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Clinical Chemistry

Clinical Chemistry

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Clinical Chemistry
Clinical Chemistry

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I.Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .140II.Sample Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .140III.Genitourinary Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .141A.Renal function and osmoregulation . . . . . . . . . . . . . . . . . . . . . . . .141B.Urinalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .142C.Figure 1.Biliverdinuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .142IV.Liver,Muscle,and Bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .143A.Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .143B.Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .145 V.Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .146 VI.Electrolytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .146 VII.Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .147A.Figure 2.Protein electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . .147 VIII.Iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .148IX.Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .148X.Table 1.Reference Intervals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .144XI.References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149
Introduction
In the past 20 years,the number of veterinarians prac-ticing companion avian medicine has increased expo-nentially in response to the increased numbers of petbirds owned in the United States.Bird owners are look-ing for veterinarians that can provide quality medicalattention.Plasma biochemistry along with hematologyis the cornerstone of medical diagnosis of disease. Veterinarians therefore have an increasing need foraccurate and useful biochemical analyses for avian andexotic animal species.Veterinary laboratories and clini-cal pathologists must meet these expectations anddevelop the techniques and services needed for avianbiochemical analysis and interpretation.Biochemical reference intervals for common speciesof psittacines (parrots),passerines (canaries and finch-es),and galliformes (turkeys and chicken) have beenestablished by specialized laboratories,eg,CaliforniaAvian Laboratory,Citrus Heights,Calif.Reference inter-vals should be developed by each laboratory analyzingavian samples,but are still lacking in some universityand private laboratories.Although a great deal now isknown regarding avian medicine,research into thediagnostic application,sensitivity,specificity,and posi-tive and negative predictive values of biochemical ana-lytes still is needed.Physiological and anatomical differences some-times require the use of different analytes in birds com-pared with mammals (eg,uric acid versus BUN).Therefore,basic knowledge of avian physiology is vitalto appropriate interpretation of blood chemistry values.This article is organized by organ system,with a reviewof applicable chemical methodology and basic aviananatomic and physiological differences.This literaturereview will likely lead to more questions than answers,but hopefully,in the next 20 years,we will be able toanswer those questions.
Sample Collection
Collection of blood volume equivalent to 1% of a bird’sbody weight is usually not associated with any adverseeffect.Illness,such as anemia or dehydration,should beconsidered when determining appropriate sample vol-ume.Adequate blood volume for laboratory testing canusually be obtained as long as the animal weighs more
Page 140
Veterinary Clinical Pathology
 Vol.31 / No.3 / 2002
Clinical Chemistry of Companion Avian Species:A Review
Kendal E.Harr
Birds have evolved alternate physiologic strategies to contend with dehydration,starvation,malnutrition,and reproduction.Basicanatomic and functional differences between birds and mammals impact clinical chemistry values and their evaluation.Interpretation of the results of standard biochemical analyses,including BUN,alanine aminotransferase,aspartate aminotrans-ferase,creatine kinase,gamma glutamyltransferase,bilirubin,ammonia,alkaline phosphatase,cholesterol,bile acids,glucose,albumin,globulins,calcium,phosphorus,prealbumin (transthyretin),fibrinogen,iron,and ferritin,is reviewed and discussed inrelation to these physiological differences.The use and interpretation of alternative analytes appropriate for avian species,such asuric acid,biliverdin,glutamate dehydrogenase,and galactose clearance,also are reviewed.Normal avian urine and appropriateuse of urinalysis,an integral part of laboratory diagnosis in mammalian species that frequently is omitted from avian diagnosticprotocols,is discussed.(
Vet Clin Pathol.
2002;31:140-151)
Key Words
: Avian,chemistry,electrolytes,enzymes,metabolites,proteins,sample collection
From Florida Veterinary Pathology,Inc,400 NW 122nd Street,Gainesville,FL 32607 (harrk@mail.vetmed.ufl.edu).
 
than 200 g,as in many common avian species.However,some of the smaller species,eg,the average 30-gbudgerigar,may not yield enough plasma for completebiochemical analysis.Modified techniques in the labo-ratory,such as the use of 10-µL microhematocrit tubes,pediatric sample cups,and dilution,can extend the sam-ple and allow more data to be collected.In the United States,many exotic animal practition-ers perform venipuncture using a needle that has beenheparinized with injectable sodium heparin to preventclot formation.Experienced avian veterinarians workingwith experienced restrainers,who minimize trauma tothe vessel wall,can collect a high quality sample withoutthe addition of heparin.Reducing the amount of heparin in the sample is desirable when using automat-ed hematology analyzers,such as the Cell Dyn 3500(Abbott Laboratories,Abbott Park,Ill) because of adecreased coefficient of variation of the WBC count
1
aswell as decreasing potential dilution and assay artifactin a biochemical sample.If most of the heparin isexpelled from the needle hub,it will minimally affectthe sample.However,the amount of heparin actuallyretained may vary among samples.Any dropletsremaining may cause dilutional effects as well as inter-fere with some analytical tests,such as sodium andalbumin.
2-4
Samples for biochemical analyses should beplaced into lithium heparin Microtainer or Vacutainertubes (Becton Dickinson,Franklin Lakes,NJ,USA) filledto the appropriate volume to avoid variable clotting timeand gelling of serum.Avian plasma samples frequently are yellow due tocarotenoid pigments,not bilirubin.
5
Pink or red plasmais usually indicative of hemolysis.When working withsmaller species of birds,tuberculin or insulin syringesare frequently used,however,not all of these syringeshave detachable needles.Ejecting blood through a 25-gauge or smaller needle may cause moderate to markedhemolysis that will invalidate many biochemical analy-ses.
6
Attached needles can easily be cut from the syringeusing a pair of large veterinary nail clippers or scissors.Insulin syringes also are available with detachable nee-dles.The collection and handling of avian blood areimportant for obtaining accurate results.Discussionwith the practitioner about appropriate materials,venipuncture technique,and sample handling can sig-nificantly improve the quality of the sample and there-fore the result.
Genitourinary Tract
Renal function and osmoregulation
There are marked differences in renal function andosmoregulation among mammalian,avian,and reptilianspecies.The avian kidney lacks a renal pelvis and dis-tinct demarcation of cortical and medullary regions ongross examination.
7,8
There are 2 distinct types of nephrons in birds: the mammalian-type nephron andthereptilian nephron”.The reptilian nephron has a lessconvoluted glomerulus and lacks a loop of Henle.Mam-malian-type nephrons are located more deeply thanreptilian types.Urine flows from the distal tubules tocollecting ducts to the ureter that empties into the uro-deum of the cloaca.Although individual nephrons inavian species have low glomerular filtration rates(GFRs),there are more nephrons per kidney in birdsthan in mammals,such that overall GFR is similar.Studying blood flow to the avian kidney is difficultdue to the complexity and inaccessibility of the renalvasculature.
7
Therefore,a reduced avian kidney modelwith a single arterial supply and accessible renal veinhas been used.This model has limitations,however,inits ability to accurately assess blood flow and clearance.Up to 50% of renal blood flow comes from the ischiacand external iliac veins that comprise the portal system.
9
The percentage of portal circulation filtered by the kid-ney varies with species,stress,and temperature.Portalcirculation is of significant clinical importance wheninjections or intravenous fluid are administered in theleg or lumbar region,because medication will be trans-ported directly to the kidneys and filtered,magnifyingany nephrotoxic effects and preventing adequate med-ication from reaching the site needing treatment.The avian kidney filters a large volume of fluid(approximately 11 times the entire body water each dayfor a 100-g bird) and then reclaims most filtered waterbytubular reabsorption.
8
The GFR decreases with dehy-dration,and with injection of arginine vasotocin,theavian analog of mammalian antidiuretic hormone.
10,11
Avian kidneys shunt blood from reptilian- to mam-malian-type nephrons when GFR is decreased,increas-ing a bird’s ability to concentrate urine.
12
The ability of the avian kidney to concentrate urine appears to varyinversely with size,ie,smaller birds have a greater abil-ity to concentrate urine.
13
Osmoregulation in birds is accomplished by contri-butions from the kidneys,intestinal tract,salt glands,and,to some extent,the skin and respiratory tract.
8,14
Urine can be actively retropulsed from the urodeum tothe coprodeum of the cloaca and then to the rectum andpotentially the large intestine,where water can be reab-sorbed and electrolytes can be modified.
10
This affectsthe specific gravity,electrolyte concentrations,and bac-terial contamination of urine.Uric acid is the major nitrogenous waste product of birds.It is hypothesized that this evolved due to ovipar-ity.
15
Embryonic and fetal development occur within a
Harr
 Vol.31 / No.3 / 2002
Veterinary Clinical Pathology
Page 141
 
Page 142
Veterinary Clinical Pathology
 Vol.31 / No.3 / 2002
closed compartment,the egg,that lacks diffusion of nut-rients and waste.Uric acid is relatively inert and sub-stantially less toxic than ammonia or urea,thus ensuringa viable hatchling.Uric acid (an oxidized form of hypox-anthine) is synthesized predominantly in the liver frompurine metabolism,with a small amount of synthesisoccurring in the renal tubules.Approximately 90% of uric acid is secreted in the proximal convoluted tubulesin the normal bird.
7,8,16
This percentage can be markedlyaltered in pathologic conditions.Due to active renal tubular secretion,blood uric acidlevels are not notably affected by dehydration until GFRis decreased to the point that uric acid is not movedthrough the tubules,which may occur in severe dehy-dration.
16
Raptors and other carnivores have higher ref-erence values for uric acid,and marked increases inplasma uric acid concentration may be observed post-prandially.
17-19
Therefore,sampling of carnivorous birdsshould be performed after a 24-hour fast.Fasting alsowill decrease the likelihood of lipemia,which frequent-lyis observed in postprandial samples.Both BUN and creatinine levels are normally low inbirds and may be below the minimum detectable limitof the assays in the laboratory (Table 1).Prerenalazotemia may be observed in dehydrated birds.
20,21
Inone study in penguins it was shown that BUN was notelevated postprandially,as was uric acid.
22
In anotherstudy in peregrine falcons (
 Falco peregrinus
) both ureaand uric acid increased above normal reference inter-vals in postprandial samples within 8 hours.
23
Azotemiaalso has been documented in birds with renal disease.
15
It may be useful to evaluate BUN and uric acid togetherto differentiate among dehydration,postprandialeffects,and renal pathology.Differentiating pathologicconditions from prerenal azotemia also should be doneusing history,physical exam,and the results of otherchemical tests and diagnostic techniques.
24
Ammonia may comprise up to 25% of the totalnitrogen in urine.Blood ammonia concentration has notbeen evaluated for diagnostic relevance in companionavian species.Acid-base alterations may result in in-creased ammonia concentration in birds.
25
Urinalysis
Urinalysis is indicated when there is azotemia,uratemia,polyuria/polydipsia,abnormal urates,or gen-itourinary masses.Birds with renal pathology frequent-lywill have polyuria,resulting in a urine sample of ade-quate volume for analysis.Avian urine is generally col-lected from a voided sample by removal of cage paperand thorough cleaning of the cage surface.A needle andsyringe or capillary tube can be used to aspirate urineand minimize fecal contamination.Ureteral catheteriza-tion has been performed but requires anesthesia and isdifficult.
26
Normal urine has a clear fluid component.There isvariation in normal urine volume among species thatare adapted to different food sources and environ-ments.
14,27
Specific gravity in most clinically normalbirds has been reported as 1.005-1.020,and avian urineis usually acidic.
19
Normal urine sediment is composedof uric acid precipitates and crystals,sloughed squa-mous epithelial cells,<3 WBC/
ϫ
40 field,<3 RBC/
ϫ
40field,and low quantities of predominantly gram-posi-tive bacteria.Bacteria in otherwise normal urine sam-ples are attributable to fecal contamination.The majority of uric acid in avian urine exists as awhite to light yellow colloidal suspension (urates) madeup of small spherical conglomerates that range indiameter from 0.5 to 15 µm.
19
The urate precipitate iscomposed of uric acid,sodium and/or potassium,andprotein.The precipitate is not measured in the specificgravity of the urine supernatant,and therefore urinespecific gravity tends to be lower in birds and reptilesthan in mammals.Any protein not reabsorbed in theproximal tubule is generally precipitated with uric acid.Assuming there is no fecal contamination,normal avianurine should not contain detectable protein on a urinedipstick.
28
Needle-shaped uric acid crystals also may beobserved in normal avian urine.
19,29
Uric acid crystalspolarize and can be tested chemically using the murex-ide test.
15
For this test,a drop of concentrated nitric acidis added to the crystals and heated to evaporation.Adrop of ammonia is then added.If uric acid is present,the liquid will turn a mauve color.Uric acid crystals canbe dissolved by adding several drops of sodiumhydroxide to a urine sample.
30
This can facilitate theidentification of casts,bacteria,and cells.Biliverdinuria is not a normal finding and is most
Avian Clinical Chemistry
Figure 1
.Biliverdinuria in excrement from an Amazon parrot.Notethe green coloration of the urates,which normally are white.

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