Complete Text (Part 15)

count- ing chamber.- 186 (1) Siugle counting chamber. From a pipette place a drop in the manner described above on the central raised ring of glass. This drop must be of such a size as to cover the entire ring after the cover slip is placed over it, but must not be large enough to cover the moat and spread over the surrounding raised glass platform. It is per- missible, but undesirable, to have a little fluid enter the moat. With the drop placed, lower the cover slip over it in such a manner that no bubbles are formed. If bubbles oc- cur, wash the counting chamber and repeat the attempt. Newton's rings. (N. B. — Bubbles cause an uneven spread of cells.) (2) The Burker Double Counting Cham- ber (with two central raised glass platforms, making it possible to make two preparations simultane- ously K Place the cover slip on the chamber and see that Newton's rings are present — do not remove the cover slip. From the pipette let a drop run under the cover glass over each central glass platform. The size of the drop must be gauged by practice. It must be sufficient^/ large to cover the whole platform and yet not large enough to cover the moat. No bubbles are permis- sible. If they occur, a fresh prepa- ration in the counting chamber must be made. 187 c. Counting cells in the counting chamber. (Re- fer to Emerson, AYebster, etc.) (1) K. B. C. (a) With high-power focus on the cen- tral portion of fine ruling. (b) Some cells will be found lying out- side the specified area, but touching the lines of division — count such cells when they lie on dividing lines to the left or above the section be- ing counted. Omit those lying on the right or beloAv. (c) Begin counting at the extreme left — move to the right over the upper row of 5 small squares, then pass to the next lower row and move to the left, then descend another row and pass to the right, and proceed in this fashion for each of the 5 rows in each of the 4 corner units. (d) Count the 4 corner squares on two preparations. (e) Calculation. 1. The area of the fine ruling rep- resents 1 square mm. of 1/10 mm. depth. 2. This area is broken up into 16 subunits, of which number the 4 corner ones in each of two preparations have been counted; therefore, 1/2 cu. mm. of diluted blood has been counted. 3. Let X ^ the sum of cells counted in the 8 corner units; therefore, X = number of cells in 1/20 cu. mm. of diluted blood. 4. In the red pipette the blood was 188 diluted 200 times ; therefore, 200 X = number of cells in 1/20 cu. mm. of undiluted blood, or 4000 x = number of cells in 1 cu, mm. of blood. Therefore, when the 4 corners in 2 preparations have been counted the number found is multiplied by 4000. The re- resulting figure represents the R. B. C. count or the number of R. B. C. found in 1 cu. mm. of blood. (2) W. B. C. (a) With the low-power focus on the ruled portion of the counting cham- ber and pick out the four large cor- ner areas of coarse ruling (each such area is the size of the central finely ruled area and represents 1 sq. mm.). (b) AVith low-power count the 4 cor- ner areas and also the whole cen- tral finely ruled area — thus count- ing 5 sq. mm. on each of 2 prepa- rations, or 10 sq. mm. in all. (c) Use the same method of including those cells which touch dividing lines to the left and above and omit those lying outside the speci- fied area, but touching dividing lines to the right and below. (d) Begin at the extreme left and work to and fro in the manner suggested for R. B. C. counting. (e) The cells counted in the 10 areas (represented by x) will represent the number found spread over a 189 surface of 10 sq. mm. with a 1/10 mm. depth; therefore, x ^ number of cells in 1 en. mm. of diluted blood. (f) The dilution in the white pipette was 20: therefore, 20x = number of cells in 1 cu. mm. of whole blood. Thus when the 10 squares have been counted, the number of cells found multiplied by 20 = the W. B. C. count or the number of W. B. C. found in 1 cu. mm. of blood, d. Kequirements for satisfactory counts. (1) K. B. C. (a) A satisfactory count requires that the difference in the number of cells counted in each of the 4 corner squares in 2 preparations shall not not exceed 25. (b) Such satisfactory counts shall be made on two successive days, with not more of a variation between the total counts than 200,000 cells. (2) W. B. C. (a) A satisfactory count requires that the dijfference in the number of cells counted in each of the 5 sq. mm. areas in 2 preparations shall not exceed 8. (b) Such satisfatcory counts shall be made on two successive days, Tvith not more of a variation between the total counts than 200. Because of the AY. B. C. of an indi- vidual vary with bathing, eating, exer- cise, etc., it is advisable to make the counts on the successive days under con- ditions as nearly constant as possible. 190 Ex. of satisfiu •torv counts. May 4tli May oth May lOth Mav nth ^^ '.B.C. ' ^Y .B.C. R.B.C. Px.B.C. Trep. 1 42 Prep. 1 32 Prep 1 149 Prep. 1 153 34 40 150 148 41 40 135 130 38 40 149 128 40 40 Prop. 2 42 Prep. 2 40 Prep 2 150 Prep. 1 155 36 40 148 135 35 40 145 150 38 39 127 131 21 33 1153 1130 387 384 1153 X 4,000 = 4.612.000 387 X 20 =3 ■; '.740 1130 X 4.000 = 4.520.000 384X20 = 7,680 IT. Haemoglobin determinations. a. Talqnist's method (see Emerson, Ed, IV, page 525, for fnll explanation). (1) Teclmiqne. With a drop of blood on a piece of provided filter paper match its color in a scale of standard colors which is to be found in the book. (2) Difficulties. The scale of standard colors does not hold its registration of color values ac- curately ; age and exposure to light tend to fade them. (X. B. — If this method is to be used, the individual color scale should be standardized from time to time by run- ning parallel determinations with it and a Sahli or Miescher instrument.) b. Sahli method (see Emerson for diagram and full description). (1) Apparatus and its use. (a) In order to insure a homogeneous shade, the sealed standard tube (filled with a standard acid hematin susnension) should be inverted sev- 1^1 eral times before a determination of Hb. is to be made. (bl The pipette is filled with blood up to the 20 cu. mm. mark (the only mark on the pipette). The filling must be done accurately and all blood adhering to the outside of the pipette must be carefully remoyed before the pipette is emptied into the calibrated tube. (c) The calibrated tube, which must be clean and dry, is first filled with a stock solution of X/10 HCl ex- actly to the 10 mark. (This is used to lake the blood Avhich is intro- duced into the tube and to trans- form the liberated haemoglobin into acid hematin.) Into this solution the pipette containing the blood is carefully introduced and the con- tents of the pipette expelled by means of careful blowing. The acid solution is cautiously drawn into the pipette and repeatedly expelled in order to remoye thoroughly all blood from the pipette. The blood is allowed to remain in the acid for exactly one minute by the watch, and then distilled water is added until the shade agrees with that in the standard tube. (N. B. — In mixing the contents of the calibrated tube the following points are of yalue : 3. If during the addition of fluids the upper part of the tube has been kept dry, it is possible, by 192 ctireful tilting, to foriii a menis- cus across the npper portion of the tuhe. When this has been acconii)lishe(l the tube may be safely inverted and the tlnid^ thoroughly mixed. 2. The fluid in the tube should never be permitted to touch linger, as a loss of fluid occurs which amounts approximately to 1 de- gree on the scale, with a result- ing inaccuracy in the ultimate reading. ( 2 ) Meihod of taking the readings. (a I Allien a reading is to be made, the tube should always be placed in the stand witli its ground glass back. ( b ) Let the light come through the back of the stand. ( c ) Readings are best made by daylight. Artiticial light makes the ditferenti- ation of shades difficult, c. Miescher method (see Emerson for diagram and full description ) . (1) Consideration of points not sufficiently emphasized. (a) The cells must be thoroughly dry and clean before the bottoms are screwed in. The groove in the glass bottom and top should be oiled each time before placing them in posi- tion. The floor must be screwed in iiglithj, so that there may be no interchange of fluids betAveen the two sides of the cell. Place dis- tilled water in one side of each cell and let the preparation stand 193 for several minutes to be sure the compartment is water tight before adding the blood preparation to the other side. (b) For normal blood a dilution of 300 is satisfactory. DraAv blood to the 2/3 mark on the pipette, and then till with 0.1% XaoCO.. (X. B.— Any trace of XaHCO. in the diluting fluid must be avoided, as it will cloud the preparation, making a satisfactory reading im- possible.) (e) Shake tlie pipette thoroughly until the solution becomes perfectly clear and the sliade homogeneous. fd) Fill tlie empty side (Avhich has been proven to be Avater tight) of both the 12 and 15 mm. cells at the same time and make readings at once on first one and then tlie other prepa-- ration. (The pipette contains enough of the preparation to per- mit of the filling of both the 12 and 15 mm. chambers at the same time.) (e) The readings are made in a dark room, prepared for the purpose. A candle flame aftords the necessary liglit for illumination of the prep- aration, and the scale may be seen by using a flash light, or turning on the electric light momentarily. Ten successive readings should be made on both the 12 and 15 cu. mm. cells. The scale should be turned alternately too high and too low and the color matched by running 194 through the higher shades down to the same shade and again by run- ning through the paler shades up to the same shade. The average of the 10 readings is the one taken as final. (f) The tables of calculations which accompany the Mieschers are not interchangeable. A preparation on a Miescher must be calculated ac- cording to the table made for that special instrument, d. Kequirements in Hb. work. On the day on which the R. B. C. count is passed (which is the day of the second successful K. B. C. count) a Sahli determi- nation shall be made and repeated again the following day. A difference of 5 is per- mitted in the readings on the two days, A Miescher reading shall be made on two suc- cessive days, the first Miescher shall be made on the day of the second Sahli determination. Readings with the two cells, both calculated for the 15 mm. cells, shall not vary over 2 points. C. ADDITIONAL BLOOD EXAMINATIONS. I. Coagulation time. (L^se of Bogg's coagulo- meter. See Emerson for full explanations and diagram.) a. The following points require emphasis : (1) Consider the time from the moment the drop appears on the skin until the radial motion in the drop is seen under the microscope. (2) Focus with the low power of the micro- scope on that part of the drop just opposite the blowing tube. 195 (3) Blow as lightly as possible on the drop, moving only 6 to 10 corpuscles. (4) Blow not oftener than at 30 sec. inter- vals— more frequently blowing produces a vicious cycle and the radial motion may never appear. b. Requirements. (1) Make three determinations in succes- sion on the same day. The difference in coagulation time should be less than -45 sec. IF. Bleeding time. a. Make a finger incision sufficiently deep so that a drop of blood 2 mm. in diameter will ai3pear spontaneously without any squeez- ing. b. Take a jnece of smooth filter paper and blot oft' the drop every 30 sec. (no nuuiipu- lation of the part is permitted). c. Divide the number of blots by 2 and the resulting figure represents the bleeding time in terms of minutes. d. Requirements : Carry out this procedure once. III. Fibrinolysis test. (Goodpasture, J. H. H. Bulletin, November, 1914.) a. Test based on the ability of the blood in certain pathological conditions to digest its own clot within a few hours at body temperature. b. Technique. Draw 3-5 cc. of blood into a small, clean test tube. Incubate 12 hours at body tem- perature. (1) Positive test. Complete digestion will have oc- curred within 12 hours. 196 d. (2) Negative test. Partial digestion or none at all within 12 hours. Action probably dne to tlie activity of nor- mal proteolytic ferments of the blood, oper- ating by virtue of a diminution or absence of normal anti-proteolytic ferments. Fibrinolysis has been found associated with chronic liver injury with severely impaired hepatic function where a hemor- rhasfic tendency exists. DIFFERENTIAL W. B. C. COUNT. Dec. 10, 1918. Wilson stain W. B. C— 8600 300 Cells Cells Counted. Ehrich or Jenner Absolute Counted. Percent Number. 1. P. M. N . 201 68 5818 9 P. ME. C) o 172 3. P.M.B 3 1 86 4. Lymbho's small . 60 20 1720 5. Lymbho's large. 3 1 86 6. Large mono's. . . 15 5 430 7. Transitionals . . 9 3 258 8. Unclassified .... . . . . •• .... 300 100 8600 197 CHART FOR SPECIAL BLOOD EXAMINATION. Patient's Name. Stain Used. Ward or Hospital. Date of Preparation. Doctor's Name. Date of Examination. I. General Comment on Preparation, Stain, etc. IL R. B. C. 1. Size. 2. Shape. 3. Color. 4. Regeneration forms : a. Nucleated R. B. C. ; type; numerous? b. Basophilia; punctate or diffuse? c. Nuclear particles. d. Cabot rings. 5. Additional Remarks. IIL W. B. C. 1. Apparent number (relative to R. B. C). 2. Apparent differential. 3. Presence of abnormal forms : a. Myelocytes; type? b. Myeloblasts. c. Irritation forms. d. Fragile W. B. C. ('^smudges") e. Pigmented W. B. C. 4. Additional Remarks. IV. Platelets. Apparent number (relative to R. B. C). Normal; increased; decreased. V. Parasites. VI. Additional Remarks, General. VII. Impression. 1. Summary of Important Evidence. 2. Probable Diagnosis. Blood Report Number. Name of Examiner. 198 HEMATOLOGY. Notp: : The following notes on blood are rather sketchy in character and are, therefore, of more value to those who attended the lectures covering this sub- ject than to those who did not. It is hoped, how- ever, that even to the latter they may prove to be of some value. The formed elements of the blood. I. Red blood cells (erythrocytes). Normal con- sideration. 1. Embryological origin of the red cells. There is a great discussion and difference of opin- ion. (a) Primary blood islands: 1. Hemoblasts. 2. Bildungszellen of Koelliker. From the outer cells arise endothelial cells. From the inner ones the primary erytJirohla'Sts arise, which contain no hemoglobin and are colorless and nu- cleated. All cells are originally of in- travascular origin. All are nucleated up until the fourth week. By the third month only one-eighth are nucleated, and at birth none are nucleated. Extravascular formation soon com- mences, and is especially active in the liver, spleen and marrow, the latter be- coming more and more the main site. Hemoglobin-free cells disappear quite early. (b) Primary erythroblasts, essentially iden- tical with megaloblasts of Ehrlich; hence the term "return to embryonal form" in anaemias. (c) Discussion of the Monistic and Dual- istic theory of origin: the discussion centers around the point as to whether 199 tlie red and white cells have a common origin. Dualists, Monists, Naegeli, Shridde, Ehrlich, Maximow, Howell, Pappenheim, et al. et al. The main mass of evidence tends to establish these points : 1. Erythroblasts occur at a time much earlier than any leucocytes. 2. Primary cells of Koelliker do not form white blood cells, otherwise it is impossible to understand why later on in the vessels one finds only ery- throblasts and neither Bildungs cells nor Avhite cells. 3. Primary erythroblasts and endothe- lial cells are sisters. 4. No direct evidence of transition stages of red blood cell into white blood cell or the reverse. Schridde and Maximow maintain this idea : Bildungzellen are in reality "large lymphocytes. From these there arise both white blood cells and red blood cells. This is said to be the "primary eiythroblast" and may be quite identical with the Myeloblast of Naegeli. 200 GENESIS R. B. C. 2. Post foetal formation of erj^throcytes. Normally from marrow only. (18(38 Neu- mann.) The marrow contains many ripe and nnripe cells, most of which are nucleated and from 6-12 microns in size. Chief variety: (a) Megaloblasts — -Definition used here: nucleated cell, densely staining, with nucleus