Complete Blood Count and Other Routine Procedures PDF

Complete Blood Count and Other Routine Procedures Richard Rupert T. Vicencio School of Medical Technology  Learning Outcomes 1 2 3 Discuss the significance Enumerate the different Explain the different...

Complete Blood Count and Other Routine Procedures Richard Rupert T. Vicencio School of Medical Technology  Learning Outcomes 1 2 3 Discuss the significance Enumerate the different Explain the different of Complete Blood Count parameters in CBC and principles and concepts of each parameter and state its function to be aware of the clinical conditions that results in variation of results from reference range  Learning Outcomes 4 5 6 Explain the different Determine the factors Discuss the significance of reticulocyte count and methods to determine affecting the results in erythrocyte sedimentation hemoglobin and the macromethod and rate and e hematocrit micromethod of concentration hematocrit determination Topic Outline 01. Complete Blood Count 05. Platelet Count 02. RBC Count 06. Hemoglobin 03. WBC Count 07. Hematocrit 04. Differential Count 08. Reticulocyte Count 09. Erythrocyte Sedimentation Rate  Complete Blood Count 01. Is the most common test performed in the hematology section Used to assess patient conditions such as 1. Infections 2. Malignancy CBC includes: Manual cell counts are performed using a hemacytometer, or counting chamber, and manual dilutions made with calibrated, automated pipettes and diluents (commercially available or laboratory prepared). — RODAK’s 6th edition Hemacytometer  RBC Count 02. - determination of the red blood cells present in 1uL (microliter) of blood Procedure 1. Diluting the blood 2. Charging the Counting Chamber 3. Counting the Cell 4. Making the Calculations  1. Diluting the blood facilitates the counting by suspending and dispersing the red cells to draw blood to mark 0.5 of the RBC pipette and the red cell diluent to mark 101 1:200 dilution dilution range that can be prepared using RBC Thoma pipette is 1:100 up to 1:1000  RBC diluting fluid 1. Dacie’s fluid - this is considered as the BEST diluent It keeps for a long time and does not alter the shape of the cells Formalin acts as a preservative The cell shape is not altered Composition: 40% formaldehyde 10 mL 3% w/v disodium citrate 990 mL 2. Hayem’s diluting fluid - this fluid is not recommended because it allows the growth of yeasts and produces clumping of cells. It can stand for long periods of time and has no corrosive effects Composition – Mercuric chloride C.P. 1.0 gram – Sodium sulfate anhydrous or 4.4 grams – 10 grams crystalline Na2SO4 – H2O – Sodium chloride C.P. 2.0 grams – Distilled water 100 mL  RBC diluting fluid 3. Gower’s solution this solution prevents ROULEAUX FORMATION Precipitates PROTEIN in cases of hemoglobinemia and hyperglobulinemia. Composition: Sodium sulfate anhydrous C.P. 12.5 grams Glacial acetic acid 33.3 mL Distilled water 200.0 mL 4. Toisson’s fluid this solution has a HIGH SPECIFIC GRAVITY and stains the WBC but supports the growth of fungi Composition Sodium chloride 1.0 gram Sodium sulfate 8.0 grams Glycerin 30.0 grams Methyl violet 0.25 grams Distilled water 180 mL  RBC diluting fluid 5. Bethell’s fluid Composition Sodium sulfate 5.0 grams Sodium chloride 1.0 gram Glycerin 20.0 mL Sodium merthriolate (1:1000) 2.0 mL Distilled water 200.0 mL 6. Normal saline solution (NSS or physiologic salt solution) this is used in EMERGENCY CASE, in the presence of ROULEAUX FORMATION and AUTOAGGLUTINATION of cells It is stable and serves as a preservative Composition Sodium chloride 0.85 gram Distilled water 100.0 mL 7. 3.8% sodium citrate Sodium citrate 3.8 grams  2. Charging the Counter Chamber representative sample of the diluted mixture is transferred to a counting chamber pipette should be mixed by shaking the pipette in any manner except along the longitudinal axis FIRST FEW DROPS coming from the capillary system SHOULD BE DISCARDED  3. Counting the Cell using the high-power objective of the microscope, count the red cells seen in 5 intermediate squares in the central ruled area.  4. Making the calculations # of RBC counted in X area c.f. X depth c.f. X dilution c.f. in 1/5 sq.mm. OR RBC count = ave. cell no. X dilution factor area counted X depth of counting chamber Reference Value Source: Clinical Hematology: Principles, Procedures and Correlations by Steininger  Physiologic Variations 1. Increased count in case of DEHYDRATION while the opposite is true in case of hydration. 2. Increased count in EXERCISE or excitement. 3. NEWBORN children have higher counts than adults. 4. Women have LOWER COUNTS than male. 5. Individuals living at HIGH ALTITUDES have higher counts than those living at sea level  Pathologic Variations 1. There is INCREASED erythrocyte count in: a. Polycythemia or polycythemia vera b. Pulmonary tuberculosis or pulmonary fibrosis c. Acute poisoning 2. There is DECREASED erythrocyte count in: a. In anemia b. After hemorrhage  WBC Count 03. determination of the white blood cells present in 1 uL (microliter) of blood.  Thoma Pipette Method This pipette is similar to the red cell pipette except that the bulb is smaller, the bore is larger, and the mark at the short limb is 11 instead of 101 a. Suck blood to mark “0.5” and diluting fluid to mark “11” b. This will give a dilution of 1 in 20 The diluent used in the count: 1. Should be HYPOTONIC 2. Should color/stain the nuclei of white blood cells  Diluent Example of Diluent: 1. 2-3 % acetic acid 2. 1% HCL added with 1 drop of methyl violet or crystal violet  Procedure 1. Diluting the blood dilution of the blood for white cell count facilitate the counting process by hemolyzing the matured red blood cells but not the nucleated (young) red cells. draw blood to mark 0.5 of the WBC pipette and the WBC diluting fluid to mark 11. 1:20 dilution dilution range that can be prepared using WBC Thoma pipette is 1:10- 1:100  Procedure 2. Charging the Counting Chamber. The procedure in erythrocyte count is also adopted  Procedure 3. Counting the cells Before counting the cells, students are advised to make a study of the uncharged counting chamber to familiarize themselves with the ruling of the chamber. After charging the counting chamber, let the cells settle for 1 to 2 minutes, so that all the cells are in the same plane. Using the low power objective, locate and scan the ruled area. If there is a good distribution of cells, start counting. Count the cells within the 4 corner squares (4sq. mm.) with 16 medium squares each Procedure  Procedure 4. Making the calculations = # of WBC counted in 4 sq. mm. X area c.f. X depth c.f. X dilution c.f. WBC count = WBC counted X 10 X 20 4 Area used in white cell count = 4 sq. mm. Area correction factor = ¼ Depth of counting chamber = 0.1 or 1/10 mm Shortcut method: Total of No. of Cells in all 4 squares X 50 Depth correction factor = 10 Ratio of Dilution = 1:20 or 1/20 Note: applicable ONLY when the dilution used is 1:20 Dilution correction factor = 20  Reminder a.Overcharging may decrease cell count b.Allow the counting chamber to stand for at least 3 minutes after charging c.Allowable difference between 2 chamber counts: - For RBCs, 15 to 16 counts - For WBCs, 10 to 12 counts  Calculation of Dilution Recommended Dilutions for Manual WBC counts (based on anticipated WBC count)  Variation in Techniques 1. Leukopenia - 1:10 dilution 2. Leukocytosis - 1:200 dilution (if blood is up to 0.5) - 1:100 (if blood is up to 1) 3. If 8 big squares are used (4 in each ruled area), change the area correction factor to 8 accordingly or first get the average of the counts made on the upper and lower chamber.  Reference Value Normal Values Conventional Unit SI Unit Adults 4,500 to 11,000/ cu.mm 4.5 – 11 X 109/L Infant 10,000 to 25, 000/ 10 -25 X 109/L cu.mm 1 year 8,000 to 15,000/ cu.mm 8-15 X 109/L  Variation on WBC count 1. There is an hourly rhythm of number of white blood cells. a. LOW LEVEL is observed in the morning and goes HIGHER in the afternoon 2. Food intake, moderate physical or emotional activity will cause a INCREASE in the number of leukocytes. 3. Highest peak is observed after meals.  Presence of nRBC? Correction of WBC count is done if there are 5 or more NRBC/per 100 white blood cells when examined in the blood smear. Formula for the corrected WBC count To correct the leukocyte count, determine the number of nucleated red blood cells (NRBC) per 100 WBC on the blood smear and proceed with the computation using the following formula: Formula for correction of WBC count Corrected WBC count = incorrected WBC count (L) X 100 No. of NRBC +100 04. WBC differential count - White blood cells are characterized and differentiated according to cell type Steps in Leukocyte Differential Count 1. Preparation of blood smear 2. Staining of blood smear 3. Differentiation of leukocytes 4. Reporting of results  1. Principle Stained blood smear is scanned microscopically to: a. Estimate leukocyte count b. Classify leukocytes into group types  2. Specimen Requirements EDTA whole blood – Skin puncture – Venous puncture 3. Reagents and Equipment Slides Hematological stains Microscope Schilling counter  Stain used in Differential Count Generally, Romanowsky stain is used in staining smear for differential leukocyte count. The following are the stains: a. Wright’s – routinely used b. Giemsa – used in searching for malarial parasites c. Leishman’s d. Other Romanowsky stain  1. Check slide identification. 2. Perform patient specimen orientation. 3. Perform low-power scan to 4. Procedure review blood film adequacy. 4. Perform differential leukocyte count. 5. Classify 100 leukocytes using the “battlement” track method 6. Report results of the 100 cells classified as percentage. 7. Keep separate count of nucleated RBCs (NRBCs). 8. Note and report the ABNORMAL leukocyte morphology. 9. Identify and report any miscellaneous non-leukocyte abnormal cells, such as endothelial cells, basket cells or NRBCs.  Criteria for Leukocyte Identification 1. Cell Size 2. Nuclear – cytoplasmic ratio a. High ratio – nucleus occupies most cell areas with only a small rim b. Low ratio – nucleus is small in relation to volume of cytoplasm. 3. Cytoplasmic characteristics a. Color of background cytoplasm b. Presence or absence of granules c. Color and size of granules 4. Nuclear characteristics a. Shape b. Color Chromatin patter c. Presence or absence of nucleus 1. Segmented Neutrophils 2. Band Neutrophil 3. Lymphocytes 4. Monocyte 5. Eosinophils 5. Basophils 6. Reference Ranges  Absolute Count Gives the number of specific WBC type per cubic millimeter of blood (mm3). more informative than the relative count. Formula: Absolute Count= Relative count (%) X WBC count Absolute Count Absolute WBC Count  Irregularities in Blood Smear Preparation Affecting Leukocytes 1. Squashed, distorted lymphocytes 2. Accumulated white cells 3. Smudge cells 4. Disintegrated or ruptured cells 5. Poorly stained leukocytes – may be caused by: 1. Incorrect pH of buffer 2. Improper mixing of stain and buffer 3. Too short staining period 6. Precipitated stain – may be caused by: 1. Unclean slides (dust, etc.) 2. Drying during the staining period 3. Inadequate washing of cells after staining period 4. Failure to hold slide horizontally during initial washing 5. Inadequate filtration of stain  7. Additional Notes A 200-cell differential should be performed when the leukocyte numbers in excess of 35.0 X 10^9/L. If the WBC ct. is less than 1 platelet/ OIO field – decrease in the number of platelets 5 – 20 platelets/OIO field (with occasional clumping) – adequate supply of platelets >25 platelets/OIO field – increase in the number of platelets.  Methods in Platelet Counting 1. Indirect 3. Dameshek’s method Brilliant cresyl blue, sodium citrate, sucrose and formalin are used the same technique as Fonio’s is used; counterstain with Wright’s stain. Procedure 1. Place a drop of diluting fluid over the puncture wound and gently press the finger so that a small amount of blood wells up into the drop of diluting fluid. The proportion of blood to the diluting fluid should use 1:5 2. Transfer the mixture into a cover slip and place it on top of a slide. 3. Allow the platelets to settle for 15-45 minutes. 4. Count the RBCs and platelets under OIO until 250 RBCs have been counted. Formula for calculation: Platelets/uL = Platelet X RBC count 1000 Thrombocyte No. Conc. = Platelet count X 0.01 = _______ X 109/L N.V. 250,000 – 500,000/uL or 250 – 500 X 109  Methods in Platelet Counting 2. Direct Method – The platelets are counted in the hemocytometer as in erythrocyte or leukocyte count. 1. Guy and Leake The diluent is made up of sodium oxalate, 40% formalin and crystal violet. Calculation: Platelet/uL = Platelet counted X 5 X 10 X 100 2. Rees and Ecker Diluent is made up of sodium oxalate, brilliant cresyl blue, formalin and distilled water.  Methods in Platelet Counting 2. Rees and Ecker Procedure 1. Draw blood up to the 0.5 mark of the RBC pipette. 2. Dilute blood with Rees-Ecker diluting fluid up to the 101 mark This makes 1:200 dilution. 3. Shake the pipette for 1-5 minutes. 4. Discard 5-6 drops and charge the counting chamber. 5. Place the counting chamber on a Petri dish with a wet filter paper to prevent evaporation. Let it stand for 10-15 minutes to allow the platelets to settle. 6. Count the platelets in all 25 tertiary squares of the central secondary square with 1 mm2 area. NOTE: Platelets are stained light blue. Calculation: Platelet count = Average platelet count x Depth factor x dilution factor x Area correction factor Platelets/uL = Platelets counted X 10 X 200 4  Methods in Platelet Counting 3. Brecher-Cronkite – Calculation is the same as Guy and Leake method. – error in this method is 11 – 15 % only – makes use of the phase contrast microscope, and the diluent is 1% ammonium oxalate – the MOST ACCURATE method because there is no difficulty in distinguishing platelets from debris because the diluting fluid swells the platelet a little and hemolyze the red cells. Formula: Platelet/uL = Platelet X 5 X 10 X 100 N.V. for direct method: 150, 000 – 450, 000/uL Or 150 – 450 X 109/L  Methods in Platelet Counting 2. Electronic Method Red cells must first be removed from whole blood, either by sedimentation or by controlled centrifugation. A. Voltage-Pulse Counting Dilution is 1:3000 (3uL of blood + 9mL of Isotonic or NSS) Variation in dilution: for platelet count of less than 250, 000/uL, the dilution is 1:300 (20 uL of blood/ 6mL of diluent) A. Electro-Optical Counting Dilution is 1:1500 – in 2M Urea In EDTA treated-blood, the following are observed in some patients. 1. Platelet satellitosis – when platelets encircle the peripheral borders of neutrophils 2. Platelet clumping – due to a type of agglutinin found in the patient.  Methods in Platelet Counting Unopette system  Methods in Platelet Counting Unopette system  Reasons why platelets are hard to count 1. They easily disintegrate. 2. Because of very small size, they could be mistaken for debris. 3. They easily clump, adhere to glass and other contact surface. 4. Uneven distribution of blood.  Sources of Error in Platelet Counting 1. Error in handling 2. Operator’s error 3. Error in equipment and reagent 4. Inherent error or field error  Causes of Platelet Clumping 1. Initiation of platelet aggregation 2. Clotting before the blood reaches the anticoagulant 3. Imperfect venipuncture 4. Delay in sampling  Physiologic Variation 1. Platelet count is slightly lower at birth than in older children and adults. 2. Platelet count may fall at the time of menstruation.  Hemoglobin 06 - Hemoglobin (Hb) is the red iron-. bearing protein contained within the erythrocytes in normal blood. - carry oxygen to and carbon dioxide from the tissues.  Methods 1. Copper Sulfate or Specific Gravity Method 2. Gasometric method (Oxygen Capacity method) 3. Chemical Method (Iron Content Method) e.g. Wong’s Method 4. Colorimetric Methods  Methods 1.Copper Sulfate or Specific Gravity Method Employ copper sulfate with a known specific gravity (1.052 and 1.054) If the drop of blood shrinks, the level of hemoglobin is ACCEPTABLE If the drop of blood floats, the level of hemoglobin is UNACCEPTABLE Specific gravity of blood = 1.053 2. Gasometric Method (Oxygen Capacity Method) Blood is lysed with SAPONIN Gas is collected and measured in a Van Slyke apparatus O2 combining capacity of blood = 1.34 mL O2/g Hb  Methods 3. Chemical Method (Iron Content Method) Iron content of Hb is 0.347%. 1g or 1000mg of Hb contains 3.47 of iron Concentration of Hb in blood = Iron Content (mg/dL) 3.47 e.g. Wong’s method 4. Colorimetric Method A. Visual 1. Direct Matching 1. Talquist Scale – based on color comparison. - uses a very simple chart consisting of varying hues of red color as a comparator  Methods 4. Colorimetric Method A. Visual 1. Direct Matching 1. Talquist Scale  Methods 4. Colorimetric Method A. Visual 1. Direct Matching 2. Dare Hemoglobinometer - consists of a glass plate and an eyepiece. It has a percentage of 20%-30%.  Methods 4. Colorimetric Method A. Visual 2. Acid Hematin - 20 uL blood + N/10 (at “20” mark) a. Sahli’s Hellige b. Haden-Hausser c. Sahli-Adams d. Haldane e. Newcomer f. Osgood  Methods 4. Colorimetric Method A. Visual 2. Alkali Hematin The use of alkaline solution for hemoglobin determination produces a true and relatively stable solution of hematin. Procedure 1. Place 5mL of NaOH on a reaction tube. 2. Deliver 0.05 mL of blood to the tube. 3. Hold the tube in a boiling water bath for 4-5 minutes. 4. Cool and read against an appropriate standard.  Methods B. Indirect Methods 1. Oxyhemoglobin A. Sodium carbonate A photometric determination of hemoglobin is done by measuring oxyhemoglobin. It is simple and quick but there is no possibility of preparing a stable HbO2 standard. B. Photoelectric oxyhemoglobin Pulse oxygen saturation and pulse rate can be measured through the finger using a photoelectric oxyhemoglobin monitor  Methods B. Indirect Methods 2. Carboxyhemoglobin test is not done as a routine examination. It is performed only when carbon monoxide poisoning is suspected. II. Gasometric Method A. Van Slyke Oxygen Capacity This method measures the amount of oxygen using a Van Slyke manometric apparatus. The level of hemoglobin is determined by computation. Note: 1gm of hemoglobin = 1.34 mL O2  1. Description of the test and principle Cyanmethemoglobin Blood is diluted in an alkaline Drabkin’s solution of: potassium cyanide potassium ferricyanide sodium bicarbonate Hemoglobin (Fe2+ ) + K3Fe (CN)6 methemoglobin (Fe3+ ) + KCN cyanmethemoglobin Read at 540 nm.  2. Materials EDTA evacuated tube Drabkin’s solution – pH 7.0 – 7.4 Potassium ferricyanide Potassium cyanide Distilled water  1. Take 5 mL of Drabkin’s reagent and add to 20 uL of blood using Sahli pipette. 2. Stopper the tube, mix by inverting several times. 3. Allow to stand for 10 minutes. 3. Procedure 4. 5. Transfer the sample to cuvette. Read the absorbance in spectrophotometer at 540 nm. 6. Also take the absorbance of the standard solution. Cyanmethemoglobin 4. Reference range Reference Values Men 140-180 g/L Women 120-160 g/L Women in late 85-140 g/L pregnancy Newborn 150 – 200 g/L  Clinical Significance 1. INCREASED (hyperchromia) in polycythemia, dehydration, in poorly compensated heart disease with cyanosis, and in changing from high to low altitude. 2. DECREASED (oligochromia) in anemias 3. Hemoglobinemia is the presence of free hemoglobin in the blood plasma. This is found in: a. Severe infection b. Severe burns and frost bite c. Poisoning with potassium chlorate and mushrooms d. Paroxysmal hemoglobinuria e. Hemolytic transfusion reactions  Sources of Error 1. Cyanmethemoglobin is sensitive to light. 2. High WBC and platelet count can cause turbidity and false increase result. 3. Lipemia can also cause turbidity  Hematocrit 07. is the volume of packed RBCs that occupies a given volume of whole blood. also known as Packed Cell Volume (PCV). reported as % (e.g., 36% or in liters per liter L/L)  Difference Between Macro and Micro Method of Hematocrit Determination Point of Difference Macro Micro 1. Method of blood Venipuncture Skin puncture collection 2. Amount of blood Larger Smaller 3. Relative centrifugal 2,000-2,300 g 10,000 – 12, 000g force 4. Time of centrifugation Longer (30 mins.) Shorter (4-5 mins.) 5. Simplicity of Not simple Simple technique 6. ESR Can be performed in the Cannot be performed wintrobe tube 7. Spilling/leakage Not common Common 8. Breakage of buffy coat Not common Common 9. Separation of buffy Complete Not complete coat 10. Cost of apparatus Expensive cheaper Clinical Importance of Hematocrit Determination 1. It gives a rough estimate of the size or erythrocytes and the concentration of erythrocytes but not the whole red cell mass. 2. It is used in the calculation of the blood indices. 3. The buffy coat obtained from the hematocrit tube has numerous uses. 4. Hematocrit is a good simple screening test for anemia. 5. Since the inherent error obtained in hematocrit is less as compared to erythrocyte count Methods of Hematocrit Determination 1. Macromethods 1. Wintrobe Method 2. Haden’s Modification 3. Van Allen Method 4. Sanford-Magath 5. Bray’s Methods of Hematocrit Determination 1. Macromethods 1. Wintrobe Method Anticoagulant used: Double oxalate Procedure 1. Fill up the Wintrobe tube to the 10 cm. Mark with oxalated blood avoiding bubble formation. 2. Centrifuge at 2500 rpm for 30 minutes. 3. Observe the following: a. Fatty layer (this is not always discernible) b. Plasma layer c. Buffy coat layer d. PCV layer Methods of Hematocrit Determination 1. Take the hematocrit layer reading at the level between the lower layer of the buffy coat and the upper layer of the packed red blood cells. 2. Report in terms of volume percent. Calculate the hematocrit value from the following formula in case of specimen which is QNS. Hematocrit (EVF) = L1 X100 L2 L1 – height of the red cell column in mm. L2 – height of the whole blood column in mm. Note: Buffy coat is not included in L1 Methods of Hematocrit Determination 1. Macromethods 2. Haden’s Modification The anticoagulant of choice is 1.1 % sodium oxalate in distilled water. The method uses a calibrated tube. 1. Place 1 mL of 1.1% sodium oxalate into the tube. 2. Add 5mL of blood. Mix well. 3. Centrifuge the mixture for 20 minutes at 3,000 rpm. 4. Read the volume of packed red blood cells Computation Hematocrit (%) = volume of PRBC’S X20 Note: If less than 5 mL of blood is used, use the following formula: Hematocrit (%) = __volume of PRBCs________X 100 Volume of whole blood used Methods of Hematocrit Determination 1. Macromethods 3. Van Allen Method The anticoagulant of choice is 1.6 sodium oxalate in distilled water. The method uses a tube with bulb and calibration of 1 to 10 cm or 10 to 100mm. Procedure: 1. Fill the tube with blood up to the 10th mark. 2. Dilute the blood with the diluting fluid up to the bulb, about half full. 3. Seal the tube and centrifuge (shaft end down) at 2,500 rpm for 15-30 minutes. 4. Read the volume of PRBCs. NOTE: Each unit of division is equal to 1% Methods of Hematocrit Determination 1. Macromethods 4. Sanford-Magath The anticoagulant of choice is 1.3% sodium oxalate; the tube is calibrated at 1 mm per division. The tube is about 5 inches long and with a funnel-like mouth. Procedure: 1. Place 1 mL of anticoagulant into the tube. 2. Add 5 mL of venous blood and mix. 3. Centrifuge the mixture at high speed for 15 minutes. 4. Read the volume of PRBCs. Normal Value: Male ---------------------------------------------------------- 2.3mL = 46-48 vol% Female ------------------------------------------------------- 2.0 mL = 40-42%  Methods of Hematocrit Determination 1. Macromethods 5. Bray’s The anticoagulant of choice is heparin, and a Bray’s tube is used. This tube is calibrated on both sides, similar to the Wintrobe tube. The calibration is from 10-15mm, each division is 1mm; and the capacity is 5 mL. Procedure: 1. Fill the tube with heparinized blood 2. Let the tube stand at a vertical position for one hour. 3. Read the volume of RBCs form the lower right side calibration. Normal Values: Male ------------------------------------------------------- 47 vol% Female -----------------------------------------------------42 vol% Methods of Hematocrit Determination 2. Micromethod Adam’s Micromethod This method uses heparinized capillary hematocrit tube of about 7cm. long and an internal diameter of 1.0mm. Heparinized (red) capilette can be used of specimen is capillary blood. Blue capillettes should be used in the case of venous blood.  Procedures 1. Fill the capillary tubes 2/3 full with blood. 2. Seal the empty end with modeling clay. (e.g. plasticine) 3. Place the tube in the radial groove of a microhematocrit centrifuge with sealed end away from the center. 4. Centrifuge for 4-5 minutes at a relative centrifugal force of 10,000 – 12, 000 against gravity. 5. The capillary tube is not calibrated and the hematocrit value is obtained by using commercially available reading device (graph)  REFERENCE VALUES Men 40-55% Women 36-48% Newborn 45 – 60% Sources of Error and other comments 1. Improper sealing of the capillary tube HCT 2. Increased concentration of anticoagulant 3. After blood loss 4. Tissue juice contamination during capillary puncture 5. Insufficient centrifugation 6. Delay in reading results HCT 7. Buffy coat should not be included in reading 8. Dehydration  RBC Indices MCV = Hct x 10 RBC ct. MCH = Hgb x10 RBC ct. MCHC = Hgb x 100 Hct  Rule of Three Hemoglobin (Hgb) X 3 = Hematocrit (HCT) +3 RBC X 3 = Hemoglobin +3 This rule applies only to specimens that have normocytic normochromic RBCs  Red Blood Cell Distribution Width (RDW) Measures the degree of anisocytosis Indicates how varied the red blood cells in terms of SIZE and VOLUME  Reticulocyte Count are young RBCs which are formed when the nucleus of the late normoblasts are lost through extrusion. is used as an index of bone marrow activity and RBC production used to monitor therapeutic measures for anemia.  Reticulocyte Count Dry Method Procedure 1. Mix equal volumes of blood and freshly filtered stain in a tube. Allow this mixture to stand at room temperature for 15-30 minutes. 2. Remix the preparation and prepare smears. Allow the smears to dry. 3. Examine under OIO. NOTE: Mature RBCs stain gray-blue while reticulocytes are identified by the presence of deep blue filamentous web or granules within the cells. Any cell that contains two or more particles of blue-stained materials is classified as a reticulocyte. 4. Count the reticulocytes seen in 10 successive fields of vision or while enumerating 1,000 mature RBCs.  Computation: 1. % Reticulocyte = No. of reticulocytes X100 1000 Ex: % Retics = _12__ X 100 = 1.2% 1000 2.  Computation: 3. Retics/mm3 = % retics x RBC count (in millions) 100 Ex: Retics/mm3 = _1.2_ X 4,000,000 100 = 48, 000, 000 retics/mm3 of blood 4. Reticulocyte Number Conc. = Retics/mm3 X.001 = ___ X 109/L Ex: 48, 000/mm3 X.001 = 48 X 109/L 5. Reticulocyte No. Fraction = % Retics X 10 ________ X 10-3/L Ex: 1.2 X 10 = 12 X 10-3/L  Computation: 6. Corrected Reticulocyte Count = % Retics X HCT (%) (normal HCT) Ex: 1.2 X 38 = 1.01 % 45 7. Reticulocyte Production Index (RPI) = ____________CRC_____________ Maturation days of retics in blood Maturation Days of Retics in Blood HCT (%) Maturation Days in Blood 45 + 5 1.0 35 + 5 1.5 25 + 5 2.0 15 + 5 2.5 to 3 days  Technique using Calibrated Miller Disk 1. A miller disk is inserted in the eyepiece of the microscope which allows rapid estimations of large number of red cells by imposing two squares (one square is nine times the area of the other square) onto the field of view. 2. Reticulocytes are counted in the large square and red cells in the small square in successive microscopic fields until at least 300 red cells are counted.  Normal Reference Ranges: % Retics: Adult: 0.5 – 1.5 % Newborn: 2-6% Ret. No. Fraction: 5 – 15 X 10-3/L Reticulocyte no. Conc: 25, 000 – 75, 000/mm3 or uL 25 – 75 X 109/L (SI) Corrected Reticulocyte Count: 1% If PCV is 35% = 2 – 3 % If PCV is 25% = 3 – 5 % RPI: 1  Techniques in Dry Method: 1. New Methylene Blue 4. Tureen 2. Cook 5. Seiverd’s 3. Mayer  Other Methods of Reticulocyte Count Other Methods of Reticulocyte Count I. Microscopic Method A. Wet Method – Rapid method of Schilling, Osgood-Wilhelm, and Sabin A film of stain is spread on a glass slide and a drop of blood is added. The preparation is covered with a coverslip. Reticulocytes are counted per 1000 red cells in the oil immersion field. The % retics is calculated in the same way as the dry method.  Other Methods of Reticulocyte Count I. Microscopic Method A. Wet Method – Rapid method of Schilling, Osgood-Wilhelm, and Sabin Both methods could be counted under the: 1. Light microscope method 2. Calibrated miller disk method Calculation for calibrated miller disk method: 1. % Retics = No. of retics counted in square A X 100 No. of retics examine in square B X 9 1. Retics/uL = % Retics X RBC count 100  Procedure 1. Place a drop of freshly filtered stain on a clean dry glass slide. 2. Add an equal volume of blood. Mix by stirring. 3. Cover the mixture with a clean coverslip line with Vaseline. 4. Proceed with counting.  Methods of Reticulocyte Count 1. Rapid Method of Schilling - The supravital stain is used in alcoholic solution of brilliant cresyl blue. 2. Sabin Method - Neutral Red, Janus Green 3. Seiverd’s Method - Brilliant Cresyl Blue 4. Osgood – Wilhelm Method - New Methylene Blue 5. New Methylene Blue Method - New Methylene Blue 6. Cook, Meyer, and Tureen Method - Brilliant Cresyl blue  Miller Disc Method Miller disc is an optical aid inserted into the eyepiece of the microscope. This allows for more accurate count. The disc ruling consists of a center square containing a secondary square ruled area that is 1/9 the area of the larger square.  Computation % reticulocyte = No. of reticulocytes in large square X 100 No. of RBC in small square X 9  Other Methods of Reticulocyte Count I. Automated Methods A. Sysmex R- 1000 Sysmex R – 1000 is an automated reticulocyte analyzer which uses flow cytometry for the determination of percent reticulocyte as well as the absolute reticulocyte count. Stains for Reticulocyte Count 1. New Methylene Blue – gives a sharp blue color. Composition: New Methylene Blue -------------------------- 1.0 gm NSS ------------------------------------------------- 80. 0 mL 3% sodium citrate ------------------------------- 20.0 mL 2. Brilliant cresyl blue  Reticulocytosis Increase in reticulocytes in blood. This is found in the following: 1. Hemolytic anemia 6. Kala-azar 2. Lead poisoning 7. Erythroblastic anemia 3. Malaria 8. Sickle cell anemia 4. Parasitic infestations 9. Relapsing fever 5. Blood intoxication 10. Leukemia 11. Splenic tumor  Reticulocytes are decreased in: 1. Aplastic anemia 2. Acute benzol poisoning 3. Chronic infections 4. Anaplastic crisis of hemolytic anemia  Physiologic Increase is observed in: 1. Pregnancy 2. At birth 3. Menstruation  Sources of Technical Error in Reticulocyte Count 1. Failure to mix the blood and stain completely. 2. Presence of refractile artifacts 3. Increased blood glucose level 4. Presence of pappenheimer bodies, Heinz bodies and Howell-Jolly bodies  Erythrocyte Sedimentation Rate classically employed as an index of the presence of active diseases like tuberculosis, tonsillitis, rheumatic fever, and rheumatic heart disease a non-specific test and results are affected by factors other than that of the blood cells and the plasma  Principle The test depends on the fact that in the blood to which anticoagulant has been added, the red corpuscles sediment until they form a packed column in the lower part of the tube or container The rate of this process depends on the number of factors like rouleaux formation, concentration of fibrinogen, alpha and beta globulin in the plasma, etc. Reported in millimeters (mm)  Stages in ESR 1. Initial period of aggregation or rouleaux formation 2. Stage of fast settling 3. Final period of packing  Importance of ESR 1. It is used as an index of the presence of an active infection. 2. It measures the suspension stability of RBCs. 3. It indicates abnormal concentration of fibrinogen, globulin, and other plasma proteins. Differences between the Two Most Commonly Used Method of ESR Points of Difference Wintrobe-Landsberg Westergren Tube Bore (diameter) 3 mm 2.5 mm Calibration Up to 100mm Up to 200 mm Length 115 mm 300 mm Anticoagulant Hellen and Paul’s double oxalate 3.8 trisodium citrate (1 part citrate + 4 parts of blood) No. of reading One reading only (after one hour) Two readings (after one hour and 2 hours) Normal values Men – 0 – 10 mm/hr Male – 0 -15 mm/hr Female – 0 -20 mm/hr Female – 0 – 17 mm/hr Dilution No dilution Involves dilution (.6mL of 3.8 sodium citrate to 1.4 mL of blood) Bottom of tube Open Flat and closed Correction for anemia Applicable Not applicable Additional tests which may be Hematocrit, microbilirubin None performed determination, icterus index Advantage/s Smaller amount of blood is needed. Most sensitive method for serial study of chronic diseases Disadvantage/s Large amount of blood is necessary Less sensitive due to shorter column Wintrobe-Landsberg Method In majority of cases, the Wintrobe- Landsberg method is quite an accurate method. Its advantages outweigh its few drawbacks. This method uses the Wintrobe tube, which is calibrated on two sides ( 0-10 and 10 – 0)  Procedure 1. Fill the Wintrobe tube with blood with a Pasteur pipette or a cannula attached to a syringe. 2. Place the tube in a vertical position on a rack. 3. After letting the tube stand for one hour, record the ESR in millimeters. Westergren Method The Westergren method is considered the most sensitive for ESR determination. It can be used for the serial study of chronic diseases like tuberculosis, carcinoma, and others.  Procedure: 1. Fill the Westergren tube with blood using a rubber aspirator. 2. Let the tube stand vertically on a Westergren rack. 3. Record ESR in millimeters after an hour.  Automated Methods A. Automated ESR System by Vega Biomedical The automated ESR system is a fully automated instrument for ESR determination One mL of blood is collected on an evacuated tube containing liquid sodium citrate Ves-Matic analyzer determination takes only 22 minutes to finish. 1. Mini-Ves – four samples at one time 2. Ves – Matic 20 – 20 samples at one time; print results 3. Ves – Matic 60- 60 samples at one time; print results; and identifies sample by barcode reader. Factors Affecting the Erythrocyte Sedimentation Rate A. Intrinsic factors a. Plasma factors b. Red cell factors B. Extrinsic factors a. Mechanical factors b. Technical factors c. Physical factors  Factors Affecting the Erythrocyte Sedimentation Rate I. Factors which INCREASE the rate of fall A. Intrinsic factors 1.Plasma factors a. Increased fibrinogen concentration b. Increased globulin concentration c. Cholesterol 2. Red cell factors a. Macrocytes b. Less number of red cell/anemia c. Hemolysis Factors Affecting the Erythrocyte Sedimentation Rate A. Extrinsic factors 1. Temperature will increase rate of ESR above 27-degree C. 2. Longer sedimentation tube. 3. Larger bore of sedimentation tube 4. Inclination of tilting of sedimentation tube. ( A 3 degree will result in an error up to 30%) 5. Increased dilution of blood. 6. Presence of air bubbles within blood column. 7. Wet glasswares 8. Hemolysis  Variations in ESR Values Physiological variations: 1. ESR is more constant in men than in women. 2. In pregnancy, ESR begins to increase at the 3rd to 4th month and does not return to normal until the 3rd or 4th week postpartum 3. In the newborn, ESR is greatly reduced, in older adults, it is rather high.  Comments and Sources of Errors 1. ESR is a non-specific indicator of tissue damage. 2. Conditions in which rouleaux formation is inhibited, such as sickle cell anemia; spherocytosis may be accompanied by a normal ESR or low ESR. 3. Blood must be fresh. Leaving the specimen for more than 2 hours at room temperature will cause RBC to become spherical and thus inhibiting rouleaux formation.  Comments and Sources of Errors 4. Whole blood specimens which were left overnight will result in lower values. 5. Slight tilting of the sedimentation tube will increase results. 6. If area of reading is hazy, read at the level where the full RBC density is apparent. 7. The presence of anemia invalidates ESR as a tool to diagnose disease process.  Zeta Sedimentation Rate (ZSR) Is performed using a zetafuge and a special capillary tubes Dependent on the concentration of fibrinogen and gamma globulins. Uses EDTA-anticoagulated venous or capillary blood. ZSR % = HCT (%) X 100 Zetacrit  Reference Range 40-51% - Normal 51-54% - borderline normal 55-59% - mildly elevated 60-64% - moderately elevated >65% - markedly elevated thank you!