Blood Cell Counting PDF - CP - M1

CLINICAL PATHOLOGY BLOOD CELL COUNTING Dr. Johari Ancheta | September 12, 2024 Blow out into the OUTLINE...

CLINICAL PATHOLOGY BLOOD CELL COUNTING Dr. Johari Ancheta | September 12, 2024 Blow out into the OUTLINE hemometer tube and rinse I. HEMOGLOBIN A. Sahli-Hellige Hemometer Method at least 2x B. Cyanmethemoglobin Method II. HEMATOCRIT A. Macromethod: Wintrobe Method B. Micromethod III.MANUAL CELL COUNTS A. Red Blood Cells B. White Blood Cells C. Platelets Add 0.1N HCl until color of D. Reticulocyte the sample in the tube E. WBC Differential Count compares with the comparator blocks IV. RED BLOOD CELL INDICES V. RED CELL DISTRIBUTION WIDTH VI. AUTOMATION Read the fluid level of the I. HEMOGLOBIN sample Main component of RBC Take the reading of the Conjugated protein that serves as the vehicle for the lower meniscus from the transportation of O2 and CO2 graduated tube → when fully saturated, each gram of Hb holds 1.34mL of 02 → red cell mass of the adult contains approximately 600 g The intensity of color is of Hb, capable of carrying 800 mL of O2 proportional to hemoglobin Consists of two pairs of polypeptide chains “globins” and content in blood four prosthetic heme groups, each containing one atom of ferrous iron. Hb based on estimation of the color of the skin and of visible mucous membranes is highly unreliable. CYANMETHEMOGLOBIN Whole blood Hemoglobin content is determined by (HEMIGLOBINCYANIDE) METHOD photometric methods All forms of Hb except sulfhemoglobin are converted to → recorded as g/dL or g/L hemiglobincyanide/cyanmethemoglobin 2 manual methods for Hb count: Reagent: Drabkin’s reagent → Sahli-Hellige Hemometer method Hgb + Drabkin’s reagent = CYANMETHEMOGLOBIN → Cyanmethemoglobin (Hemiglobincyanide) Method Potassium cyanide is a poisonous substance → Drabkin’s solution must never be pipette by mouth SAHLI-HELLIGE HEMOMETER Principle METHOD → Blood is diluted in a solution of potassium ferricyanide aka Acid Hematin Method and potassium cyanide. The potassium ferricyanide Principle oxidizes Hb to Hi (methemoglobin), and potassium → When the blood is added to dilute hydrochloric acid cyanide provides cyanide ions (CN–) to form HiCN, (HCl), hemoglobin present in the RBCs is converted which has a broad absorption maximum at a into brown-colored acid hematin. wavelength of 540 nm. The absorbance of the solution → The acid hematin solution is further diluted until its is measured in a spectrophotometer at 540 nm and is color matches exactly with the permanent standard compared with that of a standard HiCN solution brown glass compared by direct vision. Procedure → Collect blood with Sahli pipette up to 20 μL → Dilute the blood w/ 5mL Drabkin’s reagent PROCEDURE ▪ Potassium ferricyanide: oxidizes Hb to Hi Blood is collected with ▪ Potassium cyanide: provides CN- Sahli pipette up to the 20 ▪ HiCN formation μL mark → Absorbance measured at 540 nm Drabkin’s reagent composition Transcribed by: Madera (TC); Barrios, Caspe, Delmar, Manibpel, Mungcal, Pomperada, Usman (TW) NMD 2027  Blood Cell Counting Hct. The red cells must be Male: 137-180g/dL packed so that additional centrifugation does not REFERENCE Female: 120-160g/dL further reduce the packed VALUES: cell volume Child: 106-145g/dL SAMPLE - Posture, muscular activity, SOURCES OF INHERENT IN THE SAMPLE and prolonged ERROR: - improper venipuncture tourniquet-stasis can technique may introduce cause changes in Hct hemoconcentration, which - Unique to the Hct is error will make Hb due to excess EDTA concentration and cell (inadequate blood for a counts too high fixed amount of EDTA): - Improper technique in The Hct will be falsely low fingerstick or capillary as a result of cell sampling can produce shrinkage, but the Hb and errors in either direction cell counts will not be INHERENT IN THE METHOD affected - Use of the HiCN standard OTHERS: TECHNICAL for calibration of the ERRORS instrument and for the test - failure to mix the blood itself eliminates a major adequately before source of error sampling INHERENT IN THE - improper reading EQUIPMENT OPERATOR’S ERROR III. MANUAL CELL COUNTS Hemocytometer → A thick glass microscope slide with a rectangular II. HEMATOCRIT indentation that creates a chamber → This chamber is engraved with a laser-etched grid of Ratio of the volume of erythrocytes to that of the whole perpendicular lines blood. → Used to count the number of cells or particles in a Expressed as percentage (conventional) or as a specific volume of fluid decimal fraction (SI units). Measures the concentration of the red cells, not the total red cell mass. Dried heparin & EDTA are satisfactory anticoagulants. Hct may be measured directly → macromethods and micromethods Hct may be measured indirectly → MCV x RBC count Cell count formula: MACROMETHOD: → Number of red cells in 1mm3 whole blood = 50 (volume factor) x 200 (dilution factor) x number of cells counted WINTROBE METHOD Wintrobe tube (10mL) CELL COUNT centrifuge at 3,500 rpm for 30 mins RBC Count Count the five (5) small squares indicated by “R” MICROMETHOD (fig below). Each of those squares contain 16 smaller Capillary tube (7cm long; 1mm bore) squares. Use HPO (400x) Sealing clay WBC Count Count the four (4) large Hematocrit centrifuge corner squares indicated by PROCEDURE: “W” (fig below). Each of 1. Obtain blood sample & allow the tube to fill 3/4 full those squares contain 16 2. Seal one end of the tube w/ clay furnished w/ the smaller squares the same tubes as one of the red cell 3. Centrifuge for 5 mins at 10,000rpm in a squares. Use LPO (100x) microhematocrit centrifuge 4. After centrifugation, determine the hematocrit by Platelet Count the five (5) small measuring both the total height of blood and squares (yellow) on the plasma and the height of the blood cell column central large square. If the count is less than 100 then Male: 0.41 - 0.51 count more squares until it is reached. Use high power REFERENCE Female: 0.36 - 0.45 magnification ( 400X) VALUES: Note: Understand this part by looking at the next figure Child: 0.31 - 0.44 CENTRIFUGATION SOURCE OF - Adequate duration and speed of centrifugation ERROR are essential for a correct 2 of 9  Blood Cell Counting 2. Immerse the pipette tip in the red cell diluting and draw the diluting fluid exactly to the 1.01 (101 on some) mark 3. Remove and pipette from the holder and mix the contents of the pipette vigorously with a figure-of-eight motion 4. Allow cells to settle for 1-2 mins Additional Information from the book: Hemocytometer is no longer used for routine blood cell counting except for some platelet counts and low leukocyte counts General procedure for cell counts: → Dilution of blood → Sampling of diluted suspension into a measured volume → Counting of cells WHITE BLOOD CELLS EDTA should be used; heparin is unsatisfactory as an Conventional SI units anticoagulant Hemocytometer method is performed to: → Check validity of results and for calibration Erythrocytes 5.00 × 10^6/uL 5.00 × 10^12/L ▪ Leukopenia, thrombocytopenia (5.00 × Check for platelet counting interference Backup method 106/mm3) Equipment: → WBC Pipette Leukocytes (7.0 7.0 × 10^3/uL 7.0 × 10^9/L → Counting Chamber × 103/mm3) → WBC diluting fluid: (lyse RBCs) ▪ 10 mg crystal violet Platelets (300 × 300 ×10^3/uL 300 × 10^9/L ▪ 1.0 ml glacial acetic acid 103/mm3) ▪ q.s. to 100 mL with distilled H2O Unopette method → commercially available diluent system; convenient for microsampling; available with diluents for RBC, WBC, eosinophil, reticulocyte platelet counts Semi-automated methods → specimen diluted 1:250 or 1:500 1 RED BLOOD CELLS Equipment: → RBC Pipette → Counting Chamber Nucleated red blood cells → Reagents: → Cannot be distinguished at magnification used ▪ Hayem’s Solution − An isotonic fluid used for → Should be counted diluting blood samples in red blood cell counts − → Correction formula: Contains mercuric chloride, sodium sulfate and ▪ True WBC count = (Total count x 100)/(100 + sodium chloride #NRBC) ▪ Gower’s Solution − An isotonic solution for diluting ▪ Where # NRBC is number of nucleated RBCs red cell counts − Containing sodium sulfate and counted in the differential count glacial acetic acid WBC reference values Male 4-11 x 10^9/L Red blood cell count procedure Female 4-11 x 10^9/L 1. Draw blood up in Child 6-16 x 10^9/L the tube to the 0.5 mark on the Additional Information from the book: pipette Leukopenia: with a total count below 2500, the blood is diluted 1:10 3 of 9  Blood Cell Counting Leukocytosis: the dilution may be 1:100 or even 1:200. → Stable within 1-3 hrs; increases further with time d/t Sources of error change from discoid to spherical shape → Nature of the sample Platelet volume should be within 1-3 hrs after collection ▪ Decreased: partial coagulation changes distribution MPV: increased in hyperthyroidism, myeloproliferative in cells diseases ▪ Failure to mix blood Platelet count: use EDTA; platelets evenly distributed → Inaccurate equipment Phase-contrast microscopy: once considered the gold → Field errors standard but replaced by the reference method: → Nucleated nRBCs RBC/platelet ratio (use flow cytometry) Physiologic variation in leukocytes: → CD41 and CD61 → Neutrophils predominant cell after birth; lymphocytes CHECK BLOOD FILM if the platelet count was flagged become predominant after 1 week until 7 years of age by the machine → Diurnal variation: → 7 × 10^9 platelets/L lowest count that should be ▪ Neutrophil: highest levels in afternoon; lowest in reported from manual quantitation morning at rest Platelet in EDTA is satisfactory for 5 hours at 20°C; 24 ▪ Exercise produces leukocytosis hours at 4°C − Lymphocyte drainage into the blood also appears Reticulated platelets to contribute to the total increase → Estimate of thrombopoiesis − Neutrophil:shift cells from marginal to the → 3% - 20% circulating granulocyte pool → Significantly lower median levels of reticulated − Cigarette Smokers Have Higher Average platelets in frequent plateletpheresis donors than in leukocyte counts than nonsmokers. new donors suggest that repeat platelet donation ▪ Menstrual cycle might lead to relative exhaustion of thrombopoiesis − Decrease:neutrophils,monocytes → Increased reticulated platelets: − Increase:eosinophils ▪ ITP, hyperthyroidism ▪ Ovulation: decrease basophils ▪ Neonates 30 weeks younger: 2x the platelet count of full term infants PLATELETS ▪ Bone marrow recovery after chemotherapy for acute Hemocytometer method myeloid leukemia, increase in retic, platelets after → Phase-contrast microscope day 20 Equipment: → Decreased reticulated platelets → WBC pipette ▪ Aplasia, liver cirrhosis → Counting chamber The immature platelet fraction (IPF) is useful in → Diluent: 1% Ammonium Oxalate demonstrating thrombocytopenia due to increased → Unopette platelet destruction The average platelet count is slightly lower at birth than in older children and adults: 84 to 478 × 10^9/L RETICULOCYTE Immature non nucleated red blood cells that contain RNA and continue to synthesize Hb Loses RNA after a day or so in the blood When blood is briefly incubated in a solution of new methylene or brilliant cresyl blue blue, RNA is precipitated as a dye ribonucleoprotein complex. Microscopically, the complex appears as a dark blue network (reticulum or filamentous strand) or at least two dark blue granules Equipment → Test tube → Glass slides → Reagent: 1% new methylene blue in a diluent of citrate-saline ▪ 1 part 30 g/L sodium citrate plus 4 parts 9 g/L Calculation sodium chloride → Platelet count (per μL) = (Number of cells ▪ Miller disk** (to be discussed later) counted/Square s counted) x Dilution x 250 Sources of error Reticulocyte count procedure → Clumping of platelets 1. Three drops each of reagent and blood are mixed in a ▪ Initiation of platelet aggregation and clotting test tube, incubated 15 minutes at room temperature, and ▪ Skin puncture technique remixed − Capillary Gives 2x Error Than Venous 2. Two wedge films are made on glass slides and air dried ▪ Delay in sampling 3. Viewed microscopically with an oil lens, The percentage → Falsely elevated: of immersion reticulocytes is determined in at least 1000 ▪ Leukemias red cells → Falsely low: 4. A Miller disk inserted in the eyepiece allows rapid ▪ Platelet satellitism (platelet adhere to neutrophils) estimation of large numbers of red cells − Caused by EDTA 5. Reticulocytes are counted in the large square and red ▪ Agglutinins cells in the small square in successive microscopic fields ▪ Faulty blood collection until at least 300 red cells are counted This provides an estimate or reticulocytes among Additional Information from the book: at least 2700 red cells EDTA: MPV increases with time up to 1 hr in vitro 4 of 9  Blood Cell Counting Miller Disk → Elevated in persons living at high altitude Inserted into the eyepiece ▪ 1g Hb/dL @ 2km 7mmx7mm ▪ 2g Hb/dL @ 3km Small square (B) is 1/9 of the larger square (A) → A is used to count reticulocytes → B is used to count RBCs WBC DIFFERENTIAL COUNT PROCESS Process of WBC Differential Counting 1. Preparation and staining of blood smear Calculation Note: In preparing the → Reticulocytes (%) smear, a perfect smear ▪ # of reticulocytes in large squares/# red cells in small should have a “feathery squares x 100 edge” → Absolute Reticulocyte Count ▪ Reticulocyte percentage x RBC Count Reticulocyte reference values Percent 0.5 - 1.5% 2.5 - 6.5% (newborns) or 3% to 7% 2. Stain the smear in Absolute count 24-84 x10^9/L Wright’s stain or Additional Information from the book: Romanowsky stain Interpretation: provide an estimate of the rate of red cell production Absolute reticulocyte production index is more helpful than the percentage Sources of variation → Large sampling error for manual retic count → If only 1000 RBCs counted, the 95% confidence limits for: ▪ 1% count → 0.4% to 1.6% ▪ 5% count → 3.6% to 6.4% ▪ 10% count → 8.1% to 11.9% Physiologic variation in erythrocytes (read more sa 3. Mount the stained blood book) smear on the microscope → Changes in red cells values greatest during first few stage and examine at 100x weeks of life magnification (OIO) → Average capillary red counts are higher in newborns whose cords have been clamped late ▪ 0.4 × 10^12/L higher 1 hour after ▪ 0.8 × 10^12/L higher 24 hours after birth → Capillary blood: higher RBC and Hb values than venous blood; most likely due to slowing of capillary circulation and loss of fluid ▪ 0.5 × 10^12 RBC/L ▪ 3 g Hb/dL → In the full-term infant, nucleated red cells average about 0.5 × 109/L. → The normoblast count declines to about 200/μL at 24 hours, 25/μL at 48 hours, and less than 5/μL at 72 hours. By 7 days, it is rare to find circulating normoblasts Newborns → First day of life: hgb: 19.0 g/dL; cord blood: 16.8 g/dL → An initial increase in the Hb level of venous blood is seen at the end of 24 hours compared with that of cord blood Note: The indices are similar in males and females, but the Hb is 1 to 2 g/dL higher in males → Effect of androgen in stimulating erythropoietin production and its effect on the marrow Hb, hct, RBC: increase in posture and muscular activity d/t loss of plasma water; increase from recumbency to standing Hb: highest in the morning, falls during the day, and is lowest in the evening 5 of 9  Blood Cell Counting 4. Locate a region in the “feathery” end of the smear Note: You don’t do the wbc counting on the feathery edge (thin part) You basically do it on the body (thick part) FIGURE 7. White blood cells Source: Dr. Ancheta’s ppt Important: Neutrophilia Inc. in the number of neutrophils indicative of bacterial infection Leukocytosis Inc. in the number of leukocytes indicative of viral or a chronic type of infection (e.g. M. tuberculosis or in some instances in Brucellosis) Eosinophils Count the first 100 WBC encountered Inc. in allergic and parasitic (helminthic) infections Classify them by cell type and maturation Severe eosinophilia is usually seen in children having The tally is kept on a multichannel counter/ parasitic infections multichannel counting chamber specifically designed for WBC differential counting Basophilia Inc. in the number of basophils As soon as the number of 100 wbc’s is reached the bell Not normally found in PBS unless you have chronic from the multichannel counter will automatically “ring” → this myelogenous leukemia or lead poisoning will now serve as your differential count The counting can be done through battlement technique or the streak method ; depends on the preference Differential white blood cell counts of normal adult Cells predominating in the peripheral blood are white blood cells. Remember that neutrophils are the predominant cells Absolute values Percentage among white blood cells There are scenarios where one cell type inc. in proportion Neutrophils 2.0-7.0 x 10^9/L 40-75% over the others ; simply once cell type outnumber other cell types Lymphocytes 1.0-3.0 x 10^9/L 20-45% Monocytes 0.2-1.0 x 10^9/L 2-10% Eosinophils 0.02-0.5 x 10^9/L 1-6% Basophils 0.02-0.1 x 10^9/L 0-2% IV. RED BLOOD CELL (RBC) INDICES Wintrobe introduced calculations for determining the size, content, and Hb concentration of red cells Erythrocyte indices have been useful in the morphologic characterization of anemias. They may be calculated from the red cell count, Hb concentration, and Hct. Formulas: → Hct = MCV × RBC → MCH = Hb / RBC → MCHC = (Hb / Hct) × 100 Indicates: → Cell Size ▪ MCV → Cell color FIGURE 6. Multichannel counting chamber ▪ MCH and MCHC Source: Google 6 of 9  Blood Cell Counting ERYTHROCYTE INDICES V. RED CELL DISTRIBUTION WIDTH MCV: MEAN MCH: MEAN MCHC: MEAN (RDW) CELL VOLUME CELL CELL A measure of the variation of red blood cell (RBC) HEMOGLOBIN HEMOGLOBIN width that is reported as part of a standard CONCENTRATI complete blood count ON Complicated so not included in your manual counts; ONLY in results from automated system the average the content The average Normal reference range in human red blood volume of RBC (weight) of Hb of conc. of Hb in a cell: 11-15% the average RBC given volume of Formula: packed RBCs ○ RDW = (Standard deviation of MCV/mean MCV) x 100 Definition of terms: ○ Anisocytosis: Variation of size and RBC ○ Poikilocytosis: Variation in shape which MCV = Hct × MCH = Hb (in can affect RDW 1000/RBC (in g/L) / RBC (in millions per μL) millions / μL) [HENRY’s 23rd edition] Automated Differential Leukocyte Counting Differential leukocyte count is nonspecific, non precise, expressed in expressed in expressed in error prone, usually labor intensive, expensive to femtoliters or picograms g/dL perform, and of limited clinical significance to screening cubic tests micrometers Automated systems have the advantage of rapidly analyzing larger numbers of cells and significantly One femtoliter(fL) One picogram reducing the statistical error 10^-15 L = 1 (pg) = 10^-12 g Automation of the differential count eliminates some cubic micrometer of detractions the following are the requirements: (um^3) 1. The distribution of cells analyzed should be identical to that in the blood 2. All leukocytes usually found in blood diseases 80-96 fL 26-33 pg (Dr. 33-36 g/dL should be accurately identified, or detected and Ancheta) “flagged” in some way 27-33 pg 3. The speed of the process should enable a large (Henry’s 24e) number of cells to be counted to minimize statistical error 4. The instrument should be cost effective (Bentley & MCV INTERPRETATION Lewis, 1977) DISADVANTAGES Macrocytic >96 fL → categories of cells are not completely consonant with those with which we are familiar Normocytic 80-96 fL on Romanowsky’s stained films → an “unclassified” category is difficult to interpret Microcytic 33 pg > 96 g/dL VI. AUTOMATION Normochromic 26-33 pg 33-36 g/dL Flow Cytometry Hypochromic < 26 ppg