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Heart Probe—A Method for Obtaining Blood Samples from Chickens
HEART PROBE—A METHOD FOR OBTAINING BLOOD SAMPLES PROM CHICKENS H. J. SLOAN AND H. S. WILGUS, JR. From the Laboratory of Poultry Nutrition, Department of Poultry Husbandry, Cornell University Agricultural Experiment Station, Ithaca, N. Y.
MATERIAL
REQUIRED
The following materials are required: Hypodermic syringes, hypodermic needles, large centrifuge tubes (50 c c ) , glass stirring rods, vaseline, 250 or 400 cc. breakers, small erlenmeyer flasks, and a suitable box or board for holding the subjects. The size and number of these materials will vary with the number of individuals to be bled. For six-weeks old chicks (or mature rats) la/4 inch 20 gauge hypodermic needles are satisfactory. For older chicks and mature birds 1% to 2-inch 20 gauge needles work very well. It is desirable to use as small gauge needles as compatible with speed in order to lessen the possibility of damage to the birds. 10
Downloaded from http://ps.oxfordjournals.org/ at California Institute of Technology on June 23, 2015
(Received for Publication 7-1-30) The increasing use of the inorganic phosphorus and calcium content of blood plasma and sera in the diagnosis of rickets makes a simple method for obtaining blood for such analyses desirable. Though blood so obtained can no doubt be used for other kinds of analyses, the method described here was developed primarily for use in connection with studies of rickets in chicks at the Laboratory of Poultry Nutrition at the Cornell University Agricultural Experiment Station and has proved very satisfactory during the past three years in which time 3,000 chicks and about 300 hens have been bled. Though the idea was not original with the authors, it has been developed by them to the extent that the time consumed is relatively short and the mortality of the chicks is sufficiently low to make it practical. The possibility that the use of blood analyses in nutritional investigations may be limited somewhat by an unfamiliarity with a satisfactory method for obtaining blood in considerable quantities has prompted the preparation of this article. So far as the authors are aware, this method has not heretofore been described.
HEART
PROBE
11
PROCEDURE
Preparing the Subject. The operation is performed without anesthesia since it is apparently not painful, hence some provision must be made for holding the individuals. The equipment described here for holding the individuals applies only to chickens as it is not suitable for rats since they struggle a great deal more than birds, making it necessary to hold them by hand. A board or box top similar to a caponizing board can be used successfully. The bird is placed on the box with the ventral side of the bird toward the operator. The bird's head should be toward the left of the operator and the shanks toward the right, particular care being taken that the left shank lies behind the right one on the board. The birds should be stretched as much as possible as this not only makes it easier
Downloaded from http://ps.oxfordjournals.org/ at California Institute of Technology on June 23, 2015
In choosing a syringe it is desirable to have one that will hold somewhat more blood than is desired to draw from one individual; that is, if 3 ec. are to be drawn one should use a 5 cc. syringe as this may often save a second insertion of the needle if some air accidentally is drawn into the syringe. Common Luer all-glass syringes are suitable. These take the Luer Yale type needles which can be obtained in a wide variety of sizes. Several needles and two or more syringes should be available in case of breakage. The size of centrifuge tubes for containing the individual or composite samples and for centrifuging will depend on the amount of blood to be collected. More than 40 cc. of blood should not be put in one of the 50 cc. size tubes. When more than 40 cc. are to be composited, a small beaker may be used, though these are more fragile and hence more care must be exercised in mixing the sample. Conical bottom tubes are more satisfactory than round bottom ones as the clot packs better in the bottom when the blood is centrifuged. Beakers make convenient receptacles for the centrifuge tubes. Small erlenmyer flasks are used for containing the serum. Vaseline is used to lubricate the syringe plunger. Just sufficient should be used to cover completely the wall of the syringe and the tip and wall of the plunger. The greasy surface thus produced retards the rate of clotting of the blood and insures a good seal. Stirring rods are used for mixing the blood samples.
12
POULTRY
SCIENCE
Downloaded from http://ps.oxfordjournals.org/ at California Institute of Technology on June 23, 2015
to locate the heart, but it also decreases the tendency for the birds to struggle. When the bird is properly placed on the table, the feathers may be moistened over the thorax and in the ease of a bird that is fully feathered, it may be well to remove a few of the feathers around the region of the insertion of the needle. Locating the Heart. The heart in birds lies along and parallel to the median line of the sternum near the anterior end, just inside the region of the costo-sternal junctions. In chicks up to 12 weeks old, the heart is quite easily located by palpation; in older birds, due to the increased thickness of the pectoral muscles, it may be more difficult to locate definitely by palpation. The heart beat is usually, although not always, most easily discernable between the fifth and sixth ribs (there are seven in all) just above the point where they join the body of the sternum. In case the heart beat cannot readily be felt the point of insertion of the needle can be determined as follows: the next to the last or sixth rib about midway between the back and sternum is first located. The space between this and the fifth rib is the fifth intercostal space. This intercostal space is followed to the body of the sternum. Satisfactory results can usually be obtained by inserting the needle in the lower end of this space, just above the sternum and posterior to the fifth rib. After some practice, the heart can be easily and rapidly located without the necessity of counting the ribs. This may be accomplished by placing the thumb of the left hand on the anterior end of the sternum and the middle finger on the back bone just posterior to the wings. The index finger is then used to locate the heart, first by counting the ribs and finally, after practice, by drawing an imaginary line posteriorly between the thumb and forefinger, placing the index finger just back of the middle of the line and inserting the needle immediately posterior to the end of the index finger. This method may be found more convenient and time-saving if any number of birds are to be bled. Probing. The needle and syringe should be put together before the needle is inserted. The top of the syringe should lean slightly to the right of and away from the operator. Since the
HEART
PROBE
13
Downloaded from http://ps.oxfordjournals.org/ at California Institute of Technology on June 23, 2015
pectoral muscles in older birds are quite thick, it is necessary to use sharp needles for most satisfactory results. In order to prevent tissue fluids and some of the softer tissues from entering the needle while it is being inserted, the index finger of the right hand should be placed over the end of the plunger, and the syringe held firmly with the other fingers. There are two things which indicate quite plainly when the heart has been probed: (1) the beating of the heart can be felt quite distinctly through the needle, and (2) blood will appear in the base of the syringe, when the heart has been properly probed, due to the blood pressure in the heart (the pressure on the index finger should be only very slight when the point of the needle reaches the vicinity of the heart). The syringe should then be held firmly with the left hand and the plunger steadily withdrawn with the right hand until the desired amount of blood has been obtained. Rotating the needle and syringe while the sample is being taken often facilitates the drawing of blood. In removing the needle, it should not be separated from the syringe as considerable blood may be lost through the needle before it can be removed. In emptying the syringe the needle should first be removed (while the tip of the syringe is held up) as it is not uncommon for the blood to clot slightly in the needle and syringe especially when the sample has been drawn slowly. The blood is then forced out into the centrifuge tube. When the syringe is empty, the plunger is partially withdrawn, the needle replaced on the tip and held firmly between the first and second fingers, and the needle and syringe blown out by quickly forcing down the plunger. They are then cleaned by rapidly forcing in and withdrawing the plunger several times. When this is done, very little blood remains in either of them, particularly when the plunger has been properly lubricated. Care should be taken that the plunger does not strike the tip end of the syringe as the syringe can easily be broken in this manner. This procedure usually, makes it unnecessary to cleanse the syringe further before it is used on another individual especially if the blood of several individuals is to be composited. It also eliminates the necessity for the use of an anticoagulant in the syringe and needle. Not infrequently the plunger ceases to work freely. When
14
POULTRY
SCIENCE
DISCUSSION
As compared with caponizing, the operation is probably much less painful since very few if any pain sensations arise in the internal organs on stimulation. This is evidenced by the fact that most of the struggling occurs during the time that the thorax wall is being pierced. Usually the only other time that any considerable amount of struggling is experienced is after the needle has pierced the heart. The cause of death in this ease is very hard to determine, especially if post mortem examination shows only slight hemorrhage. It is probably due to the injury to the sino-auricular node, the auriculoventricular node, or the auriculoventricular bundle. Since these parts of the heart are concerned with
Downloaded from http://ps.oxfordjournals.org/ at California Institute of Technology on June 23, 2015
this occurs, it should be thoroughly cleaned and re-vaselined before further use. I t is advisable also to clean or change needles frequently. The Blood Sample. Serum may be obtained from the blood sample in either of two ways: (1) separated from the clot by breaking up the clot and then centrifuging, or (2) syneresis. The authors use the former method. In'this method, the sample of blood is agitated with a glass stirring rod as soon as a soft clot has formed. If the sample is a composite from several individuals the first portion of blood will have become quite firm, but it can be well broken up if care is used. The sample should only be agitated sufficiently to break up the clot well as excessive stirring is likely to cause some hemolysis. As soon as possible, the sample should be centrifuged in the 50 cc. centrifuge tubes (except when small samples have been taken in which case 15 cc. tubes may be satisfactory) for five to eight minutes at approximately 1500 revolutions per minute. This throws the clot to the bottom of the tube, leaving the clear serum on top. The serum can then be decanted into suitable containers. About 40 to 45% by volume of the original sample can be retrieved as serum. If the analyses are to be made immediately no preservative is necessary. If the samples are to be held for some time, they should be kept cool and some preservative used; formalin or toluene can be used in small amounts.
HEART
PROBE
15
i HAWK-BERGEIM, Practical Physiological Chemistry, 1927, Ninth Edition, Blakiston.
Downloaded from http://ps.oxfordjournals.org/ at California Institute of Technology on June 23, 2015
the origin and regulation of the heart beat, they are very susceptible to injury. Struggling at this time may not only cause considerable damage to the heart by tearing, but also may cause damage to the regulating mechanism. Mortality in chicks rarely exceeds 2% even in severely rachitic pens and in laying hens has been found to be less than 1%. It would be hard to determine how much effect the operation had on those that survived. I t might be a temporary set back or it might be negligible. However, it has never been possible for us to pick by observation any of the operated individuals from the rest of the group after the day succeeding the iperation. A study of the weights did not indicate any appreciable loss, as practically all of the individuals used for sampling grew as well during the succeeding weeks as other individuals that were comparable in weight. Little or no effect on egg production or physical well-being of laying hens was noticed when 8-10 cc. samples were withdrawn. • For some analyses it is not necessary to make the determinations immediately after the samples are drawn. When this is the case, the serum can be obtained by syneresis. This is done by simply allowing the blood to stand unstirred for 24-48 hours in a refrigerator. The clot contracts, leaving clear serum. Less serum is obtained by this method from an equal amount of whole blood than by the other method described. The amount of blood used for any composite will depend on three things: (1) nature and number of analyses to be made, (2) number of chicks per composite, and (3) the size of the chicks. Fifteen cc. o£ serum will suffice for triplicate calcium and phosphorus determinations when the Clark-Collip modification of the Kramer-Tisdall method is used for calcium, and the Fisk-Subbarrow method is used for phosphorus (as outlined by Hawk-Bergeim) 1 Though it is seldom necessary to run a triplicate and only very rarely necessary to run a quadruplicate, it is well to have sufficient serum as of course it is impossible to get the same individuals' blood again under the same conditions. This will require from 32-36 cc. of whole blood, depending on the success obtained in clotting and centrifuging.
16
POULTRY
SCIENCE
•
SUMMARY
A method has been described for obtaining blood for analyses from the hearts of chicks and mature chickens by probing the heart with a hypodermic needle. I t has the following advantages : 1. Large amounts of blood uncontaminated by the other body fluids can be obtained. 2. The birds do not have to be sacrificed. 3. Many samples can be taken in a relatively short time. 4. The mortality is very low. 5. No particular skill is necessary to perform the operation. 6. The method is very well suited to the more common methds of calcium and phosphorus analyses.
2 MACLEOD, J. J. R., Physiology and Biochemistry in Modern Medicine, 1926, p. 76, C. V. Mosby Co.
Downloaded from http://ps.oxfordjournals.org/ at California Institute of Technology on June 23, 2015
Not all of the erythrocytes will be retained in the clot, which means that not all of the serum can be poured off. If eight individuals are to be used in the composite, 4-4% ec. of whole blood are required from each. With chicks, six weeks old, 4 cc. can be drawn with no apparent damage to the chick. I t is doubtful if more should be taken at one time. Proportionately more can be drawn from older chicks. A normal six weeks old chick (Leghorn) should weigh a little over 300 grams. On the basis that 8.8% (value for humans)-' of the body weight is blood, the total blood in a six weeks old chick would weigh 26-27 grams. Four cc. then would constitute roughly one-sixth the total supply. With severely rachitic chicks which do not weigh over 200 grams at six weeks, this would constitute nearly one-fourth of the total blood. This much has frequently been drawn with no apparent permanent injury to the individual.
MATERIAL
REQUIRED
The following materials are required: Hypodermic syringes, hypodermic needles, large centrifuge tubes (50 c c ) , glass stirring rods, vaseline, 250 or 400 cc. breakers, small erlenmeyer flasks, and a suitable box or board for holding the subjects. The size and number of these materials will vary with the number of individuals to be bled. For six-weeks old chicks (or mature rats) la/4 inch 20 gauge hypodermic needles are satisfactory. For older chicks and mature birds 1% to 2-inch 20 gauge needles work very well. It is desirable to use as small gauge needles as compatible with speed in order to lessen the possibility of damage to the birds. 10
Downloaded from http://ps.oxfordjournals.org/ at California Institute of Technology on June 23, 2015
(Received for Publication 7-1-30) The increasing use of the inorganic phosphorus and calcium content of blood plasma and sera in the diagnosis of rickets makes a simple method for obtaining blood for such analyses desirable. Though blood so obtained can no doubt be used for other kinds of analyses, the method described here was developed primarily for use in connection with studies of rickets in chicks at the Laboratory of Poultry Nutrition at the Cornell University Agricultural Experiment Station and has proved very satisfactory during the past three years in which time 3,000 chicks and about 300 hens have been bled. Though the idea was not original with the authors, it has been developed by them to the extent that the time consumed is relatively short and the mortality of the chicks is sufficiently low to make it practical. The possibility that the use of blood analyses in nutritional investigations may be limited somewhat by an unfamiliarity with a satisfactory method for obtaining blood in considerable quantities has prompted the preparation of this article. So far as the authors are aware, this method has not heretofore been described.
HEART
PROBE
11
PROCEDURE
Preparing the Subject. The operation is performed without anesthesia since it is apparently not painful, hence some provision must be made for holding the individuals. The equipment described here for holding the individuals applies only to chickens as it is not suitable for rats since they struggle a great deal more than birds, making it necessary to hold them by hand. A board or box top similar to a caponizing board can be used successfully. The bird is placed on the box with the ventral side of the bird toward the operator. The bird's head should be toward the left of the operator and the shanks toward the right, particular care being taken that the left shank lies behind the right one on the board. The birds should be stretched as much as possible as this not only makes it easier
Downloaded from http://ps.oxfordjournals.org/ at California Institute of Technology on June 23, 2015
In choosing a syringe it is desirable to have one that will hold somewhat more blood than is desired to draw from one individual; that is, if 3 ec. are to be drawn one should use a 5 cc. syringe as this may often save a second insertion of the needle if some air accidentally is drawn into the syringe. Common Luer all-glass syringes are suitable. These take the Luer Yale type needles which can be obtained in a wide variety of sizes. Several needles and two or more syringes should be available in case of breakage. The size of centrifuge tubes for containing the individual or composite samples and for centrifuging will depend on the amount of blood to be collected. More than 40 cc. of blood should not be put in one of the 50 cc. size tubes. When more than 40 cc. are to be composited, a small beaker may be used, though these are more fragile and hence more care must be exercised in mixing the sample. Conical bottom tubes are more satisfactory than round bottom ones as the clot packs better in the bottom when the blood is centrifuged. Beakers make convenient receptacles for the centrifuge tubes. Small erlenmyer flasks are used for containing the serum. Vaseline is used to lubricate the syringe plunger. Just sufficient should be used to cover completely the wall of the syringe and the tip and wall of the plunger. The greasy surface thus produced retards the rate of clotting of the blood and insures a good seal. Stirring rods are used for mixing the blood samples.
12
POULTRY
SCIENCE
Downloaded from http://ps.oxfordjournals.org/ at California Institute of Technology on June 23, 2015
to locate the heart, but it also decreases the tendency for the birds to struggle. When the bird is properly placed on the table, the feathers may be moistened over the thorax and in the ease of a bird that is fully feathered, it may be well to remove a few of the feathers around the region of the insertion of the needle. Locating the Heart. The heart in birds lies along and parallel to the median line of the sternum near the anterior end, just inside the region of the costo-sternal junctions. In chicks up to 12 weeks old, the heart is quite easily located by palpation; in older birds, due to the increased thickness of the pectoral muscles, it may be more difficult to locate definitely by palpation. The heart beat is usually, although not always, most easily discernable between the fifth and sixth ribs (there are seven in all) just above the point where they join the body of the sternum. In case the heart beat cannot readily be felt the point of insertion of the needle can be determined as follows: the next to the last or sixth rib about midway between the back and sternum is first located. The space between this and the fifth rib is the fifth intercostal space. This intercostal space is followed to the body of the sternum. Satisfactory results can usually be obtained by inserting the needle in the lower end of this space, just above the sternum and posterior to the fifth rib. After some practice, the heart can be easily and rapidly located without the necessity of counting the ribs. This may be accomplished by placing the thumb of the left hand on the anterior end of the sternum and the middle finger on the back bone just posterior to the wings. The index finger is then used to locate the heart, first by counting the ribs and finally, after practice, by drawing an imaginary line posteriorly between the thumb and forefinger, placing the index finger just back of the middle of the line and inserting the needle immediately posterior to the end of the index finger. This method may be found more convenient and time-saving if any number of birds are to be bled. Probing. The needle and syringe should be put together before the needle is inserted. The top of the syringe should lean slightly to the right of and away from the operator. Since the
HEART
PROBE
13
Downloaded from http://ps.oxfordjournals.org/ at California Institute of Technology on June 23, 2015
pectoral muscles in older birds are quite thick, it is necessary to use sharp needles for most satisfactory results. In order to prevent tissue fluids and some of the softer tissues from entering the needle while it is being inserted, the index finger of the right hand should be placed over the end of the plunger, and the syringe held firmly with the other fingers. There are two things which indicate quite plainly when the heart has been probed: (1) the beating of the heart can be felt quite distinctly through the needle, and (2) blood will appear in the base of the syringe, when the heart has been properly probed, due to the blood pressure in the heart (the pressure on the index finger should be only very slight when the point of the needle reaches the vicinity of the heart). The syringe should then be held firmly with the left hand and the plunger steadily withdrawn with the right hand until the desired amount of blood has been obtained. Rotating the needle and syringe while the sample is being taken often facilitates the drawing of blood. In removing the needle, it should not be separated from the syringe as considerable blood may be lost through the needle before it can be removed. In emptying the syringe the needle should first be removed (while the tip of the syringe is held up) as it is not uncommon for the blood to clot slightly in the needle and syringe especially when the sample has been drawn slowly. The blood is then forced out into the centrifuge tube. When the syringe is empty, the plunger is partially withdrawn, the needle replaced on the tip and held firmly between the first and second fingers, and the needle and syringe blown out by quickly forcing down the plunger. They are then cleaned by rapidly forcing in and withdrawing the plunger several times. When this is done, very little blood remains in either of them, particularly when the plunger has been properly lubricated. Care should be taken that the plunger does not strike the tip end of the syringe as the syringe can easily be broken in this manner. This procedure usually, makes it unnecessary to cleanse the syringe further before it is used on another individual especially if the blood of several individuals is to be composited. It also eliminates the necessity for the use of an anticoagulant in the syringe and needle. Not infrequently the plunger ceases to work freely. When
14
POULTRY
SCIENCE
DISCUSSION
As compared with caponizing, the operation is probably much less painful since very few if any pain sensations arise in the internal organs on stimulation. This is evidenced by the fact that most of the struggling occurs during the time that the thorax wall is being pierced. Usually the only other time that any considerable amount of struggling is experienced is after the needle has pierced the heart. The cause of death in this ease is very hard to determine, especially if post mortem examination shows only slight hemorrhage. It is probably due to the injury to the sino-auricular node, the auriculoventricular node, or the auriculoventricular bundle. Since these parts of the heart are concerned with
Downloaded from http://ps.oxfordjournals.org/ at California Institute of Technology on June 23, 2015
this occurs, it should be thoroughly cleaned and re-vaselined before further use. I t is advisable also to clean or change needles frequently. The Blood Sample. Serum may be obtained from the blood sample in either of two ways: (1) separated from the clot by breaking up the clot and then centrifuging, or (2) syneresis. The authors use the former method. In'this method, the sample of blood is agitated with a glass stirring rod as soon as a soft clot has formed. If the sample is a composite from several individuals the first portion of blood will have become quite firm, but it can be well broken up if care is used. The sample should only be agitated sufficiently to break up the clot well as excessive stirring is likely to cause some hemolysis. As soon as possible, the sample should be centrifuged in the 50 cc. centrifuge tubes (except when small samples have been taken in which case 15 cc. tubes may be satisfactory) for five to eight minutes at approximately 1500 revolutions per minute. This throws the clot to the bottom of the tube, leaving the clear serum on top. The serum can then be decanted into suitable containers. About 40 to 45% by volume of the original sample can be retrieved as serum. If the analyses are to be made immediately no preservative is necessary. If the samples are to be held for some time, they should be kept cool and some preservative used; formalin or toluene can be used in small amounts.
HEART
PROBE
15
i HAWK-BERGEIM, Practical Physiological Chemistry, 1927, Ninth Edition, Blakiston.
Downloaded from http://ps.oxfordjournals.org/ at California Institute of Technology on June 23, 2015
the origin and regulation of the heart beat, they are very susceptible to injury. Struggling at this time may not only cause considerable damage to the heart by tearing, but also may cause damage to the regulating mechanism. Mortality in chicks rarely exceeds 2% even in severely rachitic pens and in laying hens has been found to be less than 1%. It would be hard to determine how much effect the operation had on those that survived. I t might be a temporary set back or it might be negligible. However, it has never been possible for us to pick by observation any of the operated individuals from the rest of the group after the day succeeding the iperation. A study of the weights did not indicate any appreciable loss, as practically all of the individuals used for sampling grew as well during the succeeding weeks as other individuals that were comparable in weight. Little or no effect on egg production or physical well-being of laying hens was noticed when 8-10 cc. samples were withdrawn. • For some analyses it is not necessary to make the determinations immediately after the samples are drawn. When this is the case, the serum can be obtained by syneresis. This is done by simply allowing the blood to stand unstirred for 24-48 hours in a refrigerator. The clot contracts, leaving clear serum. Less serum is obtained by this method from an equal amount of whole blood than by the other method described. The amount of blood used for any composite will depend on three things: (1) nature and number of analyses to be made, (2) number of chicks per composite, and (3) the size of the chicks. Fifteen cc. o£ serum will suffice for triplicate calcium and phosphorus determinations when the Clark-Collip modification of the Kramer-Tisdall method is used for calcium, and the Fisk-Subbarrow method is used for phosphorus (as outlined by Hawk-Bergeim) 1 Though it is seldom necessary to run a triplicate and only very rarely necessary to run a quadruplicate, it is well to have sufficient serum as of course it is impossible to get the same individuals' blood again under the same conditions. This will require from 32-36 cc. of whole blood, depending on the success obtained in clotting and centrifuging.
16
POULTRY
SCIENCE
•
SUMMARY
A method has been described for obtaining blood for analyses from the hearts of chicks and mature chickens by probing the heart with a hypodermic needle. I t has the following advantages : 1. Large amounts of blood uncontaminated by the other body fluids can be obtained. 2. The birds do not have to be sacrificed. 3. Many samples can be taken in a relatively short time. 4. The mortality is very low. 5. No particular skill is necessary to perform the operation. 6. The method is very well suited to the more common methds of calcium and phosphorus analyses.
2 MACLEOD, J. J. R., Physiology and Biochemistry in Modern Medicine, 1926, p. 76, C. V. Mosby Co.
Downloaded from http://ps.oxfordjournals.org/ at California Institute of Technology on June 23, 2015
Not all of the erythrocytes will be retained in the clot, which means that not all of the serum can be poured off. If eight individuals are to be used in the composite, 4-4% ec. of whole blood are required from each. With chicks, six weeks old, 4 cc. can be drawn with no apparent damage to the chick. I t is doubtful if more should be taken at one time. Proportionately more can be drawn from older chicks. A normal six weeks old chick (Leghorn) should weigh a little over 300 grams. On the basis that 8.8% (value for humans)-' of the body weight is blood, the total blood in a six weeks old chick would weigh 26-27 grams. Four cc. then would constitute roughly one-sixth the total supply. With severely rachitic chicks which do not weigh over 200 grams at six weeks, this would constitute nearly one-fourth of the total blood. This much has frequently been drawn with no apparent permanent injury to the individual.