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Simple Hematological Procedures in Office Medicine
Simple Hematological Procedures in Office Medicine EDWARD H. McGEHEE, M.D. Hematologist to the Pennsylvania Hospital; Associate Hematologist, Benjamin Franklin Clinic; Associate in Medicine, University of Pennsylvania School of Medicine; Associate1:n Medicine, The Chestnut Hill Hospital, Philadelphia
THE clinical recognition of anemia is not as easy as we practitioners are accustomed to think. Certain fair-skinned persons as well as some with olive and sallow complexions appear quite anemic but yield normal hemoglobin values. On the other hand, it is surprising how often make-up, suntan or just facial flush will totally mask a real anemia. A common source of error on the referring physician's part, as seen in a busy out-patient department and a large referral diagnostic clinic, has been the failure to do a simple hemoglobin evaluation. In conjunction with this, the passage of several years without technical help in the office has impressed me with the ease and value of performing a hemoglobin determination and a peripheral blood smear on every new patient. Leukocyte count, differential and erythrocyte sedimentation rate may also be done when indicated with little expense in time or equipment. The following are samples of simple hematological omission: A woman, 36, was referred to the Benjamin Franklin Clinie for evaluation of her anemia. She was pale and complained of constipation and easy fatigue. Her hemoglobin was 13.4 grams. No organic disease could be found. She had not had a hemoglobin determination before referral. A man, 64, was referred because of cardiac irregularity for electrocardiography and management. His hemoglobin was 5.3 grams. Carcinoma of the stomach with intestinal bleeding was found. The patient was tanned from sailing. A woman, 62, had been treated for several months for hypertensive cerebrovascular disease because of bouts of dizziness. Her hemoglobin was 8.1 grams. A gastric leiomyoma was removed, and she has had no further symptoms since her hemoglobin returned to normal range. The hypertension persists. A man, 54, had been treated with vitamin B12 for several months without any
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improvement. A peripheral smear was severely hypochromic. The hemoglobin was 5.5 grams, and the stool positive. Iron therapy was effective.
Periodic physical examinations for health maintenance are becoming increasingly frequent, and these jobs should and will fall to the practitioner in his office. The palmar crease has not been as reliable as conjunctival and oral mucosal changes in estimation of degree of anemia Such findings plus murmurs, dyspnea, edema or lightheadedness may enable us to reckon that hemoglobin is in the 4 to 6 gram range. More accurately determined initial hemoglobin levels, however, are necessary as base lines for evaluation of therapeutic response. Determination of hemoglobin level and a glance at a stained peripheral smear may also reveal the only clues to incipient disease. Normal results from such procedures should relieve anxieties on the part of the patient as much as the report of a "Pap" smear, EKG or chest film. The age of expensive electrical gadgetry has done much to stimulate a withdrawal reflex on the part of many practicing physicians. A quiet inferiority feeling has led to total cessation of laboratory work in many offices. Those who are really sick have been referred elsewhere for testing or hospital admission. Many patients moderately ill have consequently suffered from lack of any sort of determination by falling into the twilight zone between health and frank illness. This is a plea for the use of even the most meager of hematologic de terminations in every office; a few short discussions of some practical points in their accomplishment follow. THE FINGER PUNCTURE
The spring lancet and the cork with a Bard-Parker blade extending rather naughtily for 3 or 4 mm. beyond its flat undersurface are obsolete. Epidemiological studies of viral hepatitis have proved the dangers of such instruments when repeatedly used without proper sterilization. There are available several kinds of individually packaged, sterile strips of metal with one end properly sharpened and beveled. These may be discarded after one use without actually bankrupting the physician and certainly with much saving in time. Sera Sharp, Redi Lance and Meti-point are a few of these. The patient's finger should be cleansed and held firmly. The metal point in the physician's other hand is plunged into the fleshy pad of the finger, preferably a bit off cent er and with follow-through. There should be a spontaneous welling up of blood, which obviates the need for squeezing or milking the patient's finger. A well executed puncture is no more painful than one poorly done. An ample supply of blood as described yields more consistent and accurate results; smears are less distorted; pipettes clog less. Acceptance by the patient is better because a repeat wound is not necessary, and there is no fuming and difficulty
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Fig. 1. Just touch the top of the drop.
while the doctor tries to squeeze out just a little more blood while obviously not doing a job properly. After the puncture, the finger should be wiped with a dry gauze before the actual taking of each sample of blood. This will insure proper spreading, smearing and flow in pipettes. 'Whether one makes smears or uses pipettes, this should be emphasized: Just touch the top of the drop (Fig. 1). HEMOGLOBIN DETERMINATION
Visual Colorimetry
The old method of Sahli, using a graduated tube, 0.1 N hydrochloric acid, accurate hemoglobin pipettes and amber glass comparator standards, is probably the cheapest feasible method for hemoglobinometry in the average practitioner's office. Though less accurate than other methods, it will allow reasonably reliable follow-up determinations, particularly when performed by the same person week after week. Such disadvantages as a progressive increase in intensity of brownish coloring on standing during the first ten minutes after mixing and the necessity for repeating the whole procedure if too much water is added bear mentioning. The Spencer hemoglobillometer is slightly more costly but is simpler to use and somewhat more accurate. This instrument measures the intensity of green light transmitted by a thin layer of hemolyzed blood as compared with that transmitted by a standardized glass wedge. Pipetting is eliminated, as is color judging. Electrophotometry
The basic principle is that an electrophotometer measures the amount of light transmitted by a hemoglobin solution. The oxyhemoglobin
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method necessitates the pipetting of blood into a standard volume of sodium carbonate or dilute ammonium hydroxide; the cyanmethemoglobin method requires pipetting the blood sample into a solution of potassium cyanide and potassium ferricyanide. These methods are speedy and highly accurate, especially the cyanmethemoglobin method because the solution used is somewhat more stable. They do require a photoelectric colorimeter, which is costly and temperamental, necessitating calibration regularly and frequently. The Tallquist scale and the copper sulfate specific gravity method are mentioned only to advise against their use. Terminology and Normal Values
Hemoglobin should be expressed in grams per 100 milliliters of blood or, in common usage, simply in grams. This avoids the use of percentages, which confuse and disturb patients when they aren't 100 per cent, and it thereby saves the physician much time and explanation. The normal range for children is 10 to 15 grams; for women, 11.5 to 16 grams; for mfln, 12.5 to 17 grams. PERIPHERAL SMEAR
The examination of a carefully prepared peripheral blood smear remains the most significant laboratory study in patients with hematologic abnormalities. A few words to amplify "carefully prepared" seem fitting here and pertain to both coverslip and slide techniques. Preparation and Care of Glassware
The glass surfaces should be scrupulously clean and free from nicks and scratches. Washing may be done with detergent and water and should be followed by rinsing with large quantities of warm water and transfer to 95 per cent alcohol. Before use, slides and coverslips should be polished with a clean cloth free of lint, dust and grease. It has been convenient to store a small supply of slides ready for use wrapped in two's in lint-free toilet paper. Coverslips may be kept in gauze-lined jars and picked out with forceps or kept in slits in a cardboard box. They should be protected in containers with tight lids. Coverslips which are wavy, convex, or concave are useless because uniform contact between the two is necessary for proper spreading of blood. Scratched surfaces result in pooling and irregular distribution of blood. The ends of slides should be regular and smooth; any broken areas or nicks will leave a wide trail in the blood smear. A good grade of glassware should be purchased at the outset. In most instances, only new slides and coverslips ought to be used.
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Fig. 2. After the drop has spread, separate the coverslips by smooth, deft pulls in the same plane.
Making the Smear
A small drop of blood (not exceeding 3 mm. in diameter) is adequate for a good smear. The drop is placed on one coverslip, and the other is placed over it gently. Time is allowed for spread outward until the pink coloring is about 2 cm. wide. Then with a deft swish, the coverslips are slid apart, never getting out of the same plane (Fig. 2). The slide technique is more often than not ruined at the outset by too much blood. A 2 to 3 mm. drop is quite enough. As soon as the drop is on the slide, the slide is placed on a substantial surface such as a table top or desk. The end of a second slide is placed flat across the surface of the first slide with the long axis of the second slide at a 30- to 45-degree angle. The second slide is backed into the drop of blood; as soon as the blood spreads laterally about 1 cm. each way (not all the way to the edge of the slide), then smoothly and briskly the second slide is moved away from the blood droplet, the first slide being held flat and still with the other hand (Fig. 3).
Fig. 3. Back the edge of the second slide into the drop, allow the blood to spread laterally, then smoothly and briskly push the second slide away (in this sketch, from right to left); this will leave both edges available for scrutiny and room on the slide for labeling.
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Fig. 4. Wash from the side so that the surface film goes over intact.
Staining
Wright's stain iH adequate and cheap. It can be procured as solution or in the dry crystalline state. Most commercially available solutions are diluted and do not require the use of buffer, allowing for simplicity but at the expense of the finer differential features desired and expected in a good peripheral smear. Crystalline Wright's is available in vials of measured quantity which must be suspended in methyl alcohol. Like many alcoholic concoctions, it should be aged before use. It is handy to have two jars of stain so that one can be aging while the other is in use. It helps to label each as to the time of initial suspension so that at least three weeks may pass before it is used. A good shaking and resuspension every few days strengthens the solution and requires only a few seconds. Filtering is necessary to prevent deposition of minute granules of stain on the blood film. Stain poured from stock bottles should always he filtered prior to use. The solution in the drop bottle in current use should be re filtered at least weekly and the drop bottle cleansed with hot water and a methanol rinse. A buffer solution of sodium phosphate with a pH of 6.4 is readily available and cheap. It should be added to the slide in a quantity equal to that of the stain used (15 to 20 drops usually). The timing varies but the average range is 1 to 2 minutes with stain alone, 7 to 15 minutes with buffer solution added. Rinsing is simple enough, though one hint may be valuable in procuring prettier slides. The water should be accurately edged in from one side so that the scum of stain will be maintained with its surface tension intact as it is washed over the other side (Fig. 4). This prevents settling
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OIL
Fig. 5. Touch the oil droplet with a toothpick and spread a thin coating of oil over the entire blood film.
of fine granules of stain over the cells and intercellular spaces on the slide. The slide should be rinsed further in running tap water and allowed to dry in an upright position. There should not be any emergency acute enough to warrant blotting the smear, which will mar or ruin the blood film. Examining the Smear
A drop of immersion oil can be added to the stained side of a coverslip preparation, which is then placed face down on a slide. For slide preparations, a drop of immersion oil is placed at the cent er of the smear just before the trailing edges begin to narrow. Then it is spread evenly with an easy stroke of the immersion oil toothpick or an applicator stick (Fig. 5). The film is placed under the microscope (low power) and moved about until the red cells can be seen individually with space between them. The leukocytes should be seen in such an area clearly enough to differentiate monocyte, lymphocyte and neutrophil in most instances. One's first reaction to this will be, "I'm too far away to see anything." It takes a little time to become adjusted to this method of scanning blood films, but it will save the physician time and the patient much bloodshed and money in laboratory studies. It will help the physician make more diagnoses. By scanning, the general appearance of the red cells as well as the number and character of the white cells can be reckoned. With the help of oil immersion, a glance at the red cells in a few locations will settle the question of red cell shape, size and coloring. At this time an estimate of platelet population can be made by noting single platelets and collections of two to four platelets interspersed between the red cells. An incidence of one to four platelets or platelet groups per oil immersion field is normal. The white cells can then be studied and the smear discarded. (Dr.
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Fig. 6. In making a differential count, follow a regular path back and forth across the slide.
Wurzel's review of the proper paths in hematological diagnosis will be most helpful at this point. See page 1607.) Differential counts on slide preparations may be misleading if some order is not followed. I have found that the most satisfactory method is to start somewhere in the center and to move toward the side of the smear until it is reached, then to move four to six fields along parallel to the side of the slide before starting back across the middle of the smear (Fig. 6). More polymorphonuclears are seen along the edges and more lymphocytes toward the middle. This is not a problem with coverslip preparations. SEDIMENTATION RATE
For those interested in performing sedimentation rates, the Westergren method is hereby endorsed. The 200-millimeter tubes, which offer the advantage of easy filling and cleaning, are not particularly costly, and a rack can be bought or made. Sodium citrate 3.8 per cent is likewise easily available. 1.8 ml. of blood is mixed with 0.2 ml. sodium citrate. The tube is filled with blood to the "0" mark by suction and is placed in the rack. Reading is done at the end of one hour. The tube can then be drained, washed and stored. No correction for hematocrit is necessary, and this procedure provides easy reading with accuracy equal to or surpassing that of other methods. ANTICOAGULANTS AND HOUSE CALL BLOOD SAMPLES
Ammonium and potassium oxalate are the anticoagulants used by most laboratories for collecting venous blood samples for transportation, whether it is for three floors, to the next building or ten miles. Tubes or small bottles with a measured quantity of these two crystallized salts can be obtained at the nearby private and hospital laboratories which one uses or can be bought commercially. Each tube or bottle is usually adequate for 5 cc. blood. Hemoglobin, hematocrit, leukocyte count and sedimentation rate may be determined from oxalated samples. It
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should be emphasized, however, that smears should be made immediately from droplets of freshly drawn blood in order to avoid the many cellular artifacts that can result from exposure to anticoagulants. Whenever blood films are made from an oxalated sample, the slogan should be, "Don't be alarmed by odd cells seen unless smears have been repeated using fresh blood." HEMATOCRIT
The hematocrit is the most accurate procedure available for determination of anemia and polycythemia. With a standard centrifuge, Wintrobe tubes and an oxalated blood sample should be used. A sedimentation rate can be determined prior to centrifugation. The centrifuge should run at 3000 r.p.m. for 30 minutes. The volume of packed red cells is easily determined, the height of the buffy coat will give a good indication of the leukocyte count, and the thickness of the creamy white layer above the grayish-pink buffy coat will indicate the platelet count. The plasma should be observed for the increased discoloration due to jaundice or the decreased discoloration characteristic of iron deficiency. The microhematocrit methods now available deserve mention here. They yield accurate results within five minutes, since only three minutes' centrifugation is necessary. Finger puncture blood is adequate for the determination. The only deterrent is price. Microhematocrit apparatus should be considered by any group of two or more doctors equipping or refitting a small laboratory and those planning a large laboratory. LEUKOCYTE COUNTS
Certain aspects of white cell counting merit discussion. Pipettes should be kept clean and dry, and the tips should be checked frequently for nicks, cracks and other such flaws that mar the accuracy of the contents. Acetic acid 2 per cent is an adequate diluent and does not require any coloring agent to help the leukocytes stand out. The counting chamber should be cleaned after use and polished prior to use so the cells will spread evenly. Shaking can be accomplished in an automatic shaker or manually; it should be vigorous, constant and multidirectional for at least one minute to allow for proper mixing. If the count is excessively high, a red cell pipette may be used. Blood should be drawn up to the 1.0 mark and the pipette filled with WBC diluting fluid. Five large squares are counted and the total multiplied by 5000. If the count is quite low, a white cell pipette may be filled to the 1.0 mark instead of the 0.5 mark and the multiplication factor halved. A practicing physician should be able to diagnose neutrophilic leuko-
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cytosis, chronic leukemias (particularly the chronic lymphocytic) and some acute blastic leukemias in his office. "L. E." PREPARATIONS
It is feasible to demonstrate in the office laboratory the "L. E." phenomenon in clotted blood preparations or in mixtures of serum from the disseminated lupus erythematosus patient with white cells from someone else's buffy coat after some incubation. Certainly, larger laboratories should be proficient at demonstrating these cells. The physician who suspects disseminated lupus but who performs only the essentials in his small laboratory can draw 6 or 8 cc. of blood, allow it to clot and send it to a competent laboratory nearby. BONE MARROW EXAMINATION
Aspiration of bone marrow is an office or out-patient procedure. This should be clear to all practitioners, for it may save much time in a diagnostic workup and it makes hospitalization unnecessary. (Blue Shield pays the same fee whether the procedure is done in the hospital or in the office.) It is not the intention here to suggest that bone marrow aspiration be done by all practicing physicians; rather, the intention is to make clear a point that has been uncertain in the minds of many referring physicians and patients. Actually, most patients find a bone marrow aspiration no worse an ordeal than a bromsulfalein retention test and less traumatic than a glucose tolerance test. There is practically no danger. Nearly all of the twenty or so deaths from bone marrow puncture in Europe and America have resulted from trans-sternal perforatioll by untrained hands. SPECIALIZED HEMATOLOGICAL PROCEDURES
Reticulocyte counts, platelet counts, prothrombin determinations, bleeding and clotting times, RBC incubation procedures, red cell fragility studies, Coombs' antiglobulin tests and special staining techniques should not be attempted in most office laboratories since the intricacies of these procedures make them unreliable unless they are done constantly by the same personnel. COMMENT
There will be many voices raised against the suggestion that hemoglobin determinations and peripheral smears are adequate screening procedures. However, if they are used in otherwise healthy persons and
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if they are amplified for any illness or abnormality encountered, they seem to be an acceptable practical compromise. A few words against the commonly encountered defeatist attitude, "Oh, it wouldn't do any good for me to look at a slide. I wouldn't know one cell from another." This is heard from interns and first-year residents only slightly less often than from older graduates. The remedy lies not in a two-week course in Advances in Hematology offered by Antarctic U. Medical School. The remedy lies in a few hours spent with a hematologically oriented pathologist, or a hematologist, and his favorite technician. One should simply find out when slides are reviewed and be there a few times to show these people one's interest and any problem slides one might have. One should ask permission for the technician to spend a few minutes giving pointers in technique. Good hematology technicians are more numerous today and they can be of tremendous help with problems concerning red or white cell morphology as well as with more technical matters. The interpretation of peripheral blood smears should not be considered a mystery any more than should good physical diagnosis. Rales can be heard; thrills can be felt; nodes, livers and spleens can be palpated. Red cells can definitely be seen to be large, to vary remarkably in size and shape, or to be pale and lacking in hemoglobin. When doubt exists, one must repeat the smear and wait, just as with other tests. An earnest attempt to look at one's own slides will prove that hocus-pocus is not an ingredient of hematology; the main requisite is a little sincere interest. It is not my intention that these paragraphs should substitute for carefully written, stepwise methodology for the hematological investigations discussed. Ham's Syllabus of Laboratory Examinations is soundly endorsed. Certainly there are many adequate texts available. One of these should be on hand for consultation. 33 East Chestnut Hill Avenue Philadelphia 18, Pennsylvania
THE clinical recognition of anemia is not as easy as we practitioners are accustomed to think. Certain fair-skinned persons as well as some with olive and sallow complexions appear quite anemic but yield normal hemoglobin values. On the other hand, it is surprising how often make-up, suntan or just facial flush will totally mask a real anemia. A common source of error on the referring physician's part, as seen in a busy out-patient department and a large referral diagnostic clinic, has been the failure to do a simple hemoglobin evaluation. In conjunction with this, the passage of several years without technical help in the office has impressed me with the ease and value of performing a hemoglobin determination and a peripheral blood smear on every new patient. Leukocyte count, differential and erythrocyte sedimentation rate may also be done when indicated with little expense in time or equipment. The following are samples of simple hematological omission: A woman, 36, was referred to the Benjamin Franklin Clinie for evaluation of her anemia. She was pale and complained of constipation and easy fatigue. Her hemoglobin was 13.4 grams. No organic disease could be found. She had not had a hemoglobin determination before referral. A man, 64, was referred because of cardiac irregularity for electrocardiography and management. His hemoglobin was 5.3 grams. Carcinoma of the stomach with intestinal bleeding was found. The patient was tanned from sailing. A woman, 62, had been treated for several months for hypertensive cerebrovascular disease because of bouts of dizziness. Her hemoglobin was 8.1 grams. A gastric leiomyoma was removed, and she has had no further symptoms since her hemoglobin returned to normal range. The hypertension persists. A man, 54, had been treated with vitamin B12 for several months without any
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improvement. A peripheral smear was severely hypochromic. The hemoglobin was 5.5 grams, and the stool positive. Iron therapy was effective.
Periodic physical examinations for health maintenance are becoming increasingly frequent, and these jobs should and will fall to the practitioner in his office. The palmar crease has not been as reliable as conjunctival and oral mucosal changes in estimation of degree of anemia Such findings plus murmurs, dyspnea, edema or lightheadedness may enable us to reckon that hemoglobin is in the 4 to 6 gram range. More accurately determined initial hemoglobin levels, however, are necessary as base lines for evaluation of therapeutic response. Determination of hemoglobin level and a glance at a stained peripheral smear may also reveal the only clues to incipient disease. Normal results from such procedures should relieve anxieties on the part of the patient as much as the report of a "Pap" smear, EKG or chest film. The age of expensive electrical gadgetry has done much to stimulate a withdrawal reflex on the part of many practicing physicians. A quiet inferiority feeling has led to total cessation of laboratory work in many offices. Those who are really sick have been referred elsewhere for testing or hospital admission. Many patients moderately ill have consequently suffered from lack of any sort of determination by falling into the twilight zone between health and frank illness. This is a plea for the use of even the most meager of hematologic de terminations in every office; a few short discussions of some practical points in their accomplishment follow. THE FINGER PUNCTURE
The spring lancet and the cork with a Bard-Parker blade extending rather naughtily for 3 or 4 mm. beyond its flat undersurface are obsolete. Epidemiological studies of viral hepatitis have proved the dangers of such instruments when repeatedly used without proper sterilization. There are available several kinds of individually packaged, sterile strips of metal with one end properly sharpened and beveled. These may be discarded after one use without actually bankrupting the physician and certainly with much saving in time. Sera Sharp, Redi Lance and Meti-point are a few of these. The patient's finger should be cleansed and held firmly. The metal point in the physician's other hand is plunged into the fleshy pad of the finger, preferably a bit off cent er and with follow-through. There should be a spontaneous welling up of blood, which obviates the need for squeezing or milking the patient's finger. A well executed puncture is no more painful than one poorly done. An ample supply of blood as described yields more consistent and accurate results; smears are less distorted; pipettes clog less. Acceptance by the patient is better because a repeat wound is not necessary, and there is no fuming and difficulty
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Fig. 1. Just touch the top of the drop.
while the doctor tries to squeeze out just a little more blood while obviously not doing a job properly. After the puncture, the finger should be wiped with a dry gauze before the actual taking of each sample of blood. This will insure proper spreading, smearing and flow in pipettes. 'Whether one makes smears or uses pipettes, this should be emphasized: Just touch the top of the drop (Fig. 1). HEMOGLOBIN DETERMINATION
Visual Colorimetry
The old method of Sahli, using a graduated tube, 0.1 N hydrochloric acid, accurate hemoglobin pipettes and amber glass comparator standards, is probably the cheapest feasible method for hemoglobinometry in the average practitioner's office. Though less accurate than other methods, it will allow reasonably reliable follow-up determinations, particularly when performed by the same person week after week. Such disadvantages as a progressive increase in intensity of brownish coloring on standing during the first ten minutes after mixing and the necessity for repeating the whole procedure if too much water is added bear mentioning. The Spencer hemoglobillometer is slightly more costly but is simpler to use and somewhat more accurate. This instrument measures the intensity of green light transmitted by a thin layer of hemolyzed blood as compared with that transmitted by a standardized glass wedge. Pipetting is eliminated, as is color judging. Electrophotometry
The basic principle is that an electrophotometer measures the amount of light transmitted by a hemoglobin solution. The oxyhemoglobin
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method necessitates the pipetting of blood into a standard volume of sodium carbonate or dilute ammonium hydroxide; the cyanmethemoglobin method requires pipetting the blood sample into a solution of potassium cyanide and potassium ferricyanide. These methods are speedy and highly accurate, especially the cyanmethemoglobin method because the solution used is somewhat more stable. They do require a photoelectric colorimeter, which is costly and temperamental, necessitating calibration regularly and frequently. The Tallquist scale and the copper sulfate specific gravity method are mentioned only to advise against their use. Terminology and Normal Values
Hemoglobin should be expressed in grams per 100 milliliters of blood or, in common usage, simply in grams. This avoids the use of percentages, which confuse and disturb patients when they aren't 100 per cent, and it thereby saves the physician much time and explanation. The normal range for children is 10 to 15 grams; for women, 11.5 to 16 grams; for mfln, 12.5 to 17 grams. PERIPHERAL SMEAR
The examination of a carefully prepared peripheral blood smear remains the most significant laboratory study in patients with hematologic abnormalities. A few words to amplify "carefully prepared" seem fitting here and pertain to both coverslip and slide techniques. Preparation and Care of Glassware
The glass surfaces should be scrupulously clean and free from nicks and scratches. Washing may be done with detergent and water and should be followed by rinsing with large quantities of warm water and transfer to 95 per cent alcohol. Before use, slides and coverslips should be polished with a clean cloth free of lint, dust and grease. It has been convenient to store a small supply of slides ready for use wrapped in two's in lint-free toilet paper. Coverslips may be kept in gauze-lined jars and picked out with forceps or kept in slits in a cardboard box. They should be protected in containers with tight lids. Coverslips which are wavy, convex, or concave are useless because uniform contact between the two is necessary for proper spreading of blood. Scratched surfaces result in pooling and irregular distribution of blood. The ends of slides should be regular and smooth; any broken areas or nicks will leave a wide trail in the blood smear. A good grade of glassware should be purchased at the outset. In most instances, only new slides and coverslips ought to be used.
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Fig. 2. After the drop has spread, separate the coverslips by smooth, deft pulls in the same plane.
Making the Smear
A small drop of blood (not exceeding 3 mm. in diameter) is adequate for a good smear. The drop is placed on one coverslip, and the other is placed over it gently. Time is allowed for spread outward until the pink coloring is about 2 cm. wide. Then with a deft swish, the coverslips are slid apart, never getting out of the same plane (Fig. 2). The slide technique is more often than not ruined at the outset by too much blood. A 2 to 3 mm. drop is quite enough. As soon as the drop is on the slide, the slide is placed on a substantial surface such as a table top or desk. The end of a second slide is placed flat across the surface of the first slide with the long axis of the second slide at a 30- to 45-degree angle. The second slide is backed into the drop of blood; as soon as the blood spreads laterally about 1 cm. each way (not all the way to the edge of the slide), then smoothly and briskly the second slide is moved away from the blood droplet, the first slide being held flat and still with the other hand (Fig. 3).
Fig. 3. Back the edge of the second slide into the drop, allow the blood to spread laterally, then smoothly and briskly push the second slide away (in this sketch, from right to left); this will leave both edges available for scrutiny and room on the slide for labeling.
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Fig. 4. Wash from the side so that the surface film goes over intact.
Staining
Wright's stain iH adequate and cheap. It can be procured as solution or in the dry crystalline state. Most commercially available solutions are diluted and do not require the use of buffer, allowing for simplicity but at the expense of the finer differential features desired and expected in a good peripheral smear. Crystalline Wright's is available in vials of measured quantity which must be suspended in methyl alcohol. Like many alcoholic concoctions, it should be aged before use. It is handy to have two jars of stain so that one can be aging while the other is in use. It helps to label each as to the time of initial suspension so that at least three weeks may pass before it is used. A good shaking and resuspension every few days strengthens the solution and requires only a few seconds. Filtering is necessary to prevent deposition of minute granules of stain on the blood film. Stain poured from stock bottles should always he filtered prior to use. The solution in the drop bottle in current use should be re filtered at least weekly and the drop bottle cleansed with hot water and a methanol rinse. A buffer solution of sodium phosphate with a pH of 6.4 is readily available and cheap. It should be added to the slide in a quantity equal to that of the stain used (15 to 20 drops usually). The timing varies but the average range is 1 to 2 minutes with stain alone, 7 to 15 minutes with buffer solution added. Rinsing is simple enough, though one hint may be valuable in procuring prettier slides. The water should be accurately edged in from one side so that the scum of stain will be maintained with its surface tension intact as it is washed over the other side (Fig. 4). This prevents settling
Simple H ematological Procedures in Office Medicine
1501
OIL
Fig. 5. Touch the oil droplet with a toothpick and spread a thin coating of oil over the entire blood film.
of fine granules of stain over the cells and intercellular spaces on the slide. The slide should be rinsed further in running tap water and allowed to dry in an upright position. There should not be any emergency acute enough to warrant blotting the smear, which will mar or ruin the blood film. Examining the Smear
A drop of immersion oil can be added to the stained side of a coverslip preparation, which is then placed face down on a slide. For slide preparations, a drop of immersion oil is placed at the cent er of the smear just before the trailing edges begin to narrow. Then it is spread evenly with an easy stroke of the immersion oil toothpick or an applicator stick (Fig. 5). The film is placed under the microscope (low power) and moved about until the red cells can be seen individually with space between them. The leukocytes should be seen in such an area clearly enough to differentiate monocyte, lymphocyte and neutrophil in most instances. One's first reaction to this will be, "I'm too far away to see anything." It takes a little time to become adjusted to this method of scanning blood films, but it will save the physician time and the patient much bloodshed and money in laboratory studies. It will help the physician make more diagnoses. By scanning, the general appearance of the red cells as well as the number and character of the white cells can be reckoned. With the help of oil immersion, a glance at the red cells in a few locations will settle the question of red cell shape, size and coloring. At this time an estimate of platelet population can be made by noting single platelets and collections of two to four platelets interspersed between the red cells. An incidence of one to four platelets or platelet groups per oil immersion field is normal. The white cells can then be studied and the smear discarded. (Dr.
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h..--------------------..,
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Fig. 6. In making a differential count, follow a regular path back and forth across the slide.
Wurzel's review of the proper paths in hematological diagnosis will be most helpful at this point. See page 1607.) Differential counts on slide preparations may be misleading if some order is not followed. I have found that the most satisfactory method is to start somewhere in the center and to move toward the side of the smear until it is reached, then to move four to six fields along parallel to the side of the slide before starting back across the middle of the smear (Fig. 6). More polymorphonuclears are seen along the edges and more lymphocytes toward the middle. This is not a problem with coverslip preparations. SEDIMENTATION RATE
For those interested in performing sedimentation rates, the Westergren method is hereby endorsed. The 200-millimeter tubes, which offer the advantage of easy filling and cleaning, are not particularly costly, and a rack can be bought or made. Sodium citrate 3.8 per cent is likewise easily available. 1.8 ml. of blood is mixed with 0.2 ml. sodium citrate. The tube is filled with blood to the "0" mark by suction and is placed in the rack. Reading is done at the end of one hour. The tube can then be drained, washed and stored. No correction for hematocrit is necessary, and this procedure provides easy reading with accuracy equal to or surpassing that of other methods. ANTICOAGULANTS AND HOUSE CALL BLOOD SAMPLES
Ammonium and potassium oxalate are the anticoagulants used by most laboratories for collecting venous blood samples for transportation, whether it is for three floors, to the next building or ten miles. Tubes or small bottles with a measured quantity of these two crystallized salts can be obtained at the nearby private and hospital laboratories which one uses or can be bought commercially. Each tube or bottle is usually adequate for 5 cc. blood. Hemoglobin, hematocrit, leukocyte count and sedimentation rate may be determined from oxalated samples. It
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should be emphasized, however, that smears should be made immediately from droplets of freshly drawn blood in order to avoid the many cellular artifacts that can result from exposure to anticoagulants. Whenever blood films are made from an oxalated sample, the slogan should be, "Don't be alarmed by odd cells seen unless smears have been repeated using fresh blood." HEMATOCRIT
The hematocrit is the most accurate procedure available for determination of anemia and polycythemia. With a standard centrifuge, Wintrobe tubes and an oxalated blood sample should be used. A sedimentation rate can be determined prior to centrifugation. The centrifuge should run at 3000 r.p.m. for 30 minutes. The volume of packed red cells is easily determined, the height of the buffy coat will give a good indication of the leukocyte count, and the thickness of the creamy white layer above the grayish-pink buffy coat will indicate the platelet count. The plasma should be observed for the increased discoloration due to jaundice or the decreased discoloration characteristic of iron deficiency. The microhematocrit methods now available deserve mention here. They yield accurate results within five minutes, since only three minutes' centrifugation is necessary. Finger puncture blood is adequate for the determination. The only deterrent is price. Microhematocrit apparatus should be considered by any group of two or more doctors equipping or refitting a small laboratory and those planning a large laboratory. LEUKOCYTE COUNTS
Certain aspects of white cell counting merit discussion. Pipettes should be kept clean and dry, and the tips should be checked frequently for nicks, cracks and other such flaws that mar the accuracy of the contents. Acetic acid 2 per cent is an adequate diluent and does not require any coloring agent to help the leukocytes stand out. The counting chamber should be cleaned after use and polished prior to use so the cells will spread evenly. Shaking can be accomplished in an automatic shaker or manually; it should be vigorous, constant and multidirectional for at least one minute to allow for proper mixing. If the count is excessively high, a red cell pipette may be used. Blood should be drawn up to the 1.0 mark and the pipette filled with WBC diluting fluid. Five large squares are counted and the total multiplied by 5000. If the count is quite low, a white cell pipette may be filled to the 1.0 mark instead of the 0.5 mark and the multiplication factor halved. A practicing physician should be able to diagnose neutrophilic leuko-
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cytosis, chronic leukemias (particularly the chronic lymphocytic) and some acute blastic leukemias in his office. "L. E." PREPARATIONS
It is feasible to demonstrate in the office laboratory the "L. E." phenomenon in clotted blood preparations or in mixtures of serum from the disseminated lupus erythematosus patient with white cells from someone else's buffy coat after some incubation. Certainly, larger laboratories should be proficient at demonstrating these cells. The physician who suspects disseminated lupus but who performs only the essentials in his small laboratory can draw 6 or 8 cc. of blood, allow it to clot and send it to a competent laboratory nearby. BONE MARROW EXAMINATION
Aspiration of bone marrow is an office or out-patient procedure. This should be clear to all practitioners, for it may save much time in a diagnostic workup and it makes hospitalization unnecessary. (Blue Shield pays the same fee whether the procedure is done in the hospital or in the office.) It is not the intention here to suggest that bone marrow aspiration be done by all practicing physicians; rather, the intention is to make clear a point that has been uncertain in the minds of many referring physicians and patients. Actually, most patients find a bone marrow aspiration no worse an ordeal than a bromsulfalein retention test and less traumatic than a glucose tolerance test. There is practically no danger. Nearly all of the twenty or so deaths from bone marrow puncture in Europe and America have resulted from trans-sternal perforatioll by untrained hands. SPECIALIZED HEMATOLOGICAL PROCEDURES
Reticulocyte counts, platelet counts, prothrombin determinations, bleeding and clotting times, RBC incubation procedures, red cell fragility studies, Coombs' antiglobulin tests and special staining techniques should not be attempted in most office laboratories since the intricacies of these procedures make them unreliable unless they are done constantly by the same personnel. COMMENT
There will be many voices raised against the suggestion that hemoglobin determinations and peripheral smears are adequate screening procedures. However, if they are used in otherwise healthy persons and
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if they are amplified for any illness or abnormality encountered, they seem to be an acceptable practical compromise. A few words against the commonly encountered defeatist attitude, "Oh, it wouldn't do any good for me to look at a slide. I wouldn't know one cell from another." This is heard from interns and first-year residents only slightly less often than from older graduates. The remedy lies not in a two-week course in Advances in Hematology offered by Antarctic U. Medical School. The remedy lies in a few hours spent with a hematologically oriented pathologist, or a hematologist, and his favorite technician. One should simply find out when slides are reviewed and be there a few times to show these people one's interest and any problem slides one might have. One should ask permission for the technician to spend a few minutes giving pointers in technique. Good hematology technicians are more numerous today and they can be of tremendous help with problems concerning red or white cell morphology as well as with more technical matters. The interpretation of peripheral blood smears should not be considered a mystery any more than should good physical diagnosis. Rales can be heard; thrills can be felt; nodes, livers and spleens can be palpated. Red cells can definitely be seen to be large, to vary remarkably in size and shape, or to be pale and lacking in hemoglobin. When doubt exists, one must repeat the smear and wait, just as with other tests. An earnest attempt to look at one's own slides will prove that hocus-pocus is not an ingredient of hematology; the main requisite is a little sincere interest. It is not my intention that these paragraphs should substitute for carefully written, stepwise methodology for the hematological investigations discussed. Ham's Syllabus of Laboratory Examinations is soundly endorsed. Certainly there are many adequate texts available. One of these should be on hand for consultation. 33 East Chestnut Hill Avenue Philadelphia 18, Pennsylvania