clinical technique: amphibian hematology: a practitioner's guide
TRANSCRIPT
Topics in Medicine and SurgeryTopics in Medicine and Surgery
Clinical Technique: Amphibian Hematology:A Practitioner’s GuideJ. Jill Heatley, DVM, MS, Dip. ABVP (Avian), Dip. ACZM,
Mark Johnson, DVM, Dip. ACVPpling and b
14
Abstract
Amphibian hematology is challenging because of a combination of several factorsincluding small patient size, few venipuncture sites, lack of normative data, and basicvariability of the amphibian leukocyte and erythrocyte counts. This variability in am-phibian red blood cell and white blood cell counts is based on a number of extrinsic(e.g., temperature, diet, season, light cycle) and intrinsic (e.g., species, gender, lifestyle) factors. If possible, to best guide amphibian hematological interpretation, aconspecific, same gender animal can be sampled for comparison to dispel extrinsic andintrinsic variability. However, the collection of hematological measurements in thesingle amphibian patient can still provide useful clinical information to guide therapyof even the most diminutive amphibian patient. Therefore, the following brief guide-lines are presented in an attempt to guide the clinical practitioner as to collection andinterpretive techniques, which can easily be adapted to clinical practice for these fragilejewels of nature. Equipment necessary for venipuncture, venipuncture sites, a veni-puncture technique, a technique for determination of an estimated white blood cellcount and differential, and a guide for differentiation of leukocytes of the amphibianare given. This guide should by no means supplant a thorough review of the literatureor consultation with a clinical pathologist, but will provide general rules of thumb forquick interpretation. Excellent reviews of sampling and complete blood count inter-pretation are listed in the references. Copyright 2009 Elsevier Inc. All rights reserved.
Key words: amphibian; hematology; blood collection; venipuncture; clinical pathology
When collecting blood from amphibian spe-cies, one should use appropriate materialsfor venipuncture, proper handling of the
animal, and more commonly, sedation or anesthesia.Use suitable restraint including talc-free, distilledwater-washed examination gloves and a deionizedwater-washed surface; give due consideration to se-dation or light anesthesia to give the best chance ofsuccessful sample collection and the least chance ofharming the amphibian. At the minimum, be sure torecord the following information with any amphib-ian hematology sample: time, date and site of collec-tion; patient species, gender, age, and life stage; andseason, diet, and light cycle.
Recommended materials for amphibian blood sam-
lood smear preparation are given in TableJournal of Exo
1 and Figure 1. Syringes may be precoated by drawinga small amount of heparin into the syringe and thenexpelling any excess if a difficult or slow blood collec-
From the Zoological Medicine Service, Department of Small AnimalClinical Sciences, and Department of Pathobiology, College of Vet-erinary Medicine, Texas A&M University, College Station, TXUSA.
Address correspondence to: J. Jill Heatley, DVM, MS, Dip.ABVP (Avian), Clinical Associate Professor, Zoological MedicineService, Department of Small Animal Clinical Sciences, College ofVeterinary Medicine, Texas A&M University, College Station,TX 77843-4474. E-mail: [email protected].
© 2009 Elsevier Inc. All rights reserved.1557-5063/09/1801-$30.00
doi:10.1053/j.jepm.2008.10.004tic Pet Medicine, Vol 18, No 1 ( January), 2009: pp 14–19
Amphibian Hematology 15
tion procedure is expected. Additionally, if sepsis is aconcern, consider reserving one drop of blood withoutanticoagulant for placement onto a mini-tip culturette ordirectly into an enrichment broth, which then should beincubated at enclosure temperature. When collectingblood for culture, aseptic technique, which includes ade-quate patient preparation and strict surgical preparationof the skin to avoid culture contamination, is necessary.Contacting the bacteriological laboratory for appropri-ate materials, such as pediatric blood culture vials, andinstructions for sample collection and storage shouldoccur before the procedure for best diagnostic results.Although the collection of samples before antibioticadministration is preferred in a patient where sepsis isa concern, the collection of samples after antibioticadministration is not contraindicated.
Methods of blood collection vary based on the spe-cies. Most commonly used venipuncture sites includethe ventral caudal tail vein, the ventral abdominal vein,and the heart. Peripheral venipuncture may not re-quire chemical restraint and may be performed in amanner similar to that used on the tail vein of bovidsand as in reptilian species for the ventral abdominalvein. For the abdominal vein or cardiopuncture, oncethe animal is positioned appropriately, it is best tovisualize the venipuncture site. For the abdominal vein,visualization may be aided by a bright, cool fiber-opticlight source to transilluminate the vein from the side orbehind or by ultrasound guidance. The ventral abdom-inal vein may be visualized without additional aids as itcourses down the ventral midline in amphibians withminimally pigmented, translucent skin. The cardiac im-pulse can be visualized in some species without additionalaids. However, the dense skin pigmentation of many spe-
Table 1. Equipment necessary foEquipment Manu
Accusure Insulin Syringe 0.3-0.5 mL Qualitest PharmaHuntsville, AL
Selected micro cover glasses VWR Scientific InGold Seal Rite-On Microscope Slides Erie Scientific, PoDog nail trimmers, guillotine style Resco, Walled La
Heparinized hematocrit tubes Drummond ScienBroomall, PA
Lithium heparin microtainers Becton-Dickinson
cies necessitates the use of a Doppler flow probe (Fig 2),
ultrasound, or cool light transillumination to confirm theblood flow in the heart or in other vessels before veni-puncture. As in other species, cardiac puncture is notwithout risk and should be performed only in an anes-thetized animal with blood aspiration from the ventriclewith a small-gauge needle (Fig 3).
A small-gauge needle limits trauma to the amphib-ian and will not damage blood cells as long as minimalaspiration pressure is applied. Use of an insulin syringewith a zero volume hub also maximizes recovery of thesample; however, the needle must then be removed for
Figure 1. Basic equipment for amphibian hematological sampling.Pictured: Guillotine canine nail trimmers, 0.3-mL insulin syringe withzero hub needle attached, slides, cover slips, heparinized PCV tubes,and lithium heparin microtainers. Additional materials for restraint ofthe amphibian are necessary (talc-free examination gloves washedwith distilled or deionized water, large dog mask for use as inductionchamber for isoflurane anesthesia, nonbleached paper toweling or
s
phibian hematological samplingrer Comments
cals, Inc., Zero volume hub, attached needle 28gauge, 1/2’’
estchester, PA “Cover slips” for blood smear prepouth, NH 1.0-mm thicknessI Removal of hub/needle of zero
volume attached needle syringes;conserves sample volume
ompany, PCV and total solids determination;may be used to create small dropsize to facilitate amphibian bloodsmears
nklin Lakes, NJ May be used to store hematologicsamples
r amfactu
ceuti
c., Wrtsmke, M
tific C
, Fra
surface which has been moistened with distilled or deionized water).
16 Heatley and Johnson
sample processing. For needle removal, pull the sam-ple to the back of the syringe and remove the needleand hub with clean, sharp, guillotine-style dog nailtrimmers. This allows sample handling without havingto expel the blood through the small needle. Bloodmay be used for fresh smears, culture, packed cellvolume, and total solids determination. Blood smearsmay be made with a slide or cover slip technique. Theuse of heparized hematocrit tubes may facilitate mak-ing the small drops necessary for good blood smearswhen evaluating amphibian blood. The remainder canthen be placed in the lithium heparin microtainer forseparation of plasma and determination of bloodchemistries. Normal amphibian plasma varies with thespecies and may be light blue, green, or orange. In theamphibian, green plasma may or may not be due tobiliverdin elevations, and yellow or orange colorationmay be due to carotenoids or bilirubin elevations. Blueplasma has been found in apparently healthy Japanesegiant salamanders (Andrias japonicus). The clinicianshould exercise caution in the overinterpretation ofthe plasma coloration in amphibian species as thecause for coloration of serum or plasma, be it baseddisease or completely normal, only has not been inves-
Figure 2. Placement of a Doppler flow probe over the heart of aMalaysian Horned Frog (Megophrys nasuta) to verify appropriatevenipuncture site.
tigated in most species.1
For the leukocyte differential of amphibian bloodcells, a tabular (Table 2) and pictographic guide(Figs 4-10) are given. Blood cells shown were ob-tained from Malaysian Horned Frogs (Megophrys na-suta) via cardiac venipuncture. Amphibian whiteblood cells tend to vary considerably in morphologyand staining characteristics based on species. There-fore, these cells are presented only as a cursoryguide. One should consult additional texts for bestinterpretation of amphibian leukocytes.1-3
Procedural Tip I
Calculation of the Appropriate Volume ofBlood to be Sampled from an AmphibianThe guidelines below calculate a minimum safeblood draw. Conservative measures are used at eachstep to safeguard patient safety.
● Blood volume varies from 7% to 10% of bodyweight in the terrestrial amphibian and 13% to25% of body weight in the aquatic amphibian.
(Body weight, g)(0.07) � Minimal total bloodvolume (TBV)In the aquatic amphibian, the multiplication fac-tor may be increased to 10% or 0.1.
● Minimal safe blood draw (SBD) � 5% to 10% ofTBV
TBV (0.05) � SBDIn the healthy amphibian, SBD may be doubled.
● For a 35-g sick frog, SBD:
35(0.07)(0.05) � 0.12 mL
Figure 3. Blood collection via cardiac venipuncture of a MalaysianHorned Frog (Megophrys nasuta). This frog has been anesthetized
with isoflurane administered via face mask.
Amphibian Hematology 17
Table 2. Differentiation of common amphibian blood cells1,2
Cell Name*Relative Cell Size,
Shape, and Percentage Nucleus Cytoplasm/Granules
Neutrophil Predominantinflammatory cell insome species; visuallysimilar to mammaliancell
Multilobed nucleus Colorless cytoplasm, largeirregular granules withslight eosinophilia oronly slight stain uptake
Heterophil (smalleosinophilic granuleleukocyte)
30 to 32 �m; largerthan that ofmammals; 1% to 33%of leukocytes
Round, multilobed nucleus Colorless cytoplasm,distinct eosinophilic,rod-shaped, smallerthan eosinophilgranules; mayphagocytize bacteria
Eosinophil (large eosinophilicgranule leukocyte)
Same size or larger thanheterophil; 0% to 15%of leukocytes
Smooth, less lobednucleus than heterophil
Small to medium, oval toround eosinophilicgranules of variablesize
Basophil Size variable; �1% ofleukocytes of mostspecies; maypredominate in some
Nonsegmented, unlobed,slightly eccentric, oftenobscured by granules
Basophilic granules roundto oval; oftendegranulated by Diff-Quik stain
Monocyte/Azurophil Similar in appearance tomammalian cells;usually larger thangranulocytes butvaries from smaller tolarger; 0% to 20% ofleukocytes
Round, kidney, orhorseshoe shaped
Pseuodopodia may bepresent, light blue-gray,abundant cytoplasmfoamy or vacuolated;occasional small,irregular-shapedazurophilic granules(azurophils); mayphagocytize other cells
Small lymphocyte (largelymphocyte)
Similar in appearance tomammalian cells;�50% of differentialin many species;smallest leukocyte(small lymphocyte)more numerous thanlarge lymphocyte; 1/2size of erythrocyte orgranulocyte
Usually round butoccasionally cleft orbilobed, nuclearchromatin dense,clumped
Scant, small amount, paleblue to deep blue gray;less cytoplasm withmore intense color thanthrombocyte
Thrombocyte Oval to spindle shape;cells tend toaggregate
Dense to round oval Abundant colorless/palegray
Erythrocyte Larger than that ofmammals; oval cellshape
Usually nucleated, nucleusbasophilic with bulge,irregular nuclear margin
Deep eosinophilic (red topink)
*Cell name is based on amphibian cell staining characteristics and does not imply specific cell function or other similarity to mammal or othervertebrate cells.
18 Heatley and Johnson
From a minute blood sample (0.1 mL), one canexamine for hemoparasites, perform an estimatedwhite blood cell count and a blood culture, andobtain a packed cell volume and total solids deter-
Figure 4. Blood smear from the terrestrial Malaysian Horned Frog(Megophrys nasuta) stained with Diff-Quik shown at 1000� mag-nification. The leukocyte to the left of the image is a neutrophil withDöhle bodies, the leukocyte on top is a large lymphocyte with anintracellular hemoparasite, and the small cell on the bottom is athrombocyte.
Figure 5. Blood smear from the terrestrial Malaysian Horned Frog(Megophrys nasuta) stained with Diff-Quik shown at 1000� mag-nification. The eosinophil is the large cell with round cytoplasmic
granules. The remaining three leukocytes are small lymphocytes.mination with a microhematocrit tube. Obtaining anaccurate white blood cell count with the help of ahemocytometer requires only about 0.02 mL if theblood is appropriately diluted with diluent/stain.
Procedural Tip II
Estimation of the Total White Blood CellCount and Determination of theDifferential Cell Count in an Amphibian
● In the cell monolayer, count the number ofwhite blood cells in 12 high powered (400�)fields.
Figure 6. Blood smear from the terrestrial Malaysian Horned Frog(Megophrys nasuta) stained with Diff-Quik shown at 1000� mag-nification. The leukocyte shown is an azurophilic monocyte.
Figure 7. Blood smear from the terrestrial Malaysian Horned Frog(Megophrys nasuta) stained with modified Wright’s stain and shownat 1000� magnification. From the top proceeding clockwise, a smalllymphocyte, a thrombocyte, a neutrophil, and azurophilic monocyte
are shown.
Amphibian Hematology 19
● Subtract the highest and lowest number fields,then divide the remaining total by 10 to obtain X.
● Multiply X by 1000. The final number is an estimateof the amphibian total white blood cell count � LC.
● To obtain a cell differential, count 100 white bloodcells to obtain a percentage of each white blood cell
Figure 8. Blood smear from the terrestrial Malaysian Horned Frog(Megophrys nasuta) stained with a modified Wright’s stain andshown at 1000� magnification. From the top, leukocytes shown area monocyte, 3 aggregated thrombocytes, and a neutrophil.
Figure 9. Blood smear from the terrestrial Malaysian Horned Frog(Megophrys nasuta) stained with a modified Wright’s stain and shown at1000� magnification. From the top, a basophil, a small lymphocyte, a
monocyte, another basophil, and an eosinophil can be seen.type (see Table 2). Multiply each cell’s percent by LCto give an estimated leukocyte count per cell.
Summary
Amphibian hematology is still in its infancy, andsample collection and interpretation may be daunt-ing to the general practitioner. This basic summaryreference guide was prepared to facilitate interpre-tation of the amphibian complete blood count forthe veterinary clinician. By making amphibian hema-tology interpretation more uniform and accessible, itis hoped that the standard of amphibian medicinewill improve and that amphibian hematology will nolonger be regarded as a rare diagnostic procedure inthese patients. Hopefully, both amphibians andmankind will benefit from this advancement.
Acknowledgments
The author wishes to acknowledge the assistance ofDr. Sharman Hoppes for her contributions of figuresto this article.
References
1. Campbell T, Ellis CK: Avian & Exotic Animal Hematologyand Cytology (ed 3). Oxford, Blackwell Ltd, 2007
2. Wright K: Amphibian medicine and captive hus-bandry, in Wright K, Whitaker B (eds): AmphibianMedicine and Captive Husbandry. Malabar, FL,Kreiger Publishing, pp 128-146, 2001
3. Pessier A: Cytologic diagnosis of disease in amphibians. Vet
Figure 10. Blood smear from the terrestrial Malaysian Horned Frog(Megophrys nasuta) stained with Diff-Quik (Fisher Scientific, Pitts-burg, PA USA) shown at 1000� magnification. The central leukocyteis a large lymphocyte.
Clin North Am (Exotic Anim Pract) 10:187-206, 2007