ISRAEL JOURNAL OF

VETERINARY MEDICINE
MORPHOLOGICAL CHANGES IN RED BLOOD CELLS OF BIRDS AND REPTILES AND THEIR INTERPRETATION H. Pendl
ARCHEOPTERYX DIAGNOSTICSTM Hematology and Cytology in Bird and Re!tile . Ei"#ena$% Germany

SUMMARY T#e &al$e o' t#e e&al$ation o' "ell mor!#ology in a (lood 'ilm )it# !e"ial 'o"$ on a&ian and re!tilian eryt#ro"yte i demon trated. In "om(ination )it# t#e !a"*ed "ell &ol$me +!"&, t#i ra!id and lo)-"o t !ro"ed$re gi&e $ e'$l in'ormation on t#e !atient. "ondition and #el! t#e !ra"titioner to ta*e 'ir t diagno ti" and t#era!e$ti" mea $re )#ile a)aiting re $lt (eing done (y o$tdoor la(oratorie . General !rin"i!le 'or t#e di tin"tion (et)een regenerati&e and degenerati&e "#ange in a&ian and re!tilian red (lood "ell are e/!lained in "ri ! ta(le % 'ig$re % and !#otogra!# . T#ey are intended 'or dire "t $ e and #o$ld en"o$rage !ra"titioner to in"l$de t#i $ e'$l tool in t#eir daily !ra"ti"e ro$tine.

INTRODUCTION De !ite t#e te"#nologi"al !rogre in a&ian and re!tilian #ematology% time"on $ming man$al te"#ni0$e are till ne"e ary to !er'orm a "om!lete (lood "ell "o$nt% )#i"# i di''i"$lt to do in-#o$ e d$ring daily ro$tine. 1al$a(le time i lo t )it# t#e !ro"e ing o' am!le in la(oratorie % (e'ore ade0$ate diagno ti" and t#era!e$ti" te! "an (e tarted. T#e timely on et o' an ade0$ate treatment% #o)e&er% i &ital 'or t#e o$t"ome o' t#e "a e% e !e"ially in de(ilitated !atient . T#ere'ore% im!le indoor la(oratory te"#ni0$e 'or a 'ir t "#e"* $! are de ira(le. E&al$ation o' "ell mor!#ology in a (lood 'ilm i a ra!id and lo)-"o t !ro"ed$re. In "om(ination )it# t#e !a"*ed "ell &ol$me +!"&, it gi&e $ e'$l in'ormation on t#e !atient. o&erall "ondition and #el! t#e !ra"titioner to ta*e 'ir t mea $re )#ile a)aiting '$rt#er detailed re $lt 'rom t#e la(oratory. T#i !a!er 'o"$ e on t#e "#ara"teri ti" and inter!retation o' mor!#ologi"al "#ange in a&ian and re!tilian eryt#ro"yte .

TECHNICAL ASPECTS Preparation of a bloo fil! Blood mear 'rom (ird and re!tile #o$ld (e !er'ormed )it#o$t any anti"oag$lant dire"tly 'rom t#e yringe or needle and immediately a'ter "olle"tion (y a!!lying t#e 2)edge mear3 or t#e 2 lide and "o&ergla 3 te"#ni0$e +4ig. 5,. Con"erning t#e 'ir t met#od% t#e $ e o' a (e&el-edged lide i "r$"ial to a&oid arti'i"ial "ell damage. Ideally% t#e (lood 'ilm #o) a "ontin$o$ t#inning )it# a &ane at t#e end. 4or any e&al$ation% t#e monolayer +M6, i e/amined in a meandering ear"# a #o)n in 'ig$re 7a. It i t#e area% )#ere t#e "ell lie "lo e to ea"# ot#er )it#o$t o&erla!!ing t#em el&e and "an $ $ally (e 'o$nd (et)een t#e middle and t#e la t t#ird o' t#e (lood 'ilm. 4re0$ent !re!aration mi ta*e in"l$de inade0$ate &ol$me o' (lood and interr$!tion o' t#e mear mo&ement +4ig 7(-d,% )#i"# re $lt in t#e lo o' t#e monolayer area. A ro$tine !re!aration o' at lea t t#ree mear !er (lood am!ling i re"ommended to (e !re!ared 'or additional e/amination % $"# a $ !e"ted !ara itemia or 'ail$re o' !ro!er taining. T#e air-dried mear are *e!t in a d$ t'ree en&ironment $ntil t#ey are tained. D$ t !arti"le on t#e am!le get tained toget#er )it# t#e (lood "ell re $lting in !oor 0$ality o' t#e (lood 'ilm . Tr$e $("ell$lar tr$"t$re "annot (e di''erentiated 'rom arti'i"ial oiling.

Fi"# $% Te"#ni0$e 'or (lood 'ilm !re!aration

Fi"# &% Ideal (lood 'ilm and "ommon mi ta*e 8 t#e mononlayer +M6, 'or e&al$ation i 'o$nd (et)een t#e middle and t#e la t t#ird o' an ideal mear

Fi"# '% 4lo) "#art 'or t#e inter!retation o' a&ian and re!tilian #emogram

Stainin" of a bloo fil! Stain o' t#e Romano) *y ty!e% e.g. 9rig#t. % 9rig#t-Giem a% and MayGr$en)ald-Giem a are re"ommended 'or (ird and re!tile in one- or m$lti!lete!-!roto"ol 5% 7% :% ;. <n'ort$nately% t#e 0$ality o' t#e taining )it# traditional !roto"ol i in"on i tent. T#e rea on i $n*no)n:. Anot#er di ad&antage 'or daily !ra"ti"e i t#e need o' a la(or-inten i&e 're # !re!aration o' Giem a. ol$tion. E/tended e/!erimental )or* in t#i 'ield #a (een done (y =.H. Samo$r> re $lting in a 9rig#t-Giem a 'orm$la% )#i"# gi&e #ig#ly re!rod$"i(le re $lt and doe not need to (e !re!ared 're #ly +Ta(le 5,. T#e taining !roto"ol i e/"ellent 'or a&ian and re!tilian (lood. D$e to t#eir aggre i&e nat$re 'a t taining met#od li*e Di'' ?$i"* and Hema"olor tend to o&er tain or de troy 'ine $("ell$lar tr$"t$re . T#i i e !e"ially tr$e 'or re!tile (lood "ell . Ne&ert#ele % t#ey are $ e'$l a an addition to Romano) *y tain 'or do$(le "#e"* !$r!o e @ I' a $ !e"t 'inding i een (ot# in a Romano) *y and a Di'' ?$i"* tained mear% arte'a"t d$e to taining "an (e e/"l$ded. Dry te"#ni0$e #a&e (een !ro&en to (e $n $ita(le 'or (ird A% "orre !onding data 'or re!tile (lood are not a&aila(le.

Ta(le 5@ 9rig#t-Giem a Stain 'or a&ian and re!tilian (lood 'ilm Staining Stain
4lood mear% allo) to tand 'or : min Add e0$al amo$nt o' ($''er A.C; : g 9rig#t tain !o)der5 B.: g Giem a tain !o)der7 > ml gly"erol

Mi/ gently (y (lo)ing $ ing a !i!ette or a tra) $ntil metalli" green #een 'orm on t#e $r'a"e allo) to tand 'or A min

Rin e )it# ($''er allo)ing to tand 'or 5 min 'or di''erentiation 9a # "o!io$ ly )it# ($''er 9i!e t#e (a"* o' t#e mear to remo&e e/"e Pro! in ra"* $ntil dry or $ e #air dryer D gi&e (etter tain

5BBB ml a( ol$te met#anol +a"etone 'ree,: 'iltered and tored

Co&er )it# Entellan> and "o&er gla 0$ality t#an )it#o$t "o&er
5

Mer"* No 5.BE7B:.BB7>% 7 Mer"* No 5.BE7FC.BB7>% : Mer"* No 5.BABBE.5BBB% ; Mer"* No 5.55:F;.B5BB

>

Mer"* No 5.BFEA5.B5BB

E(ALUATION OF ERYTHROCYTE MORPHOLOGY In "ontra t to mo t mammalian eryt#ro"yte % a&ian and re!tilian eryt#ro"yte are tr$e n$"le$ -"ontaining "ell . Re!tilian "ell are larger% 'latter% and t#inner t#an a&ian eryt#ro"yte . T#e !#y iologi"al mor!#ology o' (ot# i "#ara"teriGed (y a more-or-le "ongr$ent o&al-to-ro$nd #a!e o' "yto!la m and n$"le$ . Mat$re a&ian red (lood "ell di !lay a (rig#t orange "olo$ration% )#erea re!tile eryt#ro"yte tend to (e !aler. Mor!#ologi" "#ange "an a''e"t "olo$ration andHor tr$"t$re o' "yto!la m and n$"le$ . In general% one #a to di ting$i # (et)een regenerati&e and degenerati&e "#ange . A regenerati&e re !on e% al o named le't #i't% i "#ara"teriGed (y an in"rea ed amo$nt o' immat$re "ell in t#e !eri!#eral (lood tream and indi"ate an in"rea ed eryt#ro!oieti" a"ti&ity. Degenerati&e "#ange in "ontra t are "#ange )#i"# do not 'it into t#e normal de&elo!mental erie o' eryt#ro!oie i . T#ere'ore% t#e in"rea ed o""$ren"e o' degenerated "ell i al)ay a ign o' a "lini"al !ro(lem. 4re0$ent mor!#ologi" "#ange and t#eir inter!retation:% F% C% E% 5B are $mmariGed in ta(le 7. In a&ian and re!tilian am!le t#e eryt#ro!oieti" a"ti&ity i e&al$ated (y e timating t#e !oly"#romato!#ili" "ell in Romano) *y tained (lood 'ilm :. 4or t#i !$r!o e a emi0$antitati&e "ale% t#e !oly"#romato!#ili" inde/ +PI, #a (een e ta(li #ed 'or (ird (y Dein55 and "an (e a!!lied al o to re!tile !e"imen )it# little modi'i"ation E% 5B +Ta(le :,.

T#e 'lo) "#art o$tlined in 'ig$re : are intended a ro$g# g$ideline 'or t#e inter!retation o' "ommon eryt#ro"yti" 'inding . Con"erning re!tile (lood% t#e !ra"titioner #a to ta*e into a""o$nt t#at "ertain "ell remain !l$ri!otent in t#e !eri!#eral (lood tream. T#rom(o"yte % 'or e/am!le% "an tran 'orm into red (lood "ell % i' t#ere i an in"rea ed demand 'or eryt#ro"yte . In t#i "a e% t#e (lood'ilm "ontain m$lti!le "ell )#i"# di !lay (ot# eryt#ro"yti" and t#rom(o"yti" "#ara"teri ti" +Colo$r !late 5% 'ig. d,. Blood !ara ite are a "ommon 'inding in re!tile (lood 'ilm % mo t o' t#em (eing intraeryt#ro"yti". T#ey are more !oradi" in (ird . < $ally t#eir "lini"al igni'i"an"e i more a"ademi" t#an #aGardo$ . <nder "ertain "ir"$m tan"e % #o)e&er% a moderate to e&ere anemia de&elo! and "an e/a"er(ate a !at#ologi"al "ondition o' di''erent etiology. Some genera $"# a Plasmodium and Haemoproteus are relati&ely ea y to re"ogniGe a !ara ite % ($t ot#er % li*e Babesia% Aegyptianella% and Rikettsia-like organi m are readily mi ta*en 'or in"l$ ion o' di''erent origin. Criteria 'or t#e di''erentiation o' !ara iti" de&elo!mental tage 'rom non-!ara iti" tr$"t$re in"l$de t#e reg$larity o' o""$ren"e% t#e e/"l$ i&ely intraeryt#ro"yti" a!!earan"e% and in many "a e t#e e&iden"e o' ome gran$lation in t#e !ara iti" in"l$ ion. T#e !e"i'i" identi'i"ation o' #ematoGoa re0$ire !e"ial *no)ledge and #o$ld (e le't to !e"ialiGed la(oratorie . In "a e o' $ !e"ted !ara itaemia% additional (lood 'ilm #o$ld (e 'i/ed in a( ol$te met#anol 'or ten min$te and tored in a d$ t 'ree en&ironment 'or '$t$re identi'i"ation57.

INTERPRETATION OF MORPHOLOGICAL CHANGES T#e re'eren"e range 'or t#e !"& in mo t (ird !e"ie i a!!ro/imately :>->>I and 7B-;BI in mo t re!tile !e"ie . T#e a(ility to J$dge t#e eryt#ro"yti" mor!#ology in a (lood mear ena(le t#e !ra"titioner to !e"i'y t#e 'inding 'or t#e !"& and to re'ine #i 'ir t diagno ti" and t#era!e$ti" a!!roa"# to)ard a "a e. It im!ortan"e (e"ome "lear )it# t#e 'ollo)ing e/am!le@ I' a !arrot di !lay a !"& o' :BI it ma*e a igni'i"ant di''eren"e% )#et#er t#e (lood mear #o) a PI o' 5% :% or >. T#e #ig#er t#e PI "ore i % t#e "lo er t#e !atient #a to (e monitored. Serial am!ling i re"ommended to 'ollo) $! t#e de&elo!ment and to ta*e '$rt#er te! in "a e t#e it$ation e/a"er(ate . In t#e 'ir t "enario )it# a PI o' 5% t#e (ird #o) a de!re i&e anemia. T#i i a "ommon 'inding )it# "#roni" di ea e. In t#e e !atient % eryt#ro!oie i i de"rea ed% a t#eir "ata(oli" tate doe not allo) normal "ell !rod$"tion. T#e anemia it el' doe not re0$ire a !e"i'i" t#era!y% alt#o$g# t#e a!!li"ation 1itamin BA% B57% 'oli" a"id and iron #a (een !ro&en to (e o' (ene'it. < $ally it )ill re ol&e )it# t#e "orre"t treatment o' t#e $nderlying "a$ e.

I' t#e PI i "ored : li*e in "enario t)o% t#e (ird i $''ering 'rom a regenerati&e anemia. In t#i "a e% t#e !ra"titioner i )ell ad&i ed to loo* 'or any "a$ e o' #emorr#age and ta*e mea $re to !re&ent '$rt#er (lood lo . T#e di''erential li t 'or t#i "enario in"l$de tra$ma +e.g. inJ$ry% (lood- $"*ing !ara ite ,% "oag$lo!at#ie +e.g. 'a"tor de'i"ien"ie % #e!ato!at#ie % to/in % #y!ere trogeni m% di eminated intra&a "$lar "oag$lo!at#y in e!ti"emi" and &iral in'e"tion ,% $l"erati&e #emorr#agi" in'lammation% and e"ondary ti $e (leeding d$e to neo!la ti" "ondition . T#e t#ird "enario )it# a PI "ore o' > #a to (e "on idered an emergen"y "a e and i mo tly een )it# #emoly i or e&ere (lood lo . T#e di''erential li t 'or #emoly i in"l$de e!ti"emia % (lood !ara ite +Plasmodium spp,% into/i"ation % a$toimm$ne rea"tion % and ($rn . In "ontra t to t#e 'ir t "a e% t#i anemia #a to (e "on idered a !rimary !ro(lem 'or t#e !atient% a $''i"ient ga e/"#ange 'or t#e maintenan"e o' elementary (ody '$n"tion i not g$aranteed. Ra!id e/a"er(ation o' t#e it$ation i "ommon in t#e e "a e . T#$ % immediate mea $re #a&e to (e ta*en to ta(iliGe t#e !atient. In "a e t#e !"& 'all (elo) 7BI tran '$ ion o' (lood or o/yglo(in #a to (e "on idered. Ba i"ally% t#e e "on ideration are al o &alid 'or re!tile % ($t ea onal in'l$en"e on t#e tat$ and rea"ti&ity o' t#e (lood y tem #a&e to (e ta*en into a""o$nt additionally. D$ring #i(ernation% (lood "ell !oie i i de"rea ed igni'i"antly and in"rea ed again a'ter a)a*ening. T#$ % a lo) !"& and an in"rea ed !oly"#romato!#ili" inde/ "an (e !#y iologi"al in a re!tile !o t #i(ernation% ($t !at#ologi" in ea on o' '$ll a"ti&ity.

DISCUSSION T#e !$r!o e o' t#i !a!er i to en"o$rage !ra"titioner to $ e a tained (lood 'ilm a a 0$i"* "#e"* te"#ni0$e to get a 'ir t im!re ion on t#e red (lood "ell !anel o' t#e "a e !re ented. It #a (een *e!t to a &ery "onden ed tyle. S!e"ial em!#a i #a (een laid on "learly arranged ta(le % 'ig$re and !#otogra!# . T#ey are de igned 'or dire"t $ e in daily !ra"ti"e and #o$ld #el! t#e e/amining !er on to 0$i"*ly ma*e a de"i ion. 9it#o$t do$(t t#i "onden ed de ign in&ol&e t#e danger o' #ortening% )#i"# may lead to 'al e "on"l$ ion . It #a to (e em!#a iGed% t#at t#e met#od !re ented "annot re!la"e a "om!lete (lood "ell "o$nt. Any !e"i'i" di ea e or etiology mentioned #a to (e $nder tood a an e/am!le% not a a de'initi&e diagno i . Ne&ert#ele % t#e e&al$ation o' t#e "ell mor!#ology in a (lood 'ilm% i a ra!id and lo) "o t !ro"ed$re and t#ere'ore !rede tined 'or indoor $ e in daily !ra"ti"e. In "om(ination )it# t#e !a"*ed "ell &ol$me +!"&, it )ill gi&e $ e'$l in'ormation on t#e !atient o&erall "ondition and #el! t#e !ra"titioner to ta*e 'ir t mea $re )#ile a)aiting re $lt (eeing done o$tdoor . E&en in "a e o' !er'orming a "om!lete (lood "ell "o$nt% mor!#ologi" "#ange o' (lood "ell "ontain im!ortant additional in'ormation and #o$ld (e e&al$ated ro$tinely. T#i i e !e"ially tr$e 'or re!tile . D$e to t#eir e"tot#ermi" nat$re "ell n$m(er are $(Je"t to an in"rea ed !#y iologi" &aria(ility. Re $lt

'rom n$meri" !arameter #a&e to (e e&al$ated e/tremely "are'$lly 7. In "a e o' e/oti" !e"ie % )#ere relia(le normati&e data i la"*ing% t#e !re en"e o' "ell )it# mor!#ologi" a(normalitie i a more relia(le inde/ o' a !at#ologi"al "#ange t#an n$meri" re $lt :.

Referen)e* 5. Alleman A.R% =a"o( on E.R% Ra *in R.E. Mor!#ologi"% "yto"#emi"al taining% and $ltra tr$"t$ral "#ara"teri ti" o' (lood "ell 'rom ea tern diamond(a"* rattle na*e +Crotal$ adamante$ ,. Am. =. 1et. Re . 8AB@>BF->5;% 5EEE. 7. Cam!(ell T.9. Clini"al Pat#ology. In@ Mader DR. Re!tile Medi"ine and S$rgery. P#iladel!#ia% 6ondon% Toronto% Montreal% Sydney% To*io@ 9B Sa$nder Com! @ 7;C7>F%5EEA. :. Ha)*ey C.M% Dennett T.B. A Colo$r Atla o' Com!arati&e 1eterinary Hematology. 6ondon@ 9ol'e Medi"al P$(li"ation 6td 5ECE. ;. M$ro =% C$en"a R% Pa tor =% 1ina 6% 6a&in S. E''e"t o' lit#i$m #e!arin and tri!ota i$m EDTA on #ematologi" &al$e o' HermannK tortoi e +Te t$do

#ermanni,. =. Loo 9ildl. Med. 87E@;B-;;% 5EEC. >. Samo$r =H. $n!$(li #ed data% !er . "omm. 7BB7 A. Ha$ *a H% S"o!e A% 1a i"e* 6.% Rea$G B. 1erglei"#ende <nter $"#$ngen G$r 4Mr($ng A&iMrer Bl$tGellen. TierMrGtl. Pra/. 8 7F +N,@ 7CB-7CF% 5EEE. F. Cam!(ell T.9.@ A&ian #ematology and "ytology. Ame % Io)a >BB5;@ Io)a State <ni&er ity Pre 8 F-5B% 5EE>. C. Pendl H% Re(all H. Hy!er"#rome NormMmien (ei AmaGonen - #Mmatologi "#e Bild. X1I. Tag$ng O(er 1ogel*ran*#eiten% De$t "#e 1eterinMrmediGini "#e Ge ell "#a't% 4a"#gr$!!e Ge'lOgel*ran*#eiten +91PA, am In tit$t 'Or Ge'lOgel*ran*#eiten der 6$d)ig-Ma/imilian -<ni&er itMt MOn"#en% 5;-7;% .7BB;. E. Ra *in R.E. Re!tilian Com!lete Blood Co$nt. In@ 4$dge A.M. +ed,@ 6a(oratory Medi"ine A&ian and E/oti" Pet . P#ilade!#ia% 6ondon% Toronto% Montreal% Sydney% To*yo@ 9.B. Sa$nder Com! @ 5E-7F% 7BBB. 5B. ROSSNOP4 9R% =r. Di order o' Re!tilian 6e$*o"yte and Eryt#ro"yte . In@ 4$dge A.M. +ed,@ 6a(oratory Medi"ine A&ian and E/oti" Pet . P#ilade!#ia% 6ondon% Toronto% Montreal% Sydney% To*yo@ 9.B. Sa$nder Com! @ 5E-7F% 7BBB. 55. Dein 4.=. A&ian #ematology@ eryt#ro"yte and anemia. Pro". Ann. Meet. A . A&ian 1et.% 5B-7:% 5EC:. 57. Samo$r =.H. !er . "omm. 7BB:.