B r. veL. }. ( 1977 ), 133, 585
OBSERVATIONS ON THE USE OF A SEMI-AUTOMATIC SYSTEM FOR HAEMATOLOGICAL MEASUREMENTS IN BIRDS By I. M. SMITH
AND
S. T.
LI CENCE
Department of Pathology, Royal Veterinary College, University of London, London NW 1 OTU
SUMMA RY
Haematological values in avian blood sampl es were determined by conventional techniques a nd by slight adaptations to one of the semi-automatic, electronic systems which are now widely employed in the examination of mammalian blood . With the major exceptions of the alkaline haematin estimate of haemoglobin concentration a nd the white-cell counts, both procedures gave results that were in reaso nable, practical agreement. I NT ROD UCTI O N
Many haematological parameters in mammals are now measured routinely by partly or completely automatic electronic instruments. No detailed examina tion of the app licabi lity of any of these systems to th e routine examination of avian blood samples appears to have been recorded . One such system was, therefore, assessed in comparison with the o lder, conventional methods recommended (e. g. by Lucas & Jamroz, 196 J) for the examination of avian blood samples. MATERIALS AND METHODS
Birds The blood samples were from 19-day-o ld males (Gallus domesticus) of the Tho rnber 909 Line (New House Hatcheries, Mytholmroyd , Yorks). The chicks were kept in isolation from the day of hatching. Standard rearing me thods were used and food and water were available ad libitum. The diet was nutritionally equivalent to those available commercially. Samples Blood was withdrawn from the right jugular vein using a separate syringe and needle for each of 20 chicks. A smear of each sample was made immediately and airdried . The balance of the sample was mixed continuously-on a rotating device in vials containing di-sodium e thylenediaminetetra-acetate as anticoagulatn (1·5 mg/ I ml of blood ).
586
BRITISH V ETERINARY JO URNAL, 133,6
Erythrocyte (RBC) counts Two erythrocyte counting techniques were used. (I ) 0·01 ml blood was diluted in 2·0 ml of phosphate- buffered saline (pH 7·2 ), mixed thorough ly a nd the red cells counted in a haemocytometer with Neubauer rul ing. Each co unt was performed separately and in duplicate by two observers and the average count recorded . (2) Each sample was diluted to 1/50000 in Isoton (Coulter Electroni cs Ltd , Harpenden, H erts) a nd the number of cells determi ned in a Co unter (Fn Mod el : Coulter Electro ni cs Ltd ). These co unts were also made in dupli cate, and the mean of'each pair was corrected for co incidence. The manufacturer's instructions for sample dilution and instrument operation were followed and the instrument was calibrated with standard blood 4C (Coulter Electronics Ltd ). Preliminary observations with avian red cells showed that good ' plateaux' occurred with th e following settings: attenuation O· 354, aperture 32, threshold 12. Leucocyte and thrombocyte counts Only the cells of the myeloid and lymphoid series are call ed leucocytes (WBC ); the thrombocytes are referred to as TBC. WBC and TBC co unts were determined by two methods. (I ) 0 ·01 ml blood was diluted in 2·0 ml of a staining fluid (Natt & H errick, 1952 ) and th e cells counted in the haemocytom eter. These counts were performed separately and in duplicate by two observers and the means recorded. (2) A total white-cell (WBC plus TBC) count was obtained electronically after lysis of the erythrocytes in a 1/50000 dilution o f the b lood with Zap-oglobin (Coulter Electronics Ltd ). The same instrument settings as for the RBC count were employed and the m ean of' duplicated readings for each sample was taken and corrected for coincidence. Differential WBC co unts and the WBC :TBC ratio were ob tained b y classifying about 400 cells in each smear after staining by May-Grunwald Giemsa (Lucas & Jamroz, 196 I). This WBC :TBC ratio was used to estimate the number of' WBC and TBC from the total white-cell count.
Haemoglobin (Hb) estimations Hb was es timated as cyanmethaemoglobin and as acid and alkaline haematin. Cyanmethaemoglobin was prepared by mixing 0·02 ml blood with 10 ml of Drabkin 's reagent (B.D.H . Ltd , Pool e, Dorset). After centrifugation (2000 rev./min for 15 min ) to remove the red-cell nuclei, the absorbance was read in a colorimeter Wford 625 filter) against a blank of the reagent. The haemoglobin concentratio n was calculated by reference to the absorbance of two standard cyanmethaemoglobi n solutions. These were (i) a cyanmethaemoglobin standard, B.S. 3985 (B.D.H. Ltd ) and (ii ) a standard so lution of avian cyanmethaemoglobin. The latter was obtained by dissolving avian haemoglobin, prepared from avian red cells by a slight modification of the technique of Moss & Thompson (1969 ), in Drabkin 's reagent. The Hb concentration of each blood sample was also determined as cyanmethaemoglobin after adding Zap-oglobin to a 1/500 dilution of the sample and, after centrifugation as above, read directly with a Haemoglobinome ter (Coulter Electronics Ltd ). The acid and alkaline haematin methods for the estimation of haemoglobin concentration were essentially the same (see Table II ) as those described by Bankowski ( 1942) and Assoku , Penhale & Buxton (1970). The spectro-photometric absorbances of the acid haematins were compared with those of two standard acid haematin
HAEMATOLOGICAL MEASUREMENTS IN BIRDS
58 7
solutions, prepared as described by Bankowski (1942), from (i) an eq uine haemin (Sigma Chemicals Ltd , Kingston, Surrey) and (ii ) an avian haemin, prepared fi'om avian blood by the method of Elvehjem (1931 ). In ashed preparations of the equine and avian haemins, their iron content, as determined by th e 2,2 ' -b ipyridyl method (Ramsay, 1957), was 7·51 and 7·49%, respectively. The ca lculati on of each Hb concentration from its acid haematin reading (Bankowski , 1942) was adjusted to take into account the sligh tl y different iron content of the haem ins . The spectrophotometric absorbances of the alkaline haematins were compared with those o f two standard alkaline haematin solutions (Tab le II) prepared from the equ ine a nd avian haemins respectively. Packed cell volume ( PC V) and red-cell indices
rcv was determined by the micro-haematocrit method given by Cohen (196 7) for avian blood samples. Mean corpuscular vo lume (MCV), mean corp uscular haemoglobin (MCH ) and mean corpuscu lar haemoglobin co ncentration (MCHC) were calculated in the standard way (Schalm, 1965). Statistical methods
The values of the following statistics F, D (Studentized range) a nd Student's t, were calculated as detailed by Snedecor & Cochran (J 967). RESULTS
REC counts
As estimated by the haemocytome ter or the Counter the RBC counts were (Tabl e I) virtually identical (P> 0 ·5 ), provided that the electronic counts were made with in a minute or so of preparation of the 1/50 000 dilution . If the electronic counts were delayed, the es timates fell linearly by approximately 13000 cells/min (Fig. I), d esp ite additional mixing of the suspension. According to the manufacturer of the Counter,
TABLE I MEAN VALUES OF HAEMATOLOGICAL PARAMETERS IN BLOOD SA MPLE S FROM 20 CH I CKS AGED 19 DAYS
Statistic
Mean Standard deviation Standard error
t P
RB C x 10 12//
WBC
x 109/ /
C
H
CQ
H
CQ
H
C
H
28 ·60
2·36
2·33
43·5
28·0
69·4
68·6
121·4
123 ·6
1·35
O· 14
0·22
10·0
6· 7
18·2
19· 3
8·0
12·9
0·30
0·03
0·05
2· 7
1·5
5·1
4·3
1·8
2·9
TBCx 109//
MCV (A)
PCV'/o
0·64 >0·5
3·69 0·5
0·89 0·20
• Calculated from the total electronic white-cell counts using WBC: TBC ralios in blood smears. C = Electronic count . H = Haemocytometer count.
588
BRITISH VETERINARY JO UR AL , 133,6
2·4
A
R=2'365 -0'013T
2·2
U
OBSERVATIONS ON THE USE OF A SEMI-AUTOMATIC SYSTEM FOR HAEMATOLOGICAL MEASUREMENTS IN BIRDS By I. M. SMITH
AND
S. T.
LI CENCE
Department of Pathology, Royal Veterinary College, University of London, London NW 1 OTU
SUMMA RY
Haematological values in avian blood sampl es were determined by conventional techniques a nd by slight adaptations to one of the semi-automatic, electronic systems which are now widely employed in the examination of mammalian blood . With the major exceptions of the alkaline haematin estimate of haemoglobin concentration a nd the white-cell counts, both procedures gave results that were in reaso nable, practical agreement. I NT ROD UCTI O N
Many haematological parameters in mammals are now measured routinely by partly or completely automatic electronic instruments. No detailed examina tion of the app licabi lity of any of these systems to th e routine examination of avian blood samples appears to have been recorded . One such system was, therefore, assessed in comparison with the o lder, conventional methods recommended (e. g. by Lucas & Jamroz, 196 J) for the examination of avian blood samples. MATERIALS AND METHODS
Birds The blood samples were from 19-day-o ld males (Gallus domesticus) of the Tho rnber 909 Line (New House Hatcheries, Mytholmroyd , Yorks). The chicks were kept in isolation from the day of hatching. Standard rearing me thods were used and food and water were available ad libitum. The diet was nutritionally equivalent to those available commercially. Samples Blood was withdrawn from the right jugular vein using a separate syringe and needle for each of 20 chicks. A smear of each sample was made immediately and airdried . The balance of the sample was mixed continuously-on a rotating device in vials containing di-sodium e thylenediaminetetra-acetate as anticoagulatn (1·5 mg/ I ml of blood ).
586
BRITISH V ETERINARY JO URNAL, 133,6
Erythrocyte (RBC) counts Two erythrocyte counting techniques were used. (I ) 0·01 ml blood was diluted in 2·0 ml of phosphate- buffered saline (pH 7·2 ), mixed thorough ly a nd the red cells counted in a haemocytometer with Neubauer rul ing. Each co unt was performed separately and in duplicate by two observers and the average count recorded . (2) Each sample was diluted to 1/50000 in Isoton (Coulter Electroni cs Ltd , Harpenden, H erts) a nd the number of cells determi ned in a Co unter (Fn Mod el : Coulter Electro ni cs Ltd ). These co unts were also made in dupli cate, and the mean of'each pair was corrected for co incidence. The manufacturer's instructions for sample dilution and instrument operation were followed and the instrument was calibrated with standard blood 4C (Coulter Electronics Ltd ). Preliminary observations with avian red cells showed that good ' plateaux' occurred with th e following settings: attenuation O· 354, aperture 32, threshold 12. Leucocyte and thrombocyte counts Only the cells of the myeloid and lymphoid series are call ed leucocytes (WBC ); the thrombocytes are referred to as TBC. WBC and TBC co unts were determined by two methods. (I ) 0 ·01 ml blood was diluted in 2·0 ml of a staining fluid (Natt & H errick, 1952 ) and th e cells counted in the haemocytom eter. These counts were performed separately and in duplicate by two observers and the means recorded. (2) A total white-cell (WBC plus TBC) count was obtained electronically after lysis of the erythrocytes in a 1/50000 dilution o f the b lood with Zap-oglobin (Coulter Electronics Ltd ). The same instrument settings as for the RBC count were employed and the m ean of' duplicated readings for each sample was taken and corrected for coincidence. Differential WBC co unts and the WBC :TBC ratio were ob tained b y classifying about 400 cells in each smear after staining by May-Grunwald Giemsa (Lucas & Jamroz, 196 I). This WBC :TBC ratio was used to estimate the number of' WBC and TBC from the total white-cell count.
Haemoglobin (Hb) estimations Hb was es timated as cyanmethaemoglobin and as acid and alkaline haematin. Cyanmethaemoglobin was prepared by mixing 0·02 ml blood with 10 ml of Drabkin 's reagent (B.D.H . Ltd , Pool e, Dorset). After centrifugation (2000 rev./min for 15 min ) to remove the red-cell nuclei, the absorbance was read in a colorimeter Wford 625 filter) against a blank of the reagent. The haemoglobin concentratio n was calculated by reference to the absorbance of two standard cyanmethaemoglobi n solutions. These were (i) a cyanmethaemoglobin standard, B.S. 3985 (B.D.H. Ltd ) and (ii ) a standard so lution of avian cyanmethaemoglobin. The latter was obtained by dissolving avian haemoglobin, prepared from avian red cells by a slight modification of the technique of Moss & Thompson (1969 ), in Drabkin 's reagent. The Hb concentration of each blood sample was also determined as cyanmethaemoglobin after adding Zap-oglobin to a 1/500 dilution of the sample and, after centrifugation as above, read directly with a Haemoglobinome ter (Coulter Electronics Ltd ). The acid and alkaline haematin methods for the estimation of haemoglobin concentration were essentially the same (see Table II ) as those described by Bankowski ( 1942) and Assoku , Penhale & Buxton (1970). The spectro-photometric absorbances of the acid haematins were compared with those of two standard acid haematin
HAEMATOLOGICAL MEASUREMENTS IN BIRDS
58 7
solutions, prepared as described by Bankowski (1942), from (i) an eq uine haemin (Sigma Chemicals Ltd , Kingston, Surrey) and (ii ) an avian haemin, prepared fi'om avian blood by the method of Elvehjem (1931 ). In ashed preparations of the equine and avian haemins, their iron content, as determined by th e 2,2 ' -b ipyridyl method (Ramsay, 1957), was 7·51 and 7·49%, respectively. The ca lculati on of each Hb concentration from its acid haematin reading (Bankowski , 1942) was adjusted to take into account the sligh tl y different iron content of the haem ins . The spectrophotometric absorbances of the alkaline haematins were compared with those o f two standard alkaline haematin solutions (Tab le II) prepared from the equ ine a nd avian haemins respectively. Packed cell volume ( PC V) and red-cell indices
rcv was determined by the micro-haematocrit method given by Cohen (196 7) for avian blood samples. Mean corpuscular vo lume (MCV), mean corp uscular haemoglobin (MCH ) and mean corpuscu lar haemoglobin co ncentration (MCHC) were calculated in the standard way (Schalm, 1965). Statistical methods
The values of the following statistics F, D (Studentized range) a nd Student's t, were calculated as detailed by Snedecor & Cochran (J 967). RESULTS
REC counts
As estimated by the haemocytome ter or the Counter the RBC counts were (Tabl e I) virtually identical (P> 0 ·5 ), provided that the electronic counts were made with in a minute or so of preparation of the 1/50 000 dilution . If the electronic counts were delayed, the es timates fell linearly by approximately 13000 cells/min (Fig. I), d esp ite additional mixing of the suspension. According to the manufacturer of the Counter,
TABLE I MEAN VALUES OF HAEMATOLOGICAL PARAMETERS IN BLOOD SA MPLE S FROM 20 CH I CKS AGED 19 DAYS
Statistic
Mean Standard deviation Standard error
t P
RB C x 10 12//
WBC
x 109/ /
C
H
CQ
H
CQ
H
C
H
28 ·60
2·36
2·33
43·5
28·0
69·4
68·6
121·4
123 ·6
1·35
O· 14
0·22
10·0
6· 7
18·2
19· 3
8·0
12·9
0·30
0·03
0·05
2· 7
1·5
5·1
4·3
1·8
2·9
TBCx 109//
MCV (A)
PCV'/o
0·64 >0·5
3·69 0·5
0·89 0·20
• Calculated from the total electronic white-cell counts using WBC: TBC ralios in blood smears. C = Electronic count . H = Haemocytometer count.
588
BRITISH VETERINARY JO UR AL , 133,6
2·4
A
R=2'365 -0'013T
2·2
U
