Hema1-Lec-1-Complete Blood Count and Other Routine Procedures pt 1 PDF

Hematology 1 AY 2024-2025 FAR EASTERN UNIVERSITY - DR. NICANOR REYES MEDICAL FOUNDATION (FEU-NRMF) Complete Blood Count and Other Routine Procedures pt 1 Sir Rupert Vicencio Topic Outline 1. Comp...

Hematology 1 AY 2024-2025 FAR EASTERN UNIVERSITY - DR. NICANOR REYES MEDICAL FOUNDATION (FEU-NRMF) Complete Blood Count and Other Routine Procedures pt 1 Sir Rupert Vicencio Topic Outline 1. Complete Blood Count 2. RBC Count 3. WBC Count 4. Differential Count 5. Platelet Count Scatter plot with Histogram 6. Hemoglobin In a normal setting, you use an automated hematology analyzer and you may encounter what 7. Hematocrit we call a scatter plot, together with a histogram (we are using principle of electronic impedance 8. Reticulocyte Count and flow cytometry) 9. Erythrocyte Sedimentation Rate How? Cells can create an electric impulse whenever Topic Outcomes they are suspended in an isotonic solution just ❖ Discuss the significance of Complete Blood like in an isotonic saline. That electrical impulse Count will create a specific resistance or electric current ❖ Enumerate the different parameters in CBC that will determine the amount or number of cells and state its function present in a solution, especially for your blood cell ❖ Explain the different principles and count and platelet. concepts of each parameter and to be For WBC, it depends on the principle of the aware of the clinical conditions that results machine but sometimes, the principle of flow in variation of results from reference range cytometry is used is utilized. ❖ Explain the different methods to determine Flow cytometry - a technology that uses a laser hemoglobin and hematocrit concentration to check for the scattering of light, it depends on ❖ Determine the factors affecting the results the lobularity of a cell. in the macromethod and micromethod of - Looking at the left side of the image then hematocrit determination on the graphical image, you can see that ❖ Discuss the significance of reticulocyte they are color-coded count and erythrocyte sedimentation rate - Say that the concentration is 64.2% for and etc neutrophils, the scatter plot or the Dot plot will represent the total population of that 1. COMPLETE BLOOD COUNT specific cell. Is the most common test performed in the Red cell parameter - consists of RBC, hematology section hemoglobin and hematocrit level, and RBC indices Used to assess the patient conditions such as In the RBC indices = concentration of mean cell 1. Infections - bacterial, viral, parasitic, volume (MCV), mean cell hemoglobin (MCH), protozoal, etc. anything that can affect the mean cell hemoglobin concentration (MCHC), red blood count cell distribution width (RDW - this reflects the 2. Malignancy - abnormality like Leukemia average varying sizes pf RBC, if there is an anisocytosis or not, this tells if there is an 1 Aly.D increased number of microcytic or macrocytic cells) 0.1 mm depth If there’s nothing sa FLAG, it means that it is normal. an ‘H’ or an ‘L’ symbol in the flag means a high or low concentration of cells on each parameters “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 2. RBC COUNT edition Determination of the red blood cell present in 1ul Hemacytometer of blood - use a thoma pipette Procedure 1. Diluting the blood 2. Charging the Counting Chamber 3. Counting the Cell - with tally c 4. Making the Calculations 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 EDTA is used, preserves the cells very well properly immerse the tip of the pipette to Neubauer chamber avoid bubbles formation wipe with a tissue to clean Anemic - lower the dilution, 1:20 na lang (pwede 1:100 or 1:10) Polycythemic - RBC thoma pipette and WBC thoma pipette = look at the color of the dot, red is for RBC while white dot is for WBC Neubauer chamber under the microscope 2 Aly.D  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 DO NOT USE MOUTH Methyl violet 0.25 grams Distilled water 180 mL RBC Diluting Fluid 1. Dacie’s fluid - also called formal citrate 5. Bethell’s fluid - this is considered as the BEST diluent - Composition: - It keeps for a long time and does not Sodium sulfate 5.0 grams alter the shape of the cells Sodium chloride 1.0 gram - Formalin acts as a preservative Glycerin 20.0 mL - The cell shape is not altered Sodium 2.0 mL - Composition: merthriolate 40% 10 mL (1:1000) formaldehyde Distilled water 200 mL 3% w/v 990 mL disodium citrate 6. Normal saline solution (NSS or physiologic salt solution) - This is used in EMERGENCY CASE, in the 2. Hayem’s diluting fluid presence of ROULEAUX FORMATION and - This fluid is not recommended because it AUTOAGGLUTINATION of cells allows the growth of yeasts and produces - It is stable and serves as a preservative clumping of cells - Composition: - It can stand for long periods of time and Sodium chloride 0.85 gram has no corrosive effects Distilled water 100.0 mL - Composition: Mercuric chloride C.P 1.0 gram Sodium sulfate 4.4 grams 7. 3.8% sodium citrate anhydrous or - Composition: 10 grams crystalline Sodium chloride 3.8 grams Na2 SO4 ALWAYS REMEMBER THAT THE DILUTION H2O FLUIDS MUST BE ISOTONIC - TO PREVENT Sodium chloride C.P. 2.0 grams SHRINKAGE AND BURST OF CELLS Distilled water 100 mL Charging the Counter Chamber Representative sample of the diluted 3. Gower’s solution mixture is transferred to a counting - This solution prevents ROULEAUX chamber FORMATION Pipette should be mixed by shaking the - Precipitates PROTEIN in cases of pipette in any manner except along the hemoglobinemia and hyperglobulinemia longitudinal axis - Composition: FIRST FEW DROPS (3-4) coming from the Sodium sulfate 12.5 grams capillary system DISCARDED - because anhydrous C.P. they do not contain cells Glacial acetic acid 33.3 mL Distilled water 200.0 mL 3 Aly.D  Reference Value 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 may vary, depending on the hospital (there’s no standard normal value) Variations Physiologic vs. Pathologic 1. Increased count in 1. There is case of INCREASED DEHYDRATION erythrocyte count while the opposite in: upper left and right, middle, lower left and right is true in case of ❖ Polycythemia or hydration polycythemia 2. Increased count in vera EXERCISE or ❖ Pulmonary excitement tuberculosis or 3. NEWBORN pulmonary children have fibrosis higher counts than ❖ Acute adults poisoning 4. Women have 2. There is LOWER COUNTS DECREASED erythrocyte count than male because look for the double border, this indicates na nasa in: a. of the hormones ❖ In anemia center ka na level (men have ❖ After Inverted ‘L’ = cells located at the right and down line high testosterone hemorrhage or are not counted, cells located at the third line of the left and upper line are not counted (to prevent double levels which severe blood counting of cells) stimulate the loss Manual counting is no longer performed except for erythropoiesis WBC directly) Calculations 5. Individuals living at # of RBC counted in X area c.f. X depth c.f. X HIGH ALTITUDES dilution c.f. in 1/5 sq.mm have higher counts OR than those living at sea level - to compensate to the level of oxygen that is available, their SHORTCUT bone marrow has a total # RBC counted x 10,000 *The shortcut method is only applicable for 1:200 tendency to dilution* produce more red blood cells 4 Aly.D  3. WBC COUNT pipette and the WBC diluting fluid to Determination of the white blood cells present in mark 11. 1 uL(microliter) of blood ❖ 1:20 dilution ❖ dilution range that can be prepared Thoma Pipette Method using WBC Thoma pipette is 1:10- This pipette is similar to the red cell pipette 1:100 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 count: - should be HYPOTONIC - to eliminate RBC - should color/stain the nuclei of white blood cells - for easy identification and characterization 2. Charging the Counting Chamber The procedure in erythrocyte count is also adopted 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 Diluent the cells settle for 1 to 2 minutes, so that Example of Diluent all the cells are in the same plane. 1. 2-3% acetic acid ❖ Using the low power objective, locate and 2. 1% HCL added with 1 drop of methyl violet scan the ruled area. If there is a good or crystal violet distribution of cells, start counting. WBC is resistant to that acid, therefore it ❖ Count the cells within the 4 corner squares will remain after the dilution (4sq. mm.) with 16 medium squares each Procedure ❖ Get the average kapag nag-count both on 1. Diluting the blood the lower and upper chamber, wag na kung ❖ dilution of the blood for white cell sa isang chamber lang. However, you need count facilitate the counting process to count both chambers by hemolyzing the matured red blood cells but not the nucleated (young) red cells. ❖ draw blood to mark 0.5 of the WBC 5 Aly.D  >30.0 1:100 RBC >100 1:200 RBC Same thoma pipette ang gagamitin, ang ia-adjust the amount of blood to put for dilution REMEMBER - what you are doing is just verifying the result from the automated Variation of Techniques same concept with the inverted ‘L’ 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 4. Making the calculations 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 Shortcut method: Total of No. of Cells in all 4 squares X 50 Reference Value Note: applicable ONLY when the dilution used is 1:20 Normal Values Conventional SI Unit Area used in white cell count = 4 sq. Unit mm. Area correction factor = ¼ Adults 4,500 to 4.5 – 11 X Depth of counting chamber = 0.1 or 11,000/ 10^9/L 1/10 mm cu.mm Depth correction factor = 10 Ratio of Dilution = 1:20 or 1/20 Infant 10,000 to 25, 10 -25 X Dilution correction factor = 20 000/ cu.mm 10^9/L Reminder 1 year 8,000 to 8-15 X a. Overcharging may decrease cell 15,000/ 10^9/L count - ulitin cu.mm b. Allow the counting chamber to stand for at least 3 minutes after charging Variation on WBC count c. Allowable difference between 2 1. There is an hourly rhythm of a number of chamber counts: white blood cells. - For RBCs, 15 to 16 counts a. LOW LEVEL is observed in the morning - For WBCs, 10 to 12 counts and goes HIGHER in the afternoon - Subtract the upper and lower 2. Food intake, and moderate physical or chamber to get the difference emotional activity will cause a INCREASE in the number of leukocytes (infants na Calculation of Dilution umiiyak sa hospital has a posibility na Recommended Dilutions for Manual WBC counts falsely increased ang WBC count kapag (based on anticipated WBC count) chineck). Anticipated Recommended Thoma 3. Highest peak is observed after meals WBC count Dilution Pipette Used How does this happen? (X10^9/L) Marginating pool - temporarily adhered to endothelial cells by selectins, WBC present in the 0.1 to 3.0 1:10 WBC marginating pool ay nagccirculate sa circulating pool which naeextract naman 3.1 to 30 1:20 WBC 6 Aly.D  Presence of nRBC? - Microscope Correction of WBC count is done if there are 5 or - Schilling counter more NRBC/per 100 white blood cells when Methanol used as a primary fixing agent examined in the blood smear (done after the Then, 10-15 dips on eosine (primary stain), then manual counting in the counting chamber, for secondary stain Lastly, is to wash the excess with distilled water counter-checking). 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 Stain used in Differential Count Generally, Romanowsky stain is used in staining Formula for correction of WBC count smear for differential leukocyte count. The following are the stains: ❖ Wright’s – routinely used ❖ Giemsa – used in searching for malarial 4. WBC differential count parasites 1. White blood cells are characterized and ❖ Leishman’s differentiated according to cell type to ❖ Other Romanowsky stain determine the origin of infection ❖ Rack method - flood the smear with the Steps in Leukocyte Differential Count stain on the rack, soak for 3 mins, then 1. Preparation of blood smear wash with water 2. Staining of blood smear 3. Differentiation of leukocytes 4. Reporting of results 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 4. Procedure - Venous puncture (preferred) ❖ Check slide identification. 3. Reagents and equipment ❖ Perform patient specimen - Slides orientation. - Hematological stains ❖ Perform low-power scan to review 7 Aly.D  blood film adequacy. ❖ Perform differential leukocyte count. ❖ Classify 100 leukocytes using the “battlement” track method Band Neutrophil ❖ Report results of the 100 cells classified as percentage. ❖ Keep separate count of nucleated RBCs (NRBCs). ❖ Note and report the ABNORMAL C shape or S shape (sausage-like appearance), no leukocyte morphology. segmentation or no constriction ❖ Identify and report any Immature neutrophil miscellaneous non-leukocyte abnormal cells, such as endothelial cells, basket cells or NRBCs. Acceptable lang ang 3-4 overlapping A. is a metamyelocyte. Observe the nucleus Criteria for Leukocyte Identification that there is slightly an indentation to Cell Size which is less than half from the farthest Nuclear – cytoplasmic ratio origin of the center of the cell, less than a. High ratio – nucleus occupies most cell half of the origin line areas with only a small rim Lymphocytes b. Low ratio – nucleus is small in relation to volume of cytoplasm. Cytoplasmic characteristics a. Color of background cytoplasm b. Presence or absence of granules c. Color and size of granules Fine chromatin structure and uniform nucleus and Nuclear characteristics sometimes the appearance of the cytoplasm a. Shape appeared as sky blue cytoplasm b. Color Chromatin patter Homogenous in structure, no indentation nor c. Presence or absence of nucleus segmentation BUT with granules (indication of Maturation - absence of nucleus indicates mature lymphocyte) undergoin maturation Monocyte Segmented Neutrophils Crumpled paper-like appearance, wrinkles Eosinophils - acidophils NORMAL mature segmented neutrophil should have atleast 3-5 lobules, outside the bracket means there’s an abnormality (hyper/hyposegmented neutrophil) REMEMBER - there should be thread-like Bright-ish orange granules connecting one lobule to the other (this also indicates that the neutrophil is a mature cell) 8 Aly.D Basophils Absolute WBC count - normal value Highly intense granules Appear blue or black SOMETIMES (basophils degranulates in case of allergic reaction) Reference Ranges WBC Differential Count Irregularities in Blood Smear Preparation Affecting Leukocytes although majority of them WBC Type Normal Reference are due to wrong smear Value (%) 1. Squashed, distorted lymphocytes 2. Accumulated white cells Neutrophil 47-79 3. Smudge cells 4. Disintegrated or ruptured cells Lymphocyte 14-47 5. Poorly stained leukocytes – may be caused by: Monocyte 2-11 ❖ Incorrect pH of buffer ❖ Improper mixing of stain and buffer Eosinophil 0-7 ❖ Too short staining period 6. Precipitated stain – may be caused by: Basophil 0-2 ❖ Unclean slides (dust, etc.) ❖ Drying during the staining period Stab or Band cell 0-7 ❖ Inadequate washing of cells after “Never Let Monket Eat Banana” staining period ❖ Failure to hold slide horizontally Diff count report during initial washing ❖ Inadequate filtration of stain Additional Notes WBC type Result A 200-cell differential should be performed when the leukocyte numbers in excess of Neutrophil 56 x 0.01 0.56 35.0 X 10^9/L. If the WBC ct. is less than 1 platelet/ OIO field – decrease in 600,000-800,000 uL Moderately increase the number of platelets - 5 – 20 platelets/OIO field (with Above 800,000 uL Marked increase occasional clumping) – adequate supply of platelets 5. Platelet Count - >25 platelets/OIO field – increase in - is the number of platelets in 1 L or 1 mL of the number of platelets whole blood - platelets are counted in the 25 small Indirect - Dameshek’s method squares in the large center square (1 mm^2) of the hemacytometer using a Brilliant cresyl blue, sodium citrate, sucrose, and phase contrast microscope in the reference formalin are used in the same technique as Fonio’s method described by Brecher and Cronk 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 Methods in Platelet Counting 4. Count the RBCs and platelets under OIO 1. Manual Method until 250 RBCs have been counted. - Indirect Formula for Calculation Fonio’s Olef’s Dameshek 2. Direct - Guy and Leake - common Direct - indirect uses a slide, direct is counted in - Rees and Ecker - common the counting chamber 3. Electronic Method The platelets are counted in the hemacytometer as - Voltage-Pulse Counting in erythrocyte or leukocyte count - Electro-optical Counting Guys and Leake The diluent is made up of sodium oxalate, Indirect - Fonio’s method 40% formalin, and crystal violet. Procedure: Calculation: 1. Place a large drop of diluting fluid on the Platelet/uL = Platelet counted X 5 X 10 X site of the puncture. Then press blood form 100 the puncture site Rees and Ecker - what is used in school 2. Mix one-part blood and five parts Diluent is made up of sodium oxalate, magnesium sulfate brilliant cresyl blue, formalin, and distilled 3. Make a smear using the mixture water 4. Allow the smear to dry, then apply staining Procedure: fluid 1. Draw blood up to the 0.5 mark of 5. Examine under OIO and count the RBCs the RBC pipette and platelets until 1,000 RBCs are counted 2. Dilute blood with Rees-Ecker diluting fluid up to the 101 mark This makes 1:200 dilution 10 Aly.D  3. Shake the pipette for 1-5 minutes. peripheral borders of 4. Discard 5-6 drops and charge the neutrophils counting chamber 2. Platelet clumping – due to a 5. Place the counting chamber on a type of agglutinin found in Petri dish with wet filter paper to the patient prevent evaporation. Let it stand for Unopette system 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 mm^2 area NOTE: Platelets are stained light blue. Calculation: Platelet count = Average platelet count x Depth factor x dilution factor x Area correction factor Brecker-Cronkite - Calculation is the same as Guy and Leake method - error in this method is 11 – 15 % only - makes use of the phase contrast Reasons why platelets are hard to count microscope, and the diluent is 1% ❖ They easily disintegrate ammonium oxalate ❖ Because of very small size, they could be - the MOST ACCURATE method mistaken for debris because there is no difficulty in ❖ They easily clump, adhere to glass and distinguishing platelets from debris other contact surface because the diluting fluid swells the ❖ Uneven distribution of blood platelet a little and hemolyze the red Sources of Error in Platelet Counting cells ❖ Error in handling - Formula: ❖ Operator’s error Platelet/uL = Platelet X 5 X 10 X ❖ Error in equipment and reagent 100 ❖ Inherent error or field error Causes of Platelet Clumping ❖ Initiation of platelet aggregation ❖ Clotting before the blood reaches the Electronic method anticoagulant Red cells must first be removed from whole blood, ❖ Imperfect venipuncture either by sedimentation or by controlled ❖ Delay in sampling centrifugation Physiologic Variation a. Voltage-Pulse Counting ❖ Platelet count is slightly lower at birth than - Dilution is 1:3000 (3uL of blood + in older children and adults 9mL of Isotonic or NSS) ❖ Platelet count may fall at the time of - Variation in dilution: for platelet menstruation count of less than 250, 000/uL, the dilution is 1:300 (20 uL of blood/ -End of Discussion- 6mL of diluent) b. Electro-Optical Counting - on light - 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 11 Aly.D