6/10/2011

BLOOD COLLECTION

 Collection Site/Method
COMPARATIVE … Cardiac
• Elevated phosphorus and 
HEMATOLOGY OF potassium
• Increased muscle enzymes
I d l
LABORATORY ANIMALS  Standardize the site
 Anesthetics
Kirstin Barnhart, DVM, PhD, DACVP … Isoflurane minimally stressful
M. D. Anderson Cancer Center … Generally CO2 and 
pentobarbital stressful

Vahl et al (2005) Am J Physiol Endocrinol Metab 289, E823-8

BLOOD COLLECTION COLLECTION SITE

Orb sin

 Anesthesia method
… CO2 administration in rodents
 Increased
Increased potassium –
potassium due
due to metabolic acidosis
to metabolic acidosis
Orb sin
 Increased lactate
 Increased pCO2
 Decreased pH
Doeing et al (2003) BMC Clinical Pathology 3:3 Nemzek et al (2001) Inflam
Res 50: 523–527

BRAINTREE SCIENTIFIC
TERMINOLOGY Blood Collection for Facial Vein
(Submandibular) Blood Samples
Clinical Biochemistry  Submandibular vein
Parameters in C57BL/6J
Mice after Blood … Facial vein MEDIPOINT, INC.
Collection from the  Retro‐orbital venous plexus The Facial Vein and the
Submandibular Vein and Submandibular Vein meet at the
Retroorbital Plexus … Retro‐orbital sinus rear end of the Mandibular Bone,
providing
p g a convenient and
consistent source of blood

Fernandez, I., et al, Jerry Silverman

JAALAS, 49:2, “Using Nomina Anatomica Veterinaria
as a guide, there is no submandibular
202-206, 2010 vein in the mouse.”

JAALAS, 49:4, p.400, 2010

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BLOOD COLLECTION

Evaluation of  Hemolysis
Saphenous
Venipuncture and
… Mild hemolysis (1+)
 generally non‐significant
Modified Tail-clip
 very common in rodent samples
Blood Collection
in Mice … Moderate to Severe (2+ to 4+)
 potassium and bilirubin elevated along with 
Abatan, O.I., et al, other analytes
JAALAS, 47:3, 8-
15, 2008

BLOOD COLLECTION EDTA

 Tube Selection and Proper filling  Advantages
… Standardize for comparison between studies … Ideal for cellular morphology
… Inhibits bacterial proliferation
… Lithium heparin
 Disadvantages
… EDTA … Cannot be used for some chemistry analytes
Cannot be used for some chemistry analytes
… Sodium citrate … Underfilled tubes – RBCs shrink due to hypertonicity
• 3.2% vs 3.8%  Types 
• Avoid underfilling … K2EDTA (Sprayed on the side)
• Ideal of routine CBC
• Will not dilute the sample
… K3EDTA (Liquid)
• Causes sample dilution and affect RBC size

LITHIUM HEPARIN SODIUM CITRATE

 Advantages
… Can also be used for clinical chemistry  3.2% vs 3.8%
… For small animals with limited blood, collection into 
a single tube minimizes loss. … Both can be used, however
… Do not have to wait for sample to clot  The milder chelation of 3.2% provides greater 
 Disadvantages
Di d t accuracy of coagulation results
… Morphology not ideal
… Interferes with some immunoassays
 Cannot compare data if collected into 
different %

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BLOOD COLLECTION RODENT ERYTHROCYTES

 As compared to Dogs
 Processing Times … More polychromasia (1‐6% circulating RBCs or 
100,000‐350,00/µl
… Serum must sit for 30 minutes, minimum to  … More anisocytosis
allow to fully clot … Occasional Howell Jolly bodies
… Delayed processing alters analytes
Delayed processing alters analytes
 e.g. Decreased glucose
 Freezing and Thawing

INTERPRETING DECREASED RBC

ERYTHROCYTE LIFE SPANS
PRODUCTION IN RODENTS
 Most species Dogs 100‐115 days (17,000‐79,000/μL)
… Indices not changed as few reticulocytes normally in 
circulation Rats 45‐50 days (135,000‐250,000/μL)
 Mice/rats
/
… Decreased MCV Mi 43 d
Mice 43 days (221,000‐370,000
(221 000 370 000/μL)
… Decreased RDW
… Increased MCHC
 Short RBC life span
… Anemia of chronic disease develops more quickly

RODENT – ERYTHROCYTE MOUSE 

PARASITES NEUTROPHIL
Mouse Dog

 Plasmodium berghei •Nuclei display more

… Many experimental mouse models hypersegmentation
… Young Wistar rats highly susceptible •Even small changes may be
 Mycoplasma
y p coccoides ((Eperythrozoon
p y significant

coccoides) •Older mice may have a marked

neutrophilia due to long-standing
… Mice and rats inflammation
… May cause anemia
•Low number of ring forms
normal, increase with
neutrophilia

P. berghei

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MOUSE  MOUSE 
EOSINOPHIL BASOPHIL

•Low numbers in peripheral •Long propagated myth that

blood mice do not have basophils
•Band-shaped nucleus that •NOT TRUE!
can form a ring
•Very rare in circulation

RODENT PLATELETS
 “Sticky” and clump easily

MOUSE – BONE MARROW

Rat Dog Mouse Macaque
Neutrophilic ring forms
893‐1340 241‐561 453‐1181 318‐672

NORMAL LEUKOCYTES IN
LEUKOCYTE COUNTS IN RODENTS
RODENTS
 Less robust in as compared to large 
MOUSE < RAT AND RABBIT < DOG AND MONKEY  animals
… Magnitude of changes (dog > rat > mouse)
 Rodent WBCs … Qualitatively similar effects with excitement and 
stress
…Mostly lymphocytes
…Mostly lymphocytes
 Inflammation in rodents
…Fewer neutrophils; mouse < rat  Increased neuts and lymphs, +/‐ monocytes
 Left shifts in rodents less common than in larger 
…Very few monocytes, eosinophils animals but morphology of the shift when 
…Basophils extremely rare present is similar

 Decreased neutrophils
 Difficult to evaluate when resting count is so low

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HISTOPATHOLOGY - SPLEEN
SPLEEN

 Output is about 50% of the bone marrow 
under normal conditions
 Reservoir for hematopoiesis
 Megakaryocytes normally in clusters in 
Megakaryocytes normally in clusters in
the red pulp

Kelly L M et al. PNAS 2002;99:8283-8288

RABBITS GUINEA PIGS

•Heterophils •Heterophils
•2-4% polychromasia
•Lymphocytes predominate
•5-30% basophils
•Kurloff cells
•Lymphocytes and neutrophils
•More common in females
approximately equal in #
•Presumed to be an NK cell
•Leukocytosis often absent in
bacterial injections

HAMSTER CASE: 2 MO, SYRIAN HAMSTER

Analyte Units Results Ref Interval

 Very similar to mouse and rat RBC x106/μL 1.7 4.0‐10.0
Hemoglobin g/dL 6.0 13.0‐19.0
 Hibernation Hematocrit % 16 39‐59

… Normal RBC lifespan 
Normal RBC lifespan ~ 50‐70 days ‐‐Increases to 160 
50 70 days Increases to 160 MCV fL 91 68‐75
MCHC g/dL 37 28‐37
days WBC x103/μL 12.0 6.5‐11.0

… RBC counts and hemoglobin levels higher Seg neutrophils x103/μL 2.3 1.9‐3.1

Lymphocytes x103/μL 9.4 4.4‐7.2
… WBC counts decrease Platelets x103/μL 573 300‐490
Plasma  Mod hemolysis

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BABESIOSIS
PIG

Rouleaux
Artifactual crenation
High sedimentation rates
Highly susceptible to
hemolysis by hypotonic
saline
Little central pallor
Band cells in normal pigs
Hamsters have been used experimentally to study Babesia spp. 
and as a tool for  diagnosing human babesiosis (Babesia
microtii) through innoculation

SHEEP SHEEP
•Mycoplasma ovis
•Small erythrocytes
•Epierythrocytic
•Small platelets
•Cause of hemolytic anemia
•Some hematology in sheep and goats
analyzers cannot
distinguish platelets from
erythrocytes

RHESUS
DEER MONKEY

•Sickled erythrocytes •Very similar to humans

Non-pathogenic •Neutrophils have visible

granules (light blue to magenta
Common depending on the stain)
•Variable reports of neut/lymph
ratios, probably due to restraint,
anesthesia, subspecies, etc.

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MACAQUE HEMATOLOGY CASE #1

Plasmodium inui
Ring trophozoites
7‐month old male rhesus
Pale haircoat
Alopecia
Seemed to be less active
PCV = 5%
… Non‐regenerative
… Normocytic, normochromic
… Morphology ‐ unremarkable
Hemazoin – malaria pigment

SQUIRREL
WHITE MONKEY DISEASE MONKEY

 Cage treated for rust several

•Blood smear morphology
months prior similar to macaques
 Zinc toxicosis •Lymphocytes reportedly
predominate, but neutrophils
 Decreased copper can be
b seen as primary
i cellll
 Severe, non-regenerative anemia •Occasional nRBCs - normal
 Hairloss and depigmentation
 Baboons, macaques – young
animals only

OWL MONKEY CHIMPANZEE

Aotus nancymae
•Blood smear morphology
•Most common species similar to rhesus monkeys
•Blood smear morphology
•Have less platelets than other
similar to macaques
NHP species
L h
•Lymphocytes d i
predominate
•Occasional nRBCs - normal
Aotus azarae
•Normally have elevated
eosinophil counts
•Heightened eosinophilopoiesis
•Decreased recruitment kinetics

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NON-HUMAN PRIMATE BONE MARROW

HEMATOLOGY CASE #2 CBC

History PCV = 26 (35-43)

Day 1 MCV = 34.8 (65.6-74.8)

Diarrhea 7 days post- MCHC = 28.5 (32-34)
weaning
T Erythromycin
Tx: E th i PO BID R ti Count
Retic C t=0
0.2%
2% (>1% = regen))
Day 4
Found recumbent in cage
Hypothermic (90°F)
Weak, staring into space Microcytic, hypochromic
TX: SQ fluids, oral non-regenerative anemia
dextrose, heat lamp,
responded rapidly

INITIAL BLOOD SMEAR ASSESSMENT

Day 6 Differential Primary Diagnoses
 Iron deficiency
* Iron levels normal to elevated
Schistocyte
 Inherited hemoglobinopathy
* Never reported in any domestic species
 Beta‐ or alpha‐ thalassemia
Ovalocyte
* Never reported in any domestic species

Secondary Considerations
 Vascular injury
Acanthocyte Hypochromic
 Immune‐mediated RBC destruction
 Infection

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CLINICAL PROGRESSION LACK OF COORDINATION

Day 7 
Tx: Prednisolone 4mg IM BID
Procrit SQ
Day 9 “thrashing around”
Not using left arm or left leg
Difficulty holding up his head
No cranial nerve deficits noted
Pale
TX:  Heparin 300U S

CLINICAL PROGRESS (CONT) REVERSE-PHASE HPLC

Buck, Sire, Dam, 1 normal 1 normal adult

weanling, 1 normal adult
Day 11 – Blood Transfusion 16.783
α
16.702
α β
31.254 31.207 44.576

P PCV 21 P PCV 32
Pre PCV = 21, Post PCV = 32 β

AU
AU

45.102
47.654 γ or X?
Femoral transfusion
TX: Rocephin 60mg IM BID
Discontinue erythromycin Minutes Minutes

POSSIBLE DIAGNOSES MICROCYTOSIS OF YOUNG RHESUS

 Not a lack of iron availability
… RBC iron utilization problem?
Sex Age Diagnosis WBC RBC HGB HCT MCV MCHC RDW PLT MPV

 Not a heritable form of hemoglobinopathy or  M Juvenile Lethargy 8.51 7.88 9.49 32.5 41.2 29.2 23.8 810 12.5

thalassemia M Infant Thin/Diarrhea 9.05 9.62 13.20 43.2 44.9 30.5 18.4 721 10

… Acquired form? M Juvenile
Diarrhea poss
26.9 6.79 11.00 35.0 51.6 31.3 31.7 723 NA
campy

 Something else? F Infant Pale 15.8 6.12 10.50 33.8 55.3 31.1 16.7 693 8.45

M Infant Thin/Diarrhea 9.05 9.62 13.20 43.2 44.9 30.5 18.4 721 10

Normal MCV = 65-73

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DAY 23 DAY 67

DAY 103 NEUROLOGIC STATUS OUTCOME

 ~200 days
… Clinically normal

 Developed into a young male adult
Developed into a young male adult
 Not used for breeding

COAGULATION IN RODENTS COAGULATION PANEL - RODENTS

 Problems and Pitfalls  Routine
… Absolute Platelet count
… Rodents are resistant to thrombus formation … Prothrombin time (PT)
… Standard coagulation biomarkers not very sensitive or  … Activated partial thromboplastin time (APTT)
specific  Variable
… Fibrinogen
… Endpoints not standard in early screening studies … MPV
… Abnormalities in longer term studies often subtle … Bleeding time
 Distal tail incision, normal less than 60 seconds
… Histopathology not sensitive  D‐dimer, ATIII can be helpful but not well characterized
… Results easily altered by collection techniques

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Spontaneous KNOCK-OUT MOUSE

Coagulopathy in FVB BACKGROUND
Inbred WAG/RijYcb
Rats
 No spontaneous hemorrhage
Booth, C., et al,
Comp Med 6):1,  Routine tail clip for genotyping
25-30, 2010
 Uncontrolled bleeding leading to death
U t ll d bl di l di t d th
Elevated APTT Elevated APTT
Normal PT Normal PT
Normal platelet Normal platelet

Gene deletion results in an intrinsic pathway defect

Hereditary defect in the intrinsic pathway

COAGULATION – PREGNANT RAT

 Suitable model for humans in late pregnancy
…
…
Increased fibrinogen
Increased platelet counts
CLINICAL CHEMISTRY OF
…
…
Decreased PT
Overall activity of vitamin‐K–dependent coagulation 
LABORATORY ANIMALS
factors
… Increased APTT
… Increased ATIII Kirstin Barnhart, DVM, PhD, DACVP
M. D. Anderson Cancer Center

GLUCOSE GLUCOSE

 Mice  Non‐human primates
… Higher in cardiac puncture as compared to jugular … Chimpanzees
… Anesthetic method has been reported to affected   Similar to humans
results
… Old World monkeys
… Normally higher than large mammals
Normally higher than large mammals  20‐30 mg/dl lower than humans
20 30 /dl l th h
 Average between 125 and 250 mg/dl after 4hr fast
 100‐126 mg/dl likely to be overt diabetes
… Diabetes – defined as greater than 300 mg/dl
 Must be persistent!
… New World monkeys
 Common to have elevations greater than this with stress
 Many species are similar to rodents  and have 
 Rats marked elevations due to stress
… Lower average fasted levels, around 100 mg/dl   Glucose greater than 200 mg/dl is common in 
squirrel and owl monkeys

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DIABETES MODELS DIABETES TESTING

 C57bl/6J ob/ob  OGTT
… Obese, hyperglycemic, hyperinsulinemic  IVGTT
 Zucker fatty rat (fa/fa)
fatty rat (fa/fa)  Euglycemic clamp
E l i l
… Obese, hyperinsulinemic, normoglycemic

 Rhesus and cynomolgus monkeys
… Insulin resistant
… Overt type II diabetes

OGTT
 Better than a fasting
blood glucose
 Best used as a
screening test
 Plagued by many
factors that cause poor
reproducibility
 20% non-diagnostic
 Must be confirmed with
a repeat test
IVGTT
 Indirect measure of insulin
sensitivity
 Calculates a glucose
disappearance rate (Kg)
 Using differential equations
(Minimal model) can
measure insulin
sensitivity/resistance
EUGLYCEMIC CLAMP
(ALSO TERMED HYPERINSULINEMIC)
 Direct Measure of Insulin Sensitivity
 General theory
… By maintaining a constant high insulin level, we can 
y g g
determine the amount of glucose that is required to 
maintain euglycemia

EUGLYCEMIC CLAMP
Advantages Disadvantages
 Insulin prime (10 minutes)
 Insulin steady state (40mU/m2/min)  Widely accepted as  Time Consuming
… Hyperinsulinemic (at this point) the reference standard
to determining insulin  Labor intensive
 20% dextrose infusion
sensitivity in humans
 Glucose testing every 5 minutes  Expensive
 Adjust glucose infusion rate to maintain euglycemia
Adj l i f i i i l i  Directly measures
whole body glucose  Experienced Operator
(80‐90 mg/dl) – this is the “clamp”
disposal at a given to manage technical
 Measure insulin every 10 minutes, every 30 minutes 
level of insulinemia difficulties
measures Potassium
 Excellent test
High Glucose infusion rate ‐ SENSITIVE characteristics
Low Glucose infusion rate ‐ RESISTANT

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PROCEDURE

 Place 3 catheters
… 1 Peripherally inserted central catheter (PICC)
… 1 18g peripheral, 1 22g peripheral

 Two infusion pumps 
 Bedside analyzer of glucose (Istat)

PROPER LENGTH

Hyperinsulinemic-euglycemic clamp studies in ob/ob mice treated with PBA or TUDCA.

INTRODUCTION

U Özcan et al. Science 2006;313:1137-1140

Published by AAAS

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IVGTT RESULTS GLUCOCORTICOIDS

Normal Insulin Resistant  Mice/rats

C D … Corticosterone predominates, 99% circulates bound to 
550 550

cortisol‐binding protein and albumiin
Glucose Glucose
500 500
Insulin Insulin
450 450
400
Glucose (mg//dl)

400

Glucose (mg//dl)
 Squirrel monkeys
ml)

ml)
S i l k
Insulin (uU/m

Insulin (uU/m
350 350
300 300
250 250
200

150
200
150
… Extremely high circulating levels
… 1/3 of cortisol circulates unbound
100 100
50 50
0 0

… Impaired clearance, decreased receptor affinity
0

10

20

30

40

50

60

10

20

30

40

50

60
Time (minutes) Time (minutes)

… High levels of ACTH

HEPATIC ENZYMES - MOUSE HEPATIC ENZYMES - RATS

Hepatocellular Injury Hepatocellular Injury
 ALT  ALT
… 1° liver but also cardiac and testicular injury  AST
… 11,000% increase with mouse hepatitis virus  Sorbitol Dehydrogenase (SDH)
… Significant elevations due to heliocbacter hepaticus … Frequently the most sensitive and specific 
 AST
… Very non‐specific
Very non specific … Requires rapid processing
q p p g
… Primarily in periportal hepatocytes  Glutamate Dehydrogenase (GLDH)
… Not affected by muscle
Cholestasis
 Alkaline phosphatase Cholestasis
… Highest levels in intestine and kidney  Alkaline phosphatase
 GGT  GGT
… Very insensitive, normal levels very low … Very insensitive, normal levels very low

HEPATIC ENZYMES - RABBIT CHEMISTRY CASE - RABBIT

Hepatocellular Injury
 ALT Clinical Pathology Differentials
… Half‐life 5 hours, 
… Liver activity similar to heart muscle • ALP – marked increase • Intestinal obstruction
 Alkaline phosphatase
• ALT- marked increase • Tyzzer’s disease
… Also less specificity
p y
 GGT • Hyperbilirubinemia • Hepatic coccidiosis
… Very insensitive, normal levels very low
• Hyperproteinemia • Hepatic lipidosis
Cholestasis
 Hyperbilirubinemia
 ALP

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HEPATIC COCCIDIOSIS HEPATIC ENZYMES - PIG

Hepatocellular Injury
Eimeria stiedae oocyst
 ALT
… Half‐life 50 hours
… Skeletal and cardiac muscle have higher activity
 Sorbitol dehydrogenase
… Half‐life ~ 2 hours
… Best for acute injury
 AST
… An indicator of non‐specific organ damage
Cholestasis
 ALP
… Decreases related to food intake
… Some utility as a hepatic marker
 GGT

HEPATIC ENZYMES - NHP GAMMA GLUTAMYLTRANSFERASE-NHP

 ALT/AST  Distribution studied in macaques and baboons
… Magnitude of the elevations not as dramatic as some species • Liver, pancreas, kidney
… Normal 10‐50 IU/L  More sensitive marker of biliary disease than ALP
… With liver disease 50‐500 IU/L  Higher in young animals and decrease with age
… Muscle Contribution  Higher in Mauritian cynos as compared to Southeast Asian
 Often greater than liver
Oft t th li  Can be significantly elevated with no change in ALP or 
 Look at the ALT/AST together with LDH and CK bilirubin
Chimp 1 Chimp 2 Chimp 3

GGT ALT AST LDH CK Chronic

Hepatic Ref
Passive Hepatitis C
33 503 2,340 16,780 234,900 Amyloidosis Interval
Congestion
GGT 269 127 1436 20
20--40
ALT 36 28 89 10
10--50
AST 31 30 37 10
10--50

ALKALINE PHOSPHATASE - NHP CREATINE KINASE ISOENZYMES

 Electrophoretic separation of isoenzymes
 Sensitivity as an indicator of cholestasis and biliary 
 Isoenzymes, rat 1 ,2 
disease is much poorer than in many other laboratory 
… CK‐MM ‐ skeletal muscle and heart
animals
… CK‐MB ‐ 1° heart but also skeletal muscle 
 Macaques can be as high as 1500 IU/L
… CK‐BB ‐ 1° heart brain but also spleen, kidney, liver, 
 Squirrel monkeys can have high levels
q y g
h
heart and skeletal muscle
d k l l l
 Similar to GGT, is higher in Mauritian cynos
 Rat heart ‐69.9 % MM, 24.43% MB, 6.38% BB
 Rat skeletal muscle ‐93.6% MM, 1.09% MB, 5.3% BB
 Of limited usefulness for cardiac diagnosis 
 Many better tests available

1. Boyd, Vet Clin Path 12:9-24, 1996

2. Fredericks et al, Clinica Chimica Acta, 304:65-74, 2001

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LACTATE DEHYDROGENASE MYOGLOBIN VS HEMOGLOBIN

 Elevates with skeletal and cardiac muscle   Both will cause a positive urine
dipstick reaction for blood
injury
 Very non‐specific  Both may or may not be
associated with intact RBCs
 Mice 
Mice – Lactate dehydrogenase elevating 
Lactate dehydrogenase elevating
 Hemoglobin quickly bound to
virus haptoglobin and retained in serum
 Unexplained elevations can be associated 
 Myoglobin unbound and clears
with tumors quickly
 Ammonium precipitate test can
confirm

CARDIAC BIOMARKERS
TROPONIN
 Troponins (I and T)
 BNP and NT‐proBNP  Protein present in the thin myofilaments
… Involved in regulation of muscle contraction
 Atrial
At i l natriuretic
t i ti peptide (ANP)
tid (ANP)
 Different isotypes present in skeletal and cardiac muscle
… Troponin I
 More widely available assays
 Most commonly used
… Troponin T
 Sensitivity and specificity equal to troponin I
 Not as many assays available

TROPONINS (CONT) BNP AND NT-PRO

 Indicate cardiac ischemia/injury  BNP is produced by the ventricles in response to 
 Assays have been validated for a wide variety of  cardiomyocyte stretching
 NT‐proBNP is the cleaved portion of the hormone, may 
species 
be less affected by degradation
 Interpret with caution in the face of renal   Strong indicator of heart failure
g
disease  Can be elevated by renal disease
 Pick either I or T but do not need to run both  Species
… Dogs – works well
… Primates – works well
… Rats – some assays have not cross‐reacted well

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 6/10/2011

BILIRUBIN BILIRUBIN
 Rodents Adult primates ‐ uncommon
… May be used for cholestasis
… Leptospirosis
… Heritable deficiency in bilirubin UDP‐
glucuronosyltransferase … Hepatocellular carcinoma
 Gunn rat – severe unconjugated
… Right sided heart failure
hyperbilirubinemia
 Models of Gilbert syndrome
 Neonatal rhesus monkeys
 Rabbits  Normal destruction of fetal
… Total activity is normally low hemoglobin
… Elevations rarely seen  Immature liver

RENAL FUNCTION - RATS

Reference Values for
Serum Proteins of
 BUN/creatinine Common Laboratory
 Uniquely susceptible to nephropathy from α2‐urinary  Rodent Strains

globulin Zaias J., et al,

JAALAS 48:4
JAALAS, 48:4,
… Contributes to strange odor of rat and mouse urine
387-390, 2009
… Persistance in proximal tubule of rats leads to 
cytotoxicity of tubular epithelium
Commercially available 
agarose system

URINE PROTEINS (RAT) URINE PROTEINS (RATS)

 Alpha2u– globulin  Major constituents of urinary protein in rats (cont.)
… Albumin
… Predominant urinary protein in adult male rat  • Urine albumin ~1mg/24hr in young adult male rat 
• Synthesized in liver under influence of androgens
• Low levels in juvenile and senescent males  • Increased amounts excreted with age
• Minimal in females
… Tamm‐Horsfall glycoprotein
… LMW (16‐20 kDa) lipocalin freely filtered at glomerulus • Wide range of normal values in literature
• Associated with chemical‐induced male rat 
specific renal tumor formation  • ↑ Day‐to‐day variability in urinary excretion
• Various exogenous chemicals bind 2u– globulin 
and prevent lysosomal degradation  … Diet influences protein excretion
• Epithelial cell degeneration and renal tumor  • High‐protein diet → increased 2u– globulin excretion
formation through increased cellular proliferation
• Protein‐deficient diet → decreases in both albumin 
and 2u– globulin

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URINE PROTEINS URINALYSIS

 Mouse  Rabbits
… Proteinuria is common in mice … Urine is major route of calcium excretion – leads to 
… Small quantities of α‐ and β‐ globulin  cloudy, turbid urine that will become pasty if fed a 
high calcium diet
… Prealbumins known as mouse urinary protein 
… Calcium oxalate, calcium carbonate, struvite
Calcium oxalate calcium carbonate struvite
(MUP) – “uromucoid”
 LMW lipocalin synthesized in liver 
 Structurally similar to 2u– globulin
… Urine concentration very high in healthy mice
(1.060‐1.080)

URINALYSIS - NHP CALCIUM 

CARBONATE

 Specific gravity
… Typical range is 1.003 – 1.020
Rabbits
… No defined isosthenuric range
Horses
 Urinary crystals common in macaques
Ui t l i
Guinea pigs
… Calcium oxalate, amorphous phosphate, struvite
Goats
 Ketonuria
… Can be seen due to negative food intake
… Fecal contamination can cause a false positive

CALCIUM 
OXALATE URINARY   Proximal tubular dysfunction
BIOMARKERS –
… N-acetyl-β-D-glucosaminidase
Can be normal in
RATS MODEL OF 
POLYCYSTIC  … α-glutathione transferase
many domestic and lab
animal species KIDNEY DISEASE   Distal tubular dysfunction
Dihydrate
Pathogenic … S
S-transferase
transferase Yb1

•Increased calcium  Glomerular lesions

Wiedmeyer C.E.,
excretion and Royal, A. B. … Collagen IV
•Oxalates Comp Med, 60:6,  Glomerular and proximal tubular
448-454, 2010
•Ethylene glycol … Albumin

Monohydrate

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 6/10/2011

CALCIUM LIPIDS
 Mice
… HDL predominant
 Rabbits often have high circulating levels … No spontaneous atherosclerosis due to low levels of LDL and 
CETP
… Absorb very efficiently from the gut independent of 
 Rats
need or vitamin D levels … HDL predominant
… Overnight fast, 
Overnight fast, ~30%
30% will have hyperlipemia, but cholesterol 
will have hyperlipemia, but cholesterol
unchanged
… Up to 16 mg/dl
 Study of gerbils, hamsters, guinea pigs
… Can make interpretation of high calcium difficult … Guinea pig best natural model

 Rabbits
… Easier to induce hypercholesterolemia than the rat

 To be effective most rodent models need to be 
genetically modified

SD RATS, 7 WEEKS, NON-FASTED

CHOLESTEROL DIETS BUN Creat T.Bili ALP GGT ALT AST CK  Na  K Cl
mg/dl mg/dl mg/dl IU/L IU/L IU/L IU/L IU/L mmpl/L mmol/L mmol/L
16.2 0.48 0.09 387 3 56 124 977 141 6.1 101
15.0 0.50 0.09 412 3 57 111 688 141 5.7 104

 Induce hemolytic anemia in the guinea  18.5
17.3
15.0
0.55
0.55
0.55
0.09
0.09
0.09
285
302
306
3
3
3
48
45
42
91
91
74
643
592
161
140
140
139
5.9
5.8
5.4
101
100
101

pig 16.4
19.4
0.53
0.49
0.09
0.09
338
335
3
3
50
10
98
58
612
355
140
139
5.8
6.2
101
99
32.3 0.37 0.09 167 3 10 131 577 149 4.0 106
17.2 0.49 0.09 364 3 9 66 560 137 5.8 97
16.5 0.49 0.09 376 3 18 735 516 138 6.0 98

 Induce hemolytic anemia in the rat
12.6 0.49 0.09 432 3 16 53 211 136 5.1 95
Induce hemolytic anemia in the rat 19.6 0.47 0.09 335 3 13 76 444 140 5.4 99

… Proposed mechanism is decreased phospholipid in  Ca P Glu Chol Trig TP Alb Glob

mg/dl mg/dl mg/dl mg/dl mg/dl g/dl g/dl g/dl
RBC membrane causing decreased deformability 10.6 8.5 148 64 98 5.8 4.3 1.5
10.4 8.7 190 45 97 5.5 4.0 1.4
10.5 10.0 230 65 175 5.3 3.8 1.5
10.9 9.6 239 68 95 5.3 3.9 1.4
10.4 10.2 249 61 55 5.3 3.9 1.4
10.6 9.4 211 61 104 5.4 4.0 1.5
10.2 9.1 215 36 4 5.6 4.3 1.4
9.3 8.0 162 33 18 4.7 3.7 1.0
10.7 10.0 204 45 11 5.4 4.2 1.1
10.8 9.7 225 38 9 5.4 4.2 1.2
10.6 9.6 215 38 12 5.3 4.2 1.0
10.3 9.3 204 38 11 5.3 4.1 1.1
Irizarry
ASVCP Case Review 2006

ELEVATED TRIGLYCERIDES
HYDRAZINE ADMINISTRATION
WITH NON-LIPEMIC SERUM

 Oral gavage for 7 days  Common in NHPs

 Elevations as high as 2500
 Decreased ALT, lipids and 
 Always in non‐lipemic serum, 
globulins
 Generally animals are fasted for 12 hours
Generally, animals are fasted for 12 hours 
 Hepatic lipidosis because anesthesia required for blood 
 Inhibits pyridoxal‐dependent  collection
enzymes (e.g. AST and ALT)  May be anorexic and on a negative 
energy plane

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 6/10/2011

GLYCEROL SERUM AMYLOID

 Total triglycerides contains 5‐10 mg/dl 
glycerol  Acute Phase Reactant
 Free glycerol included in total triglycerides … Produced at high levels by the NHP liver
… Measured without lipase for selective ID  Contributes to reactive amyloidosis
Contributes to reactive amyloidosis
 Hypertriglycerolemia in humans … Common problem in NHP colonies
… Glycerol kinase deficiency
… Frequently liver only
… Diabetes, metabolic syndrome
 Martin and Rand, 2007 Vet Record (Feline  … Can be systemic
diabetes)
 Hamsters also have frequent amyloidosis

EVALUATING INFLAMMATION
HEPATIC AMYLOIDOSIS - RHESUS
IN PRIMATES
 WBC
…Much smaller elevations than dogs
…Even a mild left shift is very significant
 Sedimentation Rate
Sedimentation Rate
…Very valuable in chimpanzees
…Rarely helpful in macaques or new world monkeys
 SAA
…Strongly associated with hepatic amyloidosis in 
chimpanzees and rhesus macaques

EVALUATING INFLAMMATION
IN PRIMATES ACUTE PHASE PROTEINS - RODENTS

Rats Mice
Fibrinogen
 Fibrinogen  Fibrinogen
…Strongly associated with acute inflammation in both 
 α2-macroglobulin  Ceruloplasmin
chimpanzees and rhesus macaques
 Total α- and β-globulins  C-reactive protein
Iron/TIBC/% Saturation
 Erythrocytes  Serum amyloid P
…Acute, significant decrease in serum
 Albumin  Serum amyloid A
…Prevalent in many NHP species
 Serum iron  α1-fetoprotein

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 6/10/2011

WASTING SYNDROMES IN NEW WORLD INDEX CASE – ADULT FEMALE

MONKEYS OWL MONKEY
1/25/2010
Marmosets Owl Monkeys  Clinical signs  Pancytopenia
 Emaciated  Severe neutropenia
 Ravenous  Severe lymphopenia
 Bright and alert  Moderate anemia
 Symmetrical, non-  Borderline regenerative
pruritic alopecia
 Moderate thrombocytopenia

BLOOD SMEAR - 1/25/2010 CBC – 1/29/2010

 RBC  Pancytopenia
… 2+ anisocytosis … Severe neutropenia
… 2+ poikilocytosis  Mild left shift

… 1+ hypochromasia … Severe lymphopenia

… Rare schistocytes … Severe anemia
 Mild regeneration, exaggerated nRBC increase
 WBC
… Moderate thrombocytopenia
… NSF

WBC Count
25

BLOOD SMEAR - 1/29/2010 20

cells x103/ul

15

10

 RBC 5

… 3+ anisocytosis 0
9

0
00

00

01

01
00
5/2

4/2

6/2

5/2

9/2

… 1+ poikilocytosis
7/1

7/2

/1

1/2

1/2

p y
12

Neutrophils (Absolute Count)

Lymphocytes (Absolute Count)

… 2+ hypochromasia
3.0
20

2.5

15

 WBC
2.0
cells x103/ul

cells x103/ul

1.5
10

… Moderate Dohle bodies
1.0

5
0.5

… Mild toxic change 0.0 0
9

0
00

00

00

01

01

00

00

01

01
00
5/2

4/2

5/2

9/2
6/2

5/2

4/2

5/2

9/2
6/2
7/1

7/2

/1

1/2

1/2

7/1

7/2

/1

1/2

1/2
12

12

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 6/10/2011

Anemia
125 Mean Platelet Volume
Platelets
500 20
HCT 1.3%
100 nRBC
ells/100WBC)

400
15

75
ct (%)

300

fL
10
Hc
nRBC (ce

50 200

5
0.5%
25 100

1.2% 0
0
0

0
00

00

01

01
00
9
09

0
00

00

01

01

4/2

6/2

/2
0

5/

5/
/2

4/2

6/2

5/2

9/2

29
7/1

7/2

/1

1/2
15

1/
7/2

/1

1/2

1/2

12
9

09

7/

12
00

00

01

01
20
/2

/2

/2

/2
6/
15

24

25

29
/1
7/

7/

1/

1/
12

Normal Marrow Marrow from Wasting Animal

BONE MARROW EVALUATION

 18 gauge intraosseous catheter
… Left femur 
 Dry tap
… Right femur 
 No blood return into the syringe.
 Small fibrous clumps of marrow pushed out 
with the stylet
 Impression smears made

ERYTHROID DYSPLASIA MYELOID DYSPLASIA

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MEGAKARYOCYTIC DYSPLASIA WASTING SYNDROME TO DATE

 Approximately 25 animals affected
 Consistent clinical pathology findings
… Pancytopenia
… Profound panhypoproteinemia
… Elevated GGT
 Intestinal histopathology 
… Villous blunting and fusion

TDX: Celiac disease d/t dietary intolerance

IRON OVERLOAD IN NEW WORLD

MARMOSET WASTING SYNDROME MONKEYS

 Some similarities with owl monkey Squirrel Monkeys Owl Monkeys

… Emaciation despite strong appetite
… Vitamin D deficiency
… Alopecia

 Differences with owl monkey
… Severe diarrhea
… Paralysis and muscular weakness
… Lethargy

INDEX CASE LIVER – H&E STAINING

 Squirrel monkey necropsy Hemochromatosis Normal

… Trauma
… Poor body condition
Poor body condition
… Renal disease
… Liver – ORANGE
 No clinical pathology available

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 6/10/2011

NORMAL HEPATIC IRON IN

LIVER – PRUSSIAN BLUE STAINING
PRIMATES

IRON OVERLOAD PREVIOUS REPORTS

 Lemurs
… Identified in 1960s
… 2006 – Am J Primatol, Glenn et al.  ‐ 50% incidence
 Callitrichidae
… 1997
1997 – Miller et al, Lab Anim
Miller et al, Lab Anim Sci, common 
Sci, common
marmosets – 100% incidence
… 2008 – Am J Primatol, Smith et al. –
marmosets/tamarins – blood values correlate with 
liver histopathology

PREVALENCE ASSOCIATED PATHOLOGY

 87 squirrel monkeys  Concurrent disease in 50% of the animals 
with elevated iron
… 29 had serum iron >160 (33%) … Weight loss, lethargy
… Renal disease
 67 owl monkeys
67 owl monkeys … Cardiac disease
… 23 had serum iron >160 (34%) … Diarrhea
 Elevated GGT
 Other findings
… % saturation frequently approaching 100%
… TIBC frequently mildly to markedly elevated

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 6/10/2011

CHALLENGING CASE – ADULT RHESUS CLINICAL CHEMISTRY – H42

MONKEY (H42) Hypertriglyceridemia Chol 175
WBC ct 16.30 Trig 916
Neutrophils 9.45
Azotemia CK 1184
BUN 77
Hyperphosphatemia
Bands 4.40
Metamyelocytes 0.33 Creat 4.50
Moderate leukocytosis Lymphocytes 1.14 Hyperglobulinemia T.Prot 8.4
Moderate neutrophilia Monocytes 0.98 Albumin 4.2
Se ere left shift
Severe left shift Eosinophils 0 00
0.00 Hypcalcemia Globulin 42
4.2
RBC ct 7.32 Ca 11.7
Slight monocytosis Hgb 16.7 Hyponatremia P 5.8
Hemoconcentration HCT 50.7 Hypochloremia Na 129
K 1.6
MCV 69.2
Hypokalemima Cl 83
MCH 22.7
TCO2 16
MCHC 32.9 Titrational acidosis Anion Gap 31.6
RDW 14
Iron 46
Platelet 556 Hypoferremia TIBC 370
MPV 9.76
%Sat 12

URINALYSIS – H42 H42 - COAGULATION

Day Glu SG Blood pH SSA WBC Casts Trans

Day 5 Day 6 Day 7
Day 6 100 1.010 Large 5.0 1+ 0-2 Few Few
PT 9.5 10.4 12.0
Hematuria with hemoglobin casts Fibrinogen 440 960 1036
Increased transitional epithelial cells
APTT 15.4 24.5 37.7
Glucosuria
ATIII 52 87 69
Aciduria
Proteinuria

NECROPSY - KIDNEY KIDNEY – WARTHIN STARRY

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 6/10/2011

CHALLENGING CASE – RHESUS

CLINICAL PATHOLOGY DATA
MONKEY
 Juvenile, rhesus monkey  Serum biochemistry
… Albumin: 1.5 g/dl (2.4‐3.6 g/dl)
 Depressed
 CBC
 Thin … PCV: 31.4% (35‐45%)
 Prominent SQ pitting edema of  … WBC: 3600 cells/ul (5000‐9,000 cell/ul)
 Moderate lymphopenia and monocytopenia
dependent areas
… TP: 5.3 g/dl (6‐8 g/dl)
 Marked lymphadenomegaly

Lymph Node Cytology

CLINICAL PATHOLOGY DATA

 Urinalysis
… Specific gravity: 1.026
… 100 mg/dl protein
… Moderate bilirubin and blood

 Urine protein:creatinine ratio = 0.8
… 2 days later = 1.8

NECROPSY: GROSS FINDINGS

 Mild peripheral SQ edema in extremities
 Moderate to marked peripheral and 
visceral lymphadenomegaly
i ll h d l
 Multiple 1‐2 mm nodules in the spleen

TDX: Leishmaniasis

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 6/10/2011

ADDITIONAL DIAGNOSTICS
Lymph Node

 Serology: 
… CDC: T. cruzi titer stronger than 
Leishmania titers
… Heska Corp: Leishmania
Corp: Leishmania negative
 Immunohistochemistry: 
… Brazilian Leishmania specific assay:
 Monkey ‐ positive
 Confirmed T. cruzi case ‐ negative

Heart Pseudocyst
CELL CULTURE

T. cruzi epimastigotes
100x

Kinetoplast adjacent to nucleus

T. CRUZI IN MONKEYS
Undulating membrane

 KCCMR ‐ Texas
… 50 adult male/female rhesus monkeys
… Blood collected – sent to CDC for blood culture – gold 
standard for T. cruzi infection
… 1/60 positive culture
/ p
… 1 previous death confirmed on necropsy

 Southwest Foundation – Texas
… 4 baboons (Williams et al, J Med Primatol, 38(2), 107‐
113, 2009
Modified Wrights 100x Diff-Quik

27
 6/10/2011

QUESTIONS?

28