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WellBalancedRadiance8883

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Chattahoochee Technical College

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hematology manual cell counts blood analysis

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This document provides information on manual cell counts in a hematology laboratory. The document presents content on methods and calculations for leukocytes, erythrocytes, and platelets. It introduces the concepts of blood counts, including quantitative and semi-quantitative methods.

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6/28/2024 CHAPTER 10 Basic Laboratory Assessment of Erythrocytes, Leukocytes and Platelets PREAMBLE  PowerPoints are a general overview and are provided to help students take notes over...

6/28/2024 CHAPTER 10 Basic Laboratory Assessment of Erythrocytes, Leukocytes and Platelets PREAMBLE  PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. o PowerPoints DO NOT cover the details needed for the Unit exam  Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES  Unit Objectives are your study guide (not this PowerPoint)  Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! 1 6/28/2024 THE COMPLETE BLOOD COUNT (CBC) Foundation procedure performed in a hematology laboratory. Consists of quantitative measurements of: Hemoglobin Hematocrit Red and white blood cell counts Platelet count Leukocyte differential (discussed in another unit) Morphologic evaluation of a peripheral blood smear (discussed in another unit) OVERALL QUANTITATIVE MEASUREMENTS Erythrocytes, leukocytes, and platelets are measured quantitatively using automated instrumentation. Although automated, occasionally manual counts may need to be performed due to low counts or quality issues. Manual determinations of hemoglobin and centrifuge-based measurement of the microhematocrit can be a quality control strategy or backup method of analysis. RBC indices are a standard part of the CBC, specifically MCV and MCHC, but more have been included such as: Reticulocyte information Red cell distribution width Blood cell histograms 2 6/28/2024 MANUAL ERYTHROCYTE, LEUKOCYTE, AND PLATELET COUNTS Blood specimens are diluted to an exact ratio with specific diluents, and the cells are counted in a hemacytometer, an accurately ruled chamber in areas of square millimeters. Since RBC counts surpass WBC and PLT counts, they need to be eliminated to count WBC and PLT counts accurately. Most of the lysing agents use the principle of osmotic pressure; placing an aliquot of blood in a hypotonic solution will cause the cell to lyse. HEMOGLOBIN MEASUREMENT IN THE LABORATORY #1 Hemoglobin (Hb) determination can be performed separately or as part of a CBC. It is usually analyzed with instrumentation but can be determined manually as well. Manual determinations can be a quality measure or backup. Manual and automated determination is most commonly performed using the cyanmethemoglobin method. Modified Drabkin’s reagent that contains potassium cyanide is mixed with blood specimen, incubate to induce lysing, and measured using a spectrophotometer at 540 nm. 3 6/28/2024 HEMOGLOBIN MEASUREMENT IN THE LABORATORY #2 Sources of error in the Hb measurement result from problems with specimen integrity. Elevations in Hb measurement are seen in lipemic, icteric, or hemolyzed blood samples. Elevations in Hb measurement are also seen in specimens with high WBC counts or RBCs containing Hb S or C. Strategies for problem solving include the following: Saline (plasma) replacement for icteric, lipemic, or hemolyzed plasma. Specimen can be centrifuged, and clear supernatant be transferred to a cuvette and measured on a spectrophotometer to address high WBC count interference. Specimens with Hb C or S can be diluted 1:2 with distilled water and run with the result multiplied by the dilution factor. HEMATOCRIT (PACKED CELL VOLUME) #1 The hematocrit (Hct) or packed cell volume is a macroscopic observation of volume of the packed RBCs in a sample of whole blood, if measured manually. When whole blood is centrifuged, the heavier particles fall to the bottom of the tube, and the lighter particles settle on top of the heavier cells. The Hct is the percentage of RBCs in a volume of whole blood. It is expressed as units of percent or as a ratio in the SI system. 4 6/28/2024 HEMATOCRIT (PACKED CELL VOLUME) #2 An automated hematocrit result is obtained when multiparameter instruments are used. The results are computed from individual MCVs and the red cell count and is not affected by the trapped plasma that is left in the RBC column. Sources of error on manual Hcts can include specimen and technical errors. Specimen errors can falsely decrease Hct values due to inadequate filling of EDTA tube, which causes RBC shrinkage. Technical errors include overcentrifugation or improper sealing of the test capillary tube. Patients with clinical RBC disorders such as macrocytic or sickle cell anemia produce falsely elevated Hct levels. RULE OF THREE Used to determine accuracy between Hb, Hct, and RBC counts. The rule states: The microhematocrit should be 3× the value of the hemoglobin (+/− 3%). The hemoglobin should be 3× the value of the RBC count. The microhematocrit should be 9× the value of the RBC count. This rule only applies to normocytic normochromic red cells, and discrepant values may be seen in other conditions. 5 6/28/2024 BLOOD VOLUME MEASUREMENT In most cases, the total number of RBCs is closely related to the red cell concentration, but in situations such as severe hemorrhage, dehydration, or overhydration, plasma volume or red cell mass or volume must be determined. Plasma volume is measured by dilution methods. MANUAL ERYTHROCYTE COUNT Cells are suspended in a fixed volume of isotonic saline with a specified dilution and counted in a hemacytometer grid with a specified area. Dilution for Manual RBC: 1/200 Diluting fluids: Hayem’s (HgCl3 + NaCl + NaSO4 + H2O) Counted on a Neubauer hemocytometer 3 mm x 3 mm x 0.1 mm = 0.9 mm3 To calculate the final cell count: Final RBC count = [average total of RBCs in 5 squares × dilution correction factor × volume correction factor] Sources of error: Increased or erratic results may be seen if contaminated diluting fluid, wet or dirty pipettes, a dirty hematocytometer, or drying of the dilution in the chamber occurs. 6 6/28/2024 HEMOCYTOMETER Counting chamber MANUAL ERYTHROCYTE COUNT Counting Factors Dilution for manual RBC counting is 1:200 Count 5 “R” sections of the 25 sections in the middle portion of the hemocytometer (see grid) 1/25 or 0.04 mm2 0.04 mm2 x 0.1mm (depth) = 0.004 mm3 7 6/28/2024 MANUAL ERYTHROCYTE COUNT Volume is number of squares counted = 5 times the volume of 1 “R” section is: 5 x 0.004 mm3 = 0.02 mm3 Volume used if 5 “R” sections counted Volume Correction factor Volume desired 1.0 mm3 = 50 Volume used 0.02 mm3 Note: If a different # of squares were counted, the volume correction factor would change *For example, if you counted 4 squares the volume correction factor would change to: 4 x 0.004 mm3 = 0.016 mm3 Volume desired 1.0 mm3 = 62.5 Volume used 0.016 mm3 MANUAL ERYTHROCYTE COUNT Calculate final RBC count by the following formula Dilution factor x Volume Correction factor x cells counted 200 x 50 x # of cells counted 200 x 50 = 10,000 is the multiplication factor therefore, the formula if 10,000 x # of cells counted Example: counted 400 cells (200 x 50) x 400 - or – 10,000 x 400 = 4,000,000  reported as 4.00 x 106/mm3 8 6/28/2024 RED BLOOD CELL INDICES #1 Mean corpuscular volume Mean corpuscular hemoglobin Mean corpuscular hemoglobin (MCV): reflects the average (MCH): reflects the weight of content (MCHC): reflects the volume or RBC size in Hb in picograms (pg) percentage of hemoglobin in femtoliters (fL) one RBC in g/dL or % Calculation: MCV = Calculation: MCH = Calculation: MCHC = [Hct × 10]/RBC count = fL [Hb × 10]/RBC count = pg [Hb × 100]/Hct = % Normal values are 80-96 Normal values are 28-33 Normal values are 33- femtoliters. picograms. 36% Terms that are associated Terms associated with with MCV: normocytic, MCHC: normochromic, microcytic, and macrocytic. hypochromic, and hyperchromic (which can only be spherocytosis or a problem with the Hgb or Hct values) RED BLOOD CELL INDICES #2 Sources of error: MCV: Autoagglutination of RBCs as in cold agglutinin disease or paraproteinemia falsely elevates MCV and falsely lowers Hct values. Hyperglycemia causes osmotic swelling of the RBCs, which leads to a falsely elevated MCV. Leukocytosis spuriously elevates MCV. MCH: Hyperlipidemia falsely elevates MCH and increases Hb values MCHC: Hyperlipidemia, autoagglutination, leukocytosis, hereditary spherocytosis, hemolysis, and ictericia all falsely elevate MCHC values. 9 6/28/2024 RED BLOOD CELL INDICES #3 Calculations Range/Cell Range/Cell Range/Cell Morphology Morphology Morphology MCV=Hct x 10 96 fl / RBC Microcytic Normocytic Macrocytic MCH=Hgb x 10 33 pg/ RBC N/A N/A N/A MCHC=Hgb x 100 36% / Hct Hypochromic Normochromic Hyperchromic RED BLOOD CELL INDICES #5 ASSOCIATED WITH MCV ASSOCIATED WITH MCHC MACROCYTIC MICROCYTIC SPHEROCYTOSIS HYPOCHROMIC (INCREASED MCHC) (DECREASED MCHC) (INCREASED MCV) ( DECREASED MCV) 10 6/28/2024 RED CELL DISTRIBUTION WIDTH (RDW) Quantitates cellular volume heterogeneity reflecting the range of RBC sizes within a sample. Useful in early classification of anemia as it first starts to become abnormal earlier in nutritional deficiencies such as iron deficiency anemia. Useful in characterizing microcytic anemias, allowing to distinguish between iron deficiency anemia and beta thalassemia. RDW can also be helpful in identify RBC fragmentation. RETICULOCYTE COUNT PB smears of normal blood stained with Wright’s stain may demonstrate a slight blue tint in some erythrocytes, referred to as polychromatophilia or polychromasia. Supravital staining is important in reticulocyte detection and counting because the RBCs have to be alive to visualize the RNA present in them. The reticulocyte count is used as an indicator of the rate of erythrocyte production, reflected as a percentage (%). Adult reference range = 0.5% to 1.5% Neonatal reference range = 2.5% to 6.5% 11 6/28/2024 CALCULATING AND EXPRESSING TRADITIONAL RETICULOCYTE VALUES Corrected reticulocyte count (CRC): to account for an anemia already present. Calculation: [reticulocyte count (%)] × [patient’s hematocrit/normal hematocrit by age and gender] Reticulocyte production index (RPI): to determine erythropoietic activity in the marrow when anemia is present. Calculation: [corrected reticulocyte count in %]/maturation time in days (see table) HCT Mat Time (days) 45 1.0 35 1.5 25 2.0 15 2.5 ABSOLUTE RETICULOCYTE COUNT The actual number of reticulocytes in 1 L or 1 µL of blood. Calculation: Absolute reticulocyte count = [%reticulocyte count × total erythrocyte count]/100 If the absolute reticulocyte value is less than 100 x 109/L, the patient has an inappropriately low erythropoietic response to an anemia. 12 6/28/2024 NEW RETICULOCYTE ASSAYS Immature reticulocyte fraction (IRF) Younger reticulocytes have more RNA to them. A dye specific for that RNA can be added to determine the quantity of low-emitting (more mature; LFR), mid-emitting (MFR), and high-emitting (youngest; HFR) reticulocytes. From those values, an IRF can be determined using the following formula: (LFR) − (MFR + HFR) = IRF Reticulocyte hemoglobin: Measurement of hemoglobin content of reticulocytes that reflects the availability of functional iron for the RBC and the incorporation of iron in the synthesis of the hemoglobin molecule over the last several days Indicator of response to iron therapy Reticulocyte hemoglobin content (CHr) Equivalent to the MCH of RBCs ASSESSMENT OF BONE MARROW RESPONSE Normal bone marrow activity = RPI = 1 Increased hemolysis or destruction = RPI = 3 to 7 Bone marrow damage or suppression = RPI < 2 13 6/28/2024 QUANTITATIVE ASSESSMENT OF LEUKOCYTES #1 Total leukocyte count: Calculation: total WBC count = average total WBCs × dilutional correction factor × volume correction factor The number of total leukocytes is the average of the two sides of the hemacytometer. The dilutional factor is 20 based on the dilution of 1:20 of the blood specimen. The volume correction is 2.5. This represents the volume desired (1.0 µL) divided by the volume counted (0.4 µL). QUANTITATIVE ASSESSMENT OF LEUKOCYTES #2 Sources of error include the following: Contaminated diluting fluid, incorrect diluting or loading of the hemacytometer, and an uneven distribution of leukocytes in the counting chamber. Normal reference range = 4.5−11.0 × 109/L Leukocytosis: values above the reference range Leukopenia: values below the reference range Leukocytosis can be seen in acute bacterial infections, inflammation, malaria, hemorrhage, pregnancy, some anemias, and in some carcinomas, including leukemia. 14 6/28/2024 METHODS OF COUNTING WBC’S Automated ACT diff 2 Manual Neubauer hemocytometer 3 mm x 3 mm x 0.1 mm = 0.9 mm3 Fochs-Rosenthal hemocytometer 4 mm x 4 mm x 0.2 mm = 3.2 mm3 Reagents LeukoCount Solution Acetic buffer Gentian violet Pipetting for WBC EDTA whole blood 1:20 dilution MANUAL WBC COUNTS Hemocytometer Read on 10x Read 4 “W” sections For good distribution the variation in the count from both W W sides should be no more than 10 cells Calculations Each “W” section has an area of 1 square mm and a depth of 0.1 mm volume of 1 “W” section: area x depth 1 mm2 x 0.1 mm = 0.1 mm3 Count 4 “W” sections for a total volume counted of W W 4 x 0.1 mm3 = 0.4 mm3 15 6/28/2024 MANUAL WBC COUNTS Calculations Figure the volume correction factor by. Volume Desired always want to report as 1 mm3 Volume used 1.0 mm3 = 2.5 0.4 mm3 Note: If a different # of squares were counted, the correction factor would change! For example, if you counted 5 squares it would change to: 1.0 mm3 = 2.0 0.5 mm3 MANUAL WBC COUNTS To calculate the WBC use the following Correction Factor (CF) = 2.5 Dilution factor (DF) = 20 # of cells counted in the 4 “W” section or 2.5 x 20 x # of cells counted = WBC count 2.5 x 20 = 50 is the multiplication factor (CF x DF) For example, if 90 cells were counted in 4 squares: (2.5 x 20_ x 90 –or- 50 x 90 = 4,500  Reported out as 4.5 x 103 / mm3  Note: If you change the # of squares counted, you will change the MF (see previous slide) Notes All numbers MUST have units or they are counted wrong Count both sides of hemocytometer and average counts Count cells on the left and top lines, but not on the right or bottom lines 16 6/28/2024 MANUEL WBC COUNTS Sources of Error Failure to mix blood specimen properly Dilution error Failure to wipe off pipette Failure to mix Failure to discard first drop of dilution Improper charging of chamber Dirty hemocytometer ABSOLUTE CELL COUNTS The absolute number of segmented neutrophils and bands is considered to be a less specific index of inflammation than other tests because the total leukocyte count drops in many patients with overwhelming infection. This condition results from the movement of circulating granulocytes into the tissue sites of infection. An absolute cell count may be valuable in other cases of inflammation. 17 6/28/2024 ASSESSMENT OF EOSINOPHILS AND BASOPHILS On average, a differential of a peripheral blood smear reveals less than 4% eosinophils. Eosinophilia, or an increase in eosinophils, can be seen in allergies and some parasitic infections. Because this method is semiquantitative, an absolute eosinophil count is preferred. The basophil is the least numerous of the granulocytes, containing only around 1% in peripheral circulation. Basophilia, or an increase in basophils, is very significant and is seen in malignancies such as CML or PV. NEUTROPHILIC HYPERSEGMENTATION INDEX #1 Mature segmented neutrophils have two to five nuclear lobes (segments). Counting the number of lobes can be performed to determine the neutrophilic hypersegmentation index (NHI). A right shift or increase in the number of lobes to five or more occurs in various conditions, for example, sepsis, chronic nephritis. The NHI is clinically useful in vitamin B12 deficiency (pernicious anemia) and folic acid diagnosis. Three methods exist for calculating the (NHI): Lobe average. This is determined by counting the number of lobes in a number of neutrophils, for example, 200, and dividing by the total number of neutrophils for the average number of lobes. The reference value is 2.5 to 3.3. Percentage of neutrophils with five or more lobes. Count the number of lobes in randomly selected segmented neutrophils, for example, 200. Add up the total number of lobes for each segmented neutrophil counted and divide by the total number of cells counted. The reference range is greater than 3%. Hypersegmentation index. To calculate this index, use a minimum of 200 segmented cells. Number of neutrophils with 5 or more lobes × 100 Number of neutrophils with 4 lobes Values greater than 16.9 are considered to indicate hypersegmentation. This method is considered to be the most sensitive method. 18 6/28/2024 NEUTROPHILIC FUNCTION A number of diseases are associated with leukocyte dysfunctions related to locomotion, chemotaxis, adhesion, or the ability of cells to destroy infectious organisms. A defect in cell adhesiveness, for example, leukocyte adhesion defect (LAD), can lead to decreased cell locomotion. A test that assesses the killing ability of granulocytes is the nitroblue tetrazolium (NBT) test. In the routine clinical laboratory, this procedure is infrequently performed. ERYTHROCYTE SEDIMENTATION RATE (ESR) The ESR continues to be an established test for inflammation with the Westergren method selected as the gold standard. The test is a nonspecific indicator of disease. Historical context: Ancient Greek medicine was based on the sedimentation of blood. Nasse recognized that increased proteins caused increased sinking speed of erythrocytes. Reference value for this tests varies with age and sex Less than 50 years of age: 10 mm/h in males and 13 mm/h in females Greater than 50 years of age: 13 mm/h in males and 20 mm/h in females The premise behind the test is rouleaux formation. Erythrocytes with abnormal or irregular shapes hinder rouleaux formation and lower the ESR. If rouleaux is naturally observed in the patients because of increased paraproteins, it will raise the ESR. 19 6/28/2024 QUANTITATIVE ASSESSMENT OF PLATELETS Platelets or thrombocytes are produced in the bone marrow by cells called megakaryocytes, which are large and multinucleated. Platelets do not have a nucleus are not actually cells; they are portions of cytoplasm pinched off the megakaryocytes and released into the bloodstream. Mature platelets are small bodies, ranging from 1.5 to 4 µm in diameter. Most platelet counts are performed using automated instruments, but manual counts can be performed when there is interference present. PLATELET COUNT The traditional manual platelet count using a premeasured reservoir mixes, and the whole blood is diluted 1:100 with 1% oxalate buffer solution. The diluent, ammonium oxalate lyses the RBCs and disaggregates the platelets. Calculation: Platelets/µL = total average number of platelets in 5 squares x dilution correction factor × volume correction factor. Sources of error: age of the specimen, clumping of platelets, debris in the diluting fluid, platelet adherence to glass, incorrect dilution of the specimen, and uneven distribution of platelets. 20 6/28/2024 PERIPHERAL BLOOD FILM EVALUATION Microscopic examination of PB smear: Low power (10×): Evaluate blood smear quality, color, and distribution of cells. Inspection for rouleaux or agglutination can be observed. Large immature or abnormal cells or disintegrating lymphocytes, and smudge cells can be seen. Platelet clumping or satellitism may also be seen. Microscopic examination of PB smear: High dry power (40×) WBC estimates can be assessed. Oil immersion lens (100×): Leukocyte differential can be performed here. RBC morphology can also be graded and evaluated here. SEMIQUANTITATIVE GRADING OF ERYTHROCYTE MORPHOLOGY Variations in RBC size = anisocytosis Variations in RBC shape = poikilocytosis Changes are commonly reported using either a numerical scale or descriptive terms, such as few, moderate, or marked. 21 6/28/2024 SEMIQUANTITATIVE ASSESSMENT OF LEUKOCYTES SEMIQUANTITATIVE ASSESSMENT OF PLATELETS Calculation = Platelet estimate = [Average number of platelets/field × total RBC count]/200 RBCs/field Normally 8 to 20 platelets are present in an oil immersion field for a normal estimate. 22 6/28/2024 LEUKOCYTE DIFFERENTIAL COUNT Begin with a correctly prepared and stained smear. Focus on 10x objective, scan the smear to check for cell distribution, clumping, and abnormal cells. Add a drop of immersion oil and switch to 100×. Find a suitable area and begin counting the manual leukocyte differential. A total of at least 100 leukocytes should be counted. Express the results as a percentage of total leukocytes counted. Abnormalities of leukocytes, erythrocytes, and platelets should be noted. CORRECTION FOR NRBC PRESENCE 23 6/28/2024 SHIFT TO THE LEFT When the percentage of band forms and other immature neutrophils like metamyelocytes and myelocytes increases, the condition is sometimes referred to as a left shift. Could be an indicator of infection or malignancy. Some authorities advocate doing away with the identification of band forms on the differential because of individual variability in cell identification and limited usefulness. POSTAMBLE  READ the TEXTBOOK for the details to answer the UNIT OBJECTIVES.  USE THE UNIT OBJECTIVES AS A STUDY GUIDE  All test questions come from detailed material found in the TEXTBOOK (Not this PowerPoint) and relate back to the Unit Objectives 24

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