Section 13.

HEMATOLOGY

Hematology is one of the major sections in the Department of Laboratory Services that offers the following services: 1. Complete Blood Count (Automated) 1.1 WBC 1.2 RBC 1.3 HGB 1.4 HCT 1.5 MCV 1.6 MCH 1.7 MCHC 1.8 Platelet 2. Special Examinations such as: 2.1 ESR 2.2 CTBT 2.3 Reticulocyte Count 2.4 LE cell 2.5 Peripheral Blood Smear This section is manned by competent Medical Technologists. 1. 2. 3. 4. 5. 6. Pathologist Senior Medical Technologists Medical Technician Laboratory Aid Phlebotomist Medical Technology Interns

ASSEMBLING OF EQUIPMENT AND COLLECTION OF BLOOD I. Assembling of Equipment: Materials: Cottonballs or gauze pads 70% alcohol or similar antiseptic, hgb, pipette Rubber sucking tubes 3 or 4 glass slides White cell diluting fluid Pencil or ballpen or requisition slip Procedures: 1. 2. 3. 4. Assemble all equipment listed above. Place equipment within easy reach. If someone else uses the rubber sucking tube, dip the mouthpiece in 70% alcohol. Attach pipette in rubber sucking tube on the table ready for use.

II. Collection of Blood Samples: A. By Skin Puncture Materials: Blood Lancet 70% alcohol Cottonballs Procedures: 1. Rub the site of puncture with an alcohol to remove dirt and epithelial debris and to increase the amount of circulation of blood in the part. 2. After allowing sufficient time for the circulation to flow 3. After withdrawing the lancet, the first drop of blood will appear and is wiped away. 4. The second drop of blood is the one used for the test. 5. After the specimen has been taken, have the patient apply sterile cotton to the punctured area (site) until bleeding stops. B. By Venipuncture Materials: Tourniquet 70% alcohol Sterile dry syringe and needle Cottonballs Procedure: 1. Apply tourniquet and palpate the anticubital vein for a suitable vein. 2. After locating the pertinent vein and firmly anchored vein, clean the area with an alcohol sponge, a circulatory motion should be used. 3. Hold the skin taut with the left hand, grasp the syringe with the right hand and place the needle point in position at the site to be punctured, placing the forefinger on the needle hub and side in controlling the needle insertion. 4. Penetrate skin and move the needle forward parallel to and alongside the vein. A slight movement toward the vein should place it in lumen. After entry into the vein, blood will appear into the syringe. 5. Aspiration of blood is accomplished gently pulling upon the syringe plunger. The syringe barrel should be held steady during the process. Withdraw the desired quantity of blood. 6. Remove the tourniquet. This must be done before withdrawing the entire needle from the vein. 7. Place a sterile gauze pad or cotton over the point where the needle entered the skin and swiftly withdraw the needle.

8. Let the patient extend his arm and maintain light pressure on the gauze pad over the venipuncture site. Remove the needle from the syringe and apply gentle pressure to the plunger and discharge the blood against the tube margin. 9. Blood specimens, thus collected may be prepared (detribrinated, oxalated or allowed to coagulate) depending upon the examinations required. SETTING UP A QUALITY CONTROL IN HEMATOLOGY It is imperative for the laboratory to have a set of regulations and workable quality control programs in order to maintain a smooth operation and to obtain accurate laboratory requests. QUALITY CONTROL IN HEMATOLORY 1. Ideal blood smear-tongue shape-read at the feather end. 2. Should give romanousky effect- the true picture of a cell in its near nature stats. 3. Presence of 5 nucleated RBC do specials stain or cytochemical staining Ex. Peroxidase stain, Sudan black, Periodic acid Schiff. 4. Evaluate the smear as to: a.) Red cell morphology b.) Platelet/oif c.) WBC/hpf 5. Calibrate regularly the spectrophotometer, speed of microhematocrit centrifuge. 6. Na Citrate best anticoagulant for Pro-time, EDTA for CBC. 7. WBC, RBC, Hgb and Hct are stable at 2-8 C for at least 24 hours. 8. Platelet count is stable for 5 hours at room temperature and for 24 hours at refrigeration. 9. Pro-time must be performed within 4 hours of collection. THINGS TO REMEMBER IN DOING SKIN PUNCTURE 1. The ideal skin puncture must be 2-3 mm. deep continues flow of blood since it will reach the capillary bed. 2. Massage can be applied before puncturing the skin, but much pressure and squeezing should be avoided. A gentle squeeze on the sides of the finger can be applied after puncturing. 3. Pressure and squeezing is avoided to eliminate the admixture of tissue juices with blood which will affect the accuracy of the test. 4. The first drop of the blood is usually discarded, since it may contain tissue juices or it may be contaminated with extraneous materials which have been clinging to the surface of the skin. 5. When collecting blood for hematologic test the finger must be wiped dry after each test (Platelets will begin to clump immediately in the blood at the puncture site). 6. The values for the res blood count. Hematocrit, hemoglobin, and platelets are lower in capillary blood but high leukocyte count as much as 1,000/cu mm.

EXAMINATION WHICH CAN BE DONE IN CAPILLARY BLOOD 1. WBC Count 2. RBC Count 3. Platelet Count 4. Hemoglobin Determination 5. Bleeding Time 6. Coagulation Time 7. Reticulocyte Count 8. Differential Count 9. Blood Typing 10. Clot Retraction Time 11. Blood Smear for Malaria, FIlaria, and Basophilic Aggregation (stippling of erythrocytes) DILUTION AND CALCULATIONS FOR HIGH COUNTS AND LOW COUNTS HIGH COUNTS: In Leukemia, the white cell count maybe extremely high, often as high as 100,000 to 500,000 cells per cubic millimeter. In such cases the counting maybe facilitated by making a greater dilution of the blood. This is accomplished by using a red cell pipette. The INCREASED DILUTION of course, must be compensated for in the calculation. To increase the dilution and make the proper calculation process as follows: 1. Using a red cell pipette, draw blood to the 1.0 mark. 2. Draw WBC diluting fluid to the 101 mark. 3. Now proceed exactly as in the normal white cell count, shake the pipette, discard the first 4 drops, charge the counting chamber and count the cells in 4 W section. 4. Multiply the total number of calls in the 4 W section by 250. The answer is the white cell count. 5. NOTE: Normally the blood is diluted in 1 in 20. The dilution correction factor is therefore 100. Multiplying this dilution correction factor of 100 by the volume correction factor of2.5, gives us 250. LOW COUNTS: In the malignant neutopenia and many virus diseases the white blood cell count maybe extremely low often varying between 500 and 3000 per cubic millimeters. The accuracy of the count may be increased by a lesser dilution of the blood. This increased dilution of the blood, of course, must be compensated for in the calculation. To decrease the dilution and make the proper calculation as follows: 1. Using the white cell pipette, draw the blood to the 1.0 mark. 2. Draw WBC diluting fluid to the 101 mark. 3. Now proceed exactly as in the normal white cell count, shake the pipette, discard the first 4 drops, charge the counting chamber, and count the cells in the w section. 4. Multiply the total number of cells in the 4 W section by 25. The answer is the white cell count.

5. Note: Normally the blood is diluted in 1 in 20 and the dilution correction factor is

20. However, in the above case the blood is diluted 1 in 10. Multiplying this dilution factor od 10 by the volume correction factor of 2.5 gives us the figure 25. PREPARATION OF SOLUTIONS AND REAGENTS

ACETIC ACID 2% acetic acid Place 98 cc. of distilled water in container Add 22 cc. of glacial acetic acid ANTICOAGULANTS a. Mixture of ammonium oxalate and potassium oxalate Using the rough balance, weigh out 2.0 grams of ammonium oxalate and 8.0 grams of potassium oxalate. Place them in a liter volumetric flask, and add distilled wter to the liter mark. Mix; place 0.5 cc. of this solution in a test tube, and evaporate to dryness either at room temperature or in an oven which is not over 100C. (The oxalate decomposes at higher temperature). The powdered oxalate remaining in the test tube will serve as an anticoagulant for 5.0 cc of blood. Sequestrene solution: Using the rough balance, weigh out 10 grams of sequestrene. Place in a clean bottle and add 00 cc of distilled water. When 0.1 cc of this solution is placed in a test tube, it will serve as an anticoagulant for 10 cc of blood. The solution does not have to be dried; it may be used in the liquid form.

b.

BENZIDINE SOLUTION Using the rough balance, weigh out 0.1 grams of benzidine. Place in 100 cc of distilled water. Mix and filter. Just before use, add a few drops of hydrogen peroxide. BRILLIANT CRESYL BLUE a. 1 % physiological saline solution of brilliant cresyl blue: Using the rough balance, weigh out 1 gram of brilliant cresyl blue. Place in a container and add 99 cc of physiological (0.85%) saline. Mix and filter. % methyl alcohol solution of brilliant cresyl blue: Using the rough balance, weigh out gram of brilliant cresyl blue. Place in a container and add 99 cc of methyl alcohol. Mix and filter.

b.

CALCUIM CHLORIDE 0.025 (fortieth) molar calcium chloride: Using the analytical balance, weigh out .3875 grams of anhydrous calcium chloride. Place in 500 cc of volumetric flask and add distilled water to the 500 cc mark. Mix.

COPPER SULFATE 0.5% copper sulfate solution: Using the analytical balance, weigh out 1.0 grams of anhydrous copper sulfate. Place in 100 cc volumetric flask and add distilled water to the 00 cc mark. Mix. DRABKINS SOLUTION (for cyanmethemoglobin) This solution is poisonous! Handle with care! Using the rough balance, weigh out .0 grams of sodium bicarbonate (C.P.). Place in a liter volumetric flask. Using the analytical balance, weigh out 52 milligrams of potassium cyanide (C.P.). Add to the volumetric flask. Using the analytical balance, weigh out 198 mg of potassium ferricyanide. Add to the volumetric flask. Add distilled water to the 1-liter mark. Transfer to a brown bottle and store in the dark. GOWERS SOLUTION Using the analytical balance, weigh out 6.250 grams of crystalline sodium sulfate. Place in a 100 cc volumetric flask. Add about 50 cc of distilled water. Add 6.7 of glacial acetic acid. Dilute to the 00 cc mark with distilled water. Mix. HAYEMS SOLUTION Using the analytical balance, weigh out 0.250 grams of mercuric chloride, 0.50 grams of sodium chloride, and add 2.50 grams of crystalline sodium sulfate. Place in a 00 cc volumetric flask, and add distilled water to the 100 cc mark. Mix HYDROCHLORIC ACID a. 1% (approximately 0.1 normal) hydrochloric acid Place in a 99 cc of distilled water in a container. Add 1 cc of concentrated hydrochloric acid. Mix. 0.1 N hydrochloric acid Using a 10 ml volumetric pipette, place 10 ml of N HCl in a 100 volumetric flask. Add distilled water to the 100 ml mark. Mix.

b.

NORMAL SALINE (Physiologic saline) a. 0.85 (normal) saline solution Using the analytical balance, weigh out 8.50 grams of sodium chloride. Place in a 1-liter volumetric flask, add distilled water to the liter mark. Mix. Sterile 0.85 % (normal saline) Using the analytical balance, weigh out 8.50 grams of sodium chloride. Place in a 1-liter volumetric flask, and add distilled water to the 1-liter mark. Autoclave for 20 minutes at 05 lbs pressure.

b.

REES AND ECKER SOLUTION Using the rough balance, weigh out 0.1 grams of brilliant cresyl blue and 3.8 grams if sodium chloride. Place them in a 100 cc volumetric flask and add 0.2 cc of 40% formaldehyde. Dilute to the 100 cc mark with distilled water. Mix. SODIUM CHLORIDE a. 0.85% sodium chloride Using the analytical balance, weigh out 8.50 grams of sodium chloride. Place in a liter volumetric flask and add distilled water to the 1-liter mark. Mix. 0.5% sodium chloride Using the analytical balance, weigh out 0.50 grams of sodium chloride. Place in a 100 cc volumetric fflask and add distilled water to the 00 cc mark. Mix. WHITE BLOOD CELL COUNT NECESSARY MATERIALS 1. 2. 3. 4. 5. 6. Microscope Cotton balls wet with alcohol Puncturing equipments Diluting fluid White blood cee count pipette Counting chamber

b.

DILUTING FLUID Acetic acid with Gentian Violet Glacial Acetic Acid Gentian Violet Distilled Water 1-3% `2 cc 1 ml (Crystal Violet,Cl No.681) (1% aqueous) 100 ml

CHARACTERISTIC OF A WBC DILUTING FLUID 1. 2. 3. 4. 5. It It It It It must must must must must be be be be be a hypotonic solution easily prepared. a good preservative. cheap. available.

DILUTION FACTORS 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1:100 1:50 1:33 1:23 1:20 1:16 1:14 1:12 1:10

11 11 11 11 11 11 11 11 11

Suck blood more than 0.5 mark in leucopenia and less than 0.5 mark in leucocytosis or leukemia. LEUKOCYTOSIS is the increased of polymorphonuclear leukocytes which usually takes place as a defense mechanism in bacterial infection, may be due to chemotaxis or stimulation of blood making organs lose blood but more cells. CAUSES OF LEUCOCYTOSIS 1. malignant disease 2. post operative leukocytosis 3. post hemorrhagic leukocytosis 4. toxic leukocytosis due to drugs 5. lymphocytic leukocytosis LEUKEMIA is a disease of the blood forming organs in which the white blood cells (leukocytes) are generally increased in number (also the abnormal forms more blood) LEUCOPENIA is the reduction in the number of white blood cells below the normal count ERRORS IN WBC COUNT 1. Tendency of WBC to clump easily if it has been standing for. 2. Presence of impurities. 3. Normal error-Poissons Law = cells/mm area Using chamber, the error is + or 15% 10-12 counts is allowable difference between counts. COMPUTATIONS LONG METHOD: WBC COUNT x 10 x 20 = WBC count/cu.mm of blood 4

10 depth 20 dilution 4 number of squares where the count was made SHORTCUT METHOD WBC COUNT x 50 = WBC count/cu.mm of blood INTERPRETATION: Normal values is the same for males and females 5,000-10,000 cu.mm of blood INCREASE WBC COUNT IN: 1. 2. 3. 4. 5. 6. Inflammation (bacteria-Gram+) Strenuous exercise After mals and in the afternoon Infectios - Pulmonary TB, Appendicitis Prolonged exercise Pneumonia, tonsillitis, meningitis, menstruation, newborn, ulcers

DECREASED WBC COUNT IN: 1. Old age 40 yrs and above 2. Leucopenia- viral infection 3. Hypersplenism 4. Replacement of marrow with malignant disease 5. Drug sulfas (reaction of epilepsy) 6. Overwhelming infections- Pulmonary TB, Septimia SOURCES OF ERROR IN THE WHITE CELL COUNT 1. Failure 2. Failure 3. Failure 4. Failure to to to to have the blood exactly on the 0.5 mark shake the pipette thoroughly. discard the first 4 drops. charge the counting chambers.

TUBE TECHNIQUE: 0.02 ml of blood is taken and mixed with 0.38 ml of diluting fluid in a small tube or snap cap plastic vial. Tube technique are preferable to the Thoma pipette method because it allows greater accuracy of dilution. Reference: Lynchs Fourth Edition Medical Technology Stanley S. Raphael Bryant-Hyde-Inwood-Lor-Spencer-Thompson 1983 WB Saunders, Company

STAINING METHODS USED IN DIFFERENTIAL COUNT (ROMANOWSKYS) WRIGHTS STAIN Method I Powdered Wrights Stain------------------------------------- 3.0 gm Acetone-free methyl alcohol--------------------------------- 30.0 ml Mix, let stand for several days, mixing a few times, each day. Filter before use. Method II Powdered Wrights Stain------------------------------------- 3.0 gm Glycerol------------------------------------------------------- 30.0 ml Mix thoroughly, grinding in a mortar if necessary. Place a 3* C incubator or water bath for a few days, stirring a few times each day. Slowly, yet constantly stirring, add acetone free methyl alcohol-------------00 ml. Allow to age for a few days. Filter before use. GIEMSAS STAIN Azur II eosin ---------------------------------3.0 gm Azur II ----------------------------------------0.8 gm Glycerin reagent -----------------------------250 gm (312 ml) Methl alcohol free ----------------------------250 gm (312 ml) Note: 3.8 gms of prepared Giemsa powder may be used. Dissolve the powder in the glycerol by holding at 555-60C for 1 to 2 hours. Then add the methyl alcohol. Before use, dilute ml of the stain with 0 ml of distilled water. STAINING METHOD COMMONLY USED WRIGHTS STAIN most satisfactory method in general routine work. PRINCIPLE: A differential staining of a thin blood film can be of various blood cells. They may then be qualitatively and quantitatively studied. PROCEDURE: a. b. c. d. e. f. Cover the blood smear with a measured number of (50 drops) of Wrights Stain using a medicine dropper. After minute, add the staining fluid on the film the same number of drops of distilled water (or buffer solution- pH 6.4-6.8) Allow the mixture to stand for 3 to 4 times. Wash the slide with distilled water by flooding the diluted stain (do not pour off the stain before the flooding or scum will form on the film). Dry the film. Examine under the oil immersion objective.

Note: Mixing Stain with buffer solutiom: a. b. c. d. e. f. The blowing stain should be done from a position about 4 inches directly above the slide. The blowing should be done on several portion of the slide. The blowing should be forceful. The blowing should last for minute or longer. The solution, of course, should not be blown off the slide. When the solutions are well mixed, a shiny scum usually floats on top of it.

GIEMSAS STAIN this stain is not used only in hematology but is an excellent stain for the inclusion bodies, like the malarial parasites, etc. PROCEDURE: a. b. c. d. The blood film is first fixed in methyl alcohol (methanol) for 3 minutes. Air dry, immersed in dilute Giemsa stain (10 dips). Wash with distilled water. Air dry and examine under oil immersion objectives.

FACTORS THAT AFFECT FAILURE TO OBTAIN THE RIGHT STAINING RESULTS

1. Imperfect polychroming of cells.

2. 3. 4. 5. 6. 7. 8. 9.

Incorrect reaction of staining fluid pH 6-7 with buffer pH 7-7.4. The solution is too acidic RBC colors red, nuclei are pale to sky blue or colorless. The solution is too alkaline - -RBC is deep blue. During fixation some cells are burst off. Operators mistakes, technique and manipulation. Unclean slides and other equipment. Evaporation of stain precipitate will appear on the slide. Incorrect preparation of stain. DIFFERENTIAL COUNT (STAINING OF SMEARS)

Differential count is an estimation of the number or percent of each variety of leukocytes in the (peripheral or venous) blood. STEPS FOR DIFFERENTIAL WHITE CELL COUNT: 1. 2. 3. 4. Making the blood smear Staining the cells. Counting the cells. Reporting the cells.

MAKING THE BLOOD SMEAR: TWO SLIDE METHOD the simplest and most popular method. Uses two slides, one for the smear and the other as spreader.

PROCEDURE: 1. Place a small drop of blood in the middle of one end of the slide. 2. Hold the second slide (spreader)above the first at an angle of 30-45 degrees. 3. Draw the spreader toward the drop of blood and contact the blood the blood should fan or spread out to the edges of the spreader. If it does not fan, wiggle the spreader a little to make it do so. (Dont let the blood get ahead of the spreader because it may cut or distort the cells) 4. Push the spreader smoothly and rapidly over the entire length of the slide at a constant angle (30-45 degree)and keep the edge of the spreader pressed firmly against the slide. 5. Air dry before staining. PRE-REQUISITES FOR PROPER BLOOD SMEAR: 1. Slides and cover glasses should be cleaned. 2. The drop of blood should not be too large nor too small since the size of the blood drop influences the thickness and thinness of the smear (large drop-thick smear, too small-thin smear). 3. The work is done quickly before coagulation. 4. Proper angle and pressure of the spreader makes an ideal smear. ERYTHROCYTE SEDIMENTATION RATE (ESR) PRINCIPLE: ESR is due to changes in the surface change of the RBC which causes them to aggregate, the larger the cells, the more it aggregates and the greater the speed rate of their fall in plasma, particularly if the plasma is in the colloidal state. ESR is slower if more cells in blood. ESR is faster if less cells in blood. IMPORTANCE OF ESR DETERMINATION:

1. Used as an index in the presence of active disease.

2. it measures the suspension stability of RBC. 3. It measures the abnormal concentration of fibrinogen and serum globulin. WESTREGREEN METHOD This method is considered the most sensitive in ESR determination for serial studyof chronic disease like TB, carcinoma, etc. 1. Anticoagulant used 3.0% sodium citrate 2. Westregreen tube: (a) Calibration is up to 200 mm It is a long tube with both endsopen or unsealed (b) The bore is 2.5 mm in diameter.

3. Procedure: (a) Fill the tube by citrated venous blood by suction method. (b) Stand the tube vertically in a rack and record the ESR at the end of hour. NORMAL VALUES: MALE: 0-15 mm/1 hr FEMALE:0-17 mm/1 hr REPORT is also expressed as normal, fast, and very fast. WINTROBE: In the vast majority of cases, this is quite an accurate method, its disadvantages outweighs its few drawbacks. 1. Anticoagulant used Paul Hellers mixture or balance mixture or Double Oxalate. 2. Wintrobe Tube: a. Flat bottomed tube (for ESR and Hematocrit determination) b. Calibration both sides are calibrated. Left Side 0 10 mm., red colored, used for ESR; reading the amount of plasma after 1 hour standing. Right Side 10 0 mm., white colored, used for hematocrit or packed red cells after 30 minutes of centrifugation at 3,500 rpm. c. The bore is 3 mm. in diameter. d. It is short with full calibration up to 100 mm. 3. PROCEDURE a. Oxalated venous blood is used b. With a capillary pipette or canula, fill the Wintrobe tube with blood up to the zero mark (left side calibration of the tube). c. Place the tube in a vertical position on a rack provided for this test and record the ESR in mm. at the end of one hour. NORMAL VALUES MALE: 0 to 9 or 0 to 10 mm/hr FEMALE: 0 to 20 mm/hr NOTE: ESR is read at the upper left position of the Wintrobe tube or the amount of plasma should be determined. Hematocrit is read at the lower right position of the Wintrobe tube, or the volume of packed red cells are determined in this test.

LAYERS SEEN IN WINTROBE TUBE AFTER 1 HOUR STANDING: 1. Plasma layer- uppermost layer or portion. 2. Buffy coat- the middle layer or portion of the blood which contains the platelets, WBC, and other cells, such as L.E. cells and other malignant cells. (1mm of buffy coat contains about 10,000 WBC.) It is whitish to creamy yellow in color. 3. Packed Red Cells layer- lowermost layer or portion of the tube which contains nucleated young red cells on top and unnucleated cells at the lower layer. HEMATOCRIT DETERMINATION Hematocrit is the volume of packed red cells after centrifugation of blood sample for 30 minutes at 3,000 to 3,500 rpm. One of the simplest, most accurate and valuable test in hematologic investigation. It is more than the RBC count, Absolute indices can be computed. The test measures the proportion of the red cells to plasma in the peripheral blood, but not in the entire circulation. The body hematocrit gives the ratio of the total erythrocytes mass to total blood volume. The hematocrit reading gives the number of mm of packed red blood cells per 100 mm of blood. MICRO METHOD This method uses capillary tubing and a finger punctured blood. PROCEDURE: a. b. c. Fill two heparinized capillary tubes about 2/3 full with blood from the finger and seal the dry end of the tube. Centrifuge for 5 minutes at 10,000 rpm (preferably in a centrifuge provided with special head for capillary tubes or rubber adapter). Measure in ml the column of packed red cells in a MICROHEMATOCRIT READER and this gives your hematocrit reading. HEMOGLOBINOMETRY Hemoglobin is the main component of the RBC, serves as the vehicle for the transportation of oxygen and of carbon dioxide. When fully saturated, each gram of hemoglobin holds approximately 1.34 ml of oxygen. The red cell mass of the adult being about 2,000 ml contains approximately 600 gm of hemoglobin capable of carrying 800 ml of oxygen. -coloring matter of the blood -is the red iron bearing protein contained within the erythrocytes in normal blood. Its major function in the normal individual is as a carrier of oxygen from the lungs to the tissues where it readily release this oxygen to the tissues and then returns to the lungs to combine with more oxygen.

PRINCIPLE: Hemoglobin is measured as oxyhemoglobin. It is first converted into one of several compounds, such as acid hematin, cyanmethemoglobin, and other less widely employed. The measurement is done by comparing the unknown with the standard. The method of comparison may be visual or photoelectric. The latter is considerably more accurate. ACID HEMATIN METHODS There are several methods that depend on converting hemoglobin into acid hematin with dilute HCl and matching the brownish yellow color of this solution with a standard in some sort of colorimeter or comparator. PRINCIPLE: The blood is diluted in an acid solution, converting the hemoglobin to acid hematin. The test solution is matched against a colored glass reference. MATERIALS: Sahli haemoglobinometer Sahli pipette (graduated to 20 mm, i.e. 0.02 ml or 20 ul) Small glass rod Dropping pipette Absorbent paper 0.1 mol/L (0.1 N) hydrochloric acid (HCl)

METHOD: 1. Fill the graduated to the20 mark (or tye mark 3g/100 ml) with 0.1 mol/1 HCl. 2. Draw venous or capillary blood to the 0.02 ml mark of the Sahli pipette. Do not allow air bubbles to eneter. With venous blood ensure that it is well mied by inverting the bottle containing it and the anticoagulant repeatedly for about 1 minute immediately before pipetting it. (Do not take the first drop of blood from the finger.) 3. Wipe the outside of the pipette with the absorbent paper. Check that the blood is still on the mark. 4. Blow the blood from the pipette into the graduated tube of the acid colution. Rinse the pipette by drawing in and blowing out the acid solution 3 times. The mixture of blood and acid gives a brownish color. Allow to stand for 5 minutes. 5. Place the graduated tube in the haemoglobinometer. Stand facing a window. Compare the color of the tube containing the diluted blood with the color of the reference tube. If the color is the same as or lighter than that of the reference tube the hemoglobin value is 40g/1 cc less. 6. If the color is darker than that of the reference tube continue to dilute by adding 0.1 mol/1 HCl drop by drop. Stir with the glass rod after adding each drop. Remove the rod and compare the colors of the 2 tubes. Top when the colos match. Distilled water can laso be used at this instead of 0.1 mol/1 HCl to continue the dilution of the blood. 7. Note the mark reached. Depending on the type of haemoglobinometer, this gives the haemoglobin concentration either 1 g/100 ml or as a percentage of normal ( the latter type, illustrated, is not recommended. To convert percentage to g/L, multiply by 1.46

Examples: a.) 14.8 g/100 ml x 10= 148 g/l b.) 85% x 1.46= 124 g/l (The Sahli method is so unreliable that factors for converting results into mol/1 are not given.) INACCURATE METHOD: The Sahli method is not an accurate way of estimating haemoglobin. Not all the form of circulating haemoglobin are changed into acid haematin; the color when viewed visually are not very true match for an acid haematin solution. A some places still use this method it is described, but it is not recommended. CYANMETHEMOGLOBIN METHOD Among the forms of hemoglobin well adapted to photometry, syanmethemoglobin has outstanding advantages:

a. All forms of hemoglobin is likely to be found in blood with the exception of

sulfahemoglobin, are qualitatively converted to cyanmethemoglobin upon the addition of a single reagent. b. Solutions of cyanmethemoglobin are the most stable of the various hemoglobin pigment. 1.) Drabkins Method Procedure: a.) Measure 5 ml of Drabkins solution into a test tube b.) With a Sahli pipette, measure 20 cu. Mm blood and transfer into the bottle containing Drabkins solution. c.) Mix let it stand for 10 minutes to allow formation of cyanmethemoglobin. d.) Read using the photometer. Filter at 550. CLINICAL SIGNIFICANCE Hemoglobin is contained in the red blood cells and is responsible for transporting oxygen. The amount of oxygen that the tissues receive depends upon the amount and the function of an available hemoglobin, on effective blood flow patterns, and on the state of the tissue and the fluid through which oxygen diffuses. Hemoglobin is elevated into polycythemia, after vigorous exercise, high altitudes excitement, haemoconcentration. Hemoglobin is DECREASED in anemia and recumbency.

METHOD PRINCIPLE: Erythrocytes are lysed by a stromatolytic agent in the presence of a surfactant and they release their hemoglobin into the solution. The iron (II) of hemo in hemoglobin is oxidized to iron (III) by potassium ferricyanide to form methemoglobin which is subsequently reacted with cyanide ions to form cyanmethemoglobin. The very stable cyamethemoglobin complex is measure photometrically at 540 nm. The method is Van Kampen-Zijlstra modification of the Drabkin cyanmethemoglobin method. MANUAL PROCEDURE: 1. Transfer 5.0 ml of HEMOGLOBIN REAGENT into vials labeled : UNKNOWN (S), CONTROL, REAGENT BLANK. 2. Add 20 ul (0.020 ml) of well mixed whole blood with anticoagulant to its respective vial and mix vigorously swirling (vortexing is recommended). 3. Let vials stand at room temperature (20-80C) for 5 minutes. 4. See the wavelength to the photometer at 540 nm. And zero the instrument with the REAGENT BLANK, read and record the absorvance of each vial. 5. Carefully transfer 5.0 ml of HEMOGLOBIN STANDARD into clean vials (same as above) read and record the absorvance of the versus the REAGENT BLANK. Use the absorvance readings of the UNKNOWN and STANDARD to calculate the hemoglobin values as follows: A= absrovance, S= standar and C= concentration. QUALITY CONTROL: It is recommended that the normal and abnormal samples be used routinely. Refer the manufacture as package insert for analyze satiability and acceptable performance limits. If you do not have local access to control materials. EXPECTED VALUES: 13-18 11-16 14-23 12-16 10-14 Gm/dl Gm/dl Gm/dl Gm/dl Gm/dl adult males adult females infants (at birth) infants (one month) infants (one year)

Gradual Increase to Adult Levels for Children (2 years to adulthood) 15.0-21.7 Gm/dl 17.3-24.2 Gm/dl adult females adult males

Individual residing at altitudes of 12-15,000 feet.

L.E. Cells Lupus Erythematosus is a disease of the skin which is characterized by reuptions on the surface of the body. Its cause is unknown. The disease is sometimes difficult to diagnose. A recent laboratory test, however, has come to the aid of the physician. This test of examining the blood for L.E. cells. The L.E. Cells derive its name from the disesase lupus erythematosus. When a patient has lupus erythomatosus, his plasma contains a substance known as L.E. factor. This L.E. factor acts upon white cells and causes a chemical change in the nucleus. The nucleus assumes the shape of a round body. This round body is then ingested by a neutrophilicsegmented cell. The neutrophilic-segmented cell containing the round body is called L.E. cell. 1. The nucleus of the nuetrophilic-segmented cell acts like a pair of pincers and surrounds or engulfs the round body. Thus, the neutrophilicsegmented cell becomes the cannibal and the round body becomes the meal. 2. Cell consists of a round body which is being attack by the neturophilicsegmented cell. Eventually one of the neutrophilic-segmented cell will win the meal. A cell known as the tart cell may be present in the blood of patients who are sensitive to various drugs. The tart cell also contains ingested material. Since the L.E. cell and the tart cell both contains ingested material, they somewhat look alike. In an L.E. cell, the ingested body is round, smooth, and eventually stained. In a tart cell, the ingested body is irregular, coarse, and unevenly stained. LUPUS ERYTHEMATOSUS (L.E.) Materials: 1. 2. 3. 4. 5. 6. 7. 8. Materials for venipuncture Serological test tube Wooden applicator stick Wintrobe tube Syringe and syringe canula or wintrobe pipette Centrifuge Glass slide Wright-Giemsa Stain

PROCEDURE: Having Clotted Blood: Withdraw few cc of blood and place in a test tube, incubate in water bath at 37C for 30 minutes. Stand at room temperature after incubation. The supernatant is withdrawn and place in wintrobe tube.

Note: Separate the big clot, then remove the remaining clots and place the remaining liquid portion in wintrobe tube. Place the test tube in a centrifuge for 0 min at 300 rpm. Smears are made from the buffy coat. Air dry and stain with wrights stain. L.E. cells are usually nutrophil polymorph spherical homogenous purlish-brown staining mass. The tubes of the polymorphic-nuclears are usually seen at the periphery of the mass. L.E. cells are numerous at the edge of the smear. A minimum of 500 polys is counted before giving a negative result. Note: Be sure o have a positive slides examined by the clinical pathologist for confirmation before reporting.

LUPUS ERYTHEMATOSUS PREPARATION PROCEDURE: 1. Draw a heparin vacutainer. An excessive amount of heparin depresses the L.E. phenomenon. Therefore, the vacutainer must be properly filled with blood. 2. Add 6 glass beads to the tube. 3. Place the tube on rotator and rotate for 30 minutes. 4. Fill disposable Wintrobe hematocrit tube with blood and centrifuge at 1,000 rpm for 10 minutes. 5. The buffy coat is then pipetted off, smeared, on 3 slides and stained with Wrights stain. The preparation is first examined under the low power in order to find areas where suspicious materials is present in greater abundance. Under oil immersion magnifications the L.E. (Lupus Erythematosus) cell is recognized by the presence of relatively smooth, homogenous nuclear mass which stains pale blue color and is less prominent than the host nucleus. The latter is compressed to the periphery of the cell by the cytoplasmic inclusion. The phagocytic cell is usually a polymorpho-nuclear neutrophil less often an eosinophil. Note: Be sure to have a positive slides examined by the clinical pathologist for confirmation before reporting.

OSMOTIC FRAGILITY TEST (The Method of Sandford) PRINCIPLES: Red cells are placed in different concentration of NaCl colutions and the extent or degree of hemolysisi is noted in each concentration. MATERIALS OR EQUIPMENT: 1. 2. 3. 4. 5. 6. 7. 8. Sterile dry syringe and needle 13 clean dry test tube (12 x 75) 1 ml pipette test tube rack 70% ethyl alcohol cotton balls 0.5% NaCl soln distilled water

PROCEDURE: 1. Prepare 3 test tube in test tube rack, and from the left number them as 25,24,23,22,21,20,19,18,17,16,15, and 14.The last test tube at the right most side label as control. 2. With a capillary pipette, place into each tube the number of drops of distilled water required to bring the content of each tube to a total of 25 drops. Mix by lateral shaking. The salt concentration in each tube is now equal to the tube number multiplied by 0.02. 3. Obtain blood by venipuncture and with the needle still attached to the syringe carefully deliver 1 gtt of blood into each tube. Mix by very slow inversion. 4. Let all tubes stand at room temperature for 2 hours. Do not disturb the tube content. 5. At the end of 2 hours, examine the tubes one after the other to right for the first evidence of hemolysis. This is indicated by pinkish supernatant layer and turbid red layer below. The first tube from left having this should be marked as having the INITIAL HEMOLYSIS. The examination of the tube is continued further to the right for COMPLETE HEMOLYSIS. The first tube from the left showing a clear red mixture and without sediment of red cells indicates this mark. This test tube as that which show COMPLETE HEMOLYSIS should then be doubt as to whether hemolysis is present, or absent. The control tube should be used. The controls contain physiologic salt solution and blood under this condition. Red cells normally will not hemolyze and the supernatant in this tube should be colorless (no hemolysis). NOTES: 1. It is very important that when delivering solution or blood from the pipettes, in order that the size of the drops being delivered be uniformed, the position of the delivering pipette should maintain in one position. Varying position will affect the size of the drop.

2. When examining the tubes for hemolysis, they should never be lifted
from the rack. Inspection should be done by holding the tubes rack to eye level and against a good source of light. Lifting the tubes individually for its content resuspends the cells. 3. Results should be reported not by tube number but rather by the concentration in percent. RESULTS: Normal values at 20C, pH 7.4 with 30% salt; there is a 97-100% hemolysis. Salt Concentration 0.35% 0.40% 0.45% 0.50% 0.55% salt salt salt salt salt % Hemolysis 90-99 50-90 5-45 0-5 0- HEMOLYSIS

Number on Test tube 25 24 23 22 21 20 19 18 17 16 15 14 Number cc (0.5% NaCl)-2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 Number cc dist H2O 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 % salt solution obtained- .50 .48 .46 .44 .42 .40 .38 .36 .34 .32 .30 .28 Normal values: Beginning of hemolysis 22 Completion of hemolyisis Hemolytic jaundice Beginning of hemolysis-24 Completion of hemolysis Obstructive Jaundice Beginning of hemolysis Completion of hemolysis 19 16 .38 .32 .48 20 .40 17 0.44 .34

CORRECTION FOR NUCLEATED RED CELLS In some anemias such as thalassemia and erythroblastosis fetalis, many nucleated red cells may be found in the blood. Since these nucleated red cells are not dissolved by the white cell diluting fluid, they are counted as white cells. This would give us an erroneously high white cell count. Consequently the count must be corrected. If you find large numbers of nucleated red cells in the stained red cell examination, correct the white cell count with the formula given below. FORMULA:

Corrected white cell count= uncorrected WBC x 600 100+A Where 100= white cell counted in the differential white count. A= number of nucleated red cells counted while counting the 100m white cell of the differential white cell count. EXAMPLE: Suppose we had a white cell count of 15,000. And we counted 50 nucleated red cells while counting the 100 white cells of the differential white cell count. Then our uncorrected white cell count would be 15,000. And A would be equal to 50. Corrected white cell count = = = = BLOOD INDICES These determinations cen be helpful in borderline or early causes of anemias. However the degrees of accuracy of this test is unjustifiable, and values must be interpreted only in the light of other findings and if the test has been carried out with meticulous care. 1. COLOR INDEX is the average amount hemoglobin in each erythrocytes compared with the average amount in a normal erythrocytes. Color Index= Percent Hemoglobin Percent Erythrocyte Example: Hemoglobin percent=9.6 x 100 = 60% Erythrocyte percent= 3,200,000 x 100 = 64% 5,000,000 Color Index = 60 = 0.94 64 Normal Value for color index varies from 0.90 to 1.10 Significance: Increased in pernicious anemia to 1% due to macrocytes and megalocytes. Decreased in secondary anemia and chlorosis. uncorrected white cell count x 100 100+A 15,000 x 100 150 15,000 x 2 3 10,000

2. VOLUME INDEX = Percent volume

Percent erythrocyte

Example: Volume percent = 32 x 100 = 68% 47 Erythrocyte percent = 3,000,000 x 100 = 60% 5,000,000 Volume Index = 68 = 1.13 60 Significance: Increased in infants in their few weeks of life. Decreased in older children , usually lower than adult 3. SATURATION INDEX = is the average amount of hemoglobin per unit volume of cell in relation to the normal. Saturation Index = Example: Color Index or Volume Index Percent hemoglobin Percent Volume

Erythrocyte count 3,500,000 = 70% Hemoglobin in grams 12 = 75% Hematocrit 31 = 66% of normal value Color Index = 75 = 1.07 70 Volume Index = 66 = 0.94 70 Saturation Index = 1.07 = 1.14 or 75 0.94 Saturation Index = 75 = 1.14 66

Normal Saturation Index varies from 0.80 to 1.20 4. MEAN CORPUSCULAR HEMOGLOBIN (MCH) is the mean of average weight of hemoglobin per erythrocytes. MCH = Hemoglobin in grams per liter of blood Erythrocyte in million per cu. mm.

Example: Hemoglobin value is 9.6 grams % Erythrocyte count is 3,200,000 Hemoglobin = 96 grams per liter Erythrocytes = 3.2 million per cu. mm. MCH = 96 = 30 micro microgram 3.2 Normal value is 29.5 adult average and varies from 27 to 33 micro micrograms Children----------------------------65 to 70 points lower Macrocyte anemia-----------------30 to 52 Normocytic anemia----------------27 to 32 Microcytic anemia------------------22 to 26 Hypochromic microcytic anemia 14 to 21

5. MEAN CORPUSCULAR VALUE (MCV) = is the mean average volume of each erythrocyte. MCV= Volume per packed cells in ml. per liter of blood Erythrocyte in million per cu. mm. Example : Hematocrit 32 Erythrocyte 3,000,000 Volume of packed cells 32 MCV= 320 = 107 cubic micron 3

Normal mean corpuscular volume of the blood averages 87 and varies from 80 to 100 cu. micron. Children---------------------------------- are about 20 points lower Macrocyte anemia----------------------- 95 to 160 Normocytic anemia---------------------- 72 to 70 Microcytic anemia------------------------ 80 to 94 Hypochromic anemia--------------------- 50 to 71 6. MEAN CORPUSCULAR DIAMETER = the mean or average diameter of erythrocytes in terms of micron. Several methods of measurements have been suggested. The Price Jones Technique: The stained smear is projected onto paper by means of a camera lucida and the maximum and minimum diameter of 104-500 RBC are recorded, ths scale being derived from a micrometer slide similarly projected. The method is time consuming and somewhat inaccurate because RBC diameters may vary greatly in different areas of the smear. Diameters of fixed and stained RBCs are about 1 micron less than those in wet preparations. A curve maybe prepared showing the distribution of cells in various diameter ranges (Price-Jones Curve). 7. Mean Corpuscular Average Thickness (MCAT)SIMPLASTIN INTENDED USE: General Diagnostic SIMPLASTIN is intended for use as a Thromboplastin Reagent for Prothrombin Time (PT) Determination. DISCUSSION General Diagnostic Simplastin contains tissue thromboplastin reagent (rabbit brain and lung) plus calcium ions in a suitable buffer. SIMPLASTIN (reconstituted with purified water) is used in a modified one-stage Prothrombin Time (PT) test.

General Diagnostic SIMPLASTIN is sensitive to deficiencies in the Extrinsic Coagulation System (Factors II, V, VII, and X). The PT is used as a presurgical screen to detect potential bleeding problems. An extended PT is usually indicative of a decreased level of one or more of the factors I nthe extrinsic system which may be caused by hereditary coagulation disorders, a vitamin K deficiency, liver disease or drug administration. It is most common laboratory assay used to monitor oral anticoagulant therapy since it is sensitive to Factors VII and X. It is not sensitive to deficiency in the Intrinsic System (Factors VIII, IX, XI, and XII). It cannot be used to monitor heparin therapy nor is it sensitive to platelet dysfunction. Activated Partial Thromboplastin Determination (General Diagnostics Automated APTT) is recommended fro monitoring heparin therapy. A Bleeding Time Test (General Diagnostics SIMPLATE) is recommended for identifying platelet dysfunction. REAGENTS Materials Provided SIMPLASTIN CONTAINS:Tissue thromboplastin from rabbit brain and lung in quantity sufficient for the number of determination indicated on the carton and vial label, together with calcium ions, barbital buffer and solution chloride. Reconstitute with the volume of purified water indicated in the label. Shake vigorously to ensure complete rehydration. Mix again immediately before use to insure homogeneity. Store in the original (tightly capped) vial at 2-8C for not more than 5 days. Record the expiration date on the vial label. Bacterial contamination which may not be visibly apparent, can cause prolonged prothrombin times. If reconstituted materials is retained for more than 24 hours, careful evaluation of control data is recommended. *Purified water- Refer to United States Pharmacopeia for specifications. SAMPLE PREPARATION Add 9 parts freshly drawn blood to 1 part anticoagulant contained in a clean tube and mix thoroughly. Store at 2-8C or in crushed ice. Centrifuge and remove plasma from cells before testing. Testing should be completed within 4 hours of sample collection. If the plasma is to be frozen, it should be frozen rapidly (-20C or lower).Rapid thawing (37C) is recommended to prevent denaturation of fibrinogen. PROTHROMBIN TIME TIME PROCEDURE The following sequence for performing a one-stage prothrombin time test is suitable for manual techniques; instruments determination should be performed according to specific instructions accompanying the instrument used. 1. Prewarm a sufficient volume of reconstituted SIMPLASTIN to 37C (0.2 ml per test). Take precaution to prevent evaporation and do not hold at 37C for longer than 60 minutes before using.

2. Label test tube for each sample (patient and control) to be tested. Duplicate
samples are recommended to insure accuracy. 3. Add 0. ml of sample or control to the appropriate tube. 4. Incubate each sample and control at 37C for 3-0 minutes 5. Forcibly add 0,2 ml of prewarned SIMPLASTIN and simultaneously begin timing for clot detection. 6. Record the time, in seconds required for clot detection. NORMAL VALUES: A normal range generally lies between 10-14 sec PROCEDURAL NOTES: Use a clean glass or plastic ware restricted specifically to coagulation is important. Consistent technique is required or for all coagulation procedures. Duplicate determinations are recommended. Plasma samples which are hemolyzed, icteric, or lipemic will usually not interfere with this procedure. However results should be evaluated carefully when testing these samples on photo-optical instruments. Although most manual or instrumental methods for clot detection may be used with SIMPLASTIN, different methods may detect slightly different endpoints. Caution must be used when comparing results. EXPECTED VALUES Clotting times are dependent on numerous factors including temperature, water quality, pH, ionic strength, test system used, the anticoagulant, the technique for specimen collection and preservation and the patient population under consideration Specific Normal Ranges for the PT test should be established by each laboratory. QUALITY CONTROL A series of control materials such as General Diagnostics VERIFY Normal Citrate and VERIFY Abnormal Citrate Levels I and II are recommended for monitoring coagulation test. Use each materials according to package insert directions for these products.

CLOT RETRACTION TIME The clot retraction time measures the ability of the blood clot to retract. In normal blood, the clot reacts as follows: After 2 hours, there is partial retraction of the clot. After 24 hours, there is complete retraction of the clot. The clot retraction time is mainly used in the diagnosis of hemorrhagic diseases. In thrombocytopenic purpura, for example, the clot retraction tie is greatly increased. In a severe case, there is no retraction of the clot- even after 24 hours.

The clot retraction time is prolonged in those conditions as follows: 1. 2. 3. 4. 5. 6. 7. thrombocytopenic purpura hemorrhagic disease of the newborn aplastic anemia pernicious anemia leukemia multiple myeloma Hodgkins disease

When the platelet count is decreased, the clot retraction time is increased. Consequently, the platelets apparently play a major role in the retraction of the clot. PROCEDURE FOR CLOT RETRACTION TIME The clot retraction time is determined by placing about 5 cc of blood in a test tube and observing the progress of clot retraction. NECESSARY REAGENTS AND EQUIPMENTS 1. One serological test tube and cork 2. Materials for venipuncture 3. 37C incubator or 37 C water bath Procedure: 1. Obtain a serological test tube, cork, and materials for venipuncture: sterile 20 gauge neddle, 10 cc syringe, wax marking pencil, tourniquet, 70% alcohol and cottonballs or gauze pads. 2. Write the patients name on the test tube. 3. Make a venipuncture, withdraw about 5 cc of blood, remove the needle fron the syringe and transfer the blood to the test tube. 4. Put the cork in the mouth of the test tube. 5. Place the test tube in either a 37C in cubator or a 37C water bath. 6. After 2 hours have elapsed, observe the clot. 7. Record your observation as no retraction, partial retraction, or complete retraction. Note: Sometimes normal blood forms a clot in which the entire outer edges cling to the walls of the test tubes. In such a clot, any retraction is not noticeable. Therefore, if the clot is sticking to the walls of the test tubes, loosen it with a wooden applicator stick. 8. Put the test tube in the 37C water bath or incubator. 9. Then make observations and recordings at 6,18,and 24 hours after the venipuncture. 10. Make your report. CLOT RETRACTION TIME TEST PRINCIPLE: The normal blood clot contracts and in so doing expressed serum. The degree of clot retraction can be measured on the basis of the amount of serum expressed. Quantitative Test estimate the degree of retraction.

STEFENIN METHOD or TEST TUBE METHOD a. b. By venipuncture, draw 3 cc of blood. Place the blood in a wasserman tube. Incubate at 37C or place at room temperature.

c.

Clot retraction begins within hour after retraction and is usually complete within 1824 hours. Record your results as follows: Normal or Complete retractivity Partial retractivity Poor retractivity Very poor retractivity NOTE: Occasionally, the blood may adhere to the wall of the test tubes (usually if the test tube is not very clean) and present a false result. In such results persist after a few hours, ring the clot round the wall of the test tube. If the blood is normal, retraction will take place in short time. CLOTTING TIME or COAGULATION TIME Coagulation time is the process of turning fibrinogens to fibrin. It is the simplest abd most informative test. PRINCIPLE: The test measures the period required for free from blood to clot or solidify after it has been removed from the body. SLIDE METHOD or DROP METHOD 1. Place a drop of blood to a glass slide. 2. Draw a sharp pin or needle across the drop of blood at 30 seconds interval. 3. Coagulation is supposed to have started the moments some fibrin threads cling to the ends or point of the pin or needle. Normal value: 2-4 minutes LEE AND WHITE METHOD or WHOLE BLOOD CLOTTING

1. Place three 3 x 100 mm clean test tube in a 37C.

2. Obtain 5 ml venous blood in a plastic syringe using the 2 syringe method. 3. Place ml blood into each of the 3 test tubes labeled 1,2, and 3. Start a stopwatch as the blood enters the tube 1.

4. Every 30 second gently tilt tube 3 test, the last to receive blood, until the blood in
it clots, tube 2 is then handled of blood in the same way, finally tube is tilted until no flow of blood is observed on tilting.

Normal values: 7-15 minutes is abnormal A clotting time over 15 minutes is abnormal NOTE: Tube used in this tesr may either be washed with saline and solution or siliconized. The sensitivity of this test can be increased by silicone coating the tubes so that the normal clotting time is prolonged to 20-40 minutes. Saline solution 6-12 minutes, Siliconized tubes discovers the hemophilic disease which will be normal clotting (coagulation) time if ordinary method is used. BLEEDING TIME PRINCIPLE: Determination of bleeding time measures the ability of small blood vessels to control bleeding after injury. This depends on the platelet pluf formation, vasoconstriction and coagulation. DUKES METHOD: 1. Cleanse the finger with an alcohol sponge and allow to dry. 2. Make a relatively deep puncture with the sterile blood lancet, and start the stopwatch. 3. Using the circular filter paper, clot the blood every 30 seconds, do not allow the filter paper to touch the wound. 4. When bleeding ceases, stop the watch, record the bleeding time. Normal values: 2-4- minutes BLOOD PLATELETS COUNTING TEST Platelets are small, colorless, moderately refractile bodies. When stained they appear azure granules with scanty light blue cytoplasm. It is 2-4 microns in diameter. Life span is 4 days. The most significant property of platelets is the readiness with which they agglutinate followed by subsequent dissolution. In general, whenever the number of thrombocytes is less than 60,000/cu mm a hemorrhagic tendency becomes evident. Reasons why platelets are hard to count: 1. Easily disintegrate. 2. Hard to distinguish from debris.

3. Has the tendency to adhere to the glass or to any foreign bodies. 4. There is evidence that that they are not evenly distributed in the blood.

Count varies inversely with bleeding time and directly with clot retraction. An increase is not so important, but a decrease may mean destruction of the megakaryocytes I nthe bone marrow or destruction of platelets in the circulation.

I. INDIRECT METHODS:
Platelets are counted in their relationship to RBC on fixed films. This method is least reliable since the results depends on the distribution of platelets and on the red blood cell count. The permanent record is an advantage because it allows study of platelets morphology. FONIOS METHOD: 1. Make a capillary puncture and make a peripheral smear, dry and stain with Wrights stain. 2. Focus on oil immersion objective. 3. Count 1,000 red blood cell and at the same time count the platelet seen. COMPUTATION: Platelet count= RBC CT (1,000) x no. of platelet counted 1,000 Normal Value: 200-400 x109/liter II. DIRECT METHOD The most accurate way of platelet counting. REES AND ECKER METHOD: Reagents: 1. 2. 3. 4. Sodium Citrate ------------------------------------ 3.8.gm Neutral formaldehyde, 40% ---------------------- 0.2 gm Brilliant cresyl blue -------------------------------- 0.1 gm Distilled water ------------------------------------- 100.0 ml

Filter and centrifuge before use. Keep stoppered in a refrigerator. Preserves RBC which may be counted with the procedure. PROCEDURE: 1. 2. 3. 4. Draw capillary or venous blood to 0.5 mark of red cell ppette. Draw centrifuged and filtered diluting fluid up to 101 mark. Shake the pipette for 3-5 minutes Charge the chamber and allow to settle for 5 minutes.

5. Count with high power objective in the 4 large corner squares (used in WBC count)

FORMULA: No of platelets x 10 x 200 = Platelets/cu. mm 4 NORMAL VALUE: 140,000-340,000/cu. mm RETICULOCYTES Reticulocytes are immature erythrocytes which show a reticulum (skein-like network) when stained by some supravital stain. Some of which appear as polychromatic cells (grayblue)on smears stained Wrights stain, and if stained with supravital dyes, they exhibit a blue staining reticulum. In older reticulocytes, the reticulum may be seen as a single bluish dot. The reticulum consists of ribonucleic acid, and with the electron microscope it can be seen to form a mitochondrial pattern. NOTE: Reticulocytes cannot be stained and demonstrated in ordinary routine stain. Reticulum may be seen in red blood cells both with or without nucleus. The younger the reticulocytes, the more filamentous and plentiful in the reticulum. NORMAL VALUES: 0.5-1.5 % IN ADULTS (Gradwohl) 1.0-2.0% in adults (Seiverds) If normal value is to be expressed per cubic millimeter the normal value is 20,00060,000/ cu. mm TYPES OF BLOOD USED FOR EXAMINATION: 1. Capillary blood usually used 2. Oxalated blood can also be used, only that it should not be more than hour old. SEIVERDS METHOD: Procedure: a. b. c. d. e. f. g. h. i. Obtain any of the solution in wet method for the stain solution. Filter about 1 ml of the staining solution. Put a drop of the filtered staining solution on a glass slides. Spread the staining solution over the same way as in making a smear. Either finger tip blood or oxalated can be used. Oxalated blood should be mixed thoroughly. Place a small drop of blood on the slide containing the stain. Put a cover slip on the small drop of blood. Then press down gently on the cover slip so the blood spread out to the edges of the cover slip Let stand for 10 minutes. This gives the cells the time to accept the stain. Examine under oil immersion objective.

COMPUTATION: Retic counted x 100 = Retics in percent 1,000 RBC counted VALUE OR IMPORTANCE OF RETICULOCYTES

1. To determine the response of the pernicious anemia patient to Vit. B12 therapy to
confirm the diagnosis of pernicious anemias by therapeutic response. 2. To aid I nthe diagnosis of lead poisoning and hemolytic icteru. 3. To determine whether regeneration of erythrocytes is proceeding normally and whether it occurs at all, which is of value in establishing a diagnosis of aplastic anemia. 4. To aid in the prognosis of acute hemorrhage, in which an increase denotes regeneration of erythrocytes. LOW COUNT OR ABSENT IN : 1. Idiopathic aplastic anemia 2. Acute benzol poisoning 3. In an aplastic crisis of hemolytic anemia INCREASE RETICULOCYTES IN: 1. Hemolytic anemias- very high 2. Kala-azar 3. Lead poisoning- very high 4. Leukemia 5. Malaria 6. Erythroblastic anemias 7. Polycythemia vera 8. Relapsing fever 9. Sickle-cell anemia 10. Splenic tumor 11. Blood intoxification 12. Parasitic infection

USER MANUAL HUMACLOT PT (Quick Test) Procedure for HEMOSTAT REAGENT Check the ISI value settings against the used reagent lot information. Prewarm the cuvette with stirrer

1. Select the program as explained before or by