Topics in Medicine and SurgeryTopics in Medicine and Surgery

Clinical Approach to the Anemic Reptile

Miguel D. Saggese, DVM, MS, PhD

hypoplasia

98

Abstract

Anemia is a relatively common blood disorder but a seemingly underreported andinvestigated condition in reptiles. Better characterization of anemia in the sickreptile could help determine which available diagnostic tools could be best utilizedto determine the cause of low red blood cell counts in these animals. There are,however, a number of limitations that exist when trying to interpret hematologicdata for the large number of reptilian species presented to the exotic animalpractitioner, including interspecific and intraspecific variation in hematologicalreference values, limited availability to specific reagents, and a lack of scientific-based studies that focus on anemic conditions in reptiles. Therefore, better docu-mentation and consistency in the scientific-based studies of reptile anemia andrapid dissemination of this information are considered necessary to increase ourknowledge in this area of reptile medicine. Collaborative research efforts betweenpractitioners, owners, reptile collection curators, pathologists, immunologists, mi-crobiologists, toxicologists, and laboratory scientists are urgently needed to aug-ment our understanding of anemia and other hematological disorders in reptiles.Copyright 2009 Elsevier Inc. All rights reserved.

Key words: anemia; blood; erythrocytes; hemoglobin; reptiles; treatment

Hematology is the medical science that stud-ies blood in health and disease. As such, thestudy of hematology has a deep impact on

our understanding of multiple diseases. Without adoubt, one of the most useful interpretations ofhematology in human and veterinary medicine is thechange in the number and morphology of erythro-cytes that occur because of pathological conditions.Among these, one of the most commonly observedpathological conditions is anemia.

The word anemia is derived from the Greek wordanaemia, which means “without (an) blood (aemia).”It can be defined as a reduction in the number ofred blood cells (RBC) or erythrocytes per volume ofblood, with the subsequent reduction in oxygen de-livery to the tissues by hemoglobin (Hb). Ultimately,anemia should be defined as a qualitative or quanti-tative deficiency in circulating Hb. Anemia can becaused by a primary blood disorder (e.g., aplasia,

) or, more frequently, a secondary com-

Journal of E

plication to other diseases. In the latter case, anemiararely constitutes a diagnosis but is rather a clinicalsign of some other pathological condition or illness.

Hb concentration and hematological indexes areinvaluable tools to investigate and diagnose reptilehematologic maladies;1-8 however, thorough investi-gation of the anemic patient appears to be rarelyconducted in reptile medicine. Identification of ane-mia in a lizard, snake, crocodile, or chelonian shouldchallenge the veterinary practitioner to perform acomprehensive investigation with the aim to deter-

From the College of Veterinary Medicine, Western University ofHealth Sciences, Pomona, CA USA.

Address correspondence to: Miguel D. Saggese, DVM, MS, PhD,College of Veterinary Medicine, Western University of HealthSciences, Pomona, CA 91766. E-mail: msaggese@westernu.edu.

© 2009 Elsevier Inc. All rights reserved.1557-5063/09/1802-$30.00

doi:10.1053/j.jepm.2009.04.003

xotic Pet Medicine, Vol 18, No 2 (April), 2009: pp 98–111

Anemia in Reptiles 99

mine the underlying cause of the condition even inthe absence of other clinical signs. Furthermore,early recognition of anemia in a reptile patient dur-ing annual or biannual examination helps to identifydiseases before they progress into life-threatening dis-orders and allows the practitioner to implement a spe-cific treatment regimen, thereby helping one establisha prognosis and monitor response to therapy.1-8

More than 400 causes of anemia have been re-ported in humans, with a smaller, although still sig-nificant, number of causes recognized in small andlarge animals.9,10 It is very likely that there are severalcauses for anemia in reptiles too, even though theseremain unidentified or not clearly defined. Becausethere are more than 8000 species of reptiles de-scribed worldwide, it is unlikely that a detailedknowledge of the erythron and its response to dis-ease for all these species will ever be achieved. How-ever, for the continued development of reptilemedicine and hematology, a more exhaustive inves-tigation of the anemic patient, including its causes andassociated changes in the erythron, is needed. Exoticanimal veterinarians may contribute to this better un-derstanding by reporting systematic investigation andtest results from their anemic reptile patients.

The goal of this article is to review and summarizegeneral aspects of reptilian hematology, samplingtechniques, and the clinical approach to the diagno-sis of anemia in reptiles. The intention is to generatemore interest from the veterinary practitioner incontributing to the study and understanding of thecauses and diagnosis of anemia in reptiles. Giventhat excellent detailed reviews about reptile hema-tology have been recently published, minimal de-scriptions of many of these basic principles are madein this article.2-5,8

Figure 1. Blood collection from the cervical sinus of a tortoise.

(Geochelone sp.) Photo courtesy of Dr. Carolina Torta.

Overview of Blood Collection in Reptiles

The method of blood collection and the use of anappropriate sampling technique are critical steps tothe proper interpretation of the reptile hemogram,especially during the investigation of an anemic pa-tient. A basic understanding of reptile anatomy, withemphasis on the topographic anatomy of venipunc-ture sites, is necessary for collecting blood and re-ducing the discomfort that the procedure may causein the patient. Excellent and detailed reviews onthese topics have been recently published.4,5,8,11-14

Blood samples can be collected from multiplelocations on a reptile patient. (Figs 1-4).4,5,8,11-14

Choosing the best site for blood collection will bedetermined by taxonomy, species anatomic charac-

Figure 2. Collecting blood from the subcarapacial vein of an Argen-tine desert tortoise (Geochelone chilensis). Photo courtesy of Dr.Guillermo Perez Jimeno.

Figure 3. Blood collection from the ventral tail vein of a python(Python sp.). Photo courtesy of Drs. Paula Moreno and Angelica

Rojas.

100 Saggese

teristics, body condition, preference and experienceof the phlebotomist, size of the patient, and amountof blood required. Selection of an adequate bloodsampling site is also essential if a significant volumeneeds to be collected.4,5,8,11-14 Peripheral blood ves-sels should be the first option for reptile blood col-lection, given that these sites are relatively moreaccessible and usually adverse to developing second-ary complications. The brachial and jugular veins(e.g., lizards, chelonians), ventral tail vein (e.g., liz-ards, snakes, chelonians, crocodilians), and ventralabdominal vein (e.g., lizards) are readily accessiblesites for most species of reptiles.4,5,12,13,15 In ophid-ians, given that these peripheral sites are not avail-able with the exception of the ventral tail vein, car-diocentesis is usually recommended.4,5,12,16 It is arelatively simple and safe procedure that allows forthe collection of a relatively large volume of blood.Another potential venipuncture site in ophidians isthe palatine veins.4,5,12,16 In crocodilians, the ventralcoccygeal veins and supravertebral vessels are safeand recommended when significant quantities ofblood are needed.12,17 In chelonians, the jugular veinis the preferred vessel because there is less risk ofcollecting lymph-diluted blood as compared with theother peripheral sites.4,5,18 Additional venipuncturesites in chelonians are the postoccipital venousplexus, femoral and coccygeal veins, and the sub-carapacial sinus.4,5,12,18

For most reptiles, the total volume of blood col-lected should not be �1% (in grams) of bodyweight.3-5,8,11 Usually, a blood volume equivalent to0.5% to 0.8% of the body weight is enough to pro-vide a diagnostically valuable sample. Ideally, theamount of blood obtained should allow the storage

Figure 4. Blood collection from the ventral tail vein of a spiny-tailedlizard (Uromastyx aegyptius). Photo courtesy Dr. Jaime Samour.

of plasma or serum if additional testing (e.g., serol-

ogy, plasma protein electrophoresis) is needed at alater date. Large volumes of blood, plasma, and/orserum can be saved at –80°C. In clinically ill patientsor those suspected of being severely ill, a smallervolume of blood should be collected (e.g., 0.5 mL isusually enough to run a complete blood count in-cluding a plasma biochemistry panel). When inves-tigating the progression of anemia using hematocrit(Ht) percentage and the RBC count, only small vol-umes of blood should be taken to avoid unnecessaryand excessive blood loss in the patient.

Veterinary clinicians must remember that reptileshave very well-developed lymphatic vessels that usu-ally run in close proximity to the main vessels used tocollect blood.3-5,12,14,15,19 As a result, contamination ofthe blood sample with lymph is a very commoncomplication, especially in chelonians. Lymph con-tamination causes hemodilution, which can signifi-cantly alter the results and diagnostic value of ablood sample.3-5,12,14,15,19 Recognition of lymph con-tamination is relatively simple if careful attention isused when collecting the sample. When entering avessel with the needle, blood will slowly fill the sy-ringe. However, when a lymphatic vessel is entered,a clear, pale yellow or whitish liquid or a change inthe color of the previously collected blood (e.g.,suddenly becoming more dilute) will be observed inthe sample when lymph is being collected instead ofblood. While the blood is being collected, carefulobservation of the syringe during blood draws willhelp to detect this contamination and consequentchange in color.4,5 When lymph contamination isdetected, the sample should be discarded and a newone obtained from a different anatomic location.

Preloading the Anticoagulant inthe SyringeAnticoagulants are routinely used for holding andstoring reptile blood samples. Blood that has beenaltered with an anticoagulant will allow for the eval-uation of blood cells or plasma components fromthe same sample. Lithium heparin is the recom-mended anticoagulant for preloading a syringe tocollect a blood sample from a reptile.4,5,8,12,19,20 How-ever, other authors report the use of sodium ethyl-enediamine tetraacetic acid (EDTA) as a better op-tion for preloading syringes when collecting bloodfrom lizards and snakes.17,21 In chelonians, heparinmay cause hemolysis.20 As in birds, the use of EDTAmay impair blood cell staining and cause changes inthe coloration of erythrocytes and hemolysis.4,5,11

Nevertheless, blood smears are better preparedwhen blood is collected without the addition of

anticoagulant in the collection syringe. Collecting a

Anemia in Reptiles 101

viable blood sample without the use of an anticoag-ulant may be difficult to achieve in small reptiles,which offer limited blood volumes.

When blood is collected without the use of ananticoagulant, it should be quickly placed in tubescontaining anticoagulant to prevent clotting. Theuse of 0.25 to 0.5 mL Microtainer blood tubes (BDVacutainer Systems; Beckton, Dickinson and Co.,Franklin Lakes, NJ USA) is recommended.4 Micro-tainer blood tubes allow for easy and fast mixing ofblood. Given that reptile erythrocytes are relativelyfragile, it is advisable to remove the needle hub fromthe syringe when transferring blood to the tube ormaking the blood film to avoid hemolysis. Once theblood film has been made, they can be stained withthe same methods used for birds, including Wright-Giemsa, May-Grünwald-Giemsa, or Diff Quick. Bothslide-to-slide and coverslide-to-coverslide techniquesare recommended when preparing a blood smear.5,12

The quality of the blood sample is always impor-tant for precise examination and diagnosis. Presenceof clots, hemolysis, excessive anticoagulant, and con-tamination with lymph will impede the collection ofblood samples and alter diagnostic values.3-5,8 It hasbeen stated that a laboratory test result is only asgood as the specimen collected. In another words,extreme caution should be used to prevent sendingthe laboratory a blood sample for analysis that willconfer misleading results.11,19

Special precautions should be taken when manip-ulating reptiles for venipuncture. They can harborseveral infectious agents that are zoonotic or poten-tially zoonotic.22 Blood samples should be treated asa potential source of blood-borne pathogens for thepersonnel as well as for other animals. Veterinariansare responsible for enforcing standard biosafety pre-cautions when working with reptiles, especially whencollecting blood samples from the diseased patient.Use of gloves, goggles, and/or a mask should alwaysbe recommended when working with reptiles thatpresent with clinical signs of disease.

Hematologic Reference ValuesAlthough published reference values are availablefor a number of reptile species, it is important toremember that both intrinsic (e.g., species, gender,age, physiological status, breeding season, hiber-nation) and extrinsic (e.g., season, temperature,environmental conditions, husbandry, diet, livingconditions) factors may cause significant variationsin hematological parameters.4,5,8,11,23 Thus, when re-ferring to a set of references values, it is important todetermine the conditions under which the blood

samples were obtained.4,5 Understanding and recog-

nizing these possible variations in reptile hemato-logic results will contribute to a judicious interpre-tation of published reference values and, at the sametime, should motivate researchers and clinicians toinvestigate these differences, obtain references val-ues adjusted to these variations, and demonstratetheir clinical significance.4-8,11,13,16-19 Ideally, at least100 individuals from the same species, age, and sex,housed under similar conditions and fed a diet thatis close in nutritional composition, should be usedwhen trying to determine normal blood referencevalues.24 Packed cell volume (PCV), RBC counts, andhematological indices vary between species andwithin individuals, making the use of published ref-erence values of limited value in most cases.4,5,8 Ul-timately, comparison of blood reference values in apatient with compiled data from multiple sourcesthat either do not identify the methodology used tocollect and test the blood sample or define the pop-ulation from which the sample is collected is notrecommended. In fact, a better approach to evaluat-ing blood work values between sick and healthy pa-tients is to obtain in situ reference values for theindividual through investigation of normal bloodvalues for the same patient by collecting and analyz-ing the samples at least two times in the year. Thisapproach can provide more valuable informationregarding what is within the normal range of healthyindividuals rather than a single value obtained froma diseased patient. For example, a reptile patientcould experience a significant drop in its PCV (e.g.,from 45% to 25%), which suggests there is an ab-normality, but if the PCV values from the animalwere captured by the reference range for that par-ticular species it may be misclassified as being nor-mal. The scenario described above reinforces theneed to compare blood reference values obtainedfrom the same patient or from a clearly definedreptile population. In cases of severe anemia whenthe PCV values fall well below the published refer-ence range, this comparison is not needed.

Evaluation of the Reptilian Erythron

Evaluation of the reptilian erythron includes thedetermination of the PCV or Ht, total RBC (TRBC)count, Hb concentration, and hematological indicessuch as mean corpuscular volume (MCV), mean cor-puscular hemoglobin (MCH), and mean corpuscu-lar hemoglobin concentration (MCHC). Calculationof these indices contributes to effectively measuringthe size of erythrocytes and calculating the contentof Hb within cells. The detailed examination of these

parameters is necessary when diagnosing, investigat-

102 Saggese

ing, and characterizing the anemic conditions thataffect reptile species.

The TRBC count can be determined by using amanual counter and the Natt Herrick or Unopettetechniques. Both methods rely in the dilution of theblood sample and further manual count of cellsthrough the use of a counting chamber.3-5,8,11,25,26

After subsequent adjustment to the dilution factor,the total number of cells/milliliter in blood are cal-culated. This can be easily performed in a veterinaryclinic without the need for expensive equipment.Other methodologies used to obtain a reptile com-plete blood count include the use of flow cytometryor automatic counters. Although equipment to per-form automated complete blood counts on reptilespecies is expensive and can be purchased, it is notavailable through most veterinary diagnostic labora-tories.3-5

Reptiles usually have a lower number of eryth-rocytes per volume of blood than mammals orbirds.27-29 An inverse relationship between size andnumber of circulating erythrocytes apparently exists.Those species with smaller erythrocytes tend to havea larger number of this cell type in their blood.27-29

The PCV or Ht measures the percentage of RBCs inthe blood. Reference PCV values in reptiles rangefrom 20% to 45%, whereas some authors report awider range of values (e.g., 15%-50%).3-5,8,16-19,28,30

Hb concentration is measured with a solution ofpotassium cyanide and potassium ferricyanide. Aspecific volume of blood is combined with cyanideforming cyanmethahemoglobin, which is read usinglight photometry.4,8,9 Reported Hb concentrationvalues for reptiles range from 5.5 to 12 g/dL.3-5,8,16-19,28,30

The above-mentioned parameters, together with thehematological indices described below, are neces-sary to effectively investigate RBC function. Hemato-logical indices allow accurate measurements oferythrocyte size and Hb concentration. When com-bined, these parameters and indices provide impor-tant information that contributes to a better diagnosis,identification of the etiology, and characterization ofanemia. The hematological indices are:

MCV (fL) correlates to the average volume of erythro-cytes. It can be calculated with the following for-mula: Ht (%) � 10/RBC count (� 1012/L). MCVvalues range from 200 to 1200 fL.3-5,8,13,14,16-19,28,30

MCH measures the average weight of Hb in the eryth-rocyte. The MCH can be calculated as follows: Hb(g/dL) � 10/RBC count (� 1012/L). MCH valuesranges from 6 to 10 g/dL.3-5,8,13,14,16-19,28,30

MCHC expresses the average concentration of Hb in

the erythrocyte. It is calculated as: Hb (g/dL) �

100/Hct (%). MCHC values reported for reptilesrange from 22% to 41%.3-5,8,13,14,16-19,28,30

Detailed descriptions of the reptilian RBC havebeen published in other articles.4,5,29,31-35 The combi-nation of basic and acid dyes allows for the differen-tial staining of blood cells and the study of theirmorphology under the light microscope (Fig 5).With practice, clinicians and technicians can be-come proficient in evaluating reptile erythrocytes.

Anemia

Reptile patients with anemia have a decreased oxy-gen-carrying capacity. This may occur because of areduction in the number of circulating erythrocytesor a decrease in the concentration of Hb within theerythrocytes. Anemias may be classified as: 1) regen-erative, when the bone marrow maintains its eryth-ropoietic function (e.g., hemorrhage, hemolysis)and the patient is capable of producing and releas-ing newly formed erythrocytes, and 2) nonregenera-tive, where no new RBCs are formed or releasedfrom the hematopoietic sites.

Regenerative AnemiaRegenerative anemias are usually caused by hemor-rhage and hemolysis. Hemorrhagic anemia in rep-tiles occurs because of blood loss after trauma andsurgery, hemathophagous external parasites, coagu-lation disorders, gastrointestinal ulcers, and neopla-sia.4,5,8,36-39

Anemia is a common finding in reptiles with

Figure 5. Blood film from a spiny-tailed lizard showing the normalshape/size of mature erythrocytes. A single teardrop erythrocyte canbe observed in the upper left quadrant. Photo courtesy of Dr. JaimeSamour.

heavy ectoparasite infestations. Ticks from the fami-

Anemia in Reptiles 103

lies Ixodidae and Argasidae are commonly identifiedas the underlying cause of anemia in reptile cases inwhich the arthropods are present (Fig 6).39,40 Theintroduction of new or recently acquired reptiles inan otherwise well-managed reptiliary could causeoutbreaks of tick infestations and anemia in pre-viously healthy individuals. Ophionyssus natricis, thesnake mite, is a common cause of anemia in captivesnakes. Juvenile animals are especially susceptible todeveloping anemia from these mite infestations.Leeches may cause significant blood loss in aquaticreptiles41 and are often a difficult problem to diag-nose if the parasites are not observed while attachedto their victims.

In chelonians, traumatic wounds caused by fight-ing, dog bites, and free falling from balconies willresult in anemia due to internal or external bloodloss (Fig 7). Gastric ulcers or trauma related to the

Figure 6. Ticks (Amblyoma argentina) on an Argentinean deserttortoise. Photo courtesy of Dr. Guillermo Perez Jimeno.

Figure 7. Traumatic hemorrhage in an Argentinean desert tortoise.

Photo courtesy of Dr. Guillermo Perez Jimeno.

ingestion of a foreign body (e.g., ingesting a fish-hook) are other possible causes of internal hemor-rhage in reptiles.42 Hemorrhagic conditions are rarein ophidians, although internal hemorrhage causedby the ingestion of rodents exposed to anticoagulantrodenticides may occur in free-ranging snakes.43 Co-agulation disorders caused by thrombocytopeniahave been described in reptile species and are oftenthe result of decreased production, accelerated use,or destruction of thrombocytes.4,5,8

Hemolytic anemia occurs when erythrocytes aredestroyed within the blood vessels (e.g., intravascularhemolysis) or outside the lumen of the vessel (e.g.,extravascular hemolysis). Hemolytic anemia can becongenital (e.g., inherited hemolytic anemia) or ac-quired. An anemia can also be classified based on themechanism of damage to the plasmatic membrane ofthe erythrocyte. This can be an immune-mediated re-sponse (e.g., antibodies) or the result of an infectiousagent, drug, or toxin on the erythrocyte membrane.Inherited hemolytic anemia has not been reported inreptiles to the author’s knowledge.

The most common cause of acquired hemolytic ane-mia in reptiles is associated with severe infestation by themalarial hemoparasites (Plasmodium).5,39,40,44,45 Haemopro-teus and Saurocytozoon are two additional genera ofhemoparasites that can also cause anemia in reptiles(Fig 8).5,45 Special considerations should be taken toidentify and differentiate these parasites from othernonpathogenic hemoparasites found in reptileblood.

Idiosyncratic drug-induced hemolysis can arisetheoretically through the administration of any med-ication prescribed for reptiles. Drug-induced hemo-

Figure 8. Blood film from a spiny-tailed lizard. An intracellularhemoparasite (Haemoproteus sp.) can be observed inside some ofthe erythrocytes. Photo courtesy of Dr. Jaime Samour.

lysis is more likely to occur with drugs well recog-

104 Saggese

nized for causing anemic conditions (e.g., nonsteroidalantiinflammatory drugs, antifungal agents, sulfon-amide antibiotics).46-48 Heavy-metal toxicity, espe-cially lead and zinc toxicosis, can induce a hemolyticanemia, in addition to other gastrointestinal andneurological clinical signs.4,5,8,43,49-51 Lead toxicosismay result from the ingestion of tainted paint,fishing sinkers, spent lead ammunition, or fromother environmental sources. Zinc toxicosis hasbeen associated with overdosing zinc supplements,ingestion of pennies coined after 1982, thermometerweights (Fig 9), and zinc-containing ointments. Useof anticoagulants containing calcium EDTA hasbeen reported to cause hemolysis in some speciesof chelonians.20 Inappropriate venipuncture tech-niques, such as excessive negative pressure, pro-longed storage, and manipulation of the sample, arecommon causes of iatrogenic hemolysis.

Nonregenerative AnemiaNonregenerative anemia is the most common man-ifestation of anemia in reptiles, although it is rarelyfully investigated and, as such, rarely reported. Inreptiles, the development of a nonregenerative ane-mia is a slow process, which is likely because of theidiosyncrasy of many chronic diseases and the longhalf-life of the reptilian erythrocyte.

The most common causes of nonregenerative ane-mia described in domestic animals and other exoticpets have been identified in reptiles.1-5,8,11 Neoplasia,systemic infectious diseases, and any moderate to se-vere chronic degenerative or inflammatory, systemic,or localized disorder that affects the liver, kidney,spleen, or lungs may be accompanied by a nonregen-erative anemia typical of chronic diseases.4,5,8,52-55

Figure 9. Ingestion of zinc pieces from a broken aquarium ther-mometer weight by a red-eared slider (Trachemys scripta). Photo

courtesy of Dr. Jennifer Burr.

Infectious diseases should always be considered asone of the top differential diagnoses and are highlyprevalent in captive reptiles.56-62 Systemic chronicinfectious diseases such as mycobacteriosis, myco-plasmosis, chlamydiosis, salmonellosis, herpesvirosis,iridovirosis, coccidiodomycosis, and aspergillosis arecommonly associated with a nonregenerative anemiawhen disseminated disease is present, or when theliver, kidney, spleen, bone marrow, and/or lungs areseriously compromised.56-62

Renal disorders, including nephritis, nephrosis,amyloidosis, and nephrocalcinosis, have been re-ported in reptiles.53,63 All of the above diseases cancause a nonregenerative form of anemia. Either in-flammatory or degenerative disease conditions canadversely affect liver and renal health, ultimatelyresulting in the development of nonregenerativeanemia (Fig 10).63 Hepatic lipidosis is a commonliver disorder in captive lizards, and many infectiousagents have been reported as underlying causes ofhepatitis.64,65 Tumors, toxins, and other liver disor-ders are rare but can also cause anemia (Fig 11).

Internal hemorrhage caused by the rupture ofblood vessels in highly vascularized or ulcerative tu-mors may cause a regenerative anemia, but mostneoplastic conditions are accompanied by nonre-generative forms of anemia.52 Chronic pneumoniacaused by bacterial, viral, or mycotic infections arecommon in lizards, snakes, and chelonians, andshould be investigated in the anemic patient.56-62

Gastrointestinal disorders associated with parasiticinfestation, chronic diarrhea, and inflammation canresult in a nonregenerative anemic condition.40

Other causes of nonregenerative anemia observed inbirds and other domestic animals may apply to the

Figure 10. Severe fungal hepatitis (caused by Schizangiella serpen-tis, a zygomycete of the order Entomophthorales, family Basidiobo-laceae) associated with a nonregenerative anemia in a snake. Photocourtesy of Dr. Tracey McNamara.

differential list of the anemic reptile, including hy-

Anemia in Reptiles 105

perestrogenism, leukemia, nutritional deficiencies,stomatitis, chronic gastritis, starvation, hypovitamin-osis A, urinary calculi, amyloidosis, gout, metabolicbone disease, chronic stress, and hypothyroidism.66

Chemotherapy for cancer and the use of certainantibiotics (e.g., chloramphenicol) may induce my-elosuppression and nonregenerative anemia.52 Pri-mary causes of bone marrow suppression that havebeen reported in mammals and birds may also occurin reptiles.4,5,8

Most nonregenerative anemias will be of the nor-mocytic normochromic type, indicating that hema-tological indices will be within reference ranges forthe species being considered. Hypochromic anemiain reptiles is rare and is classically associated withferropenic anemia. The development of ferropenicanemia has been associated with excessive iron lossdue to sequestration by bacteria, iron-deficient diets,or incomplete iron absorption as seen in malabsorp-tion syndromes and chronic diarrhea.3 Rarely, a lo-calized infection will manifest with hypochromicanemia. The primary disorders in iron metabolismthat are associated with nonregenerative hypochro-mic anemia in other classes of vertebrates have notbeen reported in reptiles. Mild anemia with hypo-chromia is also observed in reptiles coming out ofbrumation (Fig 12).

Clinical Presentation of Anemiain Reptiles

Clinical signs of anemia can be divided into twomain groups: those associated with a reduction inthe delivery of oxygen to the tissues and those thatare associated with the primary cause of the ane-

Figure 11. Hepatic lymphoma. Tumors are often associated withnonregenerative anemia in reptiles. Photo courtesy of Dr. TraceyMcNamara.

mic condition. Only those associated with a reduc-

tion in the delivery of oxygen to the tissues arebriefly discussed below. A complete description ofthe clinical signs typically associated with mostreptilian diseases that result in secondary anemiais beyond the scope of this review. The reader isencouraged to look at other sources for moredetails.1,66

Typical clinical signs of anemia in vertebratesare weakness, exercise intolerance, reluctance towalk, tachycardia, and dyspnea. However, reptiles’physiological idiosyncrasies and captive conditionscould contribute to subclinical disease signs thatare hard for even the most dedicated keeper,owner, or veterinarian to recognize. Moreover,mild to moderate decreases in the number of cir-culating RBCs and Hb concentration are not nec-essarily accompanied by clinical signs. Given theirrestricted activity, reptiles kept in small enclosuresrarely show respiratory distress, even when suffer-ing from the most severe cases of anemia. It is onlywhen the nonregenerative anemia is moderate tosevere, and when other clinical signs associatedwith the primary cause become evident, that theanemic condition will be clinically diagnosed.

Moderate to severe paleness of oral and cloacalmucosa is usually an indication of anemia in a reptilepatient. An observant owner should be able to detectthis change in the mucosal color. However, this ex-amination parameter is difficult to observe in reptilespecies with a pigmented oral mucosa. Other com-mon signs of chronic, nonregenerative anemia inreptiles, although nonspecific, are prolonged hiber-nation or brumation times, anorexia, lethargy, and

Figure 12. Blood film from a spiny-tailed lizard with erythrocytesshowing moderate hypochromasia (post-hibernation anemia). Photocourtesy of Dr. Jaime Samour.

depression.

106 Saggese

Diagnosing Anemia in a Reptile

When compared with nonreptilian species, the princi-ples and approaches used for the diagnosis of anemiain reptiles are the same. Based on the limited knowl-edge existing for reptiles, anemia rarely appears toconstitute a single or primary problem. Often, anemiais just another sign or manifestation of an associatedmedical condition or disease. Therefore, an exhaustiveexamination of the whole patient is required to identifythe etiology for the underlying disease that may becontributing to the anemic condition.

Most anemic conditions identified in reptiles willhave a traumatic, degenerative, inflammatory, neo-plastic, toxic, metabolic, nutritional, or infectiousetiology.1-5,8,11,13,14,19,36,38 In cases where one of theseetiologies is not self-evident, a complete anamnesis,evaluation of the husbandry, thorough physical ex-amination, and judicious use of appropriate comple-mentary diagnostic tests (e.g., fecal tests, radio-graphs, ultrasound, urinalysis, complete bloodcount, plasma protein electrophoresis, plasma bio-chemistry panel, bacterial cultures, fungal cultures,testing using polymerase chain reaction technology,fine-needle aspirates, biopsies, coeliotomy, laparos-copy, blood cultures, lung washes, bronchial washes)can be done to determine the underlying cause ofanemia in a reptile patient.42,53,63,64,67-70

Given the idiosyncratic physiological and behav-ioral characteristics that tend to mask signs of diseasein reptile patients, overt signs of sickness may not beevident for many owners. Without the observed pres-ence of other clinical signs, anemia is easily over-looked in its early stages. Annual or biannual physicalexaminations and diagnostic testing are recom-mended for early diagnosis of many diseases in rep-tiles, including anemia.

A complete investigation of the reptile hemogramis one of the most valuable, simple, and least expen-sive diagnostic tools available to the practitioner in-terested in identifying the cause of anemia or diseasecondition in reptile patients. A complete hematolog-ical investigation should include a review of the RBCnumbers, morphology and size, and the accompany-ing white blood cell response.3-5,8

The first specific step needed when investigatingthe cause of a reptile anemia is to estimate the totalRBC count, PCV, and Hb concentration. Unfortu-nately, reference values for these parameters areonly reported for a small percentage of reptiles, andcertain parameters, such as the total RBC count, mayvary because of intrinsic and extrinsic factors, limit-ing the value of the reference. Nevertheless, the total

RBC counts for most reptiles usually range from

300,000 to 2,500,000 erythrocytes/uL. More speci-fic range values have been provided for lizards(1,000,000 to 1,500,000 erythrocytes/�L), snakes(700,000 to 1,600,000 erythrocytes/�L), and chelo-nians (300,000 to 500,000 erythrocytes/�L).2-5,8,28-30

It becomes self-evident that for each group, whentotal RBC counts are well below the low referencerange, the RBC count can be useful in identifyinganemia. However, only a limited number of cases willbe severe enough to fall under the lower ranges.Mild to moderate physiologic changes may occurwith the total RBC count falling within the reportedoverall reference range, even if anemia is present.Therefore, cautious interpretation of published dataand evaluation of other hematological values, indi-ces, and cell morphology is required when evaluat-ing erythrocytic parameters of reptile patients.

The same considerations mentioned for RBCcounts may apply to the PCV and Hb concentration.Subsequent calculation of the hematological indicescould help to further characterize the anemia. Forexample, by measuring the average size of the eryth-rocytes (MCV), it would be possible to determinewhether the patient has a microcytic, normocytic, ormacrocytic anemia. Additional characterization ofthe anemia may be achieved through measuring theMCH to determine whether a reptile is hypochromicor normochromic. Overall, anemia in reptiles is usu-ally of the normocytic and normochromic type.1-5,8

Hypochromic anemias are less commonly observedand usually associated with iron deficiency, heavymetal toxicosis, starvation, and malnutrition.4,5

Other types of anemias (e.g., microcytic, macrocytic)have not been widely reported in reptile species.

To determine the etiologic origin of an anemiccondition requires the examination of the plasmacolor, investigation of blood films for RBC morphol-ogy and the presence of hemoparasites, and mea-surement of the reticulocyte count.2,4,5,8,9 The reticu-locyte count reflects the ability of the bone marrowto produce new blood cells. Newly produced eryth-rocytes can be easily identified and counted manu-ally when stained with vital stains (e.g., new methyl-ene blue). Given that reticulocytes are part of theregenerative response to anemia in birds and mam-mals, they are usually a good quality indicator as towhether the anemic condition is regenerative ornonregenerative in reptiles, and can also provideinformation about bone marrow function. However,it is important to note that reptiles have a very longerythrocyte half-life (up to 600 days) and a very slowturnover time for these cells.27 For most reptiles, thenormal percentage of reticulocytes is less than

2.5%.4,5,8 Therefore, an increase in the number of

Anemia in Reptiles 107

reticulocytes may not be present in reptile patientsduring the early stages of hemolytic or hemorrhagicanemia. Failing to understand the difference be-tween reptiles and other domestic and exotic petanimals can result in the inability to diagnose anonregenerative cause in the early regenerative ane-mic patient. In reptiles, it has been reported that itcan take up to 2 months to detect an increase innewly formed erythrocytes to rule out a nonregen-erative anemia without an obvious cause.5 An obvi-ous increase in the number of reticulocytes is a clearindicator of regenerative anemia.2,4,5,8

When evaluating erythrocyte polychromasia, it isimportant to recognize that up to 1% of circulatingpolychromatic cells is a normal finding in rep-tiles.4,5,8 Furthermore, a polychromatic erythrocyticpercentage of more than 1% is usually considered agood indicator of RBC regeneration. Conversely, alack of polychromasia suggests a nonregenerativeanemia. Repeated evaluation of more than 500 RBCsmay be required to estimate the true status of poly-chromasia in the reptile patient. Presence of mitoticfigures within the erythrocytes, other immature cells,and basophilic stippling of erythrocytes are goodindicators, although not exclusive, of regeneration.3-5,8

When investigating the anemic reptile, practitio-ners should keep in mind that in certain physiolog-ical stages, variations in RBC morphology may occur.Growing reptiles and those undergoing ecdysis mayhave increased numbers of polychromatic erythro-cytes. In brumating reptiles, the number of cells withmitotic figures may be more prevalent. The condi-tion described above should be considered whentrying to establish a diagnosis of anemia.5,8 On thecontrary, anemia is easily masked if blood samplesare taken shortly after brumation. The PCV and Hbconcentrations will be elevated because of the natu-ral dehydration process that occurs during the timeof brumation. A new blood sample should be taken2 to 4 weeks after the initial test to verify the results,thus taking into account normalization of the bru-mation-affected blood values if anemia is suspected.

Most regenerative causes of anemia become obvi-ous after completing the anamnesis, physical exam-ination, hematological investigation, and ancillarydiagnostic tests. For example, sudden changes inPCV are suggestive of a hemolytic or hemorrhagicdisorder. Usually, a sudden drop in PCV is accom-panied by a reddish color of the plasma or serum ifa hemolytic etiology is present. The plasma color ofreptiles is generally a yellow/clear to orange color,but may have a greenish tinge to it. Therefore, asudden drop in PCV without changes in plasma

color is more suggestive of a hemorrhagic problem.

Nevertheless, although a low PCV and reddish col-oration of plasma suggest hemolytic anemia, the testshould be repeated to rule out technical problemsthat could result in a misdiagnosis.

Because malaria is a relatively common cause ofhemolytic anemia in reptiles, blood films shouldalways be examined for the presence of pathogenichemoparasites within the RBCs.1-5,39,40,45 The pres-ence of malarial organisms will often confirm a di-agnosis of a parasite-induced hemolytic anemia.Care should be taken not to assign a parasitic causeof anemia when nonpathogenic hemoparasites areidentified (e.g., hemogregarines).4,39,40,45 Furthermore, itis important to remember that a low prevalence ofmalarial parasites is not necessarily associated withclinical signs and hemolysis. Other causes of hemo-lytic anemia in reptiles may include drug-inducedtoxicity and heavy metal toxicosis, and these shouldbe investigated following the same diagnostic proce-dures used in small animal and avian medicine.

Diagnosing internal hemorrhage in reptiles canbe challenging. In most cases, comprehensive phys-ical examination and judicious use of complimen-tary imaging techniques should be effective enoughto identify this type of anemia. Recovery of blood bygentle flushing of the coelomic cavity will be highlysupportive of a diagnosis of internal hemorrhage. Ifthese other diagnostic tests are insufficient to con-firm active internal hemorrhage, an exploratory coe-liotomy or coelioscopy can be performed to confirmthe diagnosis.

Secondary poisoning with the newer coumarinicrodenticides is a possible cause of hemorrhage inreptiles fed contaminated rodents.43 In many cases,melena and epistaxis will be present, and it is rare forinternal hemorrhage to be the only overt clinicalmanifestation. A definitive diagnosis for rodenticidetoxicity can be made in reptiles with the same tech-niques recommended for birds and mammals. Mea-suring vitamin K–dependent clotting factors is pos-sible in reptiles and may contribute to the diagnosisof a rodenticide poisoning. Investigation of the pro-thrombin, partial thromboplastine, and coagulationtimes may also have some diagnostic value if theresults are compared with those of unaffected indi-viduals from the same species, although the signifi-cance of these tests in reptiles needs further investi-gation. Primary hemostatic disorders are very rare inreptiles and constitute an area with little scientificinvestigation.

Nonregenerative causes of anemia present as acompletely opposite picture to the regenerative re-sponse. The absence of reticulocytes, polychroma-

sia, and mitotic figures is usually a good indicator

108 Saggese

of nonregenerative anemia. However, given that asmall percentage of reticulocytes (� 1%) and poly-chromatic erythrocytes (� 2.5%) are usually foundin the blood of healthy reptiles, a clinician may notbe able to use the absence of these cells as confir-mation early in the anemic process. Again, these aregeneral reference values and may not apply to in-dividual cases; therefore, careful interpretation ofthese parameters is required.

The diagnosis of nonregenerative causes of anemiarequires the combination of collecting a completepatient database, thorough physical examination,and the investigation of most common secondarycauses of nonregenerative anemia. If a diagnosiscannot be made based on this information, a bonemarrow aspirate is required.

Evaluating the Bone Marrow

A bone marrow biopsy is required to determine areptile’s status in a case of nonregenerative anemia.An examination of the cell types from the bonemarrow will allow the clinician to determine why thehematopoietic organ is not producing cells in thewake of needed production.4,5,11 Primary failure oferythropoiesis (i.e., the presence of bone marrowaplasia, hypoplasia, or both) is the most commoncause of nonregenerative anemia due to bone mar-row origin. Possible reasons for a primary failure ofbone marrow erythropoiesis include immune-medi-ated, genetic, radiation, and drug-mediated causes.Infiltration of bone marrow with fat, inflammatorycells, or fibrosis may also result in a reduction in RBCproduction. A careful anamnesis will help to rule outpossible radiation or drug-induced causes for de-creased RBC production; however, diagnostics forimmune-mediated and genetic causes are not widelyavailable for reptiles. A general lack of basic researchlimits our ability to diagnose some of these etiolo-gies, and little information about bone marrow in-terpretation has been published for reptiles.4,5

A bone marrow biopsy can easily be collected fromlizards, chelonians, and crocodilians. The preferredbiopsy sites are the femur and tibia. The landmarks forinserting the biopsy needle are the trochanteric fossae(e.g., lizards and crocodilians) and tibial crest (e.g.,lizards, chelonians, and crocodilians). The technique isconceptually similar to the biopsy technique per-formed in birds. A spinal needle or a regular hypoder-mic needle of adequate size and length can be used tocollect the sample. When using a regular needle, it isbest to use a stylet to avoid blocking the needle channel

with bone and/or cartilage. The author uses the stylet

that is part of some intravenous catheter sets whenperforming this procedure.

General anesthesia is highly recommended whenperforming a bone marrow biopsy, except in thecritical patient in which the risk of anesthesia couldcompromise the patient’s condition. In this last case,the use of a local anesthetic is recommended. Oncethe area is aseptically prepared, a small incisionshould be made on the skin and the needle insertedinto the bone with continuous and steady pressureand a slight left-right rotational movement. Once theneedle is within the medullary cavity, the styletshould be removed and a syringe attached for sam-ple collection. Bone marrow can be collected byaspirating the syringe with a slight but steady nega-tive pressure. No anticoagulant is needed. Once thesample is collected, multiple films can be preparedfor evaluating cell morphology and numbers. Sam-ples can be stained with Romanowsky or hematoxy-lin/eosin stains to evaluate the cells, and Gram’sand/or Ziehl-Neelsen stains to screen for microor-ganisms. If a bone marrow culture is desired, the hubof the needle can be swabbed and submitted forbacteriology, mycology, or virology.60,67,70

A different approach and technique are recom-mended in snakes and other reptiles lacking hind andforelimbs. The rib is the preferred site for a bonemarrow biopsy in these animals, and collecting thesample requires a surgical approach. This procedureshould be done with the patient under general anes-thesia. Once the site is aseptically prepared, a section ofa rib can be removed and submitted for testing.4,5,8

When evaluating the bone marrow biopsy, it is im-portant to review the myeloid:erythroid relationship,quantitative evaluation of different RBC precursors,morphological abnormalities, presence of inflamma-tory cells and fatty deposits, and presence of aplasia orhypoplasia. In some cases, hyperplasia of bone marrowRBC precursors is observed and indicates regenerationand normal bone marrow function. However, ineffec-tive erythropoiesis may occur if RBC precursors areobserved but not their circulating forms. Seven basicstages of erythrocyte maturation have been reported:rubriblasts, prorubricytes, basophilic rubricytes, earlypolychromatic rubricytes, late polychromatic rubri-cytes, polychromatic erythrocytes, and mature erythro-cytes.4,5,27,71,72 Relative percentages of these differentcell populations in healthy or anemic reptile patientsare not available.

When suspected, identification of infectious cau-ses of osteomyelitis should also be investigated.An increased number of inflammatory cells in thebiopsy sample may be suggestive of osteomyelitis.

Polymerase chain reaction, culture, and specific

Anemia in Reptiles 109

stains are necessary to identify fungi as well as variousbacteria in the bone marrow. Additional stainingwith Periodic acid Schiff, Congo red, and Prussianblue are useful to detect amyloid, fat deposits, andiron in the bone marrow.

Therapeutics

Very little information is available regarding treatmentof anemia in reptiles; therefore, specific therapeuticrecommendations cannot be provided. Anecdotal andclinical evidence suggests that the basic therapeuticprinciples applied to the anemic nonreptile patientmay also be of benefit to the anemic reptile. Detailedinformation about general husbandry, nutrition, andrecommended treatment for diseases such as malaria,lead toxicosis, infectious diseases, kidney and liver dis-orders, and other common causes of anemia is avail-able in other publications.16-18,66,73 In the future, re-ports of clinical experiences and additional scientificresearch about anemia, its diagnosis, characterization,and specific treatment may enhance the therapeuticoptions available to the exotic animal practitioner fortheir anemic reptile patients.

Conclusion

Anemia is a very common presentation in veterinarymedicine, but it is underreported in the scientificliterature as it relates to reptile species. Therefore,better case documentation, consistency in the inves-tigation of anemia, and rapid dissemination of theavailable information are needed to increase ourknowledge in this area of veterinary medicine. Col-laborative research efforts between practitioners,herpetologists, reptile curators, pathologists, immu-nologists, microbiologists, toxicologists, and clinicalpathologists are urgently needed to augment ourunderstanding of anemia in reptiles.

Acknowledgments

The author is deeply grateful to Drs. Janis Joslin,Jennifer Burr, Mark Mitchell, and Tom N. Tully, Jr.for their invaluable comments on early versions ofthis manuscript. Special thanks to Drs. C. Torta, D.Soler Tovar, G. Perez Jimeno, P. Moreno, A. Rojas, J.Samour, J. Burr, J. Claver, T. McNamara, and A.Quaglia for providing photographs used to illustrate

this article.

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