- Email: [email protected]
Chicken Blood Volume: The Hematocrit and Comparison of I131 and Evans Blue Methods
26
W. MORGAN AND C. W. CARLSON
in nutrition trials. Variables other than diet could be similarly evaluated. Finally, it should be pointed out that one might choose to omit K from the formula for PEL An advantage for considering the PEP = (EW) P - f - F formula is that there would be no restriction on maximum body weight. Then, the efficiency value would not heavily penalize hens which weighed more than 1800 grams (4.0 pounds). However, if PEI is to be used in selection programs designed to improve the efficiency of egg production, it is recommended that the K value be included as a part of the formula.
who are efficiency.
concerned
with
management
REFERENCES
SUMMARY A new method for evaluating performance of egg-production stocks has been proposed. This uniform measurement, which lends itself to international use, employs the metric system throughout. Determination of PEI (Production Efficiency Index) values provides a tool which may be used by breeders, by experimenters, or by those
Chicken Blood Volume: The Hematocrit and Comparison of I131 and Evans Blue Methods ANTHONY
W.
KOTULA
Market Quality Research Division, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland 20705 AND NORMAN
V.
HELBACKA
Department of Poultry Science, University of Maryland, College Park, Maryland 20740 (Received for publication May 8, 1967) INTRODUCTION
B
LOOD volumes of mammals have been 1 studied much more extensively than blood volumes of birds, therefore the procedures used for mammalian blood volume Scientific Article No. A1258. Contribution No. 3779 of the Maryland Agricultural Experiment Station (Department of Poultry Science).
determinations can be expected to serve as a basis for improvement in avian blood volume determinations. In poultry research, determination of trapped plasma in the hematocrit, the effect of blood removal during sampling on the hematocrit and absorption curves, and the use of radioactive tracers for blood volume studies have received lit-
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 5, 2015
Agricultural Research Service, 1966. 1966 Report of egg production tests in the United States and Canada. ARS 44-79-7. Card, L. E., and M. C. Nesheim, 1966. Poultry Production. Lea and Febiger, Philadelphia, Pa. 227-229. Council of American Official Poultry Tests, 1958. Production records for the period 1957-58. Report number 20. Ewing, W. R., 1963. Poultry Nutrition. The Ray Ewing Company, publisher, Pasadena, California: 166. Faulkner, J., 1965. Selling eggs by pound urged. The Poultryman, April 30, issue: 8. Frost, D. V., 1965. Logical steps to metric conversion. Poultry Sci. 44: 1227-1236. Kleiber, M., 1965. The unit for measuring food energy. World Rev. An. Prod. 2: 5-11. Morgan, W., and C. W. Carlson, 1965. Selective efficiency. Poultry Sci. 44: 1401. Quisenberry, J. H., 1965. Feed to food conversion ratios; a new method of evaluating production efficiencies. Poultry Sci. 45: 1408. Wofle, D., 1965. Adoption of the metric system. Science, 149: 139.
BLOOD
VOLUME
27
of Evans Blue Dye T-1824 and radio-iodinated (I 131 ) serum albumin (human) from the circulatory system of chickens. Feed was withheld from the birds for 24 hours and water for 4 hours prior to injection to ensure that the birds were in a post-absorptive state to preclude any error due to lipemia (Gregersen, 1944). Ten blood samples were obtained from each of 3 birds on 3 different days according to the EXPERIMENTAL METHODS following procedure: Each bird was Blood Hematocrit and Trapped Plasma Deweighed to the nearest 5 g., fastened to a termination. Radio-iodinated (I 1S1 ) serum restraining board and the feathers were realbumin (human) was injected into the bra- moved from the ventral side of both wings chial vein of 8-week-old male Arbor Acre to allow the brachial veins to be clearly visWhite Rock chickens and allowed to mix ible. The proximal end of the left wing vein with circulating blood for 10 min. before a was occluded, the needle inserted and 1 ml. 1 ml. sample was removed from the brachial of blood was withdrawn after the pressure vein of the opposite wing. Blood samples of had been released for about \ min. Syringes approximately 15 [A1. were drawn into capil- used for taking blood samples were moislary tubes. The tubes were centrifuged for tened with a few drops of heparin solution 1, 2,3, 4, 5, or 6 min. at 6.7 X 103g. The (1 ml. = 1,000 U.S.P. units) to prevent cell and plasma heights in each tube were clotting and dried to preclude errors due to determined by the method of Guest and dilution. A portion (approximately 15 jxl.) Siler (1934). Each capillary tube was of each blood sample was used for a microetched and broken £ mm. above and below hematocrit determination. Another 15 jjil the boundary between the packed blood portion from each sample was drawn accucells and the plasma. The 1 mm. portion of rately from a micropipette into a capillary to capillary containing the boundary between serve as a reference blank for a micro I 131 cells and plasma was discarded. Attempts to determination of blood-plasma volume. The separate the capillary at the precise bound- remainder of the sample was centrifuged for ary might have caused inordinate error. Ra- 30 min. at 1.8 X 103g. One-half ml. of the dioactivity from the packed-cell and plasma supernatant plasma was diluted with 9.5 ml. portions was monitored separately in a scin- of 0.85 percent saline to serve as a refertillation counter (1^-inch well). Counts ence for 100 percent transmittance in a colwere adjusted mathematically to the origi- orimeter at a wavelength of 620 mji. Zero nal hematocrit values to take into consider- and 100 percent transmittance were reset ation the 1 mm. portion that was discarded. with the undyed plasma from each bird. De The percentage of packed cells that was ac- Shazer and Weiss (1963) showed that tually plasma was (count per mm. of pooled blanks led to statistically significant packed cells X 100)/(count per mm. of errors in blood-volume determinations. free plasma). One-ml. Evans Blue-I131 solution* was
tie attention or have been neglected completely. The objectives of the present work were to adapt the radioactive tracer (I 131 ) technique of mammalian blood-volume determination for use with chickens and to compare the Evans Blue T-1824 method and the radioactive-tracer technique as indicators of chicken blood volume.
Rate of Evans Blue Dye
and
I1S1. Nine, 8-week-old male Arbor Acre White Rock chickens (1.3-2.5 kg.) were used to determine the rate of disappearance
* The 1 ml. Evans Blue T-1824 dye-I"1 solution for injection consisted of about 20 milligrams of the dye and about 3 microcuries of radio iodinated (I131) serum albumin (human) with sufficient 0.85 percent saline solution to
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 5, 2015
Disappearance
28
A. W. KOTULA AND N. V. HELBACKA
tance determinations (dye method), were used for blood volume determination in the macro I 131 method. Radioactivity of the samples was determined by counting for S min. with the aid of a scintillation well counter. Since the well counter had only a 4 cm. crystal, adjustments were necessary for loss of counts due to geometry. The average of 10 determinations showed that due to geometry, 45.17 percent of the emissions of a sample were counted. The macro procedure for determining blood volume with the tracer technique is outlined below:
Instrumentation and Calculations. A standard curve was prepared to relate transmittance at 620 mfx. to Evans Blue dye concentration in saline. The best fitting straight line was determined by regression analysis to be log tf = 2.0231-0.0100X (Y = transmittance in percent, X = [Ag. Evans dye per ml.). The correlation coefficient r, between the predicted and the actual values was 0.9995. The degree of dilution within the vascular system was determined as follows:
1. The count per minute of tracer (—background) injected into the vascular system was determined. 2. The count per minute (—background) present in 1 ml. of plasma was determined. 3. Count of step 2 above was adjusted for count loss due to geometry. Adjusted count = (actual count)/(45.17%). 4. Plasma volume (ml.) of bird = (activity injected)/(activity of 1 ml. of plasma). 5. Blood volume (ml.) = (plasma volume) /(100-hematocrit + trappel plasma).
1. The number of fxg. of Evans Blue dye injected was calculated. 2. The number of pig. of dye present in 1 ml. of plasma after dilution was determined. 3. Plasma volume (ml.) of bird — ([Jig. dye injected)/(dye in 1 ml. of plasma). 4. Blood volume (ml.) = (plasma volume) /(100-hematocrit + trapped plasma). 5. Total blood volume (ml.) = blood volume + ml. of blood removed prior to dye injection. The 10 ml. samples, used for transmitmake a volume of 1 ml. Concentrations of the dye and the I131 in the Evans-I131 solution were determined from transmittance and radioactivity measurements.
Calculations for determining blood volume by the micro I131 method differed from the macro I 131 method in only two ways. First, in the micro method the capillary tubes containing the samples were cut into pieces less than 1 cm. long so that no correction for geometry was necessary. Second, to compensate for the 15 til. sample of whole blood used in the determination, the following equation was used for the micro method. Count per min. per ml. of plasma = (sample count per min.) / (0.015-0.015 X hematocrit + 0.015 X trapped plasma). Once the count per min. per ml. of plasma was determined, steps 1, 4, and 5 of the macro I 131 method were followed.
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 5, 2015
introduced into the bird's circulatory system through the brachial vein of the left wing. One ml. samples of blood were withdrawn from the right wing at 1, 2, 5, 10, 20, 40, SO, 60, 90, and 120 min. after injection. From each of these samples, the hematocrit, the IS [xl. capillary sample for micro I131 analysis and the ^ ml. plasma in 9.S ml. saline were prepared in the same manner as the blood samples before the Evans -I 131 was injected. Transmittances of the plasma-saline solutions were determined. The same solutions were subsequently used for counting of radioactivity.
29
BLOOD VOLUME RESULTS AND DISCUSSION
0
2
3
4
5
6
CENTRIFUGATION TIME (min.) FIG. 2. Percentage of the hematocrit packed cell volume consisting of plasma that remained after centrifugation.
trapped-plasma volumes that tended to decrease with time but these differences were not significant. The mean percentages of the packed blood cell volumes that were actually plasma are shown in Figure 2 for centrifugation times of 1 through 6 min. Each of these values was based on 4 observations from one bird. An average of 3.20 ± 1.15 percent of the packed cells was really plasma. This 3.20 percent value, from 5 min. of centrifugation, was used for calculating whole blood volume from diluted plasma. Shohl and Hunter (1941), working with human blood, reported true cell volume as 4.5 percent less than the observed volume, and attributed the difference to trapped plasma. Chapin and Ross (1942) found trapped plasma to be 8.5 percent of the packed cell volume of human blood.
20 40 60 80 100 120 Disappearance Rate of Evans Blue Dye and TIME (min.)
15 6
7
9
10
II
CUMULATIVE AMOUNT OF BLOOD REMOVED (ml.l
FIG. 1. Hematocrit change when 1 ml.-blood samples were removed repeatedly at specific times.
I"1. The effect of the rate of disappearance of Evans Blue T-1824 and I 131 from the circulatory system of chickens on amount of blood calculated is presented in Figure 3. When the data were combined
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 5, 2015
Blood Hematocrit and Trapped Plasma. The mean hematocrit values of 12 chickens from which 1 ml. samples of blood had been removed at 0, 1, 2, 5, 10, 20, 40, SO, 60, 90, and 120 min. are given in Figure 1. The initial average hematocrit was 31.67 percent, whereas after 11 ml. of blood had been withdrawn the hematocrit was reduced to 25.83 percent. The findings confirm the work of Gibbs (1929) and Aramaki and Weiss (1961) who showed that drawing multiple blood samples from chickens greatly decreased hematocrit values. Because blood-volume determination by dilution techniques depends on hematocrit values, the hematocrit must be determined from the same blood sample that is used to ascertain the degree of dilution. Statistical treatment by analysis of variance of the plasma trapped in the packed cells of the hematocrit showed that centrifugation for 1 or 2 min. resulted in values that were significantly different from those obtained using the recommended centrifugation time of 5 min. Centrifugation of 1 or 2 min. were therefore considered inadequate for blood cell separation. Centrifugation for 3, 4, 5, or 6 min. resulted in
30
A. W. KOTUA AND N. V. HELBACKA
MACRO I ?»7.I513«0.0830X-0.0002506X2
<
i
'0
•
•
L
20 40 60 80 100 SAMPLING TME AFTER INJECTION (min)
FIG. 3. The effect of the disappearance of Evans Blue T-1824 and I131 from the circulatory system of chickens on the determination of blood volume expressed as a percentage of live weight.
into an overall analysis, the means for the three methods were all significantly different from each other. Separate analyses resulted in standard deviations of 10.03, 2.31 and 1.32 for the Evans Blue, Micro I131, and Macro I 131 methods respectively, thus the macro method appears to give the most precise estimate of blood volume. When the data were subjected to curvilinear regressions, the quadratic terms were found to be significant for both the macro and micro methods but not for the Evans Blue method. The best fitting equations for the three methods in which the effect of the rate of disappearance of Evans Blue T-1824 and I 131 is expressed as the amount of blood C?) and is regressed against min. of time after injection (X) are: Evans Blue Micro I 131
Y = 11.65 + 0.2388X r = .96** Y = 9.6891 4- 0.1096X - 0.0003169X2 R = .99** Macro I 131 Y = 7.1513 + 0.0830X - 0.0002506X2 R = .99** ** Significant at the 1% level.
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 5, 2015
z o z
These curves are the best fitting curves for the data and therefore do not show, as can be seen when actual values are plotted, that the blood volumes determined from the 1 min. samples were higher than those from the 2 min. samplings in the Evans Blue dye and micro I 131 determinations. Evidently, blood samples obtained 1 min. after injection reflected incomplete mixing of the dye tracer solution within the vascular system. At the 2 min. sampling time, either mixing was completed or almost completed. The 5 min. sample and all subsequent samples reflected increasing losses of the dye-tracer solution from the blood stream to the capillaries, tissues, and the excretory system. Fecal excretions contained dye and tracer as early as 10 min. after injection of the original solution. Optimum sampling time would thus be between 2 and 5 min. Miller (1947), working with dogs, indicated that mixing of dye in the circulating plasma was completed in 4 to 6 min. Pino et al. (1951), in studies of chicken blood volume, sampled at 2 min. to preclude errors due to the rapid disappearance of dye-diptheria antitoxoid from the circulatory system. Their blood-volume values were slightly lower than the 10-min. values of Newell and Shaffner (1950). The general slope of the Evans Blue dye curve in Figure 3 is much greater than that of either of the I 131 methods, indicating a greater disappearance rate. The radioactive tracer was not lost as readily as the watersoluble dye because it was already tagged to the albumin and contained less than 2 percent loosely bound iodine. Clark and Woodley (1959), working with dogs, reported similarly larger volumes when the dye-dilution technique was compared to the Cr51 tagged-cell method. Remington and Baker (1961) also reported higher plasma volumes with the Evans Blue method. Krieger et al. (1948) also compared dye and tracer techniques and showed that the iodinated
BLOOD VOLUME
protein method for determining blood volume in dogs was a simple, direct method which permitted accurate determinations of plasma volumes during a much longer period than the Evans Blue method.
ACKNOWLEDGMENTS Appreciation is expressed to Mr. E. James Koch, Agricultural Research Service, U. S. Department of Agriculture for advice and assistance with the statistical evaluation of the date. REFERENCES Anonymous, 1963. Radio-iodinated (I131) serum albumin (human) for simple accurate determinations of blood volume, brain tumor localization, circulation studies, cardiac output. Circular, 13-034/R2-15-Rev. Abbott Laboratories, North Chicago, Illinois. Aramaki, T., and H. S. Weiss, 1961. Predictability of the changes in hematocrit which follow
repeated withdrawal of blood. Proc. Soc. Exp. Biol. Med. 108: 242-244. Chapin, M. A., and J. F. Ross, 1942. The determination of the true volume by dye dilution, by protein dilution, and with radioactive iron. The error of the centrifuge hematocrit. Amer. J. Physiol. 137: 447-455. Clark, C. H., and C. H. Woodley, 19S9. A comparison of blood volumes as measured by rose bengal, T-1824 (Evans Blue), radiochromiumtagged erythrocytes, and a combination of the latter two. Amer. J. Vet. Res. 20: 1067-1068. DeShazer, J. A., and H. S. Weiss, 1963. A comparison among variations in dye dilution procedure for determining blood volume. Poultry Sci. 42: 778-780. Gibbs, O. S., 1929. The effects of drugs on the secretion of uric acid in the fowl. J. Pharm. Exp. Thera. 35: 49-62. Gregersen, M. I., 1944. A practical method for the determination of blood volume with the dye T1824. J. Lab. Clin. Med. 29: 1266-1286. Guest, G. M., and V. E. Siler, 1934. The centrifuge method for the determination of the volume of cells in blood. J. Lab. Clin. Med. 19: 757-768. Krieger, H., J. P. Storaasli, H. L. Friedell and W. D. Holden, 1948. A comparative study of blood volume in dogs. Proc. Soc. Exp. Biol. Med. 68: Sll-515. Miller, A. T., 1947. A re-evaluation of the T-1824 mixing curve. Amer. J. Physiol. 151: 234-238. Newell, G. W., and C. S. Shaffner, 1950. Blood volume determinations in chickens. Poultry Sci. 29: 78-87. Pino, J. A., H. S. Weiss and P. D. Sturkie, 1951. Blood volume determinations in the fowl using diphtheria antitoxoid. Proc. 9th World's Poultry Congress. 3 : 102-108. Remington, J. W. and C. H. Baker, 1961. Evaluation of blood volume measurement techniques. Circulation Research, 9: 60-68. Shohl, A. T. and T. H. Hunter, 1941. The measurement of cell volume of the blood by the Evans Blue dye method. J. Lab Clin. Med. 26: 1829-1837.
NEWS AND NOTES ERRATUM There is an error in Table 5 on page 1062 of the September issue in the paper entitled "The effect of photoperiodism and rearing period feed restriction on the performance of five Leghorn strains," pages 1056-1072. In test 4, light treat-
ment (6-6-18) hen day egg production should read 262 not 272. RALSTON PURINA FELLOWSHIPS Robert W. Boggs, Davis, California, has been awarded a Ralston Purina Research Fellowship
(continued on page 112)
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 5, 2015
SUMMARY The radioactive tracer (I131) technique for determination of mammalian blood volume was adapted for chickens. The effects of blood removal during sampling and trapped plasma among packed cells on the determined hematocrit were investigated. Both macro and micro measurements of tracer dilution as well as dilution of Evans Blue T-1824 in the circulatory system were studied. Both radioactive tracer techniques estimated the blood volume of chickens more precisely than the Evans Blue T-1824 method. The macro method proved superior to the micro method as indicated by a smaller standard deviation.
31
W. MORGAN AND C. W. CARLSON
in nutrition trials. Variables other than diet could be similarly evaluated. Finally, it should be pointed out that one might choose to omit K from the formula for PEL An advantage for considering the PEP = (EW) P - f - F formula is that there would be no restriction on maximum body weight. Then, the efficiency value would not heavily penalize hens which weighed more than 1800 grams (4.0 pounds). However, if PEI is to be used in selection programs designed to improve the efficiency of egg production, it is recommended that the K value be included as a part of the formula.
who are efficiency.
concerned
with
management
REFERENCES
SUMMARY A new method for evaluating performance of egg-production stocks has been proposed. This uniform measurement, which lends itself to international use, employs the metric system throughout. Determination of PEI (Production Efficiency Index) values provides a tool which may be used by breeders, by experimenters, or by those
Chicken Blood Volume: The Hematocrit and Comparison of I131 and Evans Blue Methods ANTHONY
W.
KOTULA
Market Quality Research Division, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland 20705 AND NORMAN
V.
HELBACKA
Department of Poultry Science, University of Maryland, College Park, Maryland 20740 (Received for publication May 8, 1967) INTRODUCTION
B
LOOD volumes of mammals have been 1 studied much more extensively than blood volumes of birds, therefore the procedures used for mammalian blood volume Scientific Article No. A1258. Contribution No. 3779 of the Maryland Agricultural Experiment Station (Department of Poultry Science).
determinations can be expected to serve as a basis for improvement in avian blood volume determinations. In poultry research, determination of trapped plasma in the hematocrit, the effect of blood removal during sampling on the hematocrit and absorption curves, and the use of radioactive tracers for blood volume studies have received lit-
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 5, 2015
Agricultural Research Service, 1966. 1966 Report of egg production tests in the United States and Canada. ARS 44-79-7. Card, L. E., and M. C. Nesheim, 1966. Poultry Production. Lea and Febiger, Philadelphia, Pa. 227-229. Council of American Official Poultry Tests, 1958. Production records for the period 1957-58. Report number 20. Ewing, W. R., 1963. Poultry Nutrition. The Ray Ewing Company, publisher, Pasadena, California: 166. Faulkner, J., 1965. Selling eggs by pound urged. The Poultryman, April 30, issue: 8. Frost, D. V., 1965. Logical steps to metric conversion. Poultry Sci. 44: 1227-1236. Kleiber, M., 1965. The unit for measuring food energy. World Rev. An. Prod. 2: 5-11. Morgan, W., and C. W. Carlson, 1965. Selective efficiency. Poultry Sci. 44: 1401. Quisenberry, J. H., 1965. Feed to food conversion ratios; a new method of evaluating production efficiencies. Poultry Sci. 45: 1408. Wofle, D., 1965. Adoption of the metric system. Science, 149: 139.
BLOOD
VOLUME
27
of Evans Blue Dye T-1824 and radio-iodinated (I 131 ) serum albumin (human) from the circulatory system of chickens. Feed was withheld from the birds for 24 hours and water for 4 hours prior to injection to ensure that the birds were in a post-absorptive state to preclude any error due to lipemia (Gregersen, 1944). Ten blood samples were obtained from each of 3 birds on 3 different days according to the EXPERIMENTAL METHODS following procedure: Each bird was Blood Hematocrit and Trapped Plasma Deweighed to the nearest 5 g., fastened to a termination. Radio-iodinated (I 1S1 ) serum restraining board and the feathers were realbumin (human) was injected into the bra- moved from the ventral side of both wings chial vein of 8-week-old male Arbor Acre to allow the brachial veins to be clearly visWhite Rock chickens and allowed to mix ible. The proximal end of the left wing vein with circulating blood for 10 min. before a was occluded, the needle inserted and 1 ml. 1 ml. sample was removed from the brachial of blood was withdrawn after the pressure vein of the opposite wing. Blood samples of had been released for about \ min. Syringes approximately 15 [A1. were drawn into capil- used for taking blood samples were moislary tubes. The tubes were centrifuged for tened with a few drops of heparin solution 1, 2,3, 4, 5, or 6 min. at 6.7 X 103g. The (1 ml. = 1,000 U.S.P. units) to prevent cell and plasma heights in each tube were clotting and dried to preclude errors due to determined by the method of Guest and dilution. A portion (approximately 15 jxl.) Siler (1934). Each capillary tube was of each blood sample was used for a microetched and broken £ mm. above and below hematocrit determination. Another 15 jjil the boundary between the packed blood portion from each sample was drawn accucells and the plasma. The 1 mm. portion of rately from a micropipette into a capillary to capillary containing the boundary between serve as a reference blank for a micro I 131 cells and plasma was discarded. Attempts to determination of blood-plasma volume. The separate the capillary at the precise bound- remainder of the sample was centrifuged for ary might have caused inordinate error. Ra- 30 min. at 1.8 X 103g. One-half ml. of the dioactivity from the packed-cell and plasma supernatant plasma was diluted with 9.5 ml. portions was monitored separately in a scin- of 0.85 percent saline to serve as a refertillation counter (1^-inch well). Counts ence for 100 percent transmittance in a colwere adjusted mathematically to the origi- orimeter at a wavelength of 620 mji. Zero nal hematocrit values to take into consider- and 100 percent transmittance were reset ation the 1 mm. portion that was discarded. with the undyed plasma from each bird. De The percentage of packed cells that was ac- Shazer and Weiss (1963) showed that tually plasma was (count per mm. of pooled blanks led to statistically significant packed cells X 100)/(count per mm. of errors in blood-volume determinations. free plasma). One-ml. Evans Blue-I131 solution* was
tie attention or have been neglected completely. The objectives of the present work were to adapt the radioactive tracer (I 131 ) technique of mammalian blood-volume determination for use with chickens and to compare the Evans Blue T-1824 method and the radioactive-tracer technique as indicators of chicken blood volume.
Rate of Evans Blue Dye
and
I1S1. Nine, 8-week-old male Arbor Acre White Rock chickens (1.3-2.5 kg.) were used to determine the rate of disappearance
* The 1 ml. Evans Blue T-1824 dye-I"1 solution for injection consisted of about 20 milligrams of the dye and about 3 microcuries of radio iodinated (I131) serum albumin (human) with sufficient 0.85 percent saline solution to
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 5, 2015
Disappearance
28
A. W. KOTULA AND N. V. HELBACKA
tance determinations (dye method), were used for blood volume determination in the macro I 131 method. Radioactivity of the samples was determined by counting for S min. with the aid of a scintillation well counter. Since the well counter had only a 4 cm. crystal, adjustments were necessary for loss of counts due to geometry. The average of 10 determinations showed that due to geometry, 45.17 percent of the emissions of a sample were counted. The macro procedure for determining blood volume with the tracer technique is outlined below:
Instrumentation and Calculations. A standard curve was prepared to relate transmittance at 620 mfx. to Evans Blue dye concentration in saline. The best fitting straight line was determined by regression analysis to be log tf = 2.0231-0.0100X (Y = transmittance in percent, X = [Ag. Evans dye per ml.). The correlation coefficient r, between the predicted and the actual values was 0.9995. The degree of dilution within the vascular system was determined as follows:
1. The count per minute of tracer (—background) injected into the vascular system was determined. 2. The count per minute (—background) present in 1 ml. of plasma was determined. 3. Count of step 2 above was adjusted for count loss due to geometry. Adjusted count = (actual count)/(45.17%). 4. Plasma volume (ml.) of bird = (activity injected)/(activity of 1 ml. of plasma). 5. Blood volume (ml.) = (plasma volume) /(100-hematocrit + trappel plasma).
1. The number of fxg. of Evans Blue dye injected was calculated. 2. The number of pig. of dye present in 1 ml. of plasma after dilution was determined. 3. Plasma volume (ml.) of bird — ([Jig. dye injected)/(dye in 1 ml. of plasma). 4. Blood volume (ml.) = (plasma volume) /(100-hematocrit + trapped plasma). 5. Total blood volume (ml.) = blood volume + ml. of blood removed prior to dye injection. The 10 ml. samples, used for transmitmake a volume of 1 ml. Concentrations of the dye and the I131 in the Evans-I131 solution were determined from transmittance and radioactivity measurements.
Calculations for determining blood volume by the micro I131 method differed from the macro I 131 method in only two ways. First, in the micro method the capillary tubes containing the samples were cut into pieces less than 1 cm. long so that no correction for geometry was necessary. Second, to compensate for the 15 til. sample of whole blood used in the determination, the following equation was used for the micro method. Count per min. per ml. of plasma = (sample count per min.) / (0.015-0.015 X hematocrit + 0.015 X trapped plasma). Once the count per min. per ml. of plasma was determined, steps 1, 4, and 5 of the macro I 131 method were followed.
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 5, 2015
introduced into the bird's circulatory system through the brachial vein of the left wing. One ml. samples of blood were withdrawn from the right wing at 1, 2, 5, 10, 20, 40, SO, 60, 90, and 120 min. after injection. From each of these samples, the hematocrit, the IS [xl. capillary sample for micro I131 analysis and the ^ ml. plasma in 9.S ml. saline were prepared in the same manner as the blood samples before the Evans -I 131 was injected. Transmittances of the plasma-saline solutions were determined. The same solutions were subsequently used for counting of radioactivity.
29
BLOOD VOLUME RESULTS AND DISCUSSION
0
2
3
4
5
6
CENTRIFUGATION TIME (min.) FIG. 2. Percentage of the hematocrit packed cell volume consisting of plasma that remained after centrifugation.
trapped-plasma volumes that tended to decrease with time but these differences were not significant. The mean percentages of the packed blood cell volumes that were actually plasma are shown in Figure 2 for centrifugation times of 1 through 6 min. Each of these values was based on 4 observations from one bird. An average of 3.20 ± 1.15 percent of the packed cells was really plasma. This 3.20 percent value, from 5 min. of centrifugation, was used for calculating whole blood volume from diluted plasma. Shohl and Hunter (1941), working with human blood, reported true cell volume as 4.5 percent less than the observed volume, and attributed the difference to trapped plasma. Chapin and Ross (1942) found trapped plasma to be 8.5 percent of the packed cell volume of human blood.
20 40 60 80 100 120 Disappearance Rate of Evans Blue Dye and TIME (min.)
15 6
7
9
10
II
CUMULATIVE AMOUNT OF BLOOD REMOVED (ml.l
FIG. 1. Hematocrit change when 1 ml.-blood samples were removed repeatedly at specific times.
I"1. The effect of the rate of disappearance of Evans Blue T-1824 and I 131 from the circulatory system of chickens on amount of blood calculated is presented in Figure 3. When the data were combined
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 5, 2015
Blood Hematocrit and Trapped Plasma. The mean hematocrit values of 12 chickens from which 1 ml. samples of blood had been removed at 0, 1, 2, 5, 10, 20, 40, SO, 60, 90, and 120 min. are given in Figure 1. The initial average hematocrit was 31.67 percent, whereas after 11 ml. of blood had been withdrawn the hematocrit was reduced to 25.83 percent. The findings confirm the work of Gibbs (1929) and Aramaki and Weiss (1961) who showed that drawing multiple blood samples from chickens greatly decreased hematocrit values. Because blood-volume determination by dilution techniques depends on hematocrit values, the hematocrit must be determined from the same blood sample that is used to ascertain the degree of dilution. Statistical treatment by analysis of variance of the plasma trapped in the packed cells of the hematocrit showed that centrifugation for 1 or 2 min. resulted in values that were significantly different from those obtained using the recommended centrifugation time of 5 min. Centrifugation of 1 or 2 min. were therefore considered inadequate for blood cell separation. Centrifugation for 3, 4, 5, or 6 min. resulted in
30
A. W. KOTUA AND N. V. HELBACKA
MACRO I ?»7.I513«0.0830X-0.0002506X2
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20 40 60 80 100 SAMPLING TME AFTER INJECTION (min)
FIG. 3. The effect of the disappearance of Evans Blue T-1824 and I131 from the circulatory system of chickens on the determination of blood volume expressed as a percentage of live weight.
into an overall analysis, the means for the three methods were all significantly different from each other. Separate analyses resulted in standard deviations of 10.03, 2.31 and 1.32 for the Evans Blue, Micro I131, and Macro I 131 methods respectively, thus the macro method appears to give the most precise estimate of blood volume. When the data were subjected to curvilinear regressions, the quadratic terms were found to be significant for both the macro and micro methods but not for the Evans Blue method. The best fitting equations for the three methods in which the effect of the rate of disappearance of Evans Blue T-1824 and I 131 is expressed as the amount of blood C?) and is regressed against min. of time after injection (X) are: Evans Blue Micro I 131
Y = 11.65 + 0.2388X r = .96** Y = 9.6891 4- 0.1096X - 0.0003169X2 R = .99** Macro I 131 Y = 7.1513 + 0.0830X - 0.0002506X2 R = .99** ** Significant at the 1% level.
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These curves are the best fitting curves for the data and therefore do not show, as can be seen when actual values are plotted, that the blood volumes determined from the 1 min. samples were higher than those from the 2 min. samplings in the Evans Blue dye and micro I 131 determinations. Evidently, blood samples obtained 1 min. after injection reflected incomplete mixing of the dye tracer solution within the vascular system. At the 2 min. sampling time, either mixing was completed or almost completed. The 5 min. sample and all subsequent samples reflected increasing losses of the dye-tracer solution from the blood stream to the capillaries, tissues, and the excretory system. Fecal excretions contained dye and tracer as early as 10 min. after injection of the original solution. Optimum sampling time would thus be between 2 and 5 min. Miller (1947), working with dogs, indicated that mixing of dye in the circulating plasma was completed in 4 to 6 min. Pino et al. (1951), in studies of chicken blood volume, sampled at 2 min. to preclude errors due to the rapid disappearance of dye-diptheria antitoxoid from the circulatory system. Their blood-volume values were slightly lower than the 10-min. values of Newell and Shaffner (1950). The general slope of the Evans Blue dye curve in Figure 3 is much greater than that of either of the I 131 methods, indicating a greater disappearance rate. The radioactive tracer was not lost as readily as the watersoluble dye because it was already tagged to the albumin and contained less than 2 percent loosely bound iodine. Clark and Woodley (1959), working with dogs, reported similarly larger volumes when the dye-dilution technique was compared to the Cr51 tagged-cell method. Remington and Baker (1961) also reported higher plasma volumes with the Evans Blue method. Krieger et al. (1948) also compared dye and tracer techniques and showed that the iodinated
BLOOD VOLUME
protein method for determining blood volume in dogs was a simple, direct method which permitted accurate determinations of plasma volumes during a much longer period than the Evans Blue method.
ACKNOWLEDGMENTS Appreciation is expressed to Mr. E. James Koch, Agricultural Research Service, U. S. Department of Agriculture for advice and assistance with the statistical evaluation of the date. REFERENCES Anonymous, 1963. Radio-iodinated (I131) serum albumin (human) for simple accurate determinations of blood volume, brain tumor localization, circulation studies, cardiac output. Circular, 13-034/R2-15-Rev. Abbott Laboratories, North Chicago, Illinois. Aramaki, T., and H. S. Weiss, 1961. Predictability of the changes in hematocrit which follow
repeated withdrawal of blood. Proc. Soc. Exp. Biol. Med. 108: 242-244. Chapin, M. A., and J. F. Ross, 1942. The determination of the true volume by dye dilution, by protein dilution, and with radioactive iron. The error of the centrifuge hematocrit. Amer. J. Physiol. 137: 447-455. Clark, C. H., and C. H. Woodley, 19S9. A comparison of blood volumes as measured by rose bengal, T-1824 (Evans Blue), radiochromiumtagged erythrocytes, and a combination of the latter two. Amer. J. Vet. Res. 20: 1067-1068. DeShazer, J. A., and H. S. Weiss, 1963. A comparison among variations in dye dilution procedure for determining blood volume. Poultry Sci. 42: 778-780. Gibbs, O. S., 1929. The effects of drugs on the secretion of uric acid in the fowl. J. Pharm. Exp. Thera. 35: 49-62. Gregersen, M. I., 1944. A practical method for the determination of blood volume with the dye T1824. J. Lab. Clin. Med. 29: 1266-1286. Guest, G. M., and V. E. Siler, 1934. The centrifuge method for the determination of the volume of cells in blood. J. Lab. Clin. Med. 19: 757-768. Krieger, H., J. P. Storaasli, H. L. Friedell and W. D. Holden, 1948. A comparative study of blood volume in dogs. Proc. Soc. Exp. Biol. Med. 68: Sll-515. Miller, A. T., 1947. A re-evaluation of the T-1824 mixing curve. Amer. J. Physiol. 151: 234-238. Newell, G. W., and C. S. Shaffner, 1950. Blood volume determinations in chickens. Poultry Sci. 29: 78-87. Pino, J. A., H. S. Weiss and P. D. Sturkie, 1951. Blood volume determinations in the fowl using diphtheria antitoxoid. Proc. 9th World's Poultry Congress. 3 : 102-108. Remington, J. W. and C. H. Baker, 1961. Evaluation of blood volume measurement techniques. Circulation Research, 9: 60-68. Shohl, A. T. and T. H. Hunter, 1941. The measurement of cell volume of the blood by the Evans Blue dye method. J. Lab Clin. Med. 26: 1829-1837.
NEWS AND NOTES ERRATUM There is an error in Table 5 on page 1062 of the September issue in the paper entitled "The effect of photoperiodism and rearing period feed restriction on the performance of five Leghorn strains," pages 1056-1072. In test 4, light treat-
ment (6-6-18) hen day egg production should read 262 not 272. RALSTON PURINA FELLOWSHIPS Robert W. Boggs, Davis, California, has been awarded a Ralston Purina Research Fellowship
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SUMMARY The radioactive tracer (I131) technique for determination of mammalian blood volume was adapted for chickens. The effects of blood removal during sampling and trapped plasma among packed cells on the determined hematocrit were investigated. Both macro and micro measurements of tracer dilution as well as dilution of Evans Blue T-1824 in the circulatory system were studied. Both radioactive tracer techniques estimated the blood volume of chickens more precisely than the Evans Blue T-1824 method. The macro method proved superior to the micro method as indicated by a smaller standard deviation.
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