| The amount of hemoglobin in the blood is estimated by means of some kind of color scale. Different instruments use different types of scale and different methods of preparing the blood. Older methods based upon chemical analysis for iron are much less accurate and are very cumbersome; they are not suitable for ordinary laboratory work in diagnosis. The method of securing the blood is the same for all the newer methods of determining hemoglobin. It is important to secure the drop of blood in such a manner as to prevent its changing from its condition within the vessels as little as possible. The lobe of the ear, the palmar surfaces of the fingers and the balls of the toes are almost devoid of sensory nerves. The skin at the sides of the fingers is thinner than that upon the heavier parts of the balls and is often less sensitive. In persons of certain occupations, such as piano or violin playing the finger tips may have thick skin. It is necessary to avoid any area which is more than usually liable to injury after the prick is made, the violinist might find some difficulty in playing with the injured finger for a few hours after the prick has been made, for example. The lobe of an ear is the best site in adults, the side of a toe is best in babies. Having selected the site of the puncture, the skin should be cleaned in any way that is convenient. Probably a good washing with clean water is best for ordinary conditions. Various antiseptics are sometimes employed, but these must be sufficiently dilute to prevent irritation to the skin; this means little or no antiseptic value. Any of the ordinary antiseptics strong enough to injure bacteria injure t he tissues still more seriously. But a washing with anything which removes the dust and perhaps a few desquamating epithelial cells takes away the chief source of infection and does not prevent the most speedy repair. Care must be taken in the washing that the skin is not irritated enough to cause overfilling of the vessels since this modifies the hemoglobin content. The first drop of blood must be wiped away, and the second or later drops taken up in the manner suitable for each instrument. The tissues must not be squeezed or handled roughly but the blood musts come gently and freely from the puncture. The puncture is best made with a sharp, angular lancet. The lancet which is provided with a spring makes a sudden thrust which is not painful at all. A pen which has been broken longitudinally in half, so that only one sharp point remains, is useful. A dozen or more pens can be prepared and sterilized and a new one used for each patient. Glove needles, being triangular, make a sharp, clean puncture which heals at once. Round needles are not quite so suitable because they cause more pain and leave a rougher puncture; the slight bruising which occurs may affect the concentration of the blood in the vicinity of the cut. The error due to this factor is negligible in most cases. The blood drop as it wells up from the tiny wound made for the purpose gives a general idea of its hemoglobin content, by its color. When the blood is more viscid, this drop stands up round and high, and appears to be richer in hemoglobin than it really is, when it is of low viscidity it spreads out over the surrounding skin in a thinner layer, and appears to be lower in hemoglobin content than it is. The character of the skin modifies the height of the drop, also, as does the size of the wound and the rapidity of the flow. The simplest, cheapest, and easiest method of estimating the hemoglobin is by means of the Tallquist Hemoglobin Scale. It consists of a scale of ten colored paper slips for comparison, each corresponding to a certain percentage of normal hemoglobin, and strips of filter paper of uniform thickness to take the blood. In using this scale the drop is secured by the usual means, and a strip of the filter paper laid gently and quickly over the drop, until the blood is soaked up into the paper over an area about one-fourth of an inch in diameter. This is held for a few seconds or until the first shiny look of the blood stain has disappeared, then it is compared with the color scale. Some of the scales have an opening in the center of each of the ten color-tint blocks, and the blood-stained paper is then placed beneath these openings, one after another, up and down the scale in succession until the matching tint of the scale is found. If the blood is apparently half way between two blocks as 70% and 80%, the hemoglobin may be considered as 75%. In this way, those who are experienced in the test can make estimations of the hemoglobin which are fairly useful though they cannot be considered accurate within more than 20% under the best of circumstances in the most skillful hands. The method is perhaps the most convenient of all, since there is no apparatus to wash, no special light requirements are necessary and the scale is so small that it can easily be carried in the pocket. Its lack of accuracy is the only factor against its use. The method is useful for a preliminary test or in emergency cases. Next in ease of use is Dave’s Hemoglobinometer. This instrument has a revolving color scale of glass, a telescoping tube for securing distinct vision, and two slips of glass which hold a measured film of blood between them. The slips of glass are placed in the holder and screwed closely but not too tightly together; the blood flows between them by capillary attraction. The holder than slips into its place at the back of the instrument. The telescope is pulled out until the edges of the two circles show very plainly and in good focus. The revolving color scale is moved by means of the screw at the upper part of the instrument, until the tint of the two circles appears exactly the same. The amount of hemoglobin is read off in percentages of the normal from a scale which is attached to the side of the glass circle, and which is seen through a tiny window at the side of the instrument. In using the Dave’s instrument it is necessary to have a yellowish light, not too brilliant, and to make the examination in a rather dark room. It is better to look with the eyes alternately, thus giving each eye an opportunity to rest, in turn. The correctness of the reading may be gauged by the fact that one is able to return to about the same figures several times in succession, after changing the position of the scale and resting the eyes. It is probably correct within 5% of the actual amount. This scale is made so that blood containing 137.7 grams per liter is considered as 100%, or normal. It is evident that in using this instrument correction must be made for the age of the patient. Gower’s hemoglobinometer is not now in general use. It consists of three tubes, one filled with gelatine stained with picrocarmine to be used for artificial light, one filled with gelatine stained slightly differently, to be used with daylight, and one to be filled with diluted blood, the latter graduated. The graduated tube is filled to a certain point with blood, then water is added until the tint exactly matches the tube filled with gelatine, using the one or the other according as the light is artificial (yellowish) or is daylight. The dilution is difficult; the gelatine tubes fade quickly if they are exposed to the light often (as they must be if they are used at all frequently) and the newer instruments are much more accurate and convenient. Sahli’s hemoglobinometer is somewhat like Gower’s but has certain advantages over the older instrument. It consists essentially of two tubes, one filled with a stock solution of 1% acid hematin, and one tube for the blood. Twenty cubic millimeters of blood are taken into the blood tube, and this is mixed with deci-normal hydrochloric acid. (15 c.c. HCI: 1 liter, H2O is sufficiently accurate.) The acid changes the hemoglobin of the blood into the stable acid hematin. The blood mixture is then to be diluted with water until it matches accurately the color of the stock solution in the other tube The advantages of this instrument are that it can be used in any light; since both the stock solution and the blood mixture are colored by the same compound—acid hematin,--they are equally affected by different lightings, so that yellow light, daylight, dim lights or brilliant lights all act upon both tubes in the same way. A moderate light, however, gives the most nearly accurate readings. This instrument is, in the hands of reasonably skillful observers, accurate within about five per cent. The readings are made in percentages of the normal, which is thus subject to the necessity for correction for the age of the patient. In this instrument 172 grams of hemoglobin per liter of blood are taken as 100%. The most accurate, the least convenient and the most expensive hemoglobinometer is that of Fleischl as modified by Miescher. This has for its scale a long, slender, delicate glass bar which is thick at one end and thin at the other, so colored as to be spectroscopically identical in color with hemoglobin. This has a scale connect ed with it, and is set beneath a stage which is perforated in its center. A second perforation near the edge of the stage permits the scale to be read easily. The colored prism is moved across the central perforation. Beneath the prism is a reflecting dull white surface and a candle or other dim yellowish light is placed in front of this dull reflector. The blood is taken in a pipette somewhat like those used for blood dilution for counting, except that the hemoglobinometer pipette is so graduated that dilutions of 1-200, 1-300, or 1-400 can be made. Dilution is made with 0.1% sodium carbonate; ordinary tap water is really better, in our experience. If the blood appears very pale as it emerges through the puncture use the dilution 1-200. If it appears almost or quite normal in tint use a dilution of 1-400. For milder anemias uses 1-300 dilution. In taking the blood into the pipette keep the lower end of the pipette within the blood drop but do not allow it to touch the skin. If by accident the blood should be drawn above the selected mark on the tube, or should not quite reach that mark, there are accessory graduations by means of which adequate correction can be made after the work is completed. Draw the diluting fluid into the pipette to the mark at the top of the bulb, and then immediately thoroughly shake the pipette from side to side. The shaking must be gentle, and the pipette must not be shaken endways lest some of the blood be forced into the capillary portions of the pipette. Two chambers, each divided into compartments, are used for the examination. Each chamber has a glass cover and a glass bottom, and a perforated dark metal cap to be placed over the cover glass. One chamber is 12 millimeters in depth, the other 15 millimeters, and readings are made with both chambers. Put the glass bottom into the 15 millimeter chamber and fix it in place by means of the screw-like arrangement provided for the purpose. Fill one compartment with water or the solution used for dilution. By means of a rubber bulb blow out and discard four drops from the pipette used for diluting the blood. (This removes the fluid in the capillary bore of the tube, which presumably contains no blood.) Still using the rubber bulb, fill the other compartment with the diluted blood. Be very careful that both compartments are completely filled, that none of the clear fluid reaches the blood compartment, and that none of the diluted blood reaches the clear fluid. Cover with the glass; there must not be any air drops in either compartment. Place the metal cap over the cover in such a manner that two equal squares are visible, one of the diluted blood, the other of the clear fluid. Place the chamber in the perforation provided for the purpose in the stage of the hemoglobinometer in such a manner that the clear square is exactly over the colored bar. Arrange the candle and the reflecting surface so that a dim light is thrown upward through the colored prism and the clear fluid, and through the diluted blood. Move the prism back and forth by means of the screw until the two squares have exactly the same tint. Use first one eye then the other. Do this quickly; if too much time is taken vision becomes less acute. Read and note the figures on the scale. Move the prism away from matching tint. Close the eyes for a few seconds, then repeat the test. In this way make ten different readings. If the readings do not vary more than five points the readings are sufficiently accurate. Fill the 12 millimeter chamber in exactly the same manner and make ten readings in the same manner. Make an average of the ten readings from the 12 millimeter chamber and make an average of the ten readings from the 15 millimeter chamber. Divide the first average (from the 12 millimeter chamber) by 4 and multiply by t, this should equal the average made from the ten readings from t he 15 millimeter chamber. If the figures thus secured do not agree within five points the work is not sufficiently accurate and should be repeated. Compare the figures secured, corrected for the 15 millimeter chamber, with a scale provided with the instrument. This gives the grams of hemoglobin per liter of the diluted blood. If the blood was diluted 1-200, multiply the scale reading by 200, which gives the grams of hemoglobin per liter of whole blood. If the dilution was 1-300 or 1-400, multiply by these figures. This instrument requires an absolutely dark
room; it is too cumbersome to carry about easily; its use requires
much time for the actual work and for the washing of the apparatus.
Parts are easily broken and they cannot be easily replaced.
It is by far the most accurate instrument on the market and for
this reason has an important place in hospitals and research laboratories.
We use it for checking up on other simpler instruments, and in cases
in which unusual accuracy is required. The errors that may be made in the determination of the hemoglobin are not many. By the Tallquist scale, two errors are not infrequent; the drop of blood may be small, thus the area of stained paper is too small and the reading too low. Or, in the endeavor to secure a larger area of stained paper, from an insufficient amount of blood, the paper may be pressed again and again over the wound, until the same area of paper may be soaked several times in the blood; naturally the reading is then very much higher than it should be. In severe anemias and in blood of very low viscosity, the serum soaks into the paper around the cells, leaving a rather dark center in a very pale ring. The reading is thus too high. This source of error is especially apt to occur in chlorosis, and it frequently occurs in pernicious anemia and in secondary anemias of similar type. Sometimes the examination is delayed, and the blood takes on a brown color, due, probably, to the formation of methemoglobin. When this occurs it is impossible to secure anything like an exact match of the tints of the scale; the resulting readings are certainly inaccurate, and they may be too high or too low. In using Dare’s hemoglobinometer, the slides may not be thoroughly dry, especially if it has been necessary to clean them after errors in technique. The slightest film of moisture upon the surfaces of the glass slides affects the reading by diluting the blood. Sometimes the slides are not screwed closely together; the film of blood is then too thick, and the reading too high. Sometimes the slides are screwed too tightly together, whereupon they break. The finger should be removed from the wheel which turns the scale, before the telescope-tube is removed form the eyes. Else, when the telescope is taken from the eyes, the fingers which have been turning the scale may still turn it slightly. The scale may thus be turned and the reading correspondingly inaccurate. The use of Sahli’s or Gower’s instrument depends for its accuracy upon the care with which the blood is taken, is blown into the larger diluting pipette, and with which the readings and dilutings are performed. For if the drop of blood should not be removed from the end of the tube, after the requisite 20 c.c. have been taken, a part of this is almost sure to be drawn into the tube with the diluting fluid, and the determination is increased. If the tubes are moist, the blood is unduly diluted, and the readings are too low. If the blood is not thoroughly rinsed into the mixing pipette, the readings are thus too low. In adding the last of the water to the blood in the mixing pipette, it is best to read off the hemoglobin percentage, and write it down, before the later and decisive readings are taken. Then, if by accident or by choice, the dilution should be sufficient to carry the color past that of the control, the reading can be corrected accordingly. In using the original Fleischl instrument sources of error are plentiful and for this reason it is not now used in many laboratories. The Miescher modification is subject to much less error but still it is necessary to use great care in making every step of the procedure. In very important cases for research work it is our practice to take the blood in two different dilutions and to compare these; discarding the results if the results do not agree within five points of the final computation, and making an average of the two final results if they do so agree. For each method of determining hemoglobin accuracy
is gained only by experience and carefulness; with these the hemoglobin
can be determined reasonably well, within the limits of accuracy
for the instrument employed, in any case. The enumeration of the cells by the use of a counting chamber is called the “actual” count, to distinguish it from the “differential” count, which is made upon a smear of blood. The actual count gives the actual number of cells per cubic millimeter of blood; the differential count gives the percentages of the different types of white cells, and the relative number of normoblasts, malarial corpuscles and any other items desired may also be determined. From the differential count and the actual count, the number of these various structures in an average cubic millimeter of blood can be determined. The actual count is not a complicated process but errors must be very carefully avoided or the findings may be false. Provide, for the actual count, the following articles. Microscope with mechanical stage, iris diaphragm and condenser may be of the type used in ordinary laboratory work. The binocular microscope is far better for the eyes. Have the table, the microscope and the seat to be used by the worker correctly placed so that the worker sits perfectly squarely and at a comfortable height. Counting blood takes considerable time; if the head of the worker is held at a lateral angle, or if the microscope is too high or too low there is a strain upon the neck muscles; the cervical vertebrae are held in an abnormal position and the circulation through the eyes is affected. Strain upon the intrinsic muscles of the eyes is caused by the attempt to see plainly if the eyes are not placed at right angles to the planes of the lenses of the eyepieces. In this connection it should be stated very emphatically that the condition called “eye-strain” due to the use of the microscope for considerable periods of time is much more frequently “neck-strain” and is due to carelessness in the position assumed while the work is being done. There is no reason why the use of the microscope should injure the eyes any more than reading, except that a microscope occupies a fixed position and the worker tends to accommodate his position to the needs of vision without being careful to consider the comfort of his position. In order to avoid fatigue and injury to the eyes it is only necessary to be very careful to sit comfortably, squarely facing the microscope and to have suitable lighting and proper arrangement of the iris diaphragm and the lenses. The lighting is important. In our laboratories a small electric light placed squarely beneath the condenser is used for ordinary work. A larger light placed in front of the small round reflecting mirror is good; this permits variations in lighting for low-power or high-power magnification in addition to the modifications secured by regulating the iris diaphragm. Other articles needed are a lancet for pricking
the skin, various solutions and containers, a counting chamber,
pipettes for diluting the blood and sterilizers and other articles
ordinarily present in a laboratory for clinical diagnosis. Several types of counting chamber are in use. The best arrangement is made of a single piece of glass which is so cut as to form the chamber. Lesss useful and accurate is the chamber made by cementing pieces of glass together. The chamber in which the moat is H-shaped is more convenient than the older style in which the moat is circular. In any event the counting chamber is a glass slide which has upon its upper surface several parts. In the center there is a ruled area upon which the counting is done. Several arrangements of the lines are in use but all contain lines crossing at right angles, dividing the chamber into spaces of varying sizes. The “small square” is the unit space. This is formed by lines 1-20 millimeter apart, thus squares are formed 1-20 x 1-20 millimeter in size. In order to secure ease in counting an extra line is placed midway between these lines at regular intervals thus forming smaller squares 1-40 x 1-40 millimeter; these are of no practical value in ordinary work except that they help to keep the area being counted in easy vision. The central area of the counting chamber is occupied by 144 of the squares of 1-20 millimeter. Many of these are subdivided. Around this central area many chambers are provided with larger squares for the counting of the white cells; these are made by lines 1-5 millimeter apart, with lines 1-20 millimeter apart outlining the larger spaces; sometimes three lines 1-40 millimeter apart take the place of the two lines 1-20 millimeter apart, in the formation of the larger squares. This ruled area is upon a part of the slide which is elevated above the rest of the slide; the exact amount of the elevation is of no consequence, and varies for different counting chambers. Around this ruled glass area, which is, properly, the chamber, there is a moat. This is a depression around the ruled area, itself surrounded by another elevation of glass. The glass around the moat is always 1-10 millimeter higher than the counting chamber, so that when the cover-glass is placed upon the slide a space exists between the ruled area and the cover-glass; this space is 1-10 millimeter in depth in all chambers in ordinary use. The counting chamber requires care in cleaning because the ruled lines are delicate and t he glass can be scratched easily. If the chamber is made by cementing different pieces of glass together, the chamber must never be washed or rinsed with anything but warm water. This is really all that is necessary, anyway, in ordinary cases. If the counting chamber is made of a single piece of glass, cut to form the various parts, it may be washed as any delicate glass-ware would be. The ruled area must be treated with especial
care, it must never be rubbed in drying, but only very gently mopped,
or, better, left to dry after a final rinsing in warm distilled
water. These are commonly of the Thoma-Zeiss types. They are composed of a glass bulb which receives the blood and the diluting fluid. Within this bulb is a small glass bead which facilitates mixing the blood and the fluid, and which also serve to remove deposits from the inside of the bulb in case of accident. A capillary tube receives the blood, and this is divided into ten equal parts. The entire content of the capillary tube of the erythrocyte pipette is one hundredth of the content of the bulb, and the content of one of the ten divisions is 1-1000 the content of the bulb. The fifth of these divisions is marked .5 and the division next the bulb is marked 1. At the top of the bulb another mark 101, is placed. Since the 101 includes the content of the capillary tube, and since the diluting fluid within the capillary tube is not mixed with the contents of the bulb, the dilution in the bulb can be made anything between 1 to 1,000 and 1 to 100. The most common dilutions for red cells are 1 to 100, or 1 to 200. The tube frequently used for counting the white cells has a smaller bulb, or a larger bore in its capillary tube, so the bulb contains only ten times as much fluid as the capillary tube. This tube also is divided into ten equal parts. The fifth division is marked .5 and the upper division is marked 1 as in the red cell pipette, while the upper limit of the bulb is marked 11. Thus it is possible to secure dilution of 1-10, 1020 or even 1-100 by drawing the blood to the 1 mark, to the .5 mark, or to the lowest mark, which is 12-10 the content of the bore of the capillary tube. Other markings are employed for the white cell pipettes occasionally; their significance is easily understood by a comparison of the markings with one another and with the red cell pipettes. For both pipettes there is a dilated area above
the constriction which marks the upper limit of the bulb; this receives
any superfluous amount which may be drawn up beyond the proper limit,
by accident. There is a rubber tube with a glass mouth-piece
for each blood pipette, and it is best to have these rubber tubes
a foot or more in length. The rubber tubes which come with
the pipettes are rarely long enough to permit accuracy in vision
or delicacy in manipulation of ascending columns. By having
the longer rubber tubes it is easier to see clearly the ascending
column of blood, and the increased elasticity due to the longer
rubber tube allows greater accuracy in manipulating the columns
as they are drawn upward. After the pipettes have been used rinse them in clean water immediately. Rinse thoroughly in cold water, then in hot water, then run air through them until they are perfectly dry. When no water is visible, and the glass bead rolls around freely within the bulb, the bulb is dry. It must be remembered that while the bulb is still very wet the bead rolls around freely also, but in that case the water is easily visible. If there has been some staining of the inside of the bulb with the diluting fluid, this may be removed with sulphuric, hydrochloric or nitric acid, using the acid first in a very weak solution, then increasing the strength of the solution until the stain is gone. If coagulation of blood should occur within the capillary bore or the bulb, this must be digested out. An artificial gastric juice containing both free hydrochloric acid and pepsin, or an artificial pancreatic juice containing trypsin and mildly alkaline in reaction, should be drawn into the bulb and the pipette left in an incubator over night. If the clot is not dissolved the process must be repeated, perhaps using some other artificial digestive fluid. After the clot has been digested the pipette is rinsed with cool water, then with hot water, then is dried in the usual manner. A filter pump attached to the water tap cleans the tube very easily and efficiently. Attach the pipette to the intake of the filter pump, place the other end of the pipette in cold or hot water, or in the acid solution if this is to be used. Turn on the water and allow the cleaning solution or water to flow through the pipette as long as seems desirable. Then remove the lower end of the pipette from the water and allow air to flow through until the pipette is perfectly dry. If the filter pump is not at hand, the pipettes
must be cleaned by hand. Cold water, then hot water, then
95% alcohol, then ether are used in turn and then air is blown through
the pipette until the tube is dry. Draw the fluids into the
pipettes by suction through the rubber tube, then expel them by
using a rubber bulb. Air is drawn into the pipettes and then
is expelled by the rubber bulb. The final rinsing with alcohol
and ether is intended to hasten the drying process because these
fluids evaporate rapidly. (They are not necessary when the
filter pump is used because the time question is not important to
the filter pump, and hot water is not usually limited.) The
cleaned pipettes may be kept in any place which is free from dust
and moisture. Several types of blood lancet are on the market. A glove needle, which is triangular in form, makes an excellent lancet for the purpose of securing blood. K The triangular needle makes a cleaner puncture than does the ordinary round needle, though any ordinary sewing needle can be used in emergencies, after it has been sterilized. A surgeon’s needle is convenient and easily cleaned. Half a steel pen, properly cleaned and sterilized, is very good. Lancets which are provided with a spring are rather less easily cleaned. They have the advantages of producing wounds of equal depth, and they can be set to make deeper wounds if the skin shows marked pallor, or shallower wounds if the patient is a bleeder or if the skin is very red. The needles without a spring depend upon the skill of the operator for the accuracy of gauging the depth of the wound. Needles and lancets are best sterilized by rinsing
in warm water, then dipping them into carbolic acid. After
another rinsing in sterile water they may be dried on sterile cotton
or left to dry. In order to count the blood cells it is necessary that the blood be diluted. For the white cell count acetic acid, in solutions of 3% to 8% is commonly employed. The red cells are destroyed by the acetic acid, which also causes the nuclei of the white cells to show more distinctly. Various stains may be added to the acetic acid solution and these stain the white cells. In our laboratories no stain is used; this is partly in order to allow any pigmented granules of the blood to be visible. For the red cells many solutions are employed. In our laboratories normal salt solution is tinged with a few drops of methylene blue and this is used for diluting the blood for the red cell count. The cells remain in fairly normal condition for several hours, which is all that is required for ordinary cases. If the blood must be kept in the pipette for some time before it can be counted, Hayem’s solution is excellent, and this is the one most used. The following is the formula; Distilled water, 100 c.c. For ordinary cases, a very mild soap solution at room temperature followed by sterile tap water for rinsing is preferred as mode of cleansing. These are provided in convenient small bottles. For cases requiring efficient sterilization the solutions to be employed are selected according to the nature of the infectious agent suspected. The methods of surgical procedure are followed, and this sterilization must precede the taking of the blood by an hour or by several hours, according to the manner in which the skin reacts to the sterilization process. The use of alcohol, ether or any solution which reddens or which pales the skin, or which causes any sensory irritation worthy the name is bad and interferes with the accuracy of the count. Cotton or gauze pads for applying the cleansing
solutions should be sterile and at least five pads of gauze or bits
of cotton should be ready. Very little blood is required for making ordinary cell counts, and this is, preferably, capillary blood. Capillary blood is easily secured, is alike all over the body, is modified only by vasomotor activity except as the entire blood picture changes, and the tiny wound made by securing the blood from the capillaries heals immediately. The side of a toe is the selected site for taking blood from babies or small children, for the following reasons: Vaso-constrictor control is not abundant; the skin is thin; the foot is easily held, if the child is awake, and the toe is not apt to be irritated or infected afterward. If the child is asleep the process may not awaken him. The lobe of the ear is selected for adults for the following reasons: The lobe of the ear is not visible and the patient cannot see the blood; the vaso-constrictor control is not abundant; the skin is thin, sensory nerves are scanty, and the lobe of the ear is not subject to later irritation. In the case of an adult the worker stands at the left side and to the rear of the patient. The patient sits comfortably by the side of a small table upon which the necessary equipment is placed conveniently for the worker. The patient cannot see the blood being taken. If the patient is in bed he should turn the face away from the worker, thus permitting an ear to be accessible. If really efficient sterilization of the skin should be necessary, this should have been done at least an hour before the blood is to be taken, and the skin protected by cotton or gauze in the interim. In most cases the washing which removes dust and some desquamating epithelium is all that is necessary. The ear lobe should be gently mopped with sterile water, dried, and protected against dust if any delay should occur. The lobe must not be rubbed or handled or caused to redden perceptibly by the cleansing. Such cleansing agents as alcohol are to be avoided because they dilate the capillaries and modify the counts. If the patient has come in from the street a mild soap solution may be used for cleaning, then the ear washed with sterile water and dried with sterile cotton. If the cleansing should cause visible reddening of the skin the ear should be protected with sterile cotton and the patient allowed to rest for ten minutes or more, until the normal circulation has been well established in the ear. Prick the skin with lancet or needle. Notice the emergency of the first drop, its size, color, and the manner in which it flows upward into a round drop or spreads around over the skin. Wipe away this first drop. Take the blood for the white cell count first. This is partly because the white cells stand longer without being modified, partly because the white cells are more quickly modified by the faint vasomotor change due to the slight irritation of the prick, and partly because a larger drop is used for the white cell count. (The first drops are usually rather larger than later droops of blood from so small a prick.) If the white blood cell pipette is used, draw the blood to the .5 mark, then very quickly draw the acetic acid solution to the 11 mark. Rotate the pipette gently while the solution is being drawn into the bulb. Close both ends of the pipette with thumb and finger and shake gently from side to side (not endways) to mix the blood with the solution sufficiently to prevent clotting. Lay the tube aside, or give it to an assistant who will continue the shaking, gently from side to side. If the tube is shaken vigorously the cells may be fragmented and if it is shaken endways the cells may be forced into the capillary tube, or the mixture of cells and solution may be forced into the dilatation above the bulb; if the contents of the bulb were well mixed the latter accident would be of negligible importance, but if part of the contents of the bulb were forced into the upper capillary tube of the pipette and the upper dilatation before the mixing is completed serious error would be present in the count. In our laboratories an erythrocyte pipette is used for the white cell count. Draw the blood to the 1 mark on the pipette, then draw the acetic acid solution to the 101 mark; mix the contents of the bulb by gentle shaking in a sidewise direction, as already directed. The advantage of this greater dilution lies in the absence of the debris caused by the destruction of the red cells; they are all completely destroyed by the greater amount of acetic acid and completely dissolved in the greater amount of fluid. The white cells are more easily recognizable. In cases of leukemia and leucocytosis, this method is necessary for accuracy, and we think it preferable in all cases. Next, fill the red cell pipette for the red cell count. Draw the blood to the .5 mark on the capillary tube, then draw the diluting fluid for red cells to the 101 mark. Shake as directed for the white cell pipette. Lay the tube aside until the count is to be made. The most frequent errors, in making the dilution, are these: The end of the pipette may be allowed to reach the air, either by being raised too high or by being pushed through the drop of blood. The entrance of air into the tube causes the “breaking” of the column of blood, or, if the air is admitted with the diluting fluid, bubbles are formed. In either condition a fresh drop of blood must be taken in a clean pipette and greater care observed. Sometimes the drop of blood is too small; if the blood has been secured with difficulty and if the column of blood reaches to the first mark below the .5, the count may be completed, and the correction made in the final computation. If it is practicable to secure another drop of blood this is a more satisfactory method. Sometimes the pipette is pressed against the skin too firmly, and the blood does not ascend into the tube no matter how hard the breath is drawn. In such a case, the tip is apt to be slightly lifted, and the blood is apt to ascend suddenly through the tube and into the bulb. When too great force is employed in the inspiration which draws the blood into the tube, the blood is apt to rush too rapidly upward, fill the tube, and sometimes the bulb itself. The pipettes must be rinsed immediately else the blood may coagulate within them and cleaning becomes an extremely difficult matter. Take next the blood for differential counts and for such other tests as may be indicated. The pipettes of diluted ordinary blood may rest for a time, even for an hour or two, without harm, but in cases of unusual fragility of the cells changes may occur more quickly. It is best in all cases to make the actual counts as quickly after the blood is taken as is practicable. After the blood has been taken for the various
tests indicated cleanse the ear lobe of the patient and note whether
there is any indication of persistent bleeding. The wound
should be closed by this time, if it is not handled. It may
be kept open for five minutes or more by the slight manipulations
necessary for wiping away the preceding drops of blood in preparation
for further tests. If the counting chamber has a circular moat, the following technique is to be employed. Place the counting chamber on a perfectly level surface, and the cover-glass by the chamber. Close the ends of the pipette containing the red cells and shake again, using a side-to-side staccato movement, rather gently, about one hundred times. Avoid the endwise movement in shaking. Remove the rubber tube and attach a stiff rubber bulb to the upper end of the tube, force out about four drops of the mixture and discard this. This is in order that the diluting fluid in the capillary tube, which contains no cells or, at most, only a very few cells, may not be used for counting. Let the fifth drop begin to form at the end of the capillary tube, and touch this to the surface of the counting chamber, near the rule area. Avoid allowing the end of the pipette to touch the surface of the counting chamber; if the glass should touch the ruled area the markings might be scratched. The amount of fluid necessary must be learned by experience; it is about half as much as would drop from the end of the capillary tube of the pipette if the pressure on the bulb should be increased. Holding the cover glass by its edges, lower it slowly over the counting chamber; if a bubble of air happens to be caught in the fluid clean the counting chamber and take another drop from the pipette. If the counting chamber itself is not filled with the fluid, clean the chamber and take another drop of fluid. If the amount of fluid is too great, so that the moat is filled across, the cover glass cannot fit accurately and the count will be too high. In this case clean the counting chamber and take another drop of the fluid. Only experience can win accuracy. If the counting chamber has an H-shaped moat, the procedure is somewhat less difficult. Place the cover glass on the slide first, and note that the cover glass fits the outer raised part of the chamber accurately. The test of this accurate fitting is best made by means of the phenomenon known as “Newton’s rings.” These are concentric bands of rainbow colors which appear when two glass surfaces are so closely in contact that the difference between their surfaces is not more than the distance measured by wave-lengths of light. When the slide containing the counting chamber, covered by the perfectly plane cover glass, is held slantingly to the axis of vision, in changing position, these rainbow colors should be visible. This means that the fitting is fairly accurate. If the rings are not visible press firmly upon the cover glass; this may cause the necessary approximation of the two surfaces. If the rings are then visible, and remain so when the pressure is removed, the cover-glass fits accurately. If the rings disappear when the pressure is removed, there is some dust or moisture present; clean the cover-glass and the counting chamber and repeat the process. Occasionally a counting chamber is found upon which it is impossible to secure the rings. Such a chamber may still be accurate so far as the counting is concerned. Another test for accuracy lies in the fact that two perfectly dry plane glass surfaces closely approximated adhere firmly. Having placed the cover glass I position turn the slide over gently; if the cover-glass adheres, the fitting is probably good. If the cover-glass falls off, the fitting is not good. Cleanse and dry the cover-glass and the slide and try again. This method of testing is less accurate than the finding of Newton’s rings but it is useful in the use of certain chambers. A trace of moisture causes adhesion, separates the surfaces and seriously increases the count. If Newton’s rings cannot be produced on a certain counting chamber, the chamber may be tested by another in which these rings are produced. Take the same pipette of blood and make counts of the red cells on both chambers, in one of which Newton’s rings have been produced, and in the other only the adhesion of the cover glass has been found well marked. If the counts made on the two chambers agree within the limits of accuracy permissible for the method (not more than 3% of the total number of cells counted) then the second chamber is accurate enough for all clinical purposes and its use can e continued. If the two counts do not agree, the imperfect counting chamber should be returned to the maker. Having secured the proper fitting of cover glass and counting chamber, shake the red cell pipette as before using at least 100 vibrations. Discard the first four drops, and touch the fifth drop to the edge of the chamber at the edge of the slide. The fluid runs beneath the cover glass and fills the ruled area at once, by capillary attraction. If any fluid runs out and fills the moat, thus lifting the cover glass and increasing the depth of the counting chamber, clean the cover glass and the counting chamber and take another drop from the pipette, after shaking it as before. With both types of counting chamber, the later processes are alike. Allow the filled chamber to rest for three minutes or so in order that the cells may settle to the bottom of the counting chamber. The cells of normal blood settle quickly; those of anemic blood and blood which is from persons with certain diseases settle slowly. If any cells are still floating when the count is begun, this must be deferred for a time, until all the cells rest upon the bottom of the chamber. Accurate counts are not possible when any cells are floating, because they are not in focus at the same level. Use a 1/6 objective and a one inch eyepiece on the microscope. Find the ruled area and select a large square containing five rows of five each of the small squares. Count all the cells in the upper row of five small squares and note the number found. In this count include every cell which touches the upper line and the left hand line, and the intersection of these; also at the intersection of the right line and the upper line; exclude form the count all the cells which touch the right line and the lower line, the intersection of these, and the intersection of the lower line and the left line. Repeat for each of the five rows of small cells. This makes a column consisting of five numbers, each of which indicates the number of cells found in five small squares. The sum of this column indicates the number of cells in 25 small squares. Select another area containing twenty-five small squares arranged in the same way, and repeat. For ordinary work four such columns, indicating the number of cells in 100 small squares, is sufficiently accurate. In cases of anemia, count the cells in 400 to 800 small squares, using two or more counting chambers of diluted blood. Compute as follows: Multiply the total number of cells counted by the dilution, and this by 4,000 (which is the cubic content of each square in terms of a cubic millimeter, since each small square is 1/20 by 1/20 by 1/10 millimeter in size). Divide by the number of square counted. Example, 625 cells were found in 100 small squares, the blood being diluted 200 times. The computation is: 625 X 200 X 4,000 divided by 100=5,000,000 cells per cubic millimeter. Or, the average number of cells in each square is 6.25. Each square is 1/4000 cubic millimeter in content, so that there must be 25,000 cells in each cubic millimeter of the diluted blood. Since the blood was diluted 200 times, the number of cells I one cubic millimeter of undiluted blood is 5,000,000. The counting of the white cells follows a similar process. The same tests for accuracy of fitting the cover glass and for placing the drop of fluid in the counting chamber are employed. If the white cell pipette was used, count the number of cells present in at least 800 small squares. If the red cell pipette is used count the number of cells present in 4,000 small squares. In either case the computation follows the same method,--total number of cells counted, multiplied by the dilution, multiplied by 4,000 and divided by the number of squares included in the count. For example, if the white cells pipette has been used, with a dilution of 1:10, and if 300 cells were found while counting 800 squares, the computation is: 300 X 10 X 4,000 divided by 15,000 white cells per cubic millimeter. If the red cell pipette was used, with a dilution of 1:100, and if 120 cells were counted within 4,000 small squares, the computation is: 120 X 100 X 4,000 divided by 4,000= 12,000 ells per cubic millimeter of blood. Always count at least 100 cells, even if it is necessary to cover 16,000 or more small squares in the counting. In doubtful cases count 1,000 to 5,000 cells, using several pipettes if necessary. Many counting chambers have large squares around the area of rulings for the small squares; the area of these large squares is easily understood by following the lines which form the small squares outward. By using these larger squares the white cell count is easily and quickly made, even though 8,000 small squares are necessary for accuracy. The computations are made upon a basis of the small square in all cases. This avoids any possibility of error due to the use of different units. This actual count gives the number of red cells and the number of white cells per cubic millimeter of blood. Attempts have been made to substitute estimations of the blood cell volume for studies of the blood cell count, since it is often thought more important to know the total mass of hemoglobin-containing protoplasm than the manner in which this hemoglobin is arranged in cells. On the other hand, the manner in which the hemoglobin is divided into cells is an important factor in the oxygen carrying function of the hemoglobin, since hemoglobin arranged in a comparatively large mass with relatively small surface area is exposed to the air in the lungs less efficiently than an equal mass of hemoglobin divided up into smaller masse with relatively greater surface area. The hematocrit is a form of centrifuge in which small tubes are placed in opposite arms; each tube has 100 equal divisions. The technique is simple: Secure a large drop of capillary blood by the method outlined for taking the blood for counting. Place a rubber tube over one end of the glass tube; draw the glass tube perfectly full of blood. Cover a finger with Vaseline, and cover the free end of the glass tube immediately. Remove the rubber tube; place the glass tube in the arm of the hematocrit. Place the other glass tube, filled with water, in the arm of the hematocrit, to balance the machine. If the blood of two patients is to be centrifugalized at the same time, make marks with a grease pencil upon the outside of each glass tube for identification. Start the centrifuge, gradually attaining the high speed within half a minute. Stop the centrifuge and note the height of the column of red cells at one minute intervals. When two successive examinations give identical findings, stop the centrifuge. Each division of the glass tube represents 100,000 cells, if the cells are approximately normal in size and in hemoglobin content. The column of cells is approximately equal to the column of plasma in normal blood. The blood plasma can be used for the determination of bile and other pigments. A very thin layer of fatty globules is occasionally seen at the upper end of the tube, in lipemia. In the anemias the number of cells in each division of the tube may vary very greatly, so that in cases in which accuracy of cell count is important the method has no value. As a method of determining the actual volume of hemoglobin-carrying protoplasm the method is of value. The time required for complete settling of the
red cells has been studied by many workers, and this has been shown
to vary greatly in different diseases. The differential count is made in order to determine the relative numbers of different types of white blood cells. These cannot conveniently be differentiated in the process of making the actual count of white cells. An attempt to use diluting fluids which give a differential stain in the counting chamber is not satisfactory, and in order to secure accuracy by thus combining the actual and the differential count it would be necessary to count the cells in several hundred chambers. The differential count is made of thin smears of undiluted blood, stained in some manner which affects different types of cells variably, according to their chemical constitution. By this means it is easy to recognize several different classes of white cells. Slides used for the differential count must be clean but they need not be sterile. Rather thick slides are more convenient. New slides are greasy and must be well washed, first in warm soap solution, then in hot water. A second soapy washing is often necessary. They can be kept in acid bichromate solution made approximately as follows: exact proportions are not necessary:
Potassium bichromate
10 grams In routine work take the drops which flow after the blood has been taken for an actual count. If only the differential count is to be made, prepare and prick the skin as directed for the actual count. Have ready microscope slides which are perfectly clean and perfectly dry, at least eight slides for each patient. Touch one end of a slide to the top of a drop of blood, then touch this blood to the end of another slide. Allow the drop of blood to flow along the angle between the two slides for a second or two. Push the first slide along the surface of second slide, away from the drop of blood. The blood follows the moving slide and leaves a thin, even smear upon the second slide. Never push or pull the first slide along after the drop of blood, because thus many cells are injured and there is a tendency for some cells to cling to the first slide and thus to accumulate in groups. Repeat this process for each slide. If the amount of blood is scanty it may be necessary to take only six slides; if these cannot be secured prick the skin again. Never take less than six slides of blood for a differential count. If the condition suggests leukemia, severe anemia or the need for any special study it is best to take twenty slides or more. As the smears are made lay the slides, blood side up, upon a flat surface until they are perfectly dry. Then put them into an envelope already marked with the name of the patient and the physician and the date and the hours of taking the blood. These smears keep almost indefinitely and they can be stained by different methods for special study of different structures or inclusions. If the blood arranges itself in circular areas or rings, the slide was greasy. If the blood forms stripes or bands, the motion was jerky. It is necessary that the second slide be moved along the first in a steady, even, deliberate manner. If there are threads of fibrin or small thick places in the smear, the blood was partly coagulated. If the smear is too narrow and thick, the second slide was moved before the blood spread along the edge. If the smear is too thick and spreads over the entire slide, the drop of blood was too large. If it is too thin and spreads over too small an area, the blood drop was too small. If two or more specimens are under observation
at the same time it is necessary to mark each slide for identification.
This is best done with a lead pencil. Using a pencil with
a rather soft lead, write an initial or an identifying number upon
one end of the smear, near the end of the slide, before the slide
is stained. The pencil ruins the blood cells over which it
passes and leaves a small amount of the lead; fixing the blood on
the slide makes the lines as produced permanent. By making
the marks at the end of the slide they do not interfere with the
counting, since this is done in the central area. Many different methods of staining blood smears for the differential count are in use. They include so many stains, each with so many modifications, that any satisfactory description of them all would be too long for this book. For example, about twenty different methods of using the eosin-methylene blue stain devised by Romanowsky have been described, and each method has its advocates. The method used in our laboratories is different from any of those described elsewhere, but it gives accurate and delicate staining of the structures included in an ordinary differential count, and it is easily modified so that good pictures can be secured of atypical blood. Solutions required are: Eosin yellowish, 0.5 gram in 100 c.c. methyl
alcohol. This fixes the blood and stains the acidophile structures.
The methylene blue stains the nuclei and the basophilic structures of the protoplasm. It stains also the malarial and certain other parasites. Take one of the slides already prepared and dried. Place the slide on a level surface, smear side up, and drop upon it several drops of the eosin solution. Let stand fifteen or a few more seconds; rinse gently in tap water. Drop upon it a few drops of methylene blue solution, enough to cover the smear very abundantly; let stand a minute or a little more; add a few drops of tap water and allow to stand on the slide about two minutes, rinse with tap water. Drain, allow to dry in air thoroughly, and examine, using oil immersion objective, and one inch eyepiece. Or, after rinsing with water, mount in water under a thin cover glass and examine, using a dry one-tenth objective and one inch eyepiece. The one-eighth objective does not magnify sufficiently for the finer details of cell structure to be visible. For careful study of the cell structures, a one-eighteenth objective, oil immersion, is useful. In our laboratories the dry one-tenth objective is used for ordinary work and the one-eighteenth objective for careful study of selected cells in unusual cases. See also “Other Staining Methods,” Page 325. Make a general survey of the smear in order
to note the type of blood cells present. Have ready a sheet of paper with columns arranged for each lass of blood cells,--large hyaline, small hyaline, mononuclear neutrophiles, polymorphonuclear neutrophiles, eosinophiles, basophiles, for ordinary blood. For abnormal blood other columns are required for normoblasts, megaloblasts, poikiloblasts, microblasts, reticular red cells, malarial parasites, and other peculiarities of the red cells which may be of interest in the particular case, and for myelocytes of each type found in the blood being examined. The general survey has indicated the columns required. As the count progresses other columns may be added at any time if other cell types are found. Begin the counting at one edge of the smear, move the slide, by means of the mechanical stage, so that the field is brought toward the observer (apparently) as far as the edge of the blood smear, and is carried as far to the right as the edge of the blood smear, or as far to the right as the limit of the mechanical stage permits. Then move the slide toward the left, noting each cell and making the notation in the column devoted to that cell type. When the slide cannot be moved further to the left, or when the limit of the smear in that direction has been reached, move the slide away from the eye the diameter of one field, so that some selected red cell which is barely in vision at the lower edge of the field is moved just beyond vision at the upper edge of the field. Then move the slide toward the right, counting and listing the cells as they appear in successive fields. Continue in this way, moving back and forth across the slide, until all the cells have been counted on the slide, or until the desired number of cells has been listed. With practice it becomes easy to carry the counts in mind, and to make the notations in groups of twenty, t en or five, as the case may be. In our laboratories, neutrophiles are counted in groups of twenty cells, small hyalines I groups of ten, and other cells as units. These habits are made for the sake of accuracy, convenience and speed of counting. Each worker develops his own customs. Count until the total number of cells counted is at least five hundred, in cases which present no marked variations from the normal and which show no marked irregularity of distribution of the cells. In abnormal cases at least one thousand cells should be counted, while in cases used for special study, in unusual cases, and in all the leukemias two thousand to twenty thousand cells or more should be counted. In one of our cases of leukemia, with an actual count of 250,000 leucocytes of which 80% were myelocytes of different forms, it was necessary to make a differential count of 50,000 cells in order to secure satisfactory accuracy. The number of cells to be examined depends upon the fact that successive counts of any selected number give almost or quite identical results. In normal blood successive counts of one hundred cells each give approximately identical figures for lymphocytes and for neutrophiles, but may give very different figures for eosinophiles, while basophiles may not be found at all. Successive counts of 100 cells may give eosinophiles of ten per cent in one 100, and no eosinophiles at all for another 100. (Eosinophiles have a tendency to be in groups even in the best smears of blood.) Counts of successive five hundreds of approximately normal blood give satisfactory accuracy for such blood. In acute cases in which diagnosis must be made
quickly, as in suspected pyogenic processes probably requiring speedy
surgical interference, a differential count of two hundred cells
may serve the necessary purpose and enable treatment to be initiated
quickly. But unless there is urgent need of haste, every count
should include at least five hundred cells. Even when this
haste is imperative, the count of five to ten hundred cells should
be carried on later in order that accurate findings may be kept
on record for later study and comparisons. When the total number of cells in all columns reaches one thousand, if this is the number counted, add each column and divide by ten. This gives the percentages of each column. For example, if the neutrophile column has in it 678 cells, then there is 67.8% of neutrophiles in the patient’s blood. If his actual count was 5,000 white cells per cubic millimeter, then he has 3,390 neutrophiles per cubic millimeter of blood. If there are 11 eosinophiles in that column, he has 1.1% eosinophiles, or 55 eosinophiles per cubic millimeter of blood. If the number secured by determining the actual number of any cell type from the percentage and the actual count gives a fraction of a cell, the nearest number is taken. For example, if a patient has an actual count of 5,700 leucocytes, and his large hyaline cells make up 4.3% of these, the computation gives 245.1 large hyaline. The report should indicate 245 large hyaline cells, because the limits of unavoidable error in this work are too large for us to report finding a difference of one cell in ten cubic millimeters. While making the differential count of the leucocytes, certain other structures may also be counted. Columns may be arranged for red cells containing malarial parasites, for example, or for normoblasts, megaloblasts and other atypical red cells. With careful staining the reticulated red cells may also be enumerated. A column must be arranged for each structure to be counted. As the leucocytes are counted, such other structures are also counted and the figures placed in the column allotted to them. They are not included in the sum of cells to be counted, however. Only leucocytes are to be counted in making the total of five hundred or a thousand or more upon which the percentages are to be computed. The total leucocytes must be 100%, and the other structures, not being leucocytes, must not be included. After the leucocyte count has been completed, as already directed, those other structures are considered. For example, in a patient with a total blood count of 3,000, there was 10% of large hyaline cells, that is, 300 per cubic millimeter. The differential count was based on the examination of 1,000 cells, so that 100 cells were in the column devoted to large hyaline cells. While these cells were being counted, 25 red cells were found which contained a malarial parasite, hence there were 75 malarial parasites within red blood cells per cubic millimeter of blood. That is, the amount of blood which contained 300 large hyaline cells also contained 75 cells containing malarial parasites within red cells. Extracellular malarial parasites were not included in this count. In another case, with an actual leucocyte count of 2,500 cells, the neutrophiles included one half, or 50% of the total count. The differential count was based on 1,000 cells examined. While making the differential count thirty-four megaloblasts and twenty normoblasts were noted. That is, there were fifty normoblasts and eighty-five megaloblasts per cubic millimeter in this blood from a pernicious anemia patient, taken three days before his death. In this same way, the number of several other
structures can be computed on the basis of the differential leucocyte
count, and much useful information gained thereby. In making a differential count of the blood in certain leukemias and leucocytosis, when sometimes a great predominance of one type of cell is present, it may facilitate the process and increase the accuracy of the differential counting if the work is done in two stages. First, make a differential count of two groups only, the predominant type and all others. Examine and list 1,000 cells or more in this first stage. Stain another slide and make a differential count of all cells except the predominant type. Examine and list 500 or more of the cells for this count. Determine the percentage of the predominant type of cell by the first stage of counting, and of the other cells by the second counting. For example, in one of our cases of lymphatic leukemia the small hyaline cells made up 97% of the total blood count. The first stage of counting gave 970 small hyaline cells and 30 cells of all the others together. It is evident that this differential count of 1,000 cells could not give any accurate differential count of the 30 cells. The second stage disregarded the small hyaline cells altogether, and 500 cells of the remaining types gave accurate percentages of the granular cells and the large hyaline cells. Of the 500 cells examined in the second count, there were 40 large hyaline, 52 mononuclear neutrophiles, 200 polymorphonuclear neutrophiles, 120 eosinophiles, 88 basophiles, and when these percentages are taken for the 3% of “other cells” of the first stage of counting the final results were as follows (omitting the third decimal): Total white cell count, 120,000 In cases of marked neutrophilic leucocytosis
and in cases of monocytic angina this two-stage method of counting
is very much more accurate and more convenient than the ordinary
method. Iodophilia is of little significance when taken alone. When employed with other clinical and laboratory findings, it may give very useful information. The older method of staining with iodine-gum preparations has been superseded by the staining with the vapor of iodine. A wide-mouthed, closely stoppered jar is kept for this purpose. About one gram of iodine crystals is placed in this jar. Blood smears freshly made after the manner already described for the differential count are placed in the jar, smear side exposed to the vapor of the iodine, and allowed to remain for five hours or more. The slides do not over-stain, and they may be left for several days without harm. One hundred leucocytes should be examined, and if iodophilic granules are not found, the reaction is negative. Note whether the protoplasm of the white cells
is diffusely stained and list such stained cells as iodophilic.
Note whether granules are free in the plasma or are within white
cells; if so, whether they are most abundant within the hyaline
cells or the granular cells. One of the slides prepared for the differential count can be employed. If the nuclei are not perfectly distinct, the slide should be floated with a watery solution of methylene blue for two minutes, then washed and again mounted in water. If the nuclei are still not distinct, the smear may be washed in N/100 solution of sodium bicarbonate, then the methylene blue stain repeated. The smear should be quite thin for accurate and convenient counting. Have a sheet of paper with columns numbered from 1 to 5. Rarely columns 6 and 7 will be required. Begin at one edge of the smear, as in the differential count, noting the number of nuclei in each neutrophile, but disregarding all other blood cells. Count in this way the nuclei in 100 neutrophiles. If a cell contains two nuclei, place a mark under column 2, if it has four nuclei, place a mark under column 4, and so on, until 100 cells have been counted. (Plate XIV). In counting the nuclei, a ring-shaped nucleus, even if slightly beaded in appearance, counts as a single nucleus. If the nuclear masses are united by a band, they should be counted as one. If they are united by a very thin filament of nuclear substance, they should be counted as two. If any cell has its nuclei piled one above another, so that it is impossible to determine the number of nuclei within it, it may be passed without counting; but if more than two or three such cells are found, the count must be repeated, using a thinner smear, for the higher counts will be those most often passed, under such circumstances, and the findings will thus be lower than the correct number. Add each column. The sum of the cells of column 1, plus twice the cells in column 2, plus three times the cells in column 3, plus four times the cells in column 4, plus five times the cells in column 5 and six times the cells in column 6, if any, equal the sum of the nuclei in 100 cells. This divided by 100 gives the average number of nuclei for each neutrophile. For example, in a certain specimen of blood there are: 10 cells having 1 nucleus;
or 10 nuclei in all; These 100 cells have, altogether, 157 nuclei; or an average of 2.57 nuclei per cell. The neutrophilic nuclear average of this blood
is 2.57. The nuclear average in normal adult human
blood is between 2.45 and 2.55. In normal children the nuclear
average varies from 2.00 to 2.4, according to age. Endothelial cells. A stain for differentiating between mononuclear neutrophiles and monocytes supposed to be from the reticulo-endothelial system outside of the bone marrow is as follows: Solution: 80 c.c. 100 alcohol Uses dried blood films prepared as directed for the differential count. The films should not be more than a few hours old. Place the slide on a level surface and cover with six to eight drops of the solution. Allow to stain and fix for half a minute. Add an equal amount of distilled water and allow to stain for five minutes. Rinse several times with water. Cover the slide with basic fuchsin solution (0.01%) to counter-stain for 2 minutes. Rinse with water; remove water with filter paper; dry in air; examine with oil immersion lens or mount in balsam. Basophilic elements, including nuclei, basophilic granules, basophilic protoplasm, erythrocytes and platelets take various shades of red and pink. Eosinophile granules show a peculiar circular staining so that the granules look like rings. Neutrophilic granules and the finer granules of the endothelial cells show bluish tints. The difference between the neutrophiles and the endothelial cells lies in the characteristic nuclear structure and larger granules of the neutrophiles, and the characteristic nucleus and the smaller blue granules in the protoplasm of the endothelial cells. The stain is useful for its purpose. Pappenheim’s solution is adapted especially to a study of nucleated red cells. Solution:--Take a saturated solution of methyl
green.
30 c.c. The nuclei of the normoblasts and nuclear fragments stain a clear blue, while basophilic granules within the red cells stain bright red. Ehrlich’s traced stain is now little used. It consists of equal parts of saturated solutions of indulin, nigrosin and aurantia, mixed together after a difficult and tedious technique. It can be purchased in powdered form. Ehrlich’s triple stain also is somewhat difficult
to prepare. It may be purchased ready made up, though the
commercial preparations are not usually very successful. Grubler’s
stains are commonly used. The solution is made as follows:
Saturated aqueous solution orange G
13.0 c.c. This mixture can be used at once, but it seems to improve during the few days following preparation. It deteriorates within a few weeks, more rapidly in the light or if the bottle is shaken. To stain—Fix slides with heat and place on a level surface. Cover with solution taken from about the center of the bottle containing the stain, using a glass pipette for the purpose. Never shake the bottle. Leave stain on slide for three minutes or more; the slides do not over-stain if left twenty minutes. Rinse with water, remove excess water with filter or blotting paper, dry, examine with oil immersion or mount in balsam. Erythrocytes stain yellow or buff. Normoblast nuclei stain a very dark green, almost black. Nuclei of leucocytes stain dark green but not so deep a color as the normoblast nuclei. Fine granules of the neutrophiles and the endothelial cells stain lilac or pale purple. Coarse granules of these cells and of the eosinophiles stain crimson. Basophilic granules do not stain. This stain is useful for distinguishing certain types of granules but it is not useful for general work. It is very difficult to secure good stains in cases of leukemia. Occasionally a patient appears whose blood refuses to take the Ehrlich triple stain, for no perceptible reason. Leishman’s stain is best purchased in powder form. For use make a solution of 150 mgs. of the powder in 100 c.c. of pure methyl alcohol. Smears are best made on cover glasses for this stain. Use the technique given for making smears on slides. Place a cover glass, smear side down, in a watchglass. Drop the stain into the watchglass until the cover glass floats. Allow to fix and stain for three minutes. Add an equal amount of distilled water, and allow to stain further for one minute. Remove the coverglass and wash in water, drain on edge until dry, examine with oil immersion lens. Or, mount in water and examine with dry one-tenth objective. The stain gives a fairly good picture when freshly made. After about ten days standing it gives a differential stain for the azur granules also. Red cells take a coppery tint; in polychromasia some cells are pinkish. Nuclei are in shades of reddish purple or purplish red. Cytoplasm is bluish or blue. Eosinophile granules are coppery red. Neutrophile granules take a pinkish color. Basophile granules stain purplish or reddish purple. Azur granules are cherry red. The stain is fairly good for general differentiation. Leishman’s stain has bee simplified and modified in many ways. Wright’s stain has been developed from Leishman’s stain. Its preparation is rather difficult and the resulting powder not always successful. The powdered stain, which is a precipitate formed by combining eosin yellowish with methylene blue under certain conditions, can best be purchased. This powder is to be dissolved in methyl alcohol, 1.5 gm. powder to 100 c.c. methyl alcohol. The solution keeps for a month or more. Wright’s stain is useful for general differentiation. The technique of staining is: Place the dried slide on a level surface. Flood with the methyl alcohol solution, which fixes and stains the slide at the same time. Allow the stain to stand on the slide one minute. Add an equal amount of distilled water, and allow to stand for two or three minutes,--the longer period giving a deeper blue stain, but eosinophilic granules are more deeply stained in the shorter period of time. Longer standing than three minutes may cause a precipitate to be formed. Rinse in water for about half a minute. The thinner areas should be pinkish or yellowish in tint. Experience is necessary to determine the exact degree of differentiation which gives best results for each blood specimen. Mount in water and examine by means of a dry one-tenth objective, or dry and examine by means of an oil immersion lens. Red cells show pinkish or yellowish. All nuclei are blue or purplish blue, varying in shading for different types of cells. Neutrophile granules are pinkish or pale purplish in color. Eosinophiles are brilliant reddish pink or cerise. Hyaline cells show blue protoplasm which may be very dark or rather pale. Platelets are blue or purplish. Malarial parasites are blue with darker purplish, reddish or bright red chromatin. Mast cells show deep blue granules. The stain is fairly useful in general differentiation. Tap water used for differentiation increases the blue tints and this is often desirable. Giemsa stain is much simpler than the ;methods described, and it gives excellent differentiation. The formula is simple and the stain constant in quality. The powdered stain may be purchased or it may be made up as follows:
Azur II eosin
3.0 gms. Grind up the stains in the alcohol, using a small amount of the alcohol first When thoroughly mixed add: Glycerine, c.p. 175 gms. The solution keeps for several months, and sometimes much longer. The technique of staining is simple also. Place slides on a level surface, flood with
methyl alcohol for five minutes, drain but do not rinse. Put
fifteen drops of the stain on the slide, then add ten drops of distilled
water; stain for fifteen minutes. Rinse, drain, mount in water
and examine s usual. Or dry and examine with oil immersion
lens. They fade quickly in cedar oil; paraffin oil may be
used instead. One drop of half-saturated sodium carbonate
increases the staining of the basophilic elements. The use
of tap water instead of distilled water gives better differentiation
in our laboratories. This method of staining differentiates cells derived from lymphoid tissue from those derived from the red bone marrow. Three solutions are required: A. 100 c.c. distilled water 5 drops saturated aqueous solution sodium hydroxide 1 gram alpha-naphthol Boil this solution, cool, decant fluid from any residue which may be present. Allow to stand three days or more before using. This solution will keep a month or more. B. 100 c.c. distilled water. .5 gram basic paraphenylenediamine Mix without heat. Allow to stand at least twenty-four hours before using. This solution will keep a month or more. C. 100 c.c. distilled water 5 c.c. formalin Technique of staining. First mix together equal parts of A and B and filter. This mixture must be used within an hour or so. Fix dried blood smear in solution C for five minutes. Stain with the mixture of A and B for five minutes. Rinse, mount in water and examine, using dry one-tenth lens, or dry and use oil immersion lens. Cells derived from lymphoid tissue show no granules. Cells derived from bone marrow show blue granules. The reagents are difficult to secure and preparation of the solution is cumbersome. It is rarely of value in diagnosis but it has given some good results in research work. Merck offers simpler reagents; beta-naphthol-sodium
is sold in sealed glass ampoules as Mikrozidin. Solution A
is a 2% solution of Mikrozidin in distilled water. He supplies
also dimethylyaraphenylene-hydrochloride in similar ampoules.
Solution B is a 1% solution of this in distilled water. Equal
parts of the two solutions are mixed and the resulting greenish
precipitate filtered off. The further technique is the same
as in the original method. The oxidase granules appear brownish
or blackish by this method, instead of blue; the significance is
identical. Examination of the cells from bone marrow is easily made. Take a piece of rib or of any bone containing red marrow; make a fresh break if the bone has not been removed immediately before the smear is to be made. With forceps press upon the bone just beyond the break until a drop exudes from the broken end. Very quickly make smears from this drop upon slides, following the method used for making blood smears. Dry in air, and stain after any of the methods used in the study of blood smears. Vital,--or supra-vital,--staining methods are employed in the same way. If much fat happens to be present it may be necessary to remove this by flooding the slide several times with warm alcohol, ether and alcohol, and ether alone until the fatty globules are washed away. Bone marrow from adults is usually very fatty, while bone marrow from still-born babies, human fetuses and certain laboratory animals is usually free enough from fat to stain readily and easily. To demonstrate the nerve endings in bone marrow
it is best to use histological methods, for which see any text-book
on histology. The methods employed are too long for discussion
in this chapter. Very useful information can be secured from a study of the blood in the vital state; that is, during the lifetime of the blood cells on a warm slide. The conditions of the warm slide approach those of normal blood in the capillaries and the behavior of the cells and the formation of fibrin threads present pathognomonic variations in many instances. This study requires only a small amount of time and no expensive or complicated apparatus. The only difficulty is that the microscope and the patient must be brought together and that the examination must be made immediately after the blood is taken. If the patient is too ill to go to the laboratory the microscope must be taken to his bedside. Since there is no noise, odor or confusion associated with the work of warm-slide examinations it is not often annoying to the patient or to anyone else to have the work done at a small table beside the bed. The technique is simple but considerable practice
is necessary to attain skill in securing the correct amount of blood,
speed in beginning the observations, accuracy in watching several
factors at the same time and ability to distinguish between important
and unimportant variations from the normal conditions. Have ready a microscope with a one inch eye piece and a dry objective of high power, preferably a one-tenth, though the one-eighth can be used fairly well. The light must be strong but the field must be well limited by the iris diaphragm. Electric lighting is steady and is usually accessible. Gas light or daylight can be used efficiently, and the person who makes these examinations should be constantly in the habit of using these lights interchangeably whenever there is any probability that it may be necessary to rely upon them for emergency work. Several types of warm stage are on the market. These are commonly kept at the selected temperature by electricity. If electricity is not available at the bedside a heavy slide can be selected, warmed by water of the selected temperature, dried quickly and placed upon the stage of the microscope. The stage of the microscope can be warmed by placing any heated object on it for a few minutes before the examination is made. The cover-glass is best warmed by allowing it to lie upon the warmed stage of the microscope or by holding it between the palms for a few minuets. In using these make-shift methods it is necessary to be very careful to avoid too great heat. Connect the warm stage and place it on the microscope stage several minutes before the blood is taken. Have the slide and the cover-glass perfectly clean; put them on the warm stage. There is some difference in the time required to reach the select ed heat in different warm stages. Be sure that the slide and the cover glass are thoroughly warmed before the blood is taken. Have the light in place and be sure that the iris diaphragm is correctly adjusted for the study. The iris diaphragm requires delicate adjustment, because these living blood structures are not stained and are visible only on account of the variations in their refrangibility. Cleanse the lobe of the ear of the patient, using only sterile water without any unnecessary handling. Prick the skin with the usual sterile needle and wipe away the first drop of blood. Touch the flat side of the warm; cover-glass very quickly to the top of a drop of blood just exuding from the wound, place this on the warm slide, blood side down, put it on the warm stage and examine immediately. Let another person attend to the wound in the ear; if no other person is at hand allow the wound to remain untended until the first examination of the warm slide has been made. After a minute or so the observer may leave the microscope for the few seconds necessary to wipe off the lobe of the ear and thereafter the observations may be interrupted for a few seconds at a time if necessary. In observing the changes on the warm slide many factors are to be kept in mind and all of these must be watched all the time. Note the presence or absence of fibrin threads when the slide is first seen. These are lines of highly refractive material, best seen in the spaces between blood cells. If they are present when the slide is first examined note that fact and note any later time at which new threads are first visible. Note the amount of fibrin, the size and length of the threads and their contour. They may be even and regular, or swollen at long intervals or swollen at shorter intervals, presenting a beaded appearance. Note whether the threads lie in straight lines, apparently unrelated, or whether they radiate from cells or from groups of platelets. Note whether the fibrin is formed in net-like masses or in the normal straight threads. Note when the fibrin ceases to be increased in amount. With practice all these factors can be seen at a glance. Continue the observations until no further fibrin appears. Normal blood shows a few fibrin threads within four to six minutes, and the fibrin continues to form, slowly, for about five minutes longer. The condition characterized by very scanty or absent fibrin is called hypinosis, and this is present during starvation and in certain forms of malnutrition. Excessive fibrin formation is called hyperinosis, and this condition is present during pneumonia, acute rheumatism and in certain other inflammatory states. Hyperinosis is usually marked in malignancy, and in this case the threads are formed almost at once, are very irregular, often beaded, radiate and arranged in irregular net-like tangles. In pneumonia the threads are very speedily formed, are heavy, long and abundant, and are regular in contour; they may or may not be radiating, and they do not form nets in typical cases. The test is very useful in making an earlier diagnosis of pneumonia than is possible by any other means. Note the time at which the first movement is seen in a white blood cell, and note also the classification of the cell which first moves. Usually an eosinophile or a neutrophile moves first; rarely hyaline cells are first active. Eosinophiles are recognizable by their very large granules; neutrophiles by their fine granules and their irregular nuclei; hyaline cells by their glassy protoplasm and their round, central nuclei. No attempt should be made to distinguish between the different classes of hyaline cells in ordinary cases. Note the manner of movements. Protoplasm may flow steadily or it may flow rapidly for a few seconds, then slowly or may cease moving for a time; note these conditions. Granules or intergranular protoplasm may seem to be the most active part of the cell; note these relations. Pseudopodia may be extruded slowly or rapidly; may flow from two or from several sides or angles of the cells and may seem to be purposive or purposeless and even antagonistic in their activity; note these conditions. The pseudopodia may be extruded and the protoplasm flow into them thus causing the cell to change its location on the slide. The pseudopodia may be extruded, some protoplasm may flow into them, then the protoplasm may flow back into the cell and the pseudopodia be retracted, or pseudopodia may remain present and again the protoplasm flow into them; these various activities may seem purposive or may seem erratic. Pseudopodia may vary in form being long, short, slender, broad, heavy, flat, active or inert, and may present many peculiarities of structure and of activity. All such peculiarities should be noted. The activity of the cells may change during the period the slide is under observation; cells at first normally active, inactive or excessively active may show increased or diminished activity within a few minutes, and all such variations should be noted. Note the nuclear reactions. The nucleus may follow the protoplasm in its activity, or it may remain almost or quite immovable. Hyaline cell nuclei rarely move at all in normal blood and if they do move this is of interest. Neutrophile and eosinophile nuclei tend to follow the pseudopodia fairly quickly in normal blood; if they do not move at all or if they follow the protoplasm very quickly these facts should be noted. The time when the leucocytes show diminished activity and the time when the first inactive cells appear should be noted. Pay no attention to inactive hyaline cells, since these very often fail to show activity at any time, in normal blood. Abnormally they may die with pseudopodia still visible. Normal leucocytes live at least an hour and they may live several hours on the warm slide. Keep them under occasional observation at least forty minutes. If none, or only a few, are dead after forty minutes on the warm slide they may be said to “live well on the warm slide,” which means that they have at least moderate vitality. It is not necessary to watch the individual red cells. These undergo various changes on the warm slide. With evaporation of the watery content of the blood the r ed cells crenate. First there appears a bright spot on one edge of the cell, then another, then several and many such bright spots until the surface seems covered with thorns. Crenation may occur without evaporation, but it always occurs when the osmotic tension of the fluid surrounding erythrocytes is increased. The stroma and the hemoglobin of the red cells
may undergo various degenerative changes. Parasitic and bacterial
inclusions may thus be imitated. Suitable staining methods
quickly explain these structures, and it is best to disregard them
while making the warm slide examinations. Supravital staining. Interesting facts can sometimes be learned from a study of the blood cells which have received some stain which does not visibly affect their life and which have certain affinities for living granules or other cell structures. The same technique is employed for all these stains. A few crystals of the stain may be mixed with the fresh blood, and smears made from this mixture, or a solution of the stain is allowed to dry upon a slide and the blood smear made upon this stained surface. The following stains are employed for supravital staining: Brilliant cresyl blue, Janus green, methylene blue, toluidin blue, thionin, Capri blue, Nile blue, paraphenyl blue, neutral violet, neutral red, pyronin, fuchsin and safranin. Each of these stains gives some particular reaction which may be of value in the study of cell structure or cell inclusions, but each stain is characterized by some inefficiency or some source of error for other studies than that for which it is most useful. In other words, there is no known stain which renders all cellular structures and inclusions simultaneously visible. Brilliant cresyl blue and methylene blue give useful information in certain anemias and one of these should be used in routine blood examinations in order that immature red cells may be recognized. Either of these stains demonstrates the basophilic reticulation present in immature red cells, and the number of these is an important factor in the study of anemic bloods. In our laboratories the following technique is used: Prepare the slides in advance. Put a drop or a few drops of a solution of the selected stain on about the middle of a glass slide and allow it to dry, thus leaving a precipitate of the stain on the slide. If the stain is left in unequal masses or if there are clear spots left within the stained area the stain was not completely and perfectly dissolved or the slide was not quite clean; such slides should be cleaned, the solution investigated and the preparation repeated. These slides with the stained centers should e prepared in advance; they can be kept in slide boxes, well covered, for several days or indefinitely if carefully protected against dust and moisture. Just before the examination is to be made place the stained slide on the warm stage and take the blood as before, making such observations as are desirable in connection with the special stain being used. Brilliant cresyl blue shows the reticulation of the red blood cells vividly and its chief value lies in this fact. The reticulum of the younger forms of white blood cells shows more plainly than is the case with older cells. Methylene blue is useful occasionally.
Very thin deposits are necessary for satisfactory work. Nuclear
structures and basophilic granules are shown very clearly, and the
cells retain life for a long time after being stained with methylene
blue. Methylene blue may be decolorized and its color may
be regained as successive oxidation and reduction of the stain occurs,
and thus variations in the physiological activities of the cell
may be studied. As the cells die the nuclei take the methylene
blue present in small amounts with avidity not shown during life.
If too heavy a deposit of methylene blue is present the cells do
not show these reactions but the nuclei are deeply stained at once
and the cells die rather quickly. The study of the fibrinolytic ferment is one of the newest and most interesting methods of hematological technique. This study in its present form is limited to osteopathic laboratories. The method is as follows: Prepare fibrinolysis pipettes in lots of one to several hundred at a time. Have ready glass tubing of three millimeters diameter and about forty centimeters long. Heat to redness an area about five centimeters from the end of the tube and draw out to a capillary tube five to ten centimeters in length. Repeat this heating and drawing to a capillary tube, each time leaving a space of about three centimeters of the tube unchanged in size. Continue until the entire length of glass tubing is changed into a series of small glass bulbs about three centimeters long and the diameter of the glass tubing, separated from one another by capillary tubes of five or more centimeters in length. Break the capillary tubes about half way between the bulbs. Seal the ends of the capillary tubes in flame and put into a clean dry box to keep ready for use. The interior of the pipettes is sterile, the glass having been heated to redness. The exterior is easily sterilized by passing through a flame, when the pipettes are to be used. Have ready also a supply of small vials or glass tubes. The tubes used for Wassermann tests are convenient. Small baskets in which these tubes may be supported are necessary. We use the small perforated aluminum baskets sold as coffee balls; the lids being removed they are of convenient size and shape. Each of these small baskets holds several vials or tubes, and also the identifying cards upon which notes can be written at each examination. Incubator, microscope, slides, cover-glasses and ordinary laboratory equipment, cotton and boiled water for washing the skin and a blood lancet are also required for the test. A supply of tap water which has been sterilized by boiling in a closed vessel on successive days is best for this use. Distilled water can be used but fibrinolysis proceeds best in water of the type ordinarily used for drinking,--either spring water, tap water or well water. If distilled water must be used, it should have added to it for each liter of water the following formula: Magnesium sulfate
25 mgs. When ready to take the blood pass three pipettes through a flame, break off both sealed ends of the capillary tubes, leaving about one centimeter of the capillary tube at each end of the bulb of the pipette, cleanse the skin of the lobe of the ear if the patient is an adult or a large child, or the skin of the side of a toe or of the heel, if the patient is a baby or a small child. Prick the skin deeply enough to secure several drops of blood. Wipe away the first drop, using sterile cotton (or gauze), and touch the end of a capillary tube to the next drop which oozes from the skin. Fill the bulb about half full. Repeat until three pipettes have been filled in this way. Lay the pipettes upon a sterile plate and allow to rest for an hour. Fill three of the small vials or Wassermann tubes with sterile tap water and boil for three minutes, allow to cool to about room temperature. Note on slip of paper the name of the patient, of the doctor, the date and the hour the blood was taken, the hour the blood clot was placed in the vials and into the incubator. An hour after the pipettes have been filled the blood should be thoroughly coagulated and the clot should be separated from the serum. If coagulation is not complete, let the pipettes remain longer at rest. Break off the capillary tubes at the clear end of the bulb and break the bulb-like part which is filled with blood, just at its junction with the capillary tube which is filled with blood. This leaves both ends of the blood-clot free. Catch the serum on filter paper as it oozes from the end of the broken tube. Drop the clots, still within the bulb-like part of the pipette, into the vials or Wassermann tubes already filled with sterile tap water. Close the tops of the vials and shake them until the clots drop out of the pipettes. Ordinarily three to five fairly vigorous shakes are enough to allow the clots to fall out of the pipettes. If longer shaking is necessary to free the cots, or if the clots do not fall out of the pipettes completely, the clot is said to be adherent to the tubes. Place the vials in a small basket or on a rack, and incubate until the clots are dissolved, or until three days have passed without beginning digestion. Examine the vials at twelve hour intervals until fraying of the clot is noted, then at six hour intervals until the clot is almost digested, and after that watch the progress of digestion at shorter intervals until the clot is completely digested and dissolved in the water. Note the time of beginning digestion and of completed digestion. When digestion begins in 20 to 30 hours, and is complete in 45 to 55 hours, fibrinolysis is normal. Pour the contents of two of the vials into a centrifuge tube, fill another centrifuge tube with water to balance, and centrifuge at moderate speed for ten minutes. Pour off the supernatant fluid, and examine the sediment under a 1/6 or a 1/10 objective with 1 inch eyepiece. Note whether bacteria are present; if so, contamination has occurred and the findings while not actually negligible are less accurate. If any peculiarity in the process of digestion has occurred, the test should be repeated. A few bacteria do not militate against the accuracy of the test, but absolute sterility is much to be desired. It must be remembered that the skin cannot be completely sterilized; that some bacteria are ready always present in the deeper layers of the skin and that the mixture of blood and water, at body temperature, give excellent opportunities for bacterial growth. Normally, the sediment contains debris derived from the digested fibrin; this is usually present in minute rounded masses. Normally many blood cells, both red and white, appear in the debris. When the blood cells are completely digested there is some undifferentiated proteolytic ferment present. Since this digests fibrin as well as blood cells, the repeat must e “Fibrinolysis masked by action of some undifferentiated proteolytic ferment.” If three days pass without recognizable fraying of the clot, the report must be “Fibrinolysis absent.” If the clots dissolve completely, but more rapidly or more slowly than normal, by six hours or more, the reports must e, “Fibrinolysis delayed” or “Fibrinolysis hastened,” giving the hours as noted at the different examinations of the clots. One to three pipettes filled with the blood of a person known to show normal fibrinolysis should be used as controls for every test until the technique has been standardized. The theory of fibrinolysis is discussed in chapter V of this book. Briefly, it may be said that, in terms of this theory, any person who has normal fibrinolysis has at least one factor in protection against the growth of malignant neoplasms; that any person lacking this ferment lacks this factor of protection; that non-differentiated ferments often occur during the progress of certain disease, especially during the growth of cancer in the body, an these ferments which are not differentiated digest fibrin and blood cells together. A report on the fibrinolysis test should include the following items. Name of patient and doctor in charge of the
case; date and hour of taking the blood and of placing in incubator;
or time of last examination if no digestion occurs. The report
should state also either “Fibrinolysis normal,” “Fibrinolysis absent,”
“Fibrinolysis delayed,” Fibrinolysis hastened,” or Fibrinolysis
masked by some undifferentiated proteolytic ferment. Several tests are useful under certain circumstances, but are not necessary for all cases. These are not included in routine examinations unless statistics are being accumulated for research purposes. In our laboratories, these special tests are made whenever the condition of the patient indicates that useful information might be secured thereby. Whenever special studies are being made in which the information to be secured by any special test is thought to be useful, that test is added to the routine work for all patients for as long a time s is necessary to secure the statistics desired. To secure all possible information from the
study of every blood specimen examined in our laboratories is impossible.
Some of the special tests require a considerable amount of blood,
and it is not good for sick people to yield so much blood. The time
required for making so many tests precludes their routine use. Many
of them must be made very soon after the blood is taken, so that
several persons must work at the same time if several of the special
tests are done for the same patient on the same day. For these reasons
it is best to limit the special tests to those which are indicated
in each case, plus those which can be made for research purposes
without too great demands upon the time of the laboratory staff
and without any unnecessary demands upon the blood or the strength
of the patient. Before the invention of the instruments which depend upon color changes for the estimation of hemoglobin, the determination of the specific gravity of the blood was the most important method of estimating the amount of iron in the blood. Modern hemoglobinometers give more accurate findings and the estimation of the specific gravity is now considered of little value in routine laboratory diagnosis. In research work it still have a place, and it occasionally occurs that useful information is secured by a study of the changes in the specific gravity of the blood under experimental or pathological conditions. The specific gravity of normal blood varies almost exactly with the hemoglobin and the red cell count. Daily variations in red cell count, specific gravity and hemoglobin content of the blood form practically parallel curves in normal individuals; variations in the curves fall within the percentages of unavoidable error in technique. Various methods and different students give somewhat different findings but all agree that the variations follow the blood count and the hemoglobin under normal conditions. Figures varying from 1,052 to 1,063 for healthy young people are given by different authors. For normal young women in Southern California the specific gravity of the blood varies between 1,053 and 1,055; for normal young men the specific gravity varies between 1,055 and 1059. Persons who seem perfectly normal occasionally show specific gravity as low as 1,052 or as high as 1,060. The blood of normal children has lower specific gravity than the blood of adults and may be as low as 1,048. At birth the specific gravity is much higher, corresponding to the high cell count and high hemoglobin present at birth and during the first few days of life. Pathologically the specific gravity varies considerably, though this fact has not been found useful in diagnosis. In severe anemias the specific gravity may be as low s 1,025, and in high fevers or in jaundice as high s 1,070. In certain forms of cachesia the specific gravity
does not vary wit the hemoglobin and the cell count; these may remain
almost normal while the specific gravity of the plasma may be considerably
lowered. In this case lowered osmotic tension is indicated by the
changes which occur in the red cells during the actual count. The higher specific gravity of the red cells in the plasma of lower specific gravity is associated with a common osmotic tension because the hemoglobin molecule is heavy and of large size. Since the osmotic tension varies with the number of molecules in a solution, the red cells, with heavy, iron-containing hemoglobin molecules of large size, are still isotonic with the plasma with smaller, lighter molecules of inorganic salts. When the osmotic tension of the plasma varies, the red cells imbibe or give off enough water to equalize the tension within and without the cell. Accurate determination of the specific gravity is rarely needed in diagnosis. An estimation based upon the hemoglobin percentage, the color index and the osmotic tension of the red cells is sufficient for all ordinary clinical purposes. The specific gravity of the red cells varies with the hemoglobin, almost exactly, and the specific gravity of the plasma varies with its osmotic tension. By comparing the hemoglobin, the color index and the osmotic tension it is possible to determine whether the specific gravity is normal or is increased or diminished from the normal, which is all that is useful in diagnosis. The gravimetric method is quite accurate. This requires five cubic centimeters of blood, and the blood must be taken from a vein. This is measured accurately, weighed upon delicate chemical balances, and the weight and volume compared with the weight and the volume of distilled water, with corrections for temperature and height above sea level. The gravimetric method is of value only for scientific precision in research. By using a pycnometer the specific gravity can be taken with accuracy, but a considerable amount of blood is necessary. In certain diseases the removal of ten to fifty cubic centimeters dos no harm, and normal persons can give much more than this amount without recognizable effect. The technique is as follows: Have ready the pycnometer and delicate chemical balances, also a syringe of twenty cubic centimeters capacity fitted with a medium needle, both sterile. Cleanse the skin over the vein of the patient’s elbow, washing first with water and soap, then with alcohol, they dry with sterile cotton. Leave the cotton in place until the vein is to be pricked. Weigh the perfectly dry, clean pycnometer. Fill it with distilled water at a temperature of 99 degrees F., and weigh again. Dry the pycnometer Place a firm elastic band around upper arm of the patient. Remove the cotton and take blood from the median basilic vein, enough to fill the pycnometer. Cover the wound with sterile cotton. Immediately fill the pycnometer with the blood and weigh again. Cleanse the skin of the arm from which the blood
was drawn. If bleeding persists apply ice, or cotton moistened with
alcohol, and keep under gentle pressure. Computation: Subtract the weight of the empty pycnometer from its weight filled with distilled water, also from its weight filled with blood. Thus the actual weight of the water and of the blood are determined. Divide the weight of the blood by the weight of the distilled water of the same temperature (and, of course, of the same height above sea level). This gives the specific gravity of the blood. Aremetrical methods require considerable practice but only a little blood. Have ready several perfectly clean, dry glass tubes; those used for estimating the specific gravity of urine are convenient. Fill these with varying mixtures of chloroform and benzine, or of other suitable fluids. These have the following qualities,--they must not be miscible with blood but must be freely miscible with each other; and one must have a specific gravity above and the other below the specific gravity of any blood apt to be encountered. Take five of the urinometers or other convenient tubes and fill them with mixtures of the selected fluids in proportions to produce specific gravities of 1,058, which is that of average normal blood; 1,068, which is the highest reading for normal blood; 1,051, which is the lowest reading for normal blood; 1,025, which is extremely low and 1,072, which is extremely high. Have these tubes properly marked and arranged in a row. Take a glass tube of about three microns bore and about ten centimeters long, bent at right angles about one centimeter from its lower end. Attach a rubber tube at its upper end and draw blood from a capillary puncture upward into the tube for about two centimeters. Vary quickly blow one drop of the blood into each of the urinometers at about one half the depth of the liquid. The drop will sink in the fluid whole specific gravity is lower than that of the blood and will rise in the fluid whose specific gravity is higher than that of the blood, and will remain stationary in the fluid that equals the specific gravity of the blood. If the drop does not remain stationary in any one of the fluids, prepare three other urinometers and fill with mixtures of intermediate specific gravities between that which causes the drop of blood to sink and that which causes it to rise. For example, if the drop of blood rises in the fluid with a specific gravity of 1,058 and sinks in the fluid with a specific gravity of 1,051, the fluids next selected should be of a specific gravity of 1,053, 1,055 and 1,057. If the drop sinks very slowly in the fluid with a specific gravity of 1,051, and rises rapidly in the fluid of 1,058, it may be best to prepare only one extra tube, at 1,053, for the second test. Practice in this work is necessary in order to determine what specific gravities to select for the second test, and to avoid unnecessary manipulations. If the drop of blood remains quiet in any fluid, that fluid has the specific gravity of the blood. If the blood rises in one fluid and sinks in another, some intermediate point is the specific gravity of the blood; that is, if it rises slowly at 1,055 and sinks slowly at 1,053 the specific gravity of the blood is 1,054. Exton’s immiscible balance is more convenient than the series of tubes This instrument consists of a standard which supports a glass tube supplied with a tap at the bottom through which a fluid may be added to the contents of the tube from the bottom. The top of the tube is open. The tube is large enough to permit the use of a gloat for taking the specific gravity. The tube is filled with a mixture of varnolene (petroleum ether) and carbon tetrachloride in such proportions that the specific gravity of the mixture is that to be expected in the blood to be tested. The bulb which is connected with the bottom of the tube is filled with lighter fluid (varnolene) and a supply of the carbon tetrachloride is in a dropping bottle. The lighter fluid thus can be added from the bottom of the tube, the heavier at the top. Since the two fluids are freely miscible this process causes speedy mixing of the two fluids in the tube. A drop of the blood to be tested is forced into the tube from the end of a glass tube, as in the method previously described. If the drop sinks, a drop or a few drops of varnolene are allowed to flow into the tube at the bottom. The proportions are thus varied until the drop of blood remains at about the middle of the tube. The fluids are not miscible with blood and the blood drop should remain spherical and distinct during the small amount of time necessary for the manipulation of the fluids. The specific gravity of the fluid in which the drop of blood rests is then determined by means of a float. This float must be the one provided with the instrument because this is standardized for the surface tension of the fluids mentioned. If considerable handling of the two fluids is
necessary n order to attain stability of the drop of the blood,
another mixture should be provided, of the specific gravity finally
determined upon, and a fresh drop of blood added to this. Frequently
this second drop of blood does not float freely in the mixture,
because there is some slight change in the blood when it is allowed
to remain for too long a time in the mixture of fluids. The second
determination is more quickly made, for obvious reasons. Sometimes,
of course the specific gravity first determined is found to be accurate.
There are several conditions in which the enumeration of the blood platelets give useful information in diagnosis. These structures are so fragile that it is difficult to make counts which are as nearly accurate as are those made of the red and the white blood cells, but even so the counts are useful. The simplest method is that of Wright and Knnicutt. Prepare two solutions as follows: 1. Brilliant cresyl blue 1 gram 2. Potassium cyanide .1 gram Immediately before using mix one part of solution 1 with three parts of solution 2, and filter. Take the blood as in making ordinary red cell counts, preferably making a 1 to 200 dilution, and place in the counting chamber. Allow the diluted blood to stand in the chamber for ten or fifteen minutes, in order that the platelets may settle. Platelets are stained a lavender or lilac tine; red blood cells are almost invisible and the leucocytes have dark blue nuclei. Count the platelets in 200 small squares or more. The calculations are the same as in the case of the red cell count. Because the platelets are so small it is desirable to use a higher power objective. In our work, however, an ordinary one-sixth objective with a one-inch eyepiece has been found quite satisfactory. By using a specially ground, very thin coverglass it is possible to use a one-eighth eyepiece. There is a coverglass sold which is of ordinary thickness but which has a thin area in the center; this is satisfactory but it is very easily broken. Several indirect methods are in use, and these
preserve the platelets rather better than is the case with the Wright
and Kinnicut method. Different solutions are employed by different
workers. The simplest is a 10% solution of sodium metaphosphate
in water. This preserves the platelets but does not stain them.
Water 200 c.c. Cleanse the skin as for any prick to secure blood. Place one drop of the selected fluid upon the
surface of the skin. Prick the skin through this drop, so that the
blood mixes with the fluid as it emerges from the wound. When the
drop is definitely a pink color make smears on glass slides. Dry
in air, and stain by any of the usual methods for making a differential
count. Count the red cells and the platelets on this smear and determine
the ratio of platelets to red cells. By means of this ratio and
the actual count of the red cells determine the actual number of
platelets per cubic millimeter of blood. There is little practical value in accurate estimations of the viscidity of the blood. Variations in viscidity follow variations in hemoglobin quite closely, and this is true for physiological changes and for abnormal conditions of the blood. Occasionally there is some need for the accurate determination of viscidity, and in routine reports the relative viscidity should be noted. The hemoglobinometer slides may be employed for a rough estimation of the viscidity of the blood. When the slides are separated, one slide is drawn across the other, and the amount of stickiness noted. In normal blood this stickiness is enough to delay, slightly, the separation of the slides, but not to cause any appearance of threading. Blood which has increased viscidity shows a tendency toward thread-formation when the slides are separated. Blood which is subnormal in viscidity shows no recognizable stickiness. If the viscidity is in any way abnormal, more exact methods should be employed. Viscidity is indicated by the manner in which the drop stands up as it emerges through the puncture. A normal drop of blood is practically hemispherical; blood which is deficient in viscidity spreads out over the skin; blood which is abnormally viscid stands up much higher. A more nearly exact and very satisfactory method depends upon the size and rate of dropping. For this, a pipette of certain diameter is employed; the blood taken into the pipette and allowed to drop; the number of drops falling in five seconds or ten seconds is the measure of viscidity; as 1/5 or 3/10. The normal for the method must always be noted. There is considerable variation in the viscidity
of the same blood at different times of the day and as a result
of dietetic changes. A number of methods of determining the coagulation time of the blood have been employed, but none has been altogether satisfactory. The following methods have been employed in the laboratories of the Research Institute. The blood taken in the slides of Dare’s hemoglobinometer coagulates in about five minutes, normally. The slides are allowed to remain quietly for five minutes, and are then separated. If the coagulation time is normal, the blood is found completely coagulated but the serum has not yet separated. If the blood is not coagulated the coagulation time is increased, and more exact methods should be employed. If the blood clot has begun to separate from the serum, the coagulation time is diminished, and more exact methods may or may not be employed, according to the other blood findings and the general condition of the patient. For more exact determination of the coagulation time, capillary tubes are used. These are made by drawing ordinary glass tubing out into fine capillary tubes, which should have a caliber of not more than the size of a dark hair and should be uniform in every respect. These tubes are broken into lengths of one inch. Ten of these tubes are filled for about half their length with blood, and allowed to lie for a length of time dependent upon the results of the observations made with the hemoglobinometer slides. If these have shown delayed coagulation, the capillary tubes are allowed to lie for six or even ten minutes; then one tube is broken. If the blood falls out in a cast of the tube, coagulation is complete. If it is perfectly fluid, two minutes should elapse, when another tube should be broken. If it is still perfectly fluid, two or perhaps three minutes may elapse before another tube is broken. The coagulation time is given in the number of minutes required for complete coagulation as shown by the formation of a cast of the tube when the glass is broken. This is the most accurate and satisfactory method now known. Howell’s method requires more blood. Four cubic centimeters of blood are taken from the vein at the elbow, as usual, and immediately mixed with an oxalate solution (5 cubic centimeters of 1% sodium oxalate in ten cubic centimeters of 0.9% NaCl). This prevents coagulation. The mixture is centrifugalized until the corpuscles have been thrown down. Three small tubes are provided, which contain one, two and three drops of 0.5% calcium chloride. Into each of these tubes five drops of plasma are placed; the time accurately noted, and the time of complete coagulation noted. By this method, Howell found normal coagulation time in nearly all conditions except hemophilia. Normal blood coagulates in nine to twelve minutes; hemophilic blood coagulate in one to five hours. Purpuric blood gave normal coagulation time by this test. It is evident that this method rules out all delayed coagulation due to disturbances of the calcium-content of the blood. By the method of Lee and White about 1 cubic centimeter is drawn from the bend of the elbow, as usual, using a glass hypodermic syringe which has just been rinsed with .9 NaCl solution. The blood is quickly placed in a small glass tube, about eight millimeters in diameter, which also has been rinsed in .9 NaCl solution. The tube is then turned back and forth once each half-minute and the time noted at which the blood no longer flows. This method has little to commend it, for the friction modifies the coagulability. When the coagulation time is normal but the
clot is very soft, defective fibrinogen is indicated. This is usually
due to some abnormal condition of the liver; occasionally it is
due to deficient diet. These conditions are usually easily recognized
by clinical phenomena. Cases of hemophilia may be associated with
deficient platelet count, and this is easily determined by making
an actual count of the platelets. In other cases of hemophilia the
platelets fail to agglutinate properly, and this can usually be
determined by studying warm slide specimens made with rather thick
smears of blood. In other cases of hemophilia there is deficient
calcium in the blood, and this can be determined by the following
method. Cleanse the skin of the elbow and cover with cotton or gauze wet with normal salt solution. Rinse a sterile syringe and needle with normal salt solution and leave all surfaces thoroughly wet with the solution so that the blood does not come in contact with air. Take two cubic centimeters of blood and place this in a small test tube containing one-half cubic centimeter of one percent of calcium chloride solution. Fill capillary tubes with this mixture and determine the coagulation time. Compare the coagulation time of untreated blood
with the coagulation time of the blood treated with the calcium
solution. If the blood of the hemophiliac coagulates more normally
after the addition of the calcium, the addition of calcium-containing
foods to his diet is indicated. In such cases the parathyroid glands
may be abnormal and this aspect of the case should be studied. A study of the relations between the solids and the water content of the blood is sometimes useful in research work; it is not now known to have any value in diagnosis. Have ready a perfectly clean and perfectly dry
weighing glass; weigh this accurately. Put into the glass about
five cubic centimeters of blood taken from a vein. Weigh accurately.
Tilt the weighing glass so that the blood is spread over its inner
surface, in order to increase the evaporation area. Replace the
cover at an angle so as to permit evaporation. Place the glass in
a thermostat at 60 degrees to 70 degrees C. for one or two days.
Weigh again. Compare the weight of the whole blood with the weight
of the dried blood. The dried solids make up about one-fifth the
total weight of the blood, and in men the solids are a little greater
than in women. That is, the water content of normal blood is about
four-fifths the total weight. Figures given by various investigators
vary from 19.58% to 20.53% for women and from 20.35% to 22.69% for
men. The figures given are increased by those conditions which abstract
water from the blood and decreased by hydremia. The test is not
of value in diagnosis but it is occasionally useful in research
work. The determination of the electrical resistance of the blood has been suggested as a speedy and accurate method of determining the blood cell count. It has been found that the electrical resistance varies as the amount of hemoglobin-carrying protoplasm plus the density of the blood plasma. It is a better indication of the cell-volume than of the cell count, though not very accurate at that. The technique is cumbersome and unsuited to ordinary laboratory diagnosis work. It has not, so far, been found useful in research work. The rapidity with which the red cells settle from blood whose coagulation has been prevented in some way varies in many conditions. The cells settle more rapidly in tubes which are placed at an angle than in tubes which are placed upright; no doubt this peculiarity is due to the Brownian movement of the cells. Variations in the globulin content of the blood, the salt content, the relations between the electrolytes and the non-electrolytes of the plasma and the temperature at which the test is made all affect the sedimentation rate. Cells settle more rapidly in the blood of patients with malignancy, pregnancy, fevers and inflammatory states, after severe burns and, generally speaking, whenever there is tissue destruction or increased katabolism. The technique is simple. The method of determining the blood volume by the hematocrit may be employed, except that the centrifuge is not used. The hematocrit tubes are placed at an angle of about 45 degrees in a rack and the sedimentation of the red cells timed. Normally the cells settle within about an hour. More nearly accurate findings are secured by
taking a larger amount of blood (5 cubic centimeters or more) from
a vein, citrating the blood or cooling it very quickly to 33 degrees
F., and placing it in very small tubes which are allowed to rest
at an angle of about 45 degrees until the cells have settled. D
If the cells settle within 20 minutes to 40 minutes, the sedimentation
rate is increased; if the cells settle within 50 minutes to 80 minutes,
the sedimentation rate is normal. We have not found any useful information
resulting form the test and it has been discarded in our laboratories.
Many attempts have been made to find some adequate and practical method of determining the total amount of blood in the body, but none has been altogether successful. It is evident that only qualitative results can be secured from an examination of a small portion removed for that purpose unless the total amount of blood in the body is known. The Haldane-Smith method consists in the inhalation of a measured amount of carbon monoxid, the removal of a measured amount of blood from a vein, and the determination of the amount of carbon monoxid hemoglobin by means of a spectroscope. The fraction of the carbon monoxid inhaled found as carbon monoxid hemoglobin in the measured blood indicates the amount of total blood in the body. This method is not suitable for ordinary work. Cells injured by carbon monoxid begin to be removed from the body quickly, and there is considerable unavoidable error in technique. Patients with even slight chronic carbon monoxide poisoning would give very erroneous findings. Methods based upon the injection of non-poisonous stains into a vein, and the removal of a measured amount of blood from an opposite vein, have been employed. The relative amount of the stain found in the measured blood indicates the total amount of blood in the body. The accuracy is diminished by the fact that such stains are partially removed from the blood by the liver, kidneys and other tissues of the body, and that complete mixture of the blood may not occur before this elimination has begun. Rowntree’s reports give interesting findings based on the use of Congo red and other harmless stains, injected into the blood. For the details of technique the original reports should be consulted. Briefly, the method includes the following factors: The probable plasma volume is determined for the patient by multiplying his weight in kilograms by 50, because the average plasma volume is about fifty cubic centimeters for each kilogram of body weight. The figure thus secured is divided by 200, because a dilution of one part of stain to 200 parts of plasma is practically adequate. This quotient is the amount of the dye to be injected. The dye itself is a 1.5% solution of Congo red in fresh, triple distilled water. Ten cubic centimeters of blood are taken from one elbow and the estimated amount of the dye injected into the same vein through the needle used in withdrawing the blood. Three to six minutes later 20 cubic centimeters of blood are taken from the other elbow. Both specimens are oxalated and both are centrifugalized and the hematocrit readings recorded. The plasma from the two specimens is prepared for the colorimeter tubes and the readings give the amount of the dye in the plasma taken after the injection. By comparing the amount of dye injected with the amount recovered it is a simple matter to determine the amount of blood plasma in the entire body. By comparing the hematocrit readings with the total blood plasma the total amount of blood in the body can be determined. The findings secured by Browntree and his associates have been uniform and have given valuable information. The method is the simplest and the least harmful to the patient of the various methods proposed. All of these methods have certain unavoidable
errors and all are rather dangerous in the hands of unskilled persons.
Many studies of the total volume have been made, for animals and
for human subjects, and various fractions have been given as the
relation between blood volume and body weight. These fractions vary
from 1/13 to 1/20 for both men and women. For laboratory animals
a fraction of 1/20, with a small increase during pregnancy, has
been found by nearly all those making careful tests. That is, about
5% of the total weight of the body consists of blood. In obese animals
the proportion is lower, as might be expected. In obese human subjects
the proportion of blood to body weight has also been found lower.
Pregnant women show some increase in the proportion of blood to
the body weight. The presence of parasites in the blood may be suspected when symptoms are reported such as follow such infections, or when the blood picture is atypical and is associated with eosinophilia, especially if the patient has been living in countries in which such infections are endemic. In making a differential count, fragments of parasites may be seen, or the large hyaline phagocytes may contain inclusions which arouse suspicion of parasitic infections. Inclusions found in the neutrophiles are not commonly noted but may be found occasionally. The red blood cells may show the effects of erosion due to blastomycetes, malaria or other infections. Fragments of the parasites or large hyaline bodies suggesting spores may occasionally be noted in the plasma while making a differential count, and these suggest more careful study of the blood with reference to the possibility of parasitic infection. Any findings which cannot be explained should always lead to more careful study of the blood in order that the puzzling factors may be interpreted, if possible. The first method to be employed in the search for parasites is the study of a rather thick smear of fresh blood on the warm slide. Take a warm slide on the warm stage of the microscope, cover with a warm cover glass, examine with the two-thirds objective and one inch eyepiece. Look over the entire drop of blood. Note whether the red cells show any unexplained movement; note whether any living worm-like organisms are present. Look over all thin areas with a one-sixth objective, then with one-eighth or one-tenth objective. If no organisms can be found discard the method. Use next a method of concentrating the blood.
Rub the lobe of the ear until it becomes distinctly reddened, using
some alcohol or rather hot water or some other aseptic fluid. Dilating
the blood vessels is, in this case, desirable. Have ready a centrifuge
tube containing five cubic centimeters of five per cent acetic acid
solution. If this method shows no parasites, venous blood must be used. Take ten cubic centimeters of blood from a vein in the elbow, using the technique employed for determining blood chemistry, and put four cubic centimeters of the blood into a centrifuge tube containing six cubic centimeters of five per cent solution of sodium citrate, and six cubic centimeters of blood into a tube containing four cubic centimeters of the citrate solution. Each tube then contains ten cubic centimeters of mixtures of blood and citrate solution in varying proportions. Centrifuge ten minutes at about a five hundred revolutions per minute. The red cells then occupy the lower end of the tube, the clear fluid the upper portion, while there is a thin layer of white cells at the top of the red cell column. Take one thick drop from the white cell layer form each tube, for immediate examination. Make eight or ten smears from the same layer of each tube, lay these aside to dry for staining. Examine both the thick smears, unstained, using first the lower powers and then the high powers. Note evidences of living organisms in both. Stain the thinner smears, using several different stains, as before. Note parasites and also note whether there are any cellular inclusions in the large hyaline phagocytes or in the neutrophiles. If these methods show no evidences of parasites
in the blood, repeat with blood taken at different times of the
day and night until the parasites have been found or until several
examinations several times repeated have given negative results
Take blood at about midnight, after the patient has been three or
more hours asleep at least two different times. Take blood immediately
after a heavy osteopathic treatment, especially planned to cause
increased rapidity of the circulation of the blood through the liver
and the spleen, at least two different times. Take blood in the
early morning, at about two o’clock in the afternoon and about two
hours after a heavy protein meal, at least two times each, unless
the parasites are found, or some explanation of the puzzling findings
secured, before the group of tests has been completed. Whenever
blood is taken for any of these tests, make two to six smears for
differential count, using the usual technique. Save two or three
of each of these smears, after they have been dried, for later study
after the nature of the disease has been determined. If there are enlarged lymph nodes and the surgical removal of one for the sake of diagnosis does not seem desirable, some of the juices of the gland may be taken for examination. Take the syringe used for taking blood for chemical examination, have the needle sterile but dry, plunge the needle into the substance of the gland and withdraw the plunger enough to secure a drop or a few drops of fluid. From this material make one thick smear and several thin smears, on microscope slides. Examine the thick smear at once, unstained, and preferably on the warm stage. Dry and stain the thin smear using several different methods. Inoculation of guinea pigs, white rats and rabbits
from the blood of the patient and from the glandular extracts often
determines or verifies the diagnosis. Use the technique employed
for bacterial inoculations. Examination of the unstained smears for malarial organisms is no longer a method in general use, which is, in some ways, unfortunate. The manner in which the organism moves within the cells, and the relations between cellular movements and the presence of the parasite are interesting, and in some cases are useful in the study of the efficiency of the parasiticidal activities of the phagocytes. The pigment granules of adult malarial organisms makes their recognition fairly easy. This pigment may not be present in the younger forms. Stained smears give the most rapid and accurate diagnosis of malaria. Organisms may be noted in making an ordinary differential count and their number per cubic millimeter determined. The method is described in the paragraphs which give the method of making the differential count. When malaria is suspected a thick smear may be used, in order to concentrate the organisms. Take three or four drops of blood in the center of an ordinary slide, spread the blood out over an area of about one square inch, making the smear as even as is practicable. Allow to dry at room temperature; the slide must be thoroughly dry before staining. Solutions necessary for this special method are simple. 1. Fixing fluid 2. Manson’s fluid Stand the dried thick slide in a jar containing at least fifty cubic centimeters of the fixing fluid for ten minutes. If the hemoglobin has not all disappeared by the end of ten minutes stand in a jar of tap water until all of the red or pink color has disappeared. Flood with Manson’s fluid for half a minute, rinse gently in tap water. Mount in water and examine with dry one-tenth objective, or dry and examine with oil immersion lens. It is possible to make a fairly accurate determination of the number of parasites per cubic millimeter by counting the number of parasites in several fields, then counting the number of neutrophiles or the number of small hyaline cells in the same fields. The number of neutrophiles or of small hyaline cells per cubic millimeter having already been determined by the ordinary blood counts, the number of malarial parasites per cubic millimeter can be estimated quite easily. In old cases of malaria the parasites can usually be found at almost any time. In cases in which the chills and fever follow a regular rhythm, as in tertian and quartan fevers, the smears should be taken, if possible, just before the onset of the chill. In autumnal fever the smears are most apt to show the parasite if they are taken eight or ten hours after the beginning of the fever. The patient should receive an energetic osteopathic treatment planned to secure increased speed of circulation through the spleen just before the smears are taken, if this can be done. Such a treatment tends to diminish the severity of the attack, when it is given just before the onset of the chill. In cases with irregular attacks of fever such treatments increase the number of parasites in the peripheral blood, and they also hasten recovery from the malaria itself. The different parasites which cause malaria may be distinguished as follows: Plasmodium vivax (P. tertiana; Hemameba Laverani tertiana; P. malariae tertianum) shows rather active ameboid movements. The red cells infected are enlarged somewhat, are pale, and may show basophilic degeneration, but the non-infected cells do not show basophilic degeneration. The pigment is finely granular, yellowish brown, abundant, and it remains fine throughout the life of the parasites; it may be somewhat more abundant at the periphery but some fine granules are scattered over the entire organism. The young schizonts are ring-shaped at first, later they assume rather irregular forms. They grow until they fill the entire red cell, and they may become larger than the red cells of the infected blood. These segment into fourteen to twenty or even twenty-four merozoites, and these form a rosette which is often imperfect and of irregular arrangement. The extracellular forms are not easily distinguished from those of P. malariae but they do not resemble those of P. falciparum. The macrogametocytes are large and round with abundant, finely granular pigment which is scattered over the entire parasite. The protoplasm stains deep blue with ordinary methylene blue or thionin stains; the nucleus is poor in chromatin. The microgametocytes have less abundant pigment, the cytoplasm stains rather a greenish light blue and the nucleus is rich in chromatin. Plasmodium malariae (Oscillaria malariae; P. quartana; Hemamebae malariae; H. Laverani quartana; P. malariae quartana) shows only sluggish ameboid movements and may not move at all on the warm slide. The red cells infected do not swell nor do they become paler than normal. They may assume a brownish or brassy tint, apparently due to the change of hemoglobin into methemoglobin or some related compound. Basophilic degeneration may appear in no-infected as well as in infected red cells. Pigment granules are coarse, not abundant, and are mostly arranged near the periphery of the cell. Young schizonts are ring shaped, later becoming oval or round. They have a chromatin granule which soon becomes band-shaped, and these form equatorial bands. The schizonts do not fill the red cell completely and they divide into eight to fourteen merozoites, arranged in a rosette which is nearly always perfect. Extracellular forms are somewhat smaller than is the case with P. vivax, but they have almost or quite the same structure. P. falciparum (Laverania malariae; tropical
parasite; plasmodium precox; Hemamebae malariae precox; P. immaculatum)
shows little ameboid motion but has a slight activity somewhat resembling
pulsation. The infected red cells do not swell but may shrink slightly,
and they show the peculiar brownish or brassy color noted in red
cells infected with P. vivax. The pigment granules are coarse and
dark in color. Young schizonts are small and ring-like and they
have one or two chromatin granules at the periphery. Occasionally
two or even three parasites may be found within one red cell. Larger
schizonts are not often seen in peripheral blood, and they rarely
occupy more than half or two-thirds the area of the red cell. In
larger forms the pigment granules are coarse, scanty and tend to
clump together. The grown schizonts divide into seven to twenty-four
merozoites, and these form rosettes which are not always perfect.
The extracellular macrogametes have a distinctive crescent-like
form, becoming oval and round or roundish as the parasite increases
in size. The cytoplasm stains deeply and the nucleus has scanty
chromatin. The pigment granules are coarse and clumped near the
center of the cell. Extracellular microgametocytes are also crescentic,
and become oval or round with growth. The cytoplasm stains feebly
and chromatin is fairly abundant in the nucleus. The pigment granules
are coarse and are scattered over all the cell. If filaria are suspected, the blood should be taken for examination at several different times of the day. Late in the afternoon, after the patient has rested, or better, has slept for several hours the blood may show the parasites of any variety. If the ordinary nocturnal form is present it may be necessary to take the blood at a time as near midnight as is convenient, after the patient has slept for several hours. The diurnal forms are most abundant at about noon. Other forms may be found at almost any time of day. Fresh specimens of blood are best examined. If the cover glass is ringed with vaseline the parasites may remain alive and active for several days. If the parasites are not found in ordinary thick smears, the blood should be centrifuged in order to concentrate the worms. The skin may be reddened by rubbing with the aseptic solution, then pricked, and several drops, or even several cubic centimeters, collected. This may be mixed with either 2% watery solution of sodium citrate, or with 3% acetic acid, and the mixture centrifugalized at about 500 revolutions per mite for five or ten minutes. The worms accumulate in the leucocytic layer after centrifugalization and thick smears made from this layer show them usually quite active. The two-thirds objective is usually best for examining these preparations, though the dry higher power lenses are best for studying the organisms in thin smears. The blood can be taken in small tubes and allowed to clot. The serum which separates contains nearly all of the worms. If a considerable amount of serum is collected this may be centrifuged and the sediment examined. For permanent mounts the smears should be thinner and should be dried thoroughly in the air. They are then stained with hematoxylin, Giemsa’s stain, or any of the ordinary blood stains. The hematoxylin-stained slides keep the best. In temperate zones this infection is rarely
seen. For the differentiation between the different forms a book
on tropical diseases should be consulted. These parasites are found within phagocytic cells in the blood, especially within large hyaline phagocytes. In making ordinary differential counts they may sometimes be noticed as inclusions within the large hyaline endothelial cells. In these cells occasionally great numbers are found. It is only very rarely that they are seen free in the plasma or within neutrophiles. In slides stained with Giemsa’s stain or with any of the eosin-methylene-blue preparations the cytoplasm of the parasites takes a pinkish or bluish lavender tint. The nucleus and the parabasal body stain dark blue. The parabasal body, with its long axis lying at right angles to the long axis of the cell is quite distinctive and makes the diagnosis clear when the parasites are well stained. If the organism is not found by ordinary methods of examination the blood may be concentrated. Several cubic centimeters of venous blood should be well mixed with 1% or 2% solution of sodium citrate and the mixture centrifuged at about five hundred revolutions per minute for five or ten minutes. The leucocytic layer should then be collected and made into rather thin smears, and these stained after any of the usual methods for staining blood smears. The organisms are found within large hyaline cells. Puncture of the spleen or of an enlarged lymphatic gland for the purpose of securing a few drops of the fluid for examination may be necessary for diagnosis. The removal of a small fragment of rib may be necessary in order that the red marrow may be examined. In either case the organism may be found within large hyaline cells or free in the tissue juices. Material may be secured from a local lesion of the skin quite easily. If the skin has not yet become eroded the lesion should be punctured and the fluid made into smears. If the skin has become eroded the superficial debris and bacteria should be washed away, the floor of the ulcer scraped, and deeper scrapings made into thick smears for immediate examination, and thinner smears for staining after several different methods. The organisms which produce the different types
of Leishmania cannot be differentiated from direct study of the
smears, but cultures and animal inoculations may give differentiating
features. For the differential diagnosis a book on Tropical Diseases
should be consulted. In certain forms of anemia the red blood cells are more resistant to lipolytic solutions and to hypotonic solutions. In other forms of anemia and in certain other diseases the red blood cells are less resistant to such solutions than are normal red cells. For a study of the relations mentioned it is always necessary to determine the concentration of solution which just barely destroys normal red cells, then make a series of solutions of greater concentration and of less concentration than this. The blood to be tested is t hen placed in each of the varying solutions and the manner in which the red cells behave is noted. The technique for all these solutions is about the same, and may be illustrated by the method of determining diminished resistance to hypotonic solutions. Normal blood plasma has an osmotic tension about equivalent to a 0.9% solution of sodium chloride in distilled water, and normal red cells begin to lake at about 0.4% solution of sodium chloride. In a 0.3% solution normal red cells are completely laked. The technique of determining the resistance of red cells to hypotonic salt solutions requires two racks, each containing twelve graduated centrifuge tubes. One rack is for normal blood, as control, the other for the blood to be tested. Mark the tubes in each rack as follows: .5%, .48%, .46%, .44%, .42%, .40%, .38%, .36%, .34%, .32%, .30%, and .28%. Fill the first tube to the line which indicates five cubic centimeters with 0.5% sodium chloride solution. Add distilled water to all the tubes except the first, to fill them to the line indicating five cubic centimeters. In this way the tubes all contain five cubic centimeters of solutions of sodium chloride of the strength marked upon them. Both racks are exactly alike. Mark one with the name of the control and the other with the name of the patient. Cleanse the lobe of the ear, if the patient is adult, and make a rather deep prick, so that about two cubic centimeters of blood can be taken into a graduated pipette. Drop one tenth cubic centimeter into each of the twelve tubes of sale solution. Note the time. Repeat for the control. Note this time. Allow the racks to stand for half an hour, then observe. The red blood cells should have settled to the bottoms of the tubes within a few minutes after the blood was placed in them. After half an hour there may be a faint pink color in one tube and all the tubes with weaker solutions show darker tints. In one tube the red cells may have all disappeared, and in all tubes with weaker solutions the cells also have disappeared. If these conditions are not present at the end of half an hour leave the racks for a longer time until hemolysis has begun in the tubes on the rack of the control. This is usually within an hour, but occasionally two hours may be necessary. Note the strongest solution in which hemolysis is just visible, in the control and in the patient’s blood. Note the strongest solution in which hemolysis is complete (and the red cells destroyed completely) in the control and in the patient’s blood. The report should give these four findings,
and also the time at which the blood was placed in the tubes and
the time at which the last observation was made. Since the fragility
occasionally varies during the day even in normal blood, the exact
time should always be given. Tests for bile are not, properly, included with these discussions of blood cells, except that it is often essential that the source of toxemias, which affect the blood cells, may be speedily determined. The tests devised by Van den Berg, depend upon the fact that fluids containing bile give the diazo reaction, provided the bile is not associated with the proteins of the plasma. The first is called the “direct reaction” and it is so called because of the bilirubin is free in the serum. The patient must not eat green or colored vegetables for two days beforehand. Take five cubic centimeters of blood from a vein in the elbow, after proper sterilization of the skin. Put the blood into a suitable vessel until it clots and the serum becomes separated. The serum must not be recognizably stained with hemoglobin; it may be greenish, yellowish or brownish from the bile present. Measure one cubic centimeter of the clear serum into a small tube, add one cubic centimeter of Ehrlich’s diazo reagent. A brilliant reddish or purplish color may appear immediately, in which case there is a positive direct reaction. This indicates obstructive jaundice. Sometimes the color appears only after the mixture has been standing several minutes, in which case there is a delayed positive direct reaction, and this has the same significance. The indirect reaction is so called because the bile pigment is combined with the proteins of the plasma in such a way that the reaction does not occur until these have been precipitated. Take one cubic centimeter of clear serum, add two cubic centimeters of alcohol of about 96%. Place the mixture in a centrifuge tube, balance with an equal weight of water, and centrifuge at about eight hundred revolutions per minute for ten minutes. Remove one cubic centimeter of the clear supernatant fluid and to this add one-half cubic centimeter of alcohol and one-fourth cubic centimeter of Ehrlich’s diazo reagent. The immediate appearance of reddish or purplish color indicates the positive indirect reaction, while the appearance of the same color after several minuets standing indicates the delayed positive indirect reaction. When this reaction is positive and the direct reaction negative, the jaundice is not obstructive but is due to liver injury or to blood cell destruction. Quantitative findings can be secured. One unit of bilirubin is taken to be 0.5 milligrams in one hundred cubic centimeters of blood. One-half to one-tenth of a bilirubin unit is present in normal blood. For the quantitative estimation cobalt sulphate is used for the standard. Prepare a solution of 3.92 grams of crystalline cobalt sulphate in 100 cubic centimeters of distilled water and place in one cup of the colorimeter. In the other cup place the serum which shows the positive reaction. If the color of the serum is the same as the standard solution, the blood of the patient contains five units of bilirubin. If the color is lighter or darker than that of the standard, the cups are raised or lowered until the tints match, when the concentration can be read off on the colorimeter scale. The Van den Bergh reactions are useful for distinguishing between obstructive jaundice and hemotogenous jaundice or the jaundice due to liver injury, but the reaction is not delicate and it is not useful for accurate determination of small amounts of bile. Ehrlich’s diazo reagent is made as follows: Solution A When the test is to be made take fifty parts of solution A and one part of solution B, mix and use immediately. The icteric index is better for estimating small amounts of bile. A solution of ten milligrams of potassium bichromate in one liter distilled water is taken to have an index of 1, and this solution is used for a standard. The solution keeps well in the dark. Take about ten cubic centimeters of blood from a vein of the elbow, place in a convenient vessel until the serum has separated from the clot. Take two or three cubic centimeters of serum, which must not be stained with hemoglobin, and mix with an equal amount of 0.9% sodium chloride solution. Place the diluted serum in one cup of the colorimeter, the standard solution of potassium bichromate in the other cup, and compare. The standard solution corresponds to an icteric index of 1., and normal blood has an icteric index of about 5. Icteric index of about 15, which corresponds to about two units in Van den Bergh’s reactions, is present in cases of mild jaundice. An icteric index between about 6 and 15 is present in mild cases of cholemia, in which case the blood cells show more or less marked evidences of injury. The Gmelin test is often positive for a small amount of serum. Allow the serum which is formed when the blood clot is ready to be placed in the incubator for the fibrinolysis test to soak into filter paper. Allow one or two drops of nitric acid which contains some nitrous acid to fall upon the filter paper and to touch the blood serum. Normal serum shows a brownish tint at the line of the acid, while cholemic blood gives a play of colors, including purplish or violet shades. A more delicate test is made from oxalated blood.
Take about five cubic centimeters of blood from a vein into a test
tube containing about ten milligrams powdered potassium oxalate.
Mix and centrifuge for about ten minutes. Remove the clear serum
into another tube, and underlay this with nitric acid which contains
a small amount of nitrous acid. A white coagulum will appear at
the junction of the two fluids. In this white band there will appear
a blue-green color at once in severe cholemia, or within half an
hour in less serious cases. Reports of work done in the best clinical laboratories are always given to the doctor who orders the work to be done. It should be kept clearly in mind that laboratory work is done for the doctor, and that only the doctor is responsible to the patient. A carbon copy of the report is sent also, and the doctor usually gives this to the patient, unless there is some reason why the patient should not receive it. Reports are written in technical language and the doctor should explain to the patient or to some member of his family the significance of the various items. The laboratory worker should not give such explanations, unless requested by the doctor in charge of the case to do so. K The doctor who has made the physical examinations and who has studied the symptoms and the history is the only one who is able to interpret the findings in a simple and practical manner, in the light of all the factors involved. Patients receiving copies of examinations should always be advised to keep them, and to show them to any other doctor who may be called to give treatments for any disease at a later time. This is especially important in cases of rare and chronic diseases. Much valuable time may be saved to the patient if he has such records ready at a time of later illness, in such cases. With the reports of blood examinations there is sent, upon another sheet of paper, such notes and explanations as may be useful to the doctor in explaining the significance of the findings and in determining the best methods of treatment. While the laboratory worker is rarely in active practice, still there is much useful information which he can give in selected cases, provided he is doing his work upon a professional basis. The notes are not intended for the patient and should never be given to him or to his family. The reports now being used in the clinical laboratory of The A. T. Still Research Institute are the most useful we have seen. The routine examination forms are printed upon pink paper and the special-test forms are printed upon light brown paper. This makes it easy to select from the files the reports which may be needed for special studies. The forms used for other work are of different colors; uranalyses forms are printed upon yellow paper, blood chemistry forms on green paper, and so on. The following forms are those used for blood reports in 1930:
ILLUSTRATIVE CASE REPORTS Miss R., aged twenty-eight years, presented an unusual history. Three years before coming to an osteopathic clinic for examination she had shown symptoms of pulmonary tuberculosis, and this had been treated by dietetic measures alone. Blood examination showed the usual findings in alkalosis, and after more explicit questioning a detailed account of her diet for the tubercular infection was secured. She had been given foods of the alkaline-ash type exclusively. Three times each day she was given lemon juice and soda. Twice each day she was given a soda enema. The urine was analyzed twice each month, and if it was neutral or alkaline no change was made in the diet. If the urine was acid at any time the alkalinization of the food intake was increased. Within a few weeks after the intensive alkalinization she began to complain of muscular cramps, most marked in the fingers. During the next three years the spasmodic contractions increased in frequency and in severity, and various types of paresthesia developed. During this time she consulted several other doctors of medicine. Without making any very careful study the condition was named either hysteria or acidosis by these men. Alkalinizaton was advised by every doctor consulted during this time. Colonic irrigations of soda and water were advised. The condition became gradually more severe. When she came to the osteopathic clinic the contractions were titanic in type. The muscles of the left hand were first involved and the “obstetrical hand” position assumed. The spasms then extended to the arm and shoulder, then to the neck, trunk and legs, until the entire body was involved in titanic convulsions. Attacks occurred two or three times a week. By a specially devised method the total blood alkalinity was found equivalent to 450 milligrams of sodium hydroxide per 100 cubic centimeters of blood. The diffusible alkali was much more noticeably increased than was the bound alkali. The osteopathic examination showed the rigidity of the lower thoracic region which is always present in tuberculosis, and also a definite lesion of the fifth cervical vertebra. In order that the effects of de-alkalinization might be studied, no osteopathic treatments were given during the first month. The patient acknowledge an intense craving for hot white biscuits and beef-steak. These were given her for her first meal, in moderate helpings. She was permitted other foods with acid ash until the urine became normally acid, then a wholesome, well-balanced diet was advised. The soda intake was stopped immediately. The spasms were less severe the day after the soda was stopped. By the end of the month they appeared rarely. Some paresthesias of the hands were still noticed, and the relation of the fifth cervical lesion to the innervation of the arms and hands was explained to her. This explanation induced her to carry on the treatment which had been advised. During the fifth week after the diagnosis had been made the cervical lesion was corrected and the spinal rigidity relieved. The lesions did not occur. Six weeks after the lesions were corrected a
second blood examination showed no abnormal findings. During the
subsequent nine years she has been healthy and comfortable, except
that she had colds several times, and was in an automobile accident
once which broke an arm and caused cerebral concussion of mild degree.
The attack of food poisoning was treated by the administration of large doses of soda by mouth and he was given soda enemas at frequent, though irregular intervals during the six weeks following the attack. After this time he was troubled with various gastric and intestinal disturbances, and for these increasing amounts of soda were given him. On making the routine blood examination the findings characteristic of alkalosis were noted. The urine was found to be alkaline at three examinations on successive days. The soda was stopped immediately. The spasmodic
attacks disappeared within three days and never re-appeared. The
gastric and intestinal symptoms persisted. Mr. Q., thirty-nine years old, had suffered from diabetes at the age of thirty-two years. He had been given osteopathic treatment at that time, and had been given a diet list of foods exclusively of the alkaline-ash type. He avoided all white breads, all sugars, all plums, cranberries, prunes, meats, and, indeed, every article of food said to have any tendency to cause acid reactions. He did not use soda, but he did take alkaline laxative and purgative drugs, on his own initiative, and he tested the urine occasionally with litmus paper. When the urine was acid in reaction he worried terribly and hastened to take some alkaline medicines. The only common alkaline substance which he avoided was soda. For two weeks before he came to the clinic for treatment he suffered from cramps in the muscles of his legs. These awoke him from sleep, and the pain was really quite severe. On making a blood examination the characteristic staining reactions and nuclear structure of alkalosis were recognized, and the blood was studied with reference to its alkalinity. At that time the technique of studying the reaction of blood was not so nearly accurate as is the case at this time, and it was only possible to determine that the alkalinity was considerably increased in the blood serum. In the urine the alkalinity varied, sometimes being only just recognizable sometimes definitely alkaline on voiding. Certain lesions were found and these were corrected before the blood study was completed. The lesions were not the cause of the cramping and the corrections did not affect the leg muscles. The lesions did cause some of the apathy and melancholy from which he suffered, and these symptoms were considerably relieved by the osteopathic treatments. The mental acuity was increased, and no doubt this permitted a better understanding of the conditions than might have been the case if he had not received those treatments at that time. Having the most extreme faith in the doctor who had prescribed the alkaline-ash diet, it might have been difficult to persuade him of the error of persisting in such a diet indefinitely. When the relation of his diet to his symptoms
was explained to him, he was willing to accept more nearly normal
foods. He confessed to a craving for candy and white bread, and
these foods were given him in moderation. As soon as the urine became
normally acid in reaction, a good wholesome diet was outlined and
this he employed for several weeks. The cramps diminished gradually
for ten days, then disappeared altogether. With this relief of his
symptoms he disappeared from observation for seven years. At the
end of that time he brought his little son for examination. He reported
excellent health during the interim. He had a good position, had
married a sensible wife and had not paid much attention to his diet
during the past five years, because his table was well supplied
with good, wholesome food from which he selected what he wanted
to eat. Miss T., aged twenty-five years, complained of weakness, insomnia and occasional attacks of deep breathing associated with air-hunger. Acidosis was suspected from these symptoms. The routine blood examination showed the structures usually present in acidosis. Miss T. had suffered from an attack of inflammatory rheumatism, and the medical practitioner who attended her warned her against the use of any acid fruits. He specified especially that tomatoes, lemons, grapefruit and oranges were dangerous, and he advised a diet chiefly of toasted white bread, good red meat and whatever she liked, except sour things. She was very fond of candy and pastries. With increasing weakness she avoided exertion, but she did not gain in weight. The alkalinity was diminished in the blood to the equivalent of two hundred fifty milligrams of sodium hydroxide per liter, and of this only fifteen per cent was of the diffusible type. (By the methods used twenty per cent diffusible of a total of three hundred milligrams is normal.) The spinal column showed the irregularities usually present in acidosis. These were corrected during two weeks, with no change in the diet. The symptoms diminished considerably, and the alkalinity of the blood increased to the equivalent of nearly three hundred milligrams of sodium hydroxide. The increase in the alkalinity of the blood after the osteopathic treatments, while the diet remained unchanged, was no doubt due to the fact that the correction of the lesions permitted normal circulation and innervation of the viscera, with resulting increased oxidation, the formation of katabolites more nearly neutral or, in some cases, definitely acid, and more nearly normal excretion of these from the body. In this case some exhaustive and interesting studies of the urine were made, but these are too long to be included in this report. After this study of the effects of treatment
alone, upon the reaction of the blood, the diet was changed materially.
A good, wholesome diet which included rational proportions of fruits,
vegetables and other foods was outlined. The alkalinity of the blood
returned to normal and the symptoms disappeared completely. During
the three years which intervened since that time, she has been normal
and comfortable. Mrs. N. 3. Symptoms included only persistent, dull headache and weariness for which no adequate cause could be found. On physical examination some vague tension of the cervical and upper thoracic spinal muscles was found, but no definite lesions and no recognizable evidences of visceral pathology. On blood examination the peculiar cherry-like tint suggested carbon monoxide poisoning, and by the spectroscopic examination a small, but recognizable, amount of carbon monoxide hemoglobin was determined. The patient had a closed car but drove only short distances and at intervals of several days. She used no gas for heating or cooking. Her home was in an old house on a quiet street. No manufacturing district or oil well was near. There seemed, at first, no possibility of the inhalation of fumes. On studying the plans of the house in which she lived it was found that the house had been piped for gas. On further investigation one of these old gas pipes was found beneath her bedroom, and it was leaking steadily, though only slightly. Mrs. N. was having a new house built, and in this house her bedroom was on the second floor. As soon as she moved into the new home the headaches and weariness passed away, gradually, and within a few weeks her health was fairly good. She then received a few treatments for the abnormal tension of the tissues of the neck and shoulders, and she became perfectly well again. Several weeks later another blood examination showed no evidence of carbon monoxide hemoglobin. In such a case is this it would be easy to infer that the worries inseparable from building a new home were the cause of the symptoms, and that the mental relief following the successful completion and occupancy of the new place were the cause of the recovery. The error of such an inference is obvious form the results of the blood tests. Miss K,11. This young woman, aged eighteen years, complained of languor with persistent dull headaches and some pain in the eyeballs. Physical examination showed no cause for the symptoms. No bony lesions were found, and only some slight but persistent tension of the muscles of the neck could be discovered. Relief of this tension was followed by some slight relief of the discomfort, but the tension re-appeared, together with the aches, within a few hours. She was a solicitor for a wholesale cracker house, and she spent most of her time in her little, old, closed car, driving from one hotel, grocery or eating-house to another, taking orders. She rarely had the car windows open because the breeze disturbed her papers. She lived in a house composed chiefly of wire screening, on a quiet street, and no gas pipes were in the suburb anywhere. Blood examination showed some evidence of chronic carbon monoxide poisoning and a trace of carbon monoxide hemoglobin was found on spectroscopic examination The treatment is obvious. She exchanged the old, closed car for a new roadster, and she bought some convenient cases for her papers. Within a few days the symptoms diminished, and within a few weeks she was apparently as well as could be. A blood examination made three months later showed no evidences of carbon monoxide hemoglobin. Mr. W., aged forty-three years, worked in a Los Angeles office in which smoking was habitual. His home was an hour’s ride distant, and he smoked all the time, riding with friends in the smoker. He had very severe headaches which did not yield to any treatment, and he complained of marked fatigue for which he could not find any cause. The blood showed the usual characteristics of carbon monoxide poisoning. He was persuaded to ride in the non-smoking compartment of the street-car and to have more fresh air in his office. He was also advised to diminish smoking to the lowest comfortable extent and to smoke in the open air. These changes were followed by considerable relief of the headache and the feeling of fatigue. There was recognizable improvement in the blood cells and in the color. But the discomfort of postponing smoking until he could be in the fresh air, and the lack of the usual conversations with his friends in the smoker proved too great. He returned to his old habits and accepted the headaches and other discomforts due to the bad habits. A few months later the bad habits, headaches and friendly relations were all terminated at once by a sudden attack of pneumonia. Miss J. 2. This young woman was a student in a business college. She complained of increasing weakness with dull headache and apathy, and occasional insomnia. No adequate cause for these conditions could be found on physical examination. No bony lesions and no abnormal tension of tissues could be discovered even after very careful examinations. Her school-work was done in well-ventilated rooms. No smoking was permitted in the school; she herself did not smoke, and no member of her family smoked. The house did not have gas pipes, and never had been piped for gas. She had a large, airy room and slept on a porch. No gas wells or manufacturing district was near her home. She had no automobile and rarely rode in a closed car. She walked to and from school twice each day, and sometimes she made an extra trip at night. The distance was rather more than a mile. This walk took her through a tunnel in which traffic was very heavy. The cars were forced to stop and start at intervals of a few minutes. The air was full of the fumes due to imperfect combustion of gasoline and oil. The walk through the tunnel required about twenty minutes, which meant that she spent from an hour and a half to two hours every day breathing bad air. A small amount of carbon monoxide hemoglobin was shown by spectroscopic examination of the blood, after the usual findings had been noted in the routine blood tests. The treatment was indicated by these facts. The walk through the tunnel was discontinued. The symptoms diminished gradually and two months later the blood showed no abnormal conditions. Mrs. D. 4. This woman of thirty years complained of being unable to live at high altitudes. Her home was in a city nearly nine thousand feet above sea level and her husband’s business compelled their residence there for at least ten months each year. During the two months spent at sea level and for the two weeks or so following her return home she was well. At other times she was weak, inert, with dull headaches and constant fatigue. Blood examinations made just before her return to her home, after two months at sea level, showed no abnormal findings. Blood examinations made just after her return to sea level, after ten months at her home, showed the usual evidence of chronic carbon monoxide poisoning. No abnormal findings could be discovered on physical examination. In her mountain home she enjoyed tinkering with her car. At that altitude the use of a gasoline engine presents certain problems and she enjoyed solving them in the most satisfactory manner possible. At that altitude, also, there was much cold weather, so that her work was done within the garage. She had some ventilation in the garage, but this could not be very satisfactory. On changing her habits of living, substituting
other interests for the car, and returning to her home after the
usual two months at sea level, she found herself able to go through
the winter with no ill health at all. It was not the high altitude
which affected her health, but the hours spent with her automobile
in a poorly ventilated garage. This case illustrates the danger
of superficial diagnosis. Changed environment forced changed habits,
and these caused the recovery. People who are fairly normal can
live where other normal people can live, but nobody can be well
who persistently breathes air which contains much carbon monoxide.
Patients who suffer from lesions of the eighth to the tenth thoracic vertebrae or the corresponding ribs often show moderate degrees of cholemia. This condition disappears with a week or ten days after the lesions have been corrected. Miss Y., aged twenty years, complained of dull headaches nausea, some itching of the skin. She was very slightly jaundiced. The blood cells showed the effects of some hemolytic
agent and the serum contained a moderate amount of bilirubin. The
surface tension of the serum was diminished. The urine contained
bile pigments but no recognizable amount of bile acids. There were
no other abnormal conditions of the blood or the urine. Mr. F., aged twenty-nine years, a worker in an osteopathic laboratory, was in excellent health. He used his own blood in some experimental work in the technique of testing blood serum for bile pigments and bile acids, and found the condition normal. After three days of this work he helped to lift some heavy boxes from beneath a table, and thus strained his back Thus he produced a lesion of the seventh to the ninth thoracic vertebrae, which caused pronounced discomfort but no really serious symptoms. He continued his tests for bile acids and bile pigments, and on the third day after the strain he found that his own blood contained five times the normal amount of bilirubin, according to the method he was using, and that the reactions for bile acids were definitely positive. No bile pigments or acids were present in the urine. The lesions were then corrected by means of a single, rather heavy, osteopathic treatment. Three hours later the bile acids and the bile pigments were perceptibly increased in the blood, and both bile acids and bile pigments were present abundantly in the urine. The next day, sixteen hours after the lesions had been corrected, no bile acids were present in the blood serum, the bile pigments were within normal range, and the urine was normal. The lesions did not recur and no further ill
effects were noticed. Miss J., a teacher, aged thirty-nine years, complained of increasing weakness and persistent, though slight, loss of weight. Pulmonary tuberculosis was suspected. On blood examination no evidence of tubercular or other infection could be found. There was no excess of hyaline, eosinophilic or endothelial cells. The neutrophilic cells showed the changes associated with fatigue and moderate acidosis. Immature red cells and white cells of all classes were numerous, though no anemia was present. Myelocytoid forms were also abundant, especially among the neutrophiles. A diagnosis of fatigue plus bony lesions affecting a considerable area of red bone marrow was made. This diagnosis was accepted by the osteopathic physician in charge of the case. When she was told the findings she acknowledge that her hours of work were too long. She was trying to care for an invalid mother during the nights, and was writing short stories to earn more money, while still teaching every day. She had an unusually rigid thorax; both ribs and vertebrae were involved. The treatment was evident when these facts were known. In order to study the effects of rest alone, and partly because her circumstances prevented her receiving osteopathic treatments for six weeks, she was persuaded to cease the extra writing, to have help with the invalid mother by night as well as by day, and to secure as much rest as possible in every other way. He diet was already wholesome and well balanced. With rest alone the symptoms diminished considerably The blood cells lost the evidences of toxemia but the immature forms persisted unchanged. Six weeks later she was able to have good osteopathic
treatments. With increasing flexibility of the thorax, better breathing
habits, plus the continued rest already begun as soon as the diagnosis
was definite, she began to gain in weight and to regain her normal
physical condition. A blood examination made six months later showed
no abnormal cells. Mrs. L. 7, aged sixty-four years, suffered from what seemed to be amild attack of influenza. Fever was slight but she seemed weaker than she should be if this were the correct diagnosis. A blood examination was made for this reason. There was a moderate neutrophilic leucocytosis with low nuclear average and many endothelial cells were present. The fibrin was formed abundantly and immediately upon the warm slide, and the threads were long, coarse, regular in outline, arranged in a dense felt-like mass. A diagnosis of early pneumonia was made upon these findings, though clinical symptoms were negative. Treatment for pneumonia was initiated promptly and she made a good recovery. During her convalescence the sputum was scanty and contained rusty streaks; there were abundant pneumococci in the sputum. There was only a small recognizable area of consolidation, and the speedy recovery was no doubt due to the early diagnosis. Mr. N. 5, aged seventy-three years, suffered from an attack of influenza and pneumonia was feared. No symptoms of cardiac involvement had been noted. Blood examination showed a moderate leucocytosis with low nuclear average; abundant endothelial cells were found. The blood was moderately concentrated and the leucocytes were grouped on the warm slide and in the smears for the differential counts. Splenocytes were abundant. The serum contained a trace of bilirubin. Fibrin was formed abundantly and immediately. The probability that pneumonia was complicated by cardiac inefficiency was mentioned in the notes which were sent with the report Treatment was planned for pneumonia plus cardiac inefficiency, and the patient recovered, though rather slowly. Two months later he was killed in an accident.
The autopsy showed some remaining hepatization in the lower right
lung, and the mitral and the tricuspid valves of the heart showed
the effects of an old, severe endocarditis Miss F. 11, aged seventeen years, was brought for examination because she was losing weight, and because she was becoming more and more irritable. The blood examination showed moderate secondary anemia with very slight evidences of toxemia of the type usually associated with malnutrition. The blood platelets were very low; 50,000 per cubic millimeter. The possibility that there was a lack of Vitamin A in the diet was suggested to the doctor in charge of her case. On investigation it was found that the patient displayed a marked aversion to eggs, and that other foods containing Vitamin A were avoided. Correction of the diet was followed by moderately increased weight and by the disappearance of the nervous symptoms and the irritability. The osteopathic physician reported that no definite
bony lesions were present but that there was some abnormal tension
of the spinal tissues in the upper lumbar region. This disappeared
with the correction of the diet, with no further attention. Mrs. R., aged thirty-nine years, married twenty years, no children, had been advised to submit to an operation for a rapidly growing tumor. The ordinary symptoms of pregnancy were absent. Certain atypical cells were found in the blood smear during the progress of the differential count, and on further study these appeared to be derived from the placenta. Surgical work was postponed, and a few days later an X-ray plate showed fetal vertebrae and other bones. The boy born after a rather stormy pregnancy and labor was normal, and is now nearly fifteen years old. It is rare that the placental cells are useful
in diagnosis, yet such cases have been found several times during
twenty-eight years of blood cell study. Mr. L. 22, aged fifty-two years, complained of certain vague gastric symptoms. Gastric analysis showed absent hydrochloric acid but no other important findings. Roentgenologist’s report showed some delay in the emptying time of the stomach but no evidence of gastric ulcer or cancer. Blood examination showed immediate and abundant formation of fibrin on the warm stage, with threads of irregular length and contour, often beaded, often arranged in radiating lines with a group of platelets or a lymphocyte at the center. Refractive granules were abundant, and these included iodophilic, Sudanophilic and unstained particles. Rouleaux were scanty and the red cells arranged themselves in masses. Leucocytes showed the evidences of toxemia of the type associated with disturbances of protein katabolism. Hyaline cells and eosinophiles were increased, both relatively and absolutely. The eosinophiles often showed abundant, basophilic, hyaline, intergranular protoplasm. Fibrinolysis was masked by undifferentiated
proteolysis. The probability that late malignancy was present was
noted in the extra report sent to the doctor. Surgical interference
was decided upon, and an inoperable cancer was found around the
pyloric region of the stomach. At autopsy, two months later, this
was found to be completely surrounding the pyloric antrum. It was
of the scirrhous variety, and there was no gross ulceration of the
gastric mucosa. These facts explained the erroneous roentgenological
report. Mr. O. 2, aged sixty years, suffered from severe pain in the stomach with occasional nausea; neither the pain nor the nausea seemed to bear any relation to the taking of food nor its quality. Blood examination showed the cell findings reported in the case of Mr. L, 22. Gastric analysis showed absent hydrochloric acid and also a few small masses of cells showing abundant and often irregular karyokinesis. Fibrinolysis was absent and no undifferentiated proteolytic ferment was found. Surgical interference was based upon these findings.
A small cancer was found upon the anterior aspect of the pyloric
antrum. Gastro-jejunostomy was performed, and the patient is alive
and well at this time, twelve years later. Mrs. W. 17, aged forty-three years, showed a small, hard tumor in the left breast. This was associated with vague pain in the same general region, radiating along the intercostals nerves to the spinal column. There was a history of an abscess in the place occupied by the tumor, which was present about twenty years before the tumor was noted. She was not in habit of paying very much attention to her own body, and had always been an unusually busy and active person. Blood examination gave a normal blood picture. Fibrinolysis was normal and no abnormal findings were reported for any of the special tests. Surgical interference was postponed, on these
findings. The rib lesion was corrected and the pain disappeared.
The tumor seemed unaffected by the treatment. It was kept under
observation for several years, but no increase in size ever occurred.
After her death, fifteen years later, this tumor was removed for
histological examination, and it was found to be composed of scar-like
threads of connective tissue with no evidence whatever of malignancy.
Miss C. 5, aged fifty-three years, noted slight and repeated uterine hemorrhages. On blood examination the findings characteristic of malignancy were reported, and fibrinolysis was absent. There was no evidence of an undifferentiated proteolytic ferment. Pelvic examination discovered a myofibroma of the uterus but no evidence of cancer. Uterine curettings and a bit of tissue from the region of a small cyst on the cervix were removed for microscopic examination but no evidence of malignancy was found. The uterine hemorrhage was repeated, and a second blood examination was made five weeks after the first examination. The evidences of malignancy were somewhat more marked and there was present some undifferentiated proteolytic ferment. At this time the patient complained of pain in the lower abdomen and the pelvis. Because of the myofibroma and the pain it was
decided to perform a hysterectomy. A tumor of the right ovary was
found, and on microscopical examination this proved to be a papillary
adenocarcinoma. There were several small metastatic tumors upon
the adjacent peritoneum. Radium treatment followed the removal of
the cancer and the uterus. She made an excellent recovery, and is
still in good health, eleven years later. Mrs. T. 4, aged fifty-eight years, suffered from slight but frequent uterine hemorrhage. Diagnosis of uterine cancer was made by an eminent medical surgeon, who advised immediate hysterectomy. This she refused, for the time being. She consulted an osteopathic surgeon, who found a lesion of the fifth lumbar vertebra. The patient explained this by a fall she had had about ten days before the first uterine hemorrhage. This osteopathic surgeon advised the correction of the lesion before operating. The pelvic examination discovered a heavy, edematous, congested cervix and uterus but no definitely marked tumor. Blood examination showed normal fibrinolysis with no evidences of malignancy. The lumbar lesion was corrected, the uterine hemorrhage ceased Pelvic examination three weeks later showed normal cervix and uterus, and no reason for surgery. |
