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SPEAKING MANUSCRIPT SOME NORMAL HEMATOLOGICAL VALUES' OF THE WHITE CARNEAU'PIGEONI ANIERIC&A'SSOCTATION FOR LABORATORY ANIMAL SCIENCE NEw YORK, N w ~IQg8 SEPTEMBER'2~, ly/ (REBECCA WONG) SEVERAL IINVESTI'GATORS HAVE REPORTED HEMATOLOGICAL VALUES FOR THE WHITE CARNEAU PUGEON,LITERATURE PERTAINING TO' THE DIFFERENTIAL WHITE BLOOD CELL COUNTS WAS NOT ONLY VERY SCANTY, BUT ALSOlCONFUSI'NGiREGARDI'N&THE NOMENCLATURE 0'F' THE LEUCOCYTES', NONGRANULAR LEUCOCYTES', SO CALLEDiBECAUSE STAINABLE GRA'NULES'ARE USUALLY ABSENT FROM!THE CYTOPLASM4 APPARENTLY INCLUDE . . . .. LYMPHOCYTES A'NDIMONOCYTES. THE GRANULAR' LEUCOCYTES MAY' BE CLA'SSI'FIED'AS HETEROPHILS, EOSINOPHILS, AND BASOPHI'LS, WE FOUND. THE PUBLISHED DATA, HOWEVER, TO BE H.TGHLY VARIABLE. As PART OF A STUDY'ON EXPERIMENTAL ATHEROGENESIS, WE INITIATED A'PROGRPIM TO DEFINE THE HEMATOLOGICAL VALUES OF SIX-MONTHt-OLD MALE WHITE CARNEAU PI,GEONS._ _ DIFFERENTIAL WHITE BLOOD CELL COUNTS WERE MADE WITH WR'IGHT"S STAd'N FOR'TWO~MTNUTES, PHOSPHATE'BUFFER'WAS THEN ADDED, AND' THE STAINING CONTINUED FOR TEN MORE MINUTES.THE SLIDE WAS THEN FLUSHED1WI'TH DISTILLED WATER'AND SET ASIDE TO DRY. STANDARD DIFFERENTIAL COUNTS WERE PERFORMED TO FINDiTHE RELATIVE PERCENTAGE OF EACH TYPE OF LEUCOCYTE. A TOTAL OF ONE-HUNDRED WHITE CELLS WERE COUNTED IN EACHISLID'E. MAY I HAVE THE FIRST SLIDE PLEASE?'

SLIDE 1 THIS SLIDE ILLUSTRATES A P'IGEON HETEROPH'IL, WHICH'I'S ANALOGOUS TO THE NEUTROPHIL IN MAMMALS. UNLIKE THENEUTROPHIL, ~ HOWEVER, THE HETEROPHIL HAS ANiAFFINITY FOR EOSIN DYES, IT IS CHARACTERIZED BYLONG SPINDLE SHAPED RED RODS AND A BLUE BILOBED NUCLEUS. SLIDE 2 THIS'SLIDE SHOWS AN EOSINOPHIL. EOSINOPHILS ALSO:STAIN WITH EOSIN, BUT MAY BE DISTINGUISHED FROM HETEROPHILS BY THE PRESENCE OF REACTIVITY IN GRANULES RATHERTHAN RODS. THE NUCLEUSI'S USUALLY BILOBED. SLIDE 3 TIHI'S SLIDE SHOWS A BASOPHPL. TIHEBASOPHIL CONTAINS GRANULES THAT HAVE AN AFFINITYFOR BASIC DYES. IT CAN BE IDENTIFIED BY THE PRESENCE OFPURPLE GRANULES. THE NUCLEU&IS USUALLY'PALE BLUE, SLIDE 4 ROUND IINI SHAPE, AND CENTRALLY PLA'CED., THIS SLIDE SHOWS REPRESENTATIVE LYMPHOCYTES, WE FOUND A GREAT VARIATION IN THE SIZE OF THE LYMPHOCYTES., THE SMALL LYMPHOCYTE WAS USUALLY THE MOST NUMEROUS. THE NUCLEUS IiS ROUND, BLUISH- PURPLE, AND CENTRALLY PLACED., IT ISEMCOM'PASSED BY A PALE BLUE CYTOPLASM, WHICH ISNOT READILY PERCEPTIBLE ON ALL SIDES OF THE CELL., INITHE LARGE LYMPHOCYTES, THECYTOPLASM IS MORE ABUNDANT AND THE NUCLEUS IS ECCENTRIC IN POSITION. SLIDE 5 THIS SLI~DE SHOWS A MONOCYTE. MbNOCYTES ARE THE LARGEST OF THE LEUCOCYTES. THE NUCLEUSSTAINS'PURPLE, IS RENIFORMI'N SHAPE, ANDUSUALLYIS ECCENTRIC IN'POSITION1. THE CYTOPLASM IS PROPORTIONATELY LARGER THAN IT IS IN LYMPHOCYTES. SLIDE 6 THIS SLIDE ILLUSTRATES SOME THROMBOCYTES WHICH MAY BE SEEN I'N THE DIFFERENTIAL SLIDE, THEY ARE MUCH SMALLER THAN LYMPHOCYTES,,MOREELONGATED, AND DARK PURPLE. THE THROMBOCYTE CONCENr TRATION MAY BE READILY PERFORMED FROM THIS PREPATION.

SLIDE 7 THIS SLIDE SUMMARIZED THE RESULTS' OF OUR' D1!FFERENTIAL WHITE' ~ BLOOD CELL COUNTS. THE LYMPHOCYTES WERE THE MOST ABUNDANT'CELL, COMPRISING 58% OF THE TOTAL WHITE CELL POPULATION, WITH THE HETEROPHILS THE NEXT MOST ABUNDANT CELL AT 37,3% OF THE TOTAL. THERE WERE VERY FEW MONOCYTES, EOSINOPHILS, AND BASOPHILS. SLI',DE S'' HEMA'TOCRTTS WERE DETERMINED WITH THE M'I'CROHEMATOCRIT CENTRI'FUGE', BLOOD WAS PLACED IN'A HEPAR'INI'ZED MICRO-CAPILLARY TUBE AND CENTRIFUGEDIFOR IO'MINUTES,, AT WH'I'..CH TIME.THE TUBES WERE READ ON AN INTERNATIONAL M'ICRO`CAP'ILLARY READER. MAY' I HAVE THE SLIDES OFF PL'EASE? IN OUR STUDIES, THE RED CELL CONCENTRATION WAS DETERMINED BY THE METHOD SUGGESTED BY'NATT'AND HERR11'CK'. THE DI'LUTENT UTILIZED CONSISTED OF A SINGLE SOLUTION WHICH WAS EASILY MADE 9TA'BLE, AND WAS OF'THE SAME OSMOTIC PRSSURE AS THE BI'RD!S BLOOD, THUS MIN'I'M'IZI'NG THE P03S'IBILITI'ESFORCELL DPSTORTI'AN~.~ THE BLOOD~ WAS DILUTED 1:200 WITH A RED BLOOD CELL COUNTING PIPETTE.AND ALLOWED TO STAND FOR ONE MINUTE. THE SAMPLE WAS'THEN AGITATED. FOR 30 SECONDS WITH A YANKEE PIPETTE SHAKER TO ENSURE AN, EQUAL CELL DI'STRIBUTI'ONi. ALL COUNTS'WERE MADE ON A HEMOCYTOMETER AT A MAGNIFICATION OF 1430X, THE TOTAL ERYTHROCYTE COUNT WAS MADE A'CCORDUNG TO STANDARD LABORATORY'PRQCEDURES. THE HETEROPHILS WERE THE'EASIEST WHITE CELLS TO RECOGNIZE _ ., . . .__ _ . _ _ . . . INiTHE CHA'MBER. THE NUCLEUS A'ND'CYTOPLASM STAIN A LI~GHT VdOLET'. p'READILYi DISTINGUISHABLE FEATURE OF THE CELL ARE THE BLUE- V'I'OLET, ALMOST BLAC'K, STAINIiNG GRANULES THAT FILL THE CELL. IT IS SOMETIMES NECESSARY TO VARY THE FINE ADJUSTMENT OF THE MICROSCOPE TO VISUALIZE THESE GRANULES. NETEROPHILS WERE COUNTED IN ALL SQUARES OF ONE SIDE OF THE COUNTING CHAMBER'.

l DETERMINING THE TOTAL LEUCOCYTE COUNT OF'B'IRDS WA'S. DI~FFICULT BECAUSE THE LEUCOCYTES, THROMBOCYiTES, AND ERYTHROCYTES IN THE BIRD ARE ALL NUCLEATED CELLS, THE NUCLEI OF THE'ERYTHROCYTES ARE NOT DESTROYED BY'METHODS ORDINARILY USED IN'PROCESSdiNG MA'MMALPAN BLOODy THUS MAKING IT IMPOSSIBLE TO1Dd'FFERENTIATE EA'SI.LY'BETWEENITHE LEUCOCYTES AND THE NUCLEII OF THE ERY` THROCYTES'. THUS, TOTAL WHITE BLOOD CELL CONCENTRATI~ON WAS DERIVED BY A'BACK-CALCULATION FROM!THE HETEROPHIL PERCENTAGE OBTAINED FROM THE DIFFERENTIAL WHITE CELL COUNT, AND' THE ABSO'LUTE HETEROPHIL COUNT. THE TOTAL WHiI!TE'COUNT WA'S. DETERMINED1FRpM THE FOLLOWC'NG EQUATI'.ON: TOTAL LEUCOCYTES = HET COUNT 222 ' (THE DILUTION FACTOR) X• HET % „MAY I HAVE THE NEXT SLIDE P'LEA'SE?CcLIDE 9 THI'S SLIDE SHOWS THE M'EAN VALUES OBTAI~NED IN OUR LABORATORY FOR THE RED BLOOD CELL CONCENTRATIQN, HEMATOCRIT AND TOTAL WHITE BLOOD CELL CONCENTRATION. THE AVERAGE RED BLOOD CELL COUNT! WAS 3.09 x LO6/NiM3, THE AVERAGE HEMATOCRIT 48'.6, AND THE AVERAGE WHITE BLOOD CELL T,8E X 104/MM3. THERE WA'SIA WIDE VARIATION IN THE WHITE BLOOD CELL VALUES, WE FOUND THAT OUR VALUES DIFFERED BETWEEN BIRDS, AS'WELL AS IN THE SAME Bd'RD, SLIDE 10 MEASUREMENT OF HEMOGiOBIN!WAS ALSO H'dNDERED BY THE PRESENCE OF NUCLEATED RED BLOOD CELLS.THE It C0-0XIMETER ALLOWED US TO1ACCURATELY MEASURE OXYHEMOGLOBI'N, CARBQXYHEMOGLOBIN, AND REDUCED HEMOGLOBIN FROM A S'INGLE'BLO©D SAMPLE. THIS INSTRUMENT UTILIZES~A SPECTROP'HOTOMETRI',C PR'INCIPLE BASED ON~THE FACT T!HAT'THE ABSORPTION SPECTRA OF ALL THREE SRECI'ES IS UNIQUE BUT OVERLA'PP'ING, MEASUREMENTS ARE MADE AT WAVELENGTHS' WHERE AT LEAST TWO SPECIES HAVE THE SAME ABSpRBI'TIVITY' (IiSOBESTIC

PO'INTS),, THE SPECTRA OF ALL THREE S'PECIE'S' ARE ISOBESTIC AT' 548 ' NM,' THE OXYGENATED' AND REDUCED' HEMOGLO'B'IN SPECTRA ARE I'~SOBE~ST~IC~ AT 568 N~M';, A~ND~ TH~E~ SPECTRA OF CARBQXYHIEMO~~GLOB~~I'~N AND REDUCED'HEMOGLOBIN ARE EQUAL AT 578 NM. THE TOTAL AB'SO'RBANCE AT ANY G I'VEN' WAVELENGTH I S A SUMMATION OF THE' ABSORBANCE OF ALL THREE SPEC'I'ES., SLIiDE~~ 11 BLOO~~D' SAMPL~LES, FOR' S'P'ECTR~Q~PH~OTO'METRI~C ANALYS~I'S~~ WERE PREPARED B'Y' CEN'TR I FUG I NIG THE SAMPLE AT 3Q!00 RP'M FOR' 3G MI'NIUTES, AFTER C'ENTR I FUGAT I'QN, THE PLASMA WAS D I SCARDED. AND R'EPLACED' WITH AN' EQUAL VOLUME OF THE C0-OX'IlMETER DILUTENT,, THE S'AMP'LE' W'AS VO'RTEXED FOR 9GSECOND&T0 ACHIEVE COMPLETE l M I X'I N~G AND' 'MEMO'LYS I S., TH'E' SAMPLE WAS CENTR I' F U!GED' AGA I N AT 3C)~DO' R~PM F~O~R' 30'~ M~I~NUTE~S~~. THE S~~UP~f~RNATANT~ W'AS~ITHEN US~ED~ TO~~ MEASURE THE HiEM0GL0B'I'N'. WE FOUND' THE AVE HEM'OGLOBINI VALU'E. T0~ BE~ 131.5 ~%5 l. ~ ~'iHlS~~ PROCEDURE I'S STRA~I G'~HT~ FORWARD~~ A~ND~~ R'~A~PID~LX' PERFORMED, AN~D~,ALLEV~I~~ATES~ THIE~ P~RO~~B'~LE~M~ PR'~ES~~ENTED~ BY ' THE NUCLEATED REDiBLO~~OD CE~LLS'~,~~ A~~ SAMP'LE SIZE OF THR~E~E~~ M'IL'LIlLITERS OF WHOLE BLOOD IS' SUFFICIENT FOR FIVE DETER'M'INATIONiS',i FURTTHERMORE, THE MEASUR'EMENTS MAY ALSO' BE PERFORMED ON PARTIALLY CL0'TTED BLOOD, WH I CH I S A DE F IlN I TE ADVANTAGE AS TH I S PROBLEM IS QFTEN, ENCOUNTERED WHIEN, WpRK'ING WTTH AVIAN BLOOD., SLIDE IZ' ALL OF THESE TESTS CAN' BE READILY DONE I'N! A SMALL, WELL-EQU I PP'EDiLABOR!ATORY ., B'LOQD I SID'RAWN FROM' THE ALA'R VE I'N, OF THE BIIRD'., UPON' W'ITHDRAW'ING THE NEED'LE FROM THE VEI'N, ONE MUST IMMEDIATELY AP'PLY HEAVY' PRESSURE ON THE VEI'Ni OR' A HEMATOMA WILL RESULT, MAKING IT IMPOSSIBLE TO USE THE VEINI AGAINi FaR' SEVERAL WEEKS.THREE MILLILITERS OF BL00'D IS'SUFFICIENT TO' PERFORM ALL HEMATOLOGIC TESTS, WITH A LITTLE LEFT' OVER' T0 DO

REPEATS OF ANY' QUESTIIONABLE R SULTS .WE SUGGEST THAT' THE BLOOD SMEARS AND HEM'ATOCR'IT TUBES' BE PREPARED F'I'RST. THE' RED BLOOD CELL P I P'ET BE PREPARED NEXT, AND F I'NALLY' THE BLQOD~ PLACED IN VACUTAINERS T0 BE CENTRIFUGED. AS A TIME EFFICIENT Cp'~NSID~~ERAT~ION,~ TH~E~ ERYTHROCYTE AN~D~ HETEROPHIL Cq'~U~NT&C~AN BE PERFORMED WHII LE THE BLOOD I S' BE I N'G' CENTR I FUGED . THE P'IlP'ETS' SHOULD BE A&ITATED ON THE YANKEE PI'PET SHAKER JUST PR'I'QR' T0 CHAR'G I N'G' THE HEMOCYTD'METER. ALL BLOOD SAMPLES SHb'ULD BE M'~I~XED~ WELL AND~ THE F~I R'ST FEW,D~~ROP~~&DJSCARDED~ B~EF~O~~R~E~ EACHI US~E~, WE FEEL THAT' I F ONE I''S GOING TO US'E""THE WH'I1TE CELL COUNT AS ANIINDICATOR OF INFECTION,i THEN REPEATED BLOOD SAMPL I NG, I S NECESSAR'Y' ANDiTHE RESULT I NGIVALUES VA'RY' GREATLY FRD'M THE REPORTED MEAN BEFORE INFECTION'IS SUSPECTED. THANK Y'QU.

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