tests (pH, protein, occult blood,

glucose, ketones, bilirubin, urobilinogen)Mi i f di t ( d ll• Microscopic exam of sediment (red cells, white cells, epithelial cells, casts, crystals, b t i )bacteria, sperm)

11

Additional Clinical Pathology TestsAdditional Clinical Pathology Tests• C-reactive protein, other acute phase proteins

C di t i I d/ T• Cardiac troponins I and/or T• Platelet aggregation

Ald l• Aldolase• Ionized calcium and biomarkers of bone

formation/lossformation/loss• Heinz body count and/or methemoglobin• SDH GDH bile acidsSDH, GDH, bile acids• Urine enzymes• New urine biomarkersNew urine biomarkers

12

Bone Marrow ExaminationBone Marrow Examination• Infrequently indicated

k d d ti f i l ti ll (RBC WBC– marked reduction of circulating cells (RBCs, WBCs, and/or platelets) with unknown cause

– should not be included in standard protocolsshould not be included in standard protocols

• Not indicated for chemotherapeutics• When indicated recommend qualitative• When indicated, recommend qualitative

cytologic examination first (targeted approach), rather than quantitative differential cell count or qM:E ratio

• Flow cytometry has potential for improving bone marrow evaluations when indicated

13

Tests of Limited (or No) ValueTests of Limited (or No) Value• Ornithine • Uric acid

decarboxylase• Ornithine carbamoyl

• Urine nitrite and leukocyte esterasey

transferase• Lactate

y• Serum protein

electrophoresisdehydrogenase

• Direct and indirect

p• Bone marrow M:E ratio

bilirubin• Amylase and lipasey p

14

MiceMice• 30 g x 0.07 = approximately 2 mL of blood

– usually a terminal procedure

• One mouse = hematology and short chemistry li t OR f ll h i t l (17 20 t t ) ORlist OR full chemistry panel (17-20 tests) OR coagulation testsN i l i / i h i t l ifi t t• No urinalysis/urine chemistry unless specific test– one drop = one or two tests

• Data from moribund mice often misleading; not recommended

• Nice to have at least 10/sex/group/test15

RatsRats• Baseline rarely necessary and potentially

h f l ifi t i t t PD kharmful; specific esoteric test or PD marker• Studies >13 wks and <52 wks; interim clin path

b d i blmay be desirable• Standard testing not recommended after 52 wks • Don’t use same animals for standard TK/PK• Moribund animals ok to test but not for carc studyy• Nice to have at least 10/sex/group/test

16

DogsDogs• Two baselines recommended unless long g

study or high number/sex/group• Multiple intervals recommended forMultiple intervals recommended for

studies >4 wks• Moribund animals should be tested• Moribund animals should be tested• Dogs are good

17

MonkeysMonkeys• Two baselines always recommended• Multiple intervals recommended for studies

>4 wks• Same origins strongly recommended

M ib d i l h ld b t t d• Moribund animals should be tested• If possible, avoid clinical pathology for at least

5 days after IM ketamine for other procedures (e.g., ECGs, ophthamol. exams)

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Messin’ Around/Range-FinderMessin Around/Range Finder• No need to follow guidelines (don’t do the

kitchen sink)• Coagulation assays only if indicated by test

article• Urinalysis and urine chemistry almost never

necessary or valuable– too few animals, too much normal variability

• Nonstandard tests only if indicated by test article– e.g., a pharmacodynamic marker

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Acute Single Doseg• <24 hours postdose is too soon unless indicated by a

specific objective or test article (e.g., a PD marker)– not enough time for most markers of toxicity to develop– accentuates transient, procedure-related effects

• Day 15 is too late but may use to assess recovery– most toxicities completely reversed in 2 weeks

• Day 3 is a good compromise unless other times indicated by test article (e.g., chemotherapeutic)

• Coagulation and urinalysis tests may not be necessary

• For mice, unless interim sacrifice, almost no value20

α-Naphthylisothiocyanate (ANIT) Toxicityα Naphthylisothiocyanate (ANIT) Toxicity

ALT (U/L)24 hrs

postdose48 hrs

postdose96 hrs

postdose

ALT (U/L)

postdose postdose postdose

Control ~50 ~50 ~50

Treated ~100 ~1500 ~200

Toxicologic Pathology 36:818-826, 2008.

21

α-Naphthylisothiocyanate (ANIT) Toxicityα Naphthylisothiocyanate (ANIT) Toxicity

Total bilirubin (mg/dL)24 hrs

postdose48 hrs

postdose96 hrs

postdose

Total bilirubin (mg/dL)

postdose postdose postdose

Control ~0.05 ~0.05 ~0.05

Treated ~0.8 ~8.5 ~1.0

Toxicologic Pathology 36:818-826, 2008.

22

Chemotherapeutics Affecting H t i iHematopoiesis

• To assess maximum effect and recovery ofTo assess maximum effect and recovery of different cell lines, need serial sampling for hematology

• For example, after single dose:D 3 5 7 10 14– Day 3, 5, 7, 10, 14

– reticulocyte and neutrophil count nadir, Day 3 or 5– platelet count nadir Day 7 or 10– platelet count nadir, Day 7 or 10– rebound reticulocytosis and neutrophilia, Day 7 or 10– rebound thrombocytosis, Day 14

23

Carcinogenicity/Oncogenicity Studiesg y g y• Recent CPMP (European) carcinogenicity study

guideline says chemistry and urinalysis “shouldguideline says chemistry and urinalysis should be performed” at study termination– survey of multiple companies indicates this has had nosurvey of multiple companies indicates this has had no

impact on current practices

• Common sense– make and hold blood smears from all sacrifices

(moribund and terminal)– examine only if requested by anatomic pathologist to

assist in diagnosis of possible hematopoietic neoplasia

24

Goal #1 – Reliable ResultsGoal #1 Reliable Results• Must do these things right

– study design– study conduct… every procedure– data interpretation– reporting

• Variability compromises reliability– understand sources– standard procedures– training and experience

25

Sources of VariabilitySources of Variability• Preanalytical variationy

– artifact– physiologicalp y g– procedural

• Analytical variation

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Preanalytical Variation: ArtifactPreanalytical Variation: Artifact• Sample Quality• Collection problemsCollection problems

– inexperienced phlebotomist or new technique– sick, small, or dehydrated animals (esp. mice)

i– common issues• clotted hematology sample• platelet clumpsp p• hemolysis• short fill (critical for coagulation tests)

ti l t i• anticoagulant misuse• Samples mishandled or treated differently

– fresh vs frozen or stored– uncapped, evaporation

27

Preanalytical Variation: PhysiologicalPreanalytical Variation: Physiological

• Species • DietSpecies• Strain

Age

Diet• Circadian effects

am vs pm• Age• Sex

– am vs pm

• Excitement/Fear• StressS ess• Fasted/Nonfasted

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Species: rat < dog < monkey < mouse

Parameter Rat Dog Monkey Mouse

Plt count(x 1000/uL) 895 - 1340 241 - 561 302 - 722 543 - 1481( )

UN(mg/dL) 10 - 17 8 - 19 10 - 28 19 - 40(mg/dL)

T Protein(g/dL) 5.5 – 7.4 4.9 – 6.5 5.9 – 10.4 4.3 – 5.5(g/dL)

ALT(IU/L) 25 - 52 18 - 44 17 - 142 22 - 168(IU/L) 25 52 18 44 17 142 22 168

29

Age-Related ChangesAge Related Changes• Young rodents (4-8 wks) and dogs (4-6 mos)

– increasing red cell mass (RBC count, Hgb, Hct)– increasing absolute neutrophil count

increasing total protein and globulin– increasing total protein and globulin– decreasing absolute reticulocyte count and MCV

decreasing absolute lymphocyte count– decreasing absolute lymphocyte count– decreasing alkaline phosphatase and inorganic

phosphorus• Older rodents (>20 wks) and dogs (>18 mos)

– more variability

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Excitement/Fear: Fight or FlightExcitement/Fear: Fight or Flight• Epinephrine (adrenaline) release

• Immediate but short-lived (30 minutes)– increased heart rate and blood pressure– heart and skeletal muscle vasodilatation

increased red cell mass (splenic contraction)– increased red cell mass (splenic contraction)– increased leukocyte counts (marginating pool flushed

into circulating pool)g p )– increased glucose concentration (glycogenolysis)

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Excitement/Fear• 4-week monkey study• six/sex/groupg p• WBC count (/µL)

M l P d 4 P d 10 D 24Males Predose 4 Predose 10 Day 24

Group 1 11,920 9560 7410p ,Group 2 13,330 9670 8540Group 3 15 710 12 040 10 660*Group 3 15,710 12,040 10,660Group 4 16,610 13,310 11,670*G 5 14 640 10 440 8180

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Group 5 14,640 10,440 8180

Stress• Endogenous corticosteroid release

dogs and monkeys = cortisol– dogs and monkeys = cortisol– rodents = corticosterone

• Gradual effect, longer-lasting, g g• Leukocyte counts

– mature neutrophilia– lymphopenia and eosinopenia

• Increased adrenal gland weight; decreased thymus weightthymus weight

• Lymphocyte depletion in lymphoid tissues• Corticosteroid induced alkaline phosphatase in• Corticosteroid-induced alkaline phosphatase in

dogs33

Fastingg• Fasting preferred for most species

– same starting point with respect to food intakesame starting point with respect to food intake– reduce liver glycogen

• Mice - short fast or no fast– about 3-5 hours maximum– fasted mice do not drink much

• hemoconcentration• prerenal azotemia• more difficult to bleed

• Hamster - cheek pouch• Rabbits - weight loss an issue; coprophagia

34

Fasted vs NonfastedFasted vs Nonfasted• Fasted rats vs nonfasted rats (literature)

– lower WBC count– lower urea nitrogen– lower cholesterol and triglycerides– lower alanine aminotransferase and alkaline

phosphatasephosphatase

• Changes after a meal (esp. large animals)i d d ll t i (fl id hift)– increased red cell mass, serum proteins (fluid shift)

– increased urea nitrogen (fluid shift, protein)i d l t i l id ( t i t b ti )– increased glucose, triglycerides (nutrient absorption)

35

Fasting• 14-day rat study to assess hepatotoxicity• multiple interim sacrifices• triglycerides (mg/dL)

Day 7 Day 7 Day 11 Day 14Day 7 Day 7 Day 11 Day 14

Fasted Nonfasted Fasted NonfastedFasted Nonfasted Fasted Nonfasted

Control 41 197 46 206

Treated 65 100* 44 79*

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Fasting• 14-day rat study to assess hepatotoxicity• multiple interim sacrificesp• alkaline phosphatase (U/L)

Day 7 Day 7 Day 11 Day 14

Fasted Nonfasted Fasted NonfastedFasted Nonfasted Fasted Nonfasted

Control 160 265 179 238

Treated 261* 280 240 261

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Preanalytical Variation: ProceduralPreanalytical Variation: Procedural• Blood collection site and technique• Order of sample collection/processing/analysis• Iatrogenic blood loss• Handling technique• Other study design factors

– timing of other procedures, anesthesia– route of administration– vehicle

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Sample Collection in Group OrderGroup Glucose

(mg/dL)AST(IU/L)

Na(mmol/L)

1 125 104 1461 125 104 146

2 (TA A) 121 93 146

3 127 100 147

4 132 91 148*

5 127 80 148*

6 125 87 150*

7 (TA B) 133 86 149*

8 132 80* 149*

9 140* 67* 150*

10 135* 67* 150*

11 140* 65* 151*39

Delayed Separation of Serum from ClotDelayed Separation of Serum from Clot

• Glucose consumed by red blood cells– approximately 10% per hour– serum separator tubes do not prevent this p p

completely

• Release of blood cell constituentsRelease of blood cell constituents– potassium (dependent on species) and

inorganic phosphorusinorganic phosphorus– aspartate aminotransferase (AST) and lactate

dehydrogenase (LDH)dehydrogenase (LDH)

40

Iatrogenic Blood Loss• 14-day monkey study; daily dose• seven bleeds for TK, Ab, and banking on Days 1 and 14• hematocrit (%)

Control ♀ Predose 15 Day 3 Day 14Control ♀ Predose 15 Day 3 Day 14

1 46.1 36.1 47.5

2 38.7 34.5 39.7

3 41 3 37 4 38 83 41.3 37.4 38.8

4 40.4 33.3 37.0

41

5 43.6 34.9 40.5

Handling and IM Ketamine Injection• 14-day monkey study; daily dose• IM ketamine for ECG and ophthalmic exams on Day 2• aspartate aminotransferase (AST; U/L)

Control ♀ Predose 15 Day 3 Day 14♀ y y

1 49 86 54

2 29 44 23

3 38 65 333 38 65 33

4 29 44 27

42

5 49 231 44

Intramuscular Ketamine Injection• 4-week monkey study• four/sex/group

ti ki (U/L) t “ ti i ki ”• creatine kinase (U/L); not “creatinine kinase”

Group ♂ Predose Day 17 Day 30p y y

1 1299 323

2 1453 221

3 1019 347

4 1447 245

43

5 1130 246

Handling TechniqueHandling Technique• Swaim LD et al: The effect of handling

techniques on serum ALT activity in mice. J Appl Toxicol, 5:160-162, 1985.

• Mean ALT 1 hour and 1 week afterMean ALT 1 hour and 1 week after handling, 36/group:– control (not handled): 79 0 and 46 3 IU/Lcontrol (not handled): 79.0 and 46.3 IU/L– handled by tail: 77.2 and 49.5 IU/L

handled by body: 286 7 and 54 3 IU/L– handled by body: 286.7 and 54.3 IU/L

44

Chronic Intravenous Infusion• Increased animal-to-animal variability• Findings associated with inflammation common• Findings associated with inflammation - common

– decreased red cell mass (RBC, Hgb, Hct)increased leukocyte counts– increased leukocyte counts

– increased fibrinogen, C-reactive protein, globulindecreased alb min alb min to glob lin ratio– decreased albumin, albumin-to-globulin ratio

• Findings associated with tissue damage - less commoncommon– increased aspartate and alanine aminotransferase

(AST and ALT)(AST and ALT)– increased creatine kinase or cardiac troponins

45

Analytical Variation• 39-week monkey study• six/sex/groupg p• activated partial thromboplastin time (APTT; seconds)

Males Predose 21 Day 79 Day 252

Group 1 29 5 30 2 21 4Group 1 29.5 30.2 21.4

Group 2 29.8 30.4 20.9

Group 3 30.4 30.3 21.6

Group 4 29 3 30 7 21 8

46

Group 4 29.3 30.7 21.8

Study Data InterpretationStudy Data Interpretation

• Two key questions for any finding:Two key questions for any finding:

I it l? (i l t d t t t ti l– Is it real? (i.e., related to test article administration)

– Is it bad? (i.e., adverse, toxicologically ( , , g yimportant)

47

Is It Real? Factors to Consider• How large is the difference?

Is the difference dose dependent?• Is the difference dose-dependent?• Consistent over time? Between sexes?• Statistically significant? Present before treatment?• When did the difference occur with respect to

dosing?• Correlative findings? In-life, clinical or anatomical

pathology?• What is known about the test article?• What is known about the vehicle?

48

Is It Real? Factors to Consider• How many animals were tested?• How much inter- and intra-animal variability isHow much inter and intra animal variability is

expected?– species, age– unique study design conditions

• staggered start• fasted vs nonfasted• route of administration• time bias not eliminated• IM ketamine administration• excessive blood collections• unusual site for blood collection

• How much analytical variability is expected?• How much analytical variability is expected?

49

Is It Real?• hematocrit (%)• reference interval = 40-50%

Group Study A Study B

Control 48 42

High-dose 42* 37*

50

Is It Real?• dog study; end of dosing phase• absolute neutrophil count (3700-12,900/µL)• alanine aminotransferase (18-45 U/L)• chloride (103-120 mmol/L)

Dose (mg/kg) Neutrophils ALT Chloride( g/ g)

0 4700 31 118100 5300 37 122*100 5300 37 122*300 6700 40 134*

51

500 9900* 50* 138*

2-fold Increase ALT - Is It Real?• Young vs old• Rat vs mouse• Dog vs monkey• 15/sex/group vs 2/sex/group• Day 3 vs Week 52Day 3 vs Week 52• Gavage vs infusion• Saline vs alphabet soup• Group effect vs individual outliers• Liver lesions vs “clean livers”• Correlative findings• Correlative findings• Previous studies, drug class history

Underline indicates greater likelihood of being real52

Is It Bad? Factors to Consider• Is affected analyte critical to health or a marker

for a process?• Is the direction of change clinically relevant?• Are there correlative findings?

– survival– histopathology

clinical signs general health of affected animals– clinical signs, general health of affected animals• Is it reversible?• What is the mechanism?What is the mechanism?• What is the pharmacologic activity of the test

article?article?

53

Is It Bad? Factors to Consider• How large is the effect?• How much inter- and intra-animal• How much inter- and intra-animal

variability is expected?– species agespecies, age– unique study design conditions

• staggered startgg• fasted vs nonfasted• route of administration

ti bi t li i t d• time bias not eliminated• IM ketamine administration• excessive blood collectionsexcessive blood collections• unusual site for blood collection

54

Critical to Health or a Marker?Critical to Health or a Marker?Critical to health: Marker for a process:

hemoglobinneutrophils

MCV, MCHCneutrophils

fibrinogenglucosecalcium

fibrinogenurea nitrogen, creatininealbumin to globulin ratiocalcium

potassiumalbumin-to-globulin ratioserum enzyme activitiescardiac troponinscardiac troponins

55

Is It Bad?• hematocrit (%)• reference interval = 40 50%• reference interval = 40-50%

G St d A St d B St d XGroup Study A Study B Study X

Control 48 42 45Control 48 42 45

Hi h d 42* 37* 25*High-dose 42* 37* 25*

56

3-fold Increase ALT - Is It Bad?• Day 3 vs Week 52• Primary vs secondary

if d th i bl ld b b d– if secondary, then primary problem could be bad• Liver lesions vs “clean livers”• Correlative effects on function (e.g., bilirubin, cholesterol, ( g , , ,

glucose, proteins, coagulation)• Irreversible vs reversible

P i t di d l hi t f b d• Previous studies, drug class history of badness• Individual outliers vs group effect

– this one is tough, but if you are convinced that it is real, thenthis one is tough, but if you are convinced that it is real, then individual outliers large enough to increase group mean to 3-fold control have a greater likelihood of being bad

Underline indicates greater likelihood of being bad57

Lies, Damn Lies, and Reference Intervals

• Appropriateness of reference intervals for dataAppropriateness of reference intervals for data interpretation for any given study is a function of the partitioning factors that define the reference population

• Do not replace concurrent controls or sound scientific judgment

• High potential for inappropriate use

58

Partitioning Factors for Reference Intervals

• Species and strain • Methodology• Species and strain• Sex and age• Supplier

• Methodology– instrument– reagent system• Supplier

• Site of collectionF ti t t

reagent system• Sample storage

– fresh• Fasting status• Anesthesia

S l t i

– frozen– how long?

• Sample matrix– serum

plasma

• Housing/bedding• Diet

– plasma

59

More Partitioning Factors for Controls

• Route of administrationRoute of administration– oral vs intravenous

• Vehicle or control articleVehicle or control article– corn oil vs reverse osmosis water– dextrose vs saline

• Prior blood collection– TK/PK– serial clinical pathology

• Prior anesthesia

60

Defining the Reference Population

• LumpersLumpers– few partitioning factors

lots of animals– lots of animals– wide ranges

S litt• Splitters– many partitioning factors– few animals– narrow ranges

61

If you just can’t help yourselfIf you just can t help yourself

Use reference intervals the way a d k l tdrunk uses a lamp post….

for support, not illumination.

62

Quiz Time!Quiz Time!

Which finding is most important?

63

Which Finding Is Most Important?Which Finding Is Most Important?

• Hemoglobin 10% lowerg• Neutrophil count 10% higher• Creatinine 10% higher• Creatinine 10% higher• Sodium 10% lower• Potassium 10% higher• ALT 10% higherg• Urine pH 10% lower

64

Which Finding Is Most Important?Which Finding Is Most Important?

• Hemoglobin mildly lowerg y• Neutrophil count minimally higher• Creatinine minimally higher• Creatinine minimally higher• Sodium markedly lower• Potassium mildly higher• ALT minimally highery g• Urine pH mildly lower

65

“Word” Severity ModifiersWord Severity Modifiers• No universally (or even locally) accepted

definitions for preclinical studies

• Too many variables (see factors affecting interpretation)

• If percent- or fold-differences are pe ce o o d d e e ces a ecalculated, a severity modifier should be added for perspectivep p

66

Recovery/ReversibilityRecovery/Reversibility• Key – individual recovery animal resultsy y

– variable effects within an affected group– can’t expect a complete return to controlcan t expect a complete return to control

mean or an individual’s pretreatment results• “exhibit reversibility”y

• Recovery time varies with the parameter– red blood cell indices vs leukocyte countsred blood cell indices vs leukocyte counts

• Rebound effects for cell counts

67

Poor-Doers and “ADRs”• Decreased BW, BWG, and/or FC• Clinical signs (scruffy squinty squatty)• Clinical signs (scruffy, squinty, squatty)• Mildly decreased red cell mass

– reticulocytes unaffected or mildly decreased– reticulocytes unaffected or mildly decreased• Mildly decreased total protein

– often just albumin (calcium goes with albumin)often just albumin (calcium goes with albumin)• Other possible small changes

– decreased glucose, urea nitrogen, creatinine,decreased glucose, urea nitrogen, creatinine, cholesterol, triglycerides, aminotransferases, alkaline phosphatase, inorganic phosphorusi d h l t l t i l id– increased cholesterol, triglycerides

68

Inflammation• Clinical signs vary with severity/cause• Increased leukocyte counts

– neutrophilia more pronounced in dogs and monkeys– monocytosis variable

l mphoc tosis more common in rodents– lymphocytosis more common in rodents• Protein changes

– decreased albumin (negative acute phase protein)decreased albumin (negative acute phase protein)– increased globulin

• acute phase proteins (e.g. C-reactive protein, fibrinogen)i l b li• immunoglobulin

• Mildly decreased red cell mass if longstanding– reticulocytes unaffected or mildly decreasedreticulocytes unaffected or mildly decreased

• Microscopic evidence of inflammation69

Dehydrationy• Clinical signs of dehydration if severe enough

I d d ll (RBC t H b H t)• Increased red cell mass (RBC count, Hgb, Hct)• Increased serum proteins

albumin and globulin increase proportionately– albumin and globulin increase proportionately• Increased urea nitrogen, creatinine, phosphorus

– “prerenal azotemia” due to reduced renal blood flowprerenal azotemia due to reduced renal blood flow– UN increase usually more apparent than creatinine

increaseD d i l• Decreased urine volume

• Increased urine specific gravity or osmolalityEl t l t ll ff t d l V/D• Electrolytes usually unaffected unless V/D

70

Blood Loss (Hemorrhage)Blood Loss (Hemorrhage)• Clinical signs vary with rate of onset

D d d ll (RBC t H b H t)• Decreased red cell mass (RBC count, Hgb, Hct)• Reticulocytosis within a few days

– increased MCV and RDW, polychromasia andincreased MCV and RDW, polychromasia and anisocytosis

– increased splenic weights, EMH• Decreased serum proteins (albumin & globulin)• Decreased serum proteins (albumin & globulin)• Often have increased neutrophils and platelets• Occult loss (gastrointestinal urinary)Occult loss (gastrointestinal, urinary)• Iatrogenic (most common in monkeys)• Microscopic evidence of source (e.g., GI ulcer)Microscopic evidence of source (e.g., GI ulcer)

71

Red Cell Destruction (Hemolysis)• Onset usually rapid; clinical signs more common• Decreased red cell mass (RBC count, Hgb, Hct)( g )• Reticulocytosis within a few days; tends to exceed that

for blood loss– increased MCV and RDW polychromasia and anisocytosisincreased MCV and RDW, polychromasia and anisocytosis– increased splenic weights, EMH

• May see red cell abnormalities such as Heinz bodies, spherocytes or schistocytes (mechanism)spherocytes, or schistocytes (mechanism)

• Serum proteins unaffected or ↓ albumin and ↑ globulin• Often have increased neutrophils and platelets• Possible increased serum bilirubin• Possible bilirubinuria or hemoglobinuria

Mi i id f h l i i t• Microscopic evidence of hemolysis; pigment

72

Bone Marrow Toxicity• Clinical signs often associated with gastrointestinal injury

(rapidly dividing cells)f d/li id f d bl k– nonformed/liquid feces, red or black

– decreased FC, BW• Decrease of all blood cell lines

– reticulocytes/RBCs, neutrophils/WBCs, and platelets– decrease in relationship to circulating lifespan (i.e., neutrophils

and reticulocytes first, RBCs last)– rebound increases in reticulocytes, neutrophils, and platelets

• Other clinical pathology findings dependent on severity of effects on other organs/tissuesof effects on other organs/tissues– e.g., decreased lymphocytes (variable), electrolytes (GI losses),

inorganic phosphorus (low FC)Microscopic evidence varies with timing of sacrifice• Microscopic evidence varies with timing of sacrifice

73

Hepatocellular D ti /N iDegeneration/Necrosis

• Clinical signs varied, nonspecific; icterus only if severeg , p ; y• Increased aminotransferase activities

– ALT usually exceeds ASTin absence of muscle injury notably increased AST with ALT– in absence of muscle injury, notably increased AST with ALT usually indicates more severe injury

– SDH and GDH are additional liver-specific enzymesE id f d d f ti if• Evidence of decreased function if severe– increased total bilirubin and bilirubinuria– decreased glucose, cholesterol, proteins, UN– prolonged coagulation times

• Markers of inflammation• Usually see microscopic correlate with ALT > 200 U/L• Usually see microscopic correlate with ALT > 200 U/L

74

Hepatobiliary Injury and CholestasisCholestasis

• Clinical signs varied, nonspecific• Icterus only if total bilirubin > 3 or 4 mg/dL• Increased alkaline phosphatase and/or GGT

– alkaline phosphatase very sensitive to cholestasis in dogalkaline phosphatase very sensitive to cholestasis in dog– increased GGT is also associated with microsomal enzyme

inducers and centrilobular hypertrophy• Increased total bilirubin bilirubinuria• Increased total bilirubin, bilirubinuria

– relatively insensitive• Increased cholesterol, serum bile acids• Microscopic evidence of injury/cholestasis

– biliary hyperplasia and/or degeneration/necrosis– bile pooling in canaliculi or ductulesbile pooling in canaliculi or ductules– gall bladder findings (may be macroscopic)

75

Renal Toxicity• Clinical signs varied

– oliguria or anuria with severe acute failureg– polyuria, polydipsia

• Increased urea nitrogen, creatinine, phosphorusli i l t di i b f th ti l ¾ h l– preclinical studies; increases before theoretical ¾ nephron loss

• Increased urine volume, decreased urine specific gravity or osmolality– lost concentrating ability; theoretical ⅔ nephron loss– isosthenuria (1.008 – 1.012)– sometimes hard to evaluate because of normal variability

• Urinalysis findings varied– proteinuria, hematuria, glucosuria, casts, etc…

• Microscopic evidence is varied• Microscopic evidence is varied

76

Cardiac Muscle Injuryj y• Old school

– creatine kinase MB– lactate dehydrogenase 1

• New school– cardiac troponin I and T (cTnI and cTnT)

l b l t i t i di fil t– globular proteins present in cardiac myofilaments– new “biomarkers” that actually work; in large part

because gold standard in humans to evaluate cardiacbecause gold standard in humans to evaluate cardiac injury was transferred to animals

77

Prolonged Coagulation TimesProlonged Coagulation Times• Common causes for prolonged times

diffi lt ll ti– difficult collection• poor group health with difficult collection cause appearance

of a group effectti l t– excess anticoagulant

• short sample or polycythemia– beaglesg

• inherited factor VII deficiency, increased PT

• Most clotting factors synthesized by the liveri l ti l li i j t ff t ti– requires relatively severe liver injury to affect times

• Factors II, VII, IX, and X require vitamin Kfake fats anticoagulants (dicumarol)– fake fats, anticoagulants (dicumarol)

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Clinical Pathology ReportsClinical Pathology Reports

“No passion in the world is equal to the passion to alter someone else’s draft ”passion to alter someone else s draft.

H. G. Wells

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