MANUAL-WBC-COUNTING.pdf

HEMATOLOGY 1 | LABORATORY SUMMER: MANUAL WBC COUNTING Granulocytic Series Basophilic Myelocyte Stages:  dark blue-purple specific granules 1. MYELOBLAST 2. PROMYELOCYTE 3....

HEMATOLOGY 1 | LABORATORY SUMMER: MANUAL WBC COUNTING Granulocytic Series Basophilic Myelocyte Stages:  dark blue-purple specific granules 1. MYELOBLAST 2. PROMYELOCYTE 3. MYELOCYE 4. METAMYELOCTE 5. BAND 6. MATURE GRANULOCYTE (BEN) Metamyelocyte Myeloblast  10-15 um  15- 20 um  Indented or Kidney Shaped  N:C ratio 4:1  Chromatin Pattern is coarse and clump  Cytoplasm: Moderate Blue. No granules  Nucleus: Round to Oval. Extremely Fine chromatin.  Reddish Purple. Two to Five Nucleoli  First stage: Found in the Bone Marrow Promyelocyte  Diameter: 15-21 um  Cytoplasm: Presence of primary (azurophilic) granules  Blue to reddish purple  N:C ratio: 3:1 to 2:1 Myelocyte  12-18 um  Secondary granules (Specific granules) begin to appear  Last Stage Capable of Mitosis Neutrophilic Myelocyte  pink specific granules Band  9-15 um Eosinophilic Myelocyte  Sausage shape, narrow, Horse shoe shaped  Dirty Orange to Red granules nucleus  Indention in nucleus is 1/3 the diameter of the cell  Last stage found in the bone marrow  1st stage that is found in the circulation (0-3% in the circulation) JHERLYN E. ANTIAMPO | MLS-2F HEMATOLOGY 1 | LABORATORY SUMMER: MANUAL WBC COUNTING Monocytic Series 1. Monoblast 2. Promonocyte 3. Monocyte Monoblast  Found in the bone marrow. Mature Neutrophil  Diameter: 12-20 um  9-15 um  Cytoplasm: Moderately Basophilic or bluegray  pink to rose violet granules  N:C Ratio is 4:1  Nucleus: 2-5 lobes connected by thin nuclear  Nucleus: Round or ovoid, Light Blue-purple filament  Lacey chromatin, 1-2 nucleoli  increased during Bacterial Infection Promonocyte  14-18 um  Cytoplasm: Ground glass appearance  May contain dustlike azurophilic granules  Nucleus: Oval, may have a single fold or fissure. 1 nucleoli Monocyte  14-20 um  Cytoplasm: bundant, blue-gray. Ground glass Eosinophil apearance due to many fine azurophilic  9-15 um granules.  large reddish orange granules  Irregular, presence of pseudopods  Nucleus: two lobes  Presence of vacuoles  Nucleus: Kidney shaped, brain like convolution.  Chromatin is lacey. No nucleoli present  They are phagocytic cells that will migrate in areas of inflammation. Rise during later stage of bacterial infection and in chronic infections Basophil  10-16 um  dark purple to blue black granules obscuring the nucleus  nucleus: bilobed or rarely 3-4 lobes JHERLYN E. ANTIAMPO | MLS-2F HEMATOLOGY 1 | LABORATORY SUMMER: MANUAL WBC COUNTING LYMPHOCTIC SERIES Granular Megakaryocyte  Lymphoblast  30-90 um  Prolymphocyte  Cytoplasm: Abundant, Pinkish-Blue in color.  Mature Lymphocyte Very fine and diffusely granular. Lymphoblast  Nucleus: Multiple nuclei or may show multi-  10-18 um lobulation,  Cytoplasm: No granules, more abundant than  Chromatin is coarser. No nucleoli are visible. myeloblast, moderate to dark blue.  N:C ratio: 2:1 to 1:1  Nucleus: Round to oval, contain 1-2  distinct nucleoli Mature megakaryocyte  N:C ratio: 4:1  40-120 um  The Largest cell in the bone marrow Prolymphocyte  Cytoplasm: Contains coarse clumps of  Smaller as the lymphoblast granules aggregating into little bundles, which  Cytoplasm: Moderate to dark blue. May bud off from the periphery to become contain occasional azurophilic granules platelets.  Round, ova or slightly indented. Chromatin  Nucleus: Multilobulated. No nucleoli visible. pattern is clumped  N:C Ratio less than 1:1  1-2 nucleoli Platelets (Thrombocytes) Lymphocyte  1-4 um  The WBC that will increase during cases of viral  No nucleus Infection.  Cytoplasm: very granular, Light blue to purple  8-10 um  Two parts  Cytoplasm: Light Blue, scanty to moderate, 1. Chromomere - granular and located with few azurophilic granules centrally.  Nucleus: Purple, dense, Clumped chromatin, 2. Hyalomere - Surrounds the chromomere, Round, eccentric. No nucleoli non-granular and clear to light blue. MEGAKARYOCYTIC SERIES 1. Megakaryoblast 2. Promegakaryocyte 3. Granular megakaryocyte 4. Mature megakaryocyte 5. Platelet Megakaryoblast  20-50 um  Cytoplasm: non-granular, Blue, darker than myeloblast, with small, blunt pseudopods,  Nucleus: Round, oval, multiple Nucleoli  N:C Ratio: 10:1 Promegakaryocyte  20-60 um  Cytoplasm: More abundant, less basophilc, granules begin to form  Nucleus: Multiple nucleoli are visible. Irregular in shape. May show lobulation.  N:C Ratio: 4:1 to 7:1 JHERLYN E. ANTIAMPO | MLS-2F HEMATOLOGY 1 | LABORATORY SUMMER: MANUAL WBC COUNTING WBC COUNT Computation  Refers to the routine procedure that gives an Cell/mm3 = Average cell counted x Dilution Factor approximation of the total number of Area x Depth leukocytes in the circulation Area: 4mm2  Function of the WBC is to provide immunity ; Depth: 0.1 mm the ability to resist infection  In a normal adult, WBC ranges about 4,000-  When WBC is markedly elevated (100-300 x109 /L) 11,000/mm3 the dilution can be increased to 1:200  When WBC is also below 3 x109 /L, the dilution can  A Count above normal can is called Leukocytosis be reduced to 1:10  Physiologic causes: Corrected WBC Count 1. Exercise  Nucleated RBC are resistant to lysis during blood 2. Stress dilution and is counted as WBC in the 3. Obsteric labor hemacytometer. 4. Anesthesia  If more than 5 NRBC are seen in the blood smear  Pathologic causes during differential count, the WBC count must be 1. Infection corrected. 2. Hematologic Disorder (leukemia)  Leukopenia- counts below normal Corrected WBC Count = 1. Viral Infection Uncorrected WBC count X 100 2. Ionizing Radiation (Number of NRBC per 100 wbc) + 100 3. Chemicals 4. Drugs 5. Hematologic problems (aplastic anemia)  Dilution Ratio: 1: 20  Dilution Factor: 20  Blood units: 0.5 Direct Eosinophil Counting  Bulb Units: 10  Eosinophil  Blood is diluted with either:  a granulocyte that is present at sites of allergic  2% acetic Acid reaction and parasitic  1% Hydrochloric Acid  Can be obtained indirectly by determining  Turk’s Diluting fluid (Containing Acetic WBC Count multiplied by the eosinophil Acid-3ml, 1 ml of aqueous gentian violet, differential and 100 ml distilled water)  Normal Range is: 50- 350 x106/L or 0.5-3.5 % Procedure of WBC 1. Perform blood dilution (1:20) using a WBC  Eosinophilia - Increased eosinophils above thoma pipet normal 2. Load hemacytometer (Settle for 3 min) Causes: 3. Using LPO, locate the four large corner squares  Allergic Reactions and start counting WBC excluding those  Parasitic infestation/infection touching the bottom and right borders  Brucellosis 4. All squares should agree with cells not greater  Certain leukemias than 10  Eosinopenia -decreased eosinophils count 5. Count on both Platforms and get average.  Hyperadrenalism (Cushing's Disease)  Shock  ACTH Administration JHERLYN E. ANTIAMPO | MLS-2F HEMATOLOGY 1 | LABORATORY SUMMER: MANUAL WBC COUNTING  Steroids has inhibiting effect to eosinophil production. Direct Eosinophil Counting  WBC Thoma Pipet is being used  Dilution is 1:10 (Fill blood up to 1.0 Mark, fluid up to 11 mark)  Diluting Fluid used:  A. Phyloxine Diluting Fluid: (Commonly Used)  Propylene Glycol  Distilled H2O 3. Speirs-Levy Counting  Phyloxine (1% w/v) Four platforms/ counting area:  Sodium Carbonate  Each platform has 10 Squares used  Pilot's Solution for eosinophil counting that has an  All components of Phyloxine diluting area of 1 mm2 each (10 mm2) fluid but with the addition of 100 units  Depth: 0.2 mm of Heparin.  Area: 10 mm2  Randolf’s Stain Solution 1:  Methylene Blue (0.1% w/v) in Propylene Glycol  Distilled H2O Solution 2:  Phyloxine (0.1% w/v) in Propylene Glycol  Distilled Water Functions of Diluting Fluid Components Computation  Propylene Glycol – will lyse the RBCs Cell/mm3 = Average cell counted x Dilution Factor  Phyloxine - will stain eosinophils red Area x Depth  Sodium Carbonate and water – will help lyse WBC's except Eosinophils  Heparin – (if present) will prevent WBC clumping Procedure  Same as manual WBC count  Load Hemacytometer (settle for 15 min)  Count using Low power, the get the total of each platform, get the average, then compute.  Counting Chambers used: 1. Improved Neubauer (not recommended due to small volume) depth: 0.1 mm2 2. Fuchs-rosenthal counting chamber consist of 2 platforms. Each ruled area has 16 large squares used for eosinphil count with an area of 1 mm2 each (16 mm2 all)  Depth: 0.2 mm  Area: 16 mm2 JHERLYN E. ANTIAMPO | MLS-2F