Hematology - 07 Laboratory Evaluation of WBC and Special Hematologic Tests

HEMATOLOGY • Formula: • Sample: 15 reticulocytes are counted per 1000 RBC observed. o 15 Reticulocytes o 985 RBC LABORATORY EVALUATION OF WBC AND SPECIAL HEMATOLOGIC TESTS OUTLINE • • Reticulocyte Count o Reticulocyte o Reticulocyte Count o Miller Disc Method o Clinical Significance o Absolute Reticulocyte Count o Corrected Reticulocyte Count • o Reticulocyte Production Index o Automated Reticulocyte Count o Siderocyte Count • Erythrocyte Sedimentation Rate (ESR) o Stages of ESR • o Methods • o Clinical Significance o Factors Affecting ESR Result o Automated ESR • White Blood Cell (WBC) Count o Clinical Significance o Materials o Manual WBC Count • Procedure o Computation for WBC Count o Nucleated RBC (nRBC) Count o Corrected WBC Count WBC Differential Count o Blood Smear Preparation o Microscopic Examination o WBC Differential Count o Methods o Manner of Reporting Osmotic Fragility Test o Procedure o Reading o Osmotic Fragility Curve o Incubated OFT o Clinical Significance Sucrose Hemolysis Test Automation: Automated Hematology Analyzers o Common Basic Components o Electronic Impedance o Radiofrequency o Errors encountered o Optical Scatter Automation: Histograms o RBC Histogram o WBC Histogram o Platelet Histogram RETICULOCYTE COUNT MILLER DISC METHOD • • • • RETICULOCYTE • • • • • • • • The last immature erythrocyte stage Stained darkly with RNA remnants Assess erythropoietic activity of bone marrow “Poor Man’s Bone Marrow” Spend 2-3 days in Bone marrow, and 1 day in peripheral blood before developing into a mature RBC. Specimen: EDTA Blood Reagent: Supravital/Vital Stains o New Methylene Blue o Brilliant Cresyl Blue Reference Range: o Newborn: 1.5-5.8% o Adult: 0.5-1.5% OTHER METHODS DRY METHOD • RETICULOCYTE COUNT • • Principle: Whole Blood is stained with supravital stain. Any non-nucleated erythrocyte that contains two or more particles of bluestained granulofilamentous material is a reticulocyte. Procedure: 1. Mix equal amounts of EDTA whole blood and supravital stain (2-3 drops each). 2. Incubate for 3-10 minutes at room temperature 3. Remix the preparation 4. Prepare a blood smear 5. Count 1000 RBC/Reticulocyte under OIF. Principle: A miller disc is a calibrated ocular disc that is inserted into the eyepiece of the microscope. The disk imposes two squared onto the field view: Large Square/Square A: used for reticulocyte counting o Denotes the entire field Small Square/Square B: used for RBC counting o 1/9 of Square A Procedure: 1. Count reticulocyte in the entire Square A 2. Count at least 112 RBCs in Square B (reticulocytes are counted as RBC and Retics) • Procedure: 1. Mix equal amounts of EDTA whole blood and supravital stain (2-3 drops each). 2. Incubate for 3-10 minutes at room temperature 3. Remix the preparation ▪ Reticulocyte tend to settle at the top (low sp.gv.) ▪ Inability to remix may cause false decrease 4. Prepare a blood smear ▪ Allow slide to dry 5. Count 1000 RBC/Reticulocyte under OIF. ▪ RBC and reticulocyte are counted as one Formula: WET METHOD • • Rapid method of Schilling, Osgood-Wilhelm, and Sabin o Smear is not allowed to dry Procedure: 1. Place a drop of freshly filtered stain on a clean glass slide. 2. Add an equal volume of blood. Mix by stirring. 3. Cover mixture w/ a clean coverslip lined w/ Vaseline. 4. Proceed with counting. (same counting as dry method) CLINICAL SIGNIFICANCE • • Increased Retics (Reticulocytosis): o Hemolytic anemia o Iron deficiency anemia o Thalassemia o Sideroblastic anemia o Myeloproliferative disorder o Acute and chronic blood loss Decreased Retics: o Bone marrow failure o Aplastic anemia o Decreased erythropoietin production AUTOMATED RETICULOCYTE COUNT • • ABSOLUTE RETICULOCYTE COUNT (ARC) • • Actual number of reticulocytes in 1 liter of whole blood Computation: Principle: o Method: Optical scatter or fluorescence (fluorometer) o Blood is treated with fluorescent dyes or nucleic acid stains to stain residual RNA in the reticulocytes. Advantage: o Statistically more valid than manual method (due to more cell counted compared to manual) o Results are both expressed in percentage and absolute count o Evaluates other Reticulocyte Parameters: ▪ Immature Reticulocyte Fraction (IRF) • More than 1 day in the circulation ▪ Reticulocyte Hemoglobin Concentration • Useful for early iron deficiency ▪ Reticulocyte Indices SIDEROCYTE COUNT • • • Reference Range: 25-75 x 109/L Sample: If patient’s reticulocyte count is 2% and the RBC count is 2.20 x 1012/L. What is the ARC? • • • • • Siderocytes o RBC with siderotic granules (Pappenheimer Bodies) ▪ Siderotic granules - iron granules; should not be normally visible inside RBC Reference Range: 0.5-3.0 % Two Methods: Douglas and Dacie Method: o Includes siderocyte and reticulated siderocytes (reticulocyte with siderotic granules) o Reagents: ▪ Prussian Blue (Perl’s Reagent) ▪ Safranin O - counterstain Single Stain Method o Includes Siderocytes only o Only uses one stain (New Methylene Blue, Wright Stain) CORRECTED RETICULOCYTE COUNT (CRC) • • • • o aka Reticulocyte Index or Hematocrit Correction In specimens with low Hematocrit, the percentage of retics may be falsely elevated because whole blood contains fewer RBCs. Correction Factor: 45% Computation: RETICULOCYTE PRODUCTION INDEX (RPI) • • • • aka Shift Correction Provides further refinement of the CRC Measures erythropoietic activity when stress/shift reticulocytes are present. Formula: ERYTHROCYTE SEDIMENTATION RATE (ESR) • • • • Maturation Index: HEMATOCRIT (%) 40 – 45 35 – 39 25 – 34 15 – 24 <15 • MATURATION INDEX (days) 1 1.5 2 2.5 3 • • Aka Sed Rate The distance in millimeter (mm) at which the RBCs fall in 1 hour A non-specific marker of underlying inflammation (regardless of cause of inflammation) Useful diagnostic test for temporal arteritis and polymyalgia rheumatica Directly proportional to the weight of cell aggregate Inversely proportional to the surface area, and plasma viscosity STAGES OF ESR 1. 2. 3. Agglomeration Phase/Lag Phase (10 minutes) o Initial rouleaux formation occurs o Few cells sink under gravity but majority form agglomerates Rapid/Fast Settling Phase (40 minutes) o aka Decantation phase o More rapid and constant RBC sedimentation Final Sedimentation Phase (10 minutes) o aka Final Settling Phase o RBC sedimentation is slow o Remaining RBS settle Wintrobe tube METHODS • • Macromethods (more common) o Wintrobe Landsberg o Westergren o Graphic and Cutler o Linzenmeier Micromethods: o Micro-Landau o Smith o Hellige-Volmer (Crista) o Micro-ESR WINTROBE-LANDSBERG METHOD • • • • • • Aka Wintrobe Method Wintrobe Tube: o Bore: 3mm o Calibration: up to 100mm (10cm) o Length: 115mm Specimen: Whole Blood Anticoagulant: EDTA Reading: Read after 1hr of undisturbed vertical standing on the left side (Red) calibration at room temperature Reference values: o Men: 0-7 mm/hr o Women: 0-17 mm/hr o Children: 1-15 mm/hr CLINICAL SIGNIFICANCE • • • • • • • Most commonly used method for ESR Westergren Tube: o Bore: 2.5mm o Calibration: up to 200mm o Length: 300mm Specimen: EDTA Blood Diluent: 3.8% Sodium citrate or 0.85% Sodium Chloride Dilution: 4 parts blood to 1 part diluent Reading: Read after 1hr of undisturbed vertical standing at room temperature o Only one calibration exclusive for ESR Reference value: o Adult Male: 0-10 mm/hr o Adult Female: 0-15 mm/hr High ESR o Rheumatoid arthritis o Lupus o Anemia o Endocarditis o Kidney disease o Osteomyelitis o Tuberculosis o Syphilis o Thyroid disease Low ESR o Sickle cell anemia o Polycythemia o Blood hyperviscosity o Hypofibrinogenemia o Congestive heart failure FACTORS AFFECTING ESR RESULT • MODIFIED WESTERGREN METHOD • • Westergren Tube • Intrinsic Factor o Plasma Factors ▪ Increased ESR: Increased plasma proteins, cholesterol • All plasma proteins increase ESR except for albumin (decrease ESR) ▪ Decreased ESR: Increased albumin, glucose, lecithin, phospholipids. o Red Cell Factors ▪ Increased ESR: Macrocytes, anemia, hemolysis ▪ Decreased ESR: Leukocytosis, Anisocytosis, Microcytes, Acanthocytosis, Spherocytosis, HbC, Polycythemia, Sickle cells, Thalassemia • Anisocytosis, leukocytosis - greatly affect ESC; marked increase Extrinsic Factor o Factor not present in blood sample o Temperature, etc. SPECIFIC FACTORS AFFECTING ESR Factors Old Specimen (>4hrs) Plasma Proteins Sickle Cell and Spherocytes Plasma viscosity Temperature (Optimal: 2227C) Inclination Effects ↓ ESR All Plasma Proteins: ↑ ESR Albumin: ↓ ESR ↓ ESR More viscous: ↓ ESR Less viscous: ↑ ESR <22C: ↓ ESR >27C: ↑ ESR Tilt (3°) – up to 30% error Factors Effects Bubbles ↓ ESR Anticoagulant concentration Increased AC conc: ↓ ESR Vibration ↑ ESR Pregnancy ↑ ESR Thalassemia ↓ ESR • Lifespan of Specimen o At room temp (18-25C) - within 4 hours o Refrigerated (4C) - within 24 hours provided specimen is preheated 15 mins prior to testing • High ESR when put on top of ref due to vibration & heat from backside AUTOMATED ESR • • • Advantage o Require small volume of blood o Faster determination of ESR The Ves-Matic system o Method: ▪ Optoelectronic Sensor which measures the change in opacity of a column of blood as sedimentation of blood progresses ▪ More settling = more opaque o Turn-around-time: 20 minutes Sedimat 15 o Method: Infrared measurement o Advantages: ▪ Batch testing (1-8 tests per run) ▪ Random Access capability (allow STAT testing) ▪ Turn-around-time: 15 minutes WHITE BLOOD CELL (WBC) COUNT • • • • • aka Leukocyte Count Measures the total WBC component of whole blood (hemacytometry) Used to indicate infection Specimen: EDTA Whole Reference Value: X109/L (SI) /mm3 (conventional) Adult 4.5 - 11.0 4,500 - 11,000 Newborn 5.0 - 21.0 5,000 - 21,000 MATERIALS • • • • • • Hemacytometer Diluting Fluid Coverslip WBC Pipette Suction device Cell counter DILUTING FLUIDS • • • Hypotonic Will hemolyze red blood cells except metarubricytes/orthochromic normoblast Examples: o 2% (v/v) Glacial Acetic Acid o 1-3% Acetic acid with Gentian Violet o 1% (v/v) Hydrochloric acid o Turk’s Solution – enhances leukocyte nuclear definition ▪ Acetic Acid ▪ Gentian Violet ▪ Water MANUAL WBC COUNT PROCEDURE 1. 2. 3. 4. 5. 6. 7. 8. Mix specimen thoroughly (EDTA Whole Blood) Aspirate EDTA whole blood to 0.5 mark of WBC pipet. Wipe excess blood not touching the tip of the pipet Aspirate WBC diluting fluid to 11 mark. (0.5:11 dilution) Shake RBC pipet in figure of 8 motion for 5 minutes Discard few drops (the stem of WBC pipet may contain pure diluting fluid). (0.5:10 or 1:20 dilution) Charge the counting chamber (ensure that it will not spill on the sides of the counting chamber) Stand for 3 minutes Count WBC in 4 secondary squares (four corners of the primary square). CLINICAL SIGNIFICANCE • • Increased WBC Count (Leukocytosis) o Bacterial infection o Appendicitis o Leukemia o Pregnancy o Uremia o Ulcers Decreased WBC Count (Leukopenia) o Viral infection o Brucellosis o Typhoid fever o Rheumatoid arthritis o Cirrhosis • Note: o Count WBC on both counting site of the hemacytometer and get the average. o Allowable difference of WBC count between 2 chamber is 10-12 counts COMPUTATION FOR WBC COUNT • General Formula: WBC count = # of cells counted x dilution Depth x Area o Unit: cells/mm3 o SI unit: x 109/L (cells/mm3 x 1000) o o o • Dilution Factor: 20 Depth: 0.1 mm (1/10 mm) Area: ▪ 1° square - 9 mm2 ▪ 2° square - 1 mm2 ▪ 3° squares • Corner 3° squares: 0.0625 mm2 • Central 3° squares: 0.04 mm2 ▪ 4° squares (of central square): 0.01 mm2 Simplified Formula: WHITE BLOOD CELL (WBC) DIFFERENTIAL COUNT BLOOD SMEAR PREPARATION WEDGE TECHNIQUE • • • • • • • o • Note: When dividing fractions, take the reciprocal of each fractions and change the operation to multiplication. Sample Problem 1: A manual leukocyte count was performed on an EDTA-anticoagulated specimen. The specimen was diluted 1:20, and an average of 150 leukocyte were counted in the four secondary squares of both counting sites of the hemacytometer. What is the leukocyte count? o # of cells counted: 150 o Dilution: 1:20 o Area: 1 mm2 x 4 = 4 mm2 o Depth: 0.1 mm2 (1/10 mm2) aka Double slide, spreader slide, or push smear The most convenient and most commonly used PBS preparation method. Should be made within 2-3 hours of drawing specimen o Beyond 3 hours may have RBC artifacts Requires two 3 x 1 inch glass slides (film slide, spreader/pusher slide) Specimen: EDTA blood Fixative: Methanol Procedure: 1. A drop of blood is placed 0.25 inch from one end of the film slide (2-3 mm diameter) 2. The spreader slide is held at 30-45° angle on the film slide (not touching the blood) 3. Draw back the slide until it touches the drop of blood, allow the blood to spread across the width of the slide. 4. Push the slide quickly towards the other end to create a wedge film (even pressure) 5. Allow to air dry 6. Fix the blood film in the slide using methanol NUCLEATED RBC (nRBC) COUNT • • • aka Metarubricyte/ Orthochromic normoblast/ Orthochromatic erythroblast/ acidophilic normoblast/ pyknotic erythroblast Not lysed by WBC diluting fluid hence falsely counted as WBC Reference Value: o Adult: <5 nRBC/100 WBC o Newborn: <10 nRBC/100 WBC CORRECTED WBC COUNT • • • Performed if there are ≥5 nRBC (adult), ≥10 nRBC (newborn) seen in 100 WBCs. Formula: FACTORS AFFECTING PBS THICKNESS/THINNESS Size of Blood Drop Angle of Spreader Slide Pressure exerted Speed Hematocrit Sample Problem: o WBC Count: 15,000/mm3 o 12 nRBCs seen (in 100 WBC) Thick Smear Large drop Thin Smear Small drop Increased angle Decreased angle Heavy pressure Too fast Increased Hct Light pressure Too slow Decreases Hct AUTOMATED SLIDE MAKING AND STAINING • Sysmex SP-10, Beckman Coulter o The slides are dried, loaded into a cassette, and moved to the staining position o o Advantages: ▪ Has built-in rule criteria ▪ Films are produced every 30 seconds ▪ Automatically adjusts size of blood drop based on Hematocrit level Supravital Monochrome Stain ▪ Used to stain specific cellular components ▪ No fixatives are used ▪ Example: • New Methylene Blue o Precipitates RNA in reticulocytes • Brilliant Cresyl Green with Neutral Red FEATURES OF A WELL-MADE PERIPHERAL BLOOD FILM • Macroscopic: 1. The film is 2/3 to 3⁄4 of the length of the slide 2. The film is finger shaped (very slightly rounded feathery edge) 3. The lateral edges are visible 4. The film is smooth (no irregularities, holes, streaks, etc) 5. Presence of “rainbow” appearance in the feathery edge of the film 6. Pink to purple color of smear after staining • Microscopic: o RBC – orange to salmon pink o WBC Nuclei – pink to purple ▪ Neutrophils - pink to tan with violet or lilac granules ▪ Eosinophils – bright orange refractile granules ▪ Basophil – purplish black granules ▪ Lymphocyte – robin’s egg blue cytoplasm, compact nucleus ▪ Monocyte - faint blue gray cytoplasm o Platelets – purple blue to lilac with cytoplasm containing red blue granules STAINING OF PBS • • • • Purpose: to make cells more visible for morphology evaluation Two Methods: Quick Stain Method (2-5 minutes) 1. Immerse slide in fixative 2. Methylene Blue 3. Eosin 4. Wash with water 5. Air dry Two-Step/Rack Method 1. Flood with wright stain (1-3 minutes) 2. Add equal amount of buffer (3 minutes or more) 3. Flush with distilled/deionized water 4. Residual stains are flushed from the back of the slide 5. Air dry o Metallic sheen “green scum” of stain indicated proper mixing of stain and buffer HEMATOLOGIC STAINS • • Romanowsky Stain o Non-vital Polychrome Stain o Examples: Wright, Giemsa, Leishman-Jenner, MayGrunwald ▪ Wright & Giemsa - most common o Contains: ▪ Methylene Blue • Basic dye - stains acidic components • Stains DNA/RNA • blue ▪ Eosin • Acidic dye - stains basic components • Stains hemoglobin and eosinophilic granules • Red-orange Other Stains: o Nonvital Monochrome Stain ▪ Stains specific cellular components ▪ Example: Prussian Blue • Contains: K ferrocyanide, HCl and safranin counterstain ▪ Stains: iron granules (in RBC), histiocytes, and urine epithelial cells UNACCEPTABLE & POOR BLOOD SMEAR A. B. C. D. E. Chipped or rough edge Hesitation in forward motion Spreader slide pushed too quickly Drop of blood too small Drop of blood not allowed to spread across the width of the slide F. Dirt or grease on the slide G. Uneven pressure on the spreader slide H. Drop of blood began to dry POORLY-STAINED SMEAR Problems RBCs - gray WBCs - too dark Eosinophil granules - gray WBCs - too pale or red WBCs - barely visible Causes • Stain or buffer too alkaline • Inadequate rinsing • Prolonged staining • Heparinized blood specimen • Short drying period • Too alkaline washing water • Old smear • Stains or buffer too acidic • Underbuffering • Excessive rinsing AUTOMATED SLIDE STAINER • • Method of staining is similar with manual method but with the aide to automated machine. Advantages: o Batch Staining o 5-10 minutes per batch WBC DIFFERENTIAL COUNT • • Percentage of each type of WBC in the blood: o Neutrophils o Lymphocytes o Monocytes o Eosinophils o Basophils Specific WBC type is counted using peripheral blood smear (100x objective) MICROSCOPIC EXAMINATION • • • 10x Objective Examination (LPO) o Assessment of overall film quality, color, and distribution of cells 40x Objective Examination (HPO) o WBC estimation (use 10 fields) ▪ Average WBC counted x 2000 100x Objective Examination (OIO) o WBC Differential Count (100 cells) o Battlement Method ▪ aka Back-and-forth Serpentine Granulocyte Segmented Neutrophils/ PMN (mature) Band/Stab/ Staff (youngest granulocytic precursor) Eosinophil Basophil OPTIMAL ASSESSMENT AREA • • Macroscopically: o Area between the thick area and the very thin area (feathered edge) Microscopically: o RBCs are uniformly and singly distributed, with few touching or overlapping, and have their normal biconcave appearance o Feathery edge - increased distance between RBC; RBC has no central pallor o Thick part - RBC are overlapping forming false rouleaux formation Cell Size 9-15 um 9-15 um 9-15 um 10-16 um Cytoplasm With fine lilacpink granules With coarse reddishorange granules With coarse bluish-black granules that obscure the nucleus Nucleus Multilobed (2-5) connected by thin filaments Indented nucleus (>1/2 of nuclear diameter) S, C, Z shaped Sausage-shaped Bilobed Multilobes (3-4) Agranulocyte Small Lymphocyte Cell Size 7-10 um Large/Big/ Atypical Lymphocyte 11-25 um Monocyte 14-20 um Cytoplasm Nucleus Sky-blue (Robin Compact, large egg blue) and round No granules Sky-blue (Robin Immature looking egg blue) (round, oval, or No granules slightly Indented by RBC indented) Blue-gray with Kidney-shaped , ground glass horshoe/coffee appearance bean with brainwith/out vacuoles like convolutions OSMOTIC FRAGILITY TEST (OFT) • • • • • • • Measure of resistance of RBC to hemolysis by osmotic stress o Osmotic stress - decreasing strength of saline soln ▪ 0.85 → 0.0% A traditional & non-specific test for hereditary spherocytosis Affected by cell membrane permeability and surface-tovolume ratio o Increase ratio = less internal RBC component = high resistance to hemolysis exposed to osmotic pressure o Decreased ratio = full RBC = burst when exposed to osmotic stress resulting in hemolysis Indication: for detection of hereditary spherocytosis and thalassemia Sample: Heparinized Whole Blood Incubated OFT – useful for detection of mild hereditary spherocytosis o Blood is incubated at 37C for 24 hours before performing the test ▪ Incubation enhances shape of spherocytes ▪ Allows spherocytes become more spherical o Mild hereditary spherocytosis - have normal OFT Tonicity Principle: METHODS • • • 100-cell differential – Routinely performed method of differential count 50-cell differential – performed only if WBC count is <1.0 x 109/L o Use buffy coat smear 200-cell differential – performed only if: o Leukocytosis o Eosinophil = >10% o Basophil = >2% o Monocytes = >11% o Lymphocytes > Neutrophils (among adults) • • MANNER OF REPORTING • Relative Count o The number of specific WBC type per 100 WBCs o Formula: • • Absolute Count o Gives the number of specific WBC type per cubic millimeter of blood o More informative than Relative count o Formula: PROCEDURE 1. • Reference Ranges: NEUTROPHIL BAND/STAFF/STAB LYMPHOCYTE MONOCYTE EOSINOPHIL BASOPHIL RELATIVE COUNT (%) 47 – 77% 2 – 6% 20 – 40% 2 –10% 0 – 6% 0 – 1% ABSOLUTE COUNT(/mm3) 1,600 – 7,260 350 - 700 960 – 4,400 180 – 880 45 – 440 45 – 110 Isotonic - amount of water entering the cell is equal to the water that leaves the cell; remains normal (biconcave) o Eg. 0.85% NaCl Hypertonic - fluid outside the cell has higher concentration than inside of the cell o Releases water to maintain equilibrium o Dilutes extracellular fluid o RBC become crenated o Eg. >0.85% NaCl Hypotonic - intracellular concentration is higher than extracellular concentration o Absorbs water to maintain equilibrium o Causes swelling of cells which may lead to bursting o Eg. <85% NaCl 2. 3. 4. • Heparinized whole blood is mixed with different concentration of NaCl Solutions (0.85% to 0.0% at 0.050.10% increments) Incubated the mixture (30 minutes at room temperature) Centrifuge tubes after incubation o Non-hemolyzed cells will settle on tube Determine hemolysis macroscopically or spectrophotometrically (540 nm) Normal Sample o No Hemolysis – 0.85% NaCl Solution (Isotonic) o Initial Hemolysis – 0.45% NaCl Solution (Hypotonic) o Complete Hemolysis – 0.35-0.30% NaCl Solution (Hypotonic) READING • Normal Red Blood Cells CLINICAL SIGNIFICANCE • • • Spherocytes: o Decreased surface-to-volume ratio ▪ Indicate fullness of RBC o Increased Osmotic Fragility o RBC susceptible or tend to lyse in less hypotonic soln Increased Osmotic Fragility o Hereditary Spherocytosis o Burns - external factor o Immune Hemolytic Anemia Decreased Osmotic Fragility o Thalassemia o Target Cells o Sickle Cell Anemia o Iron Deficiency Anemia Normal Hereditary Spherocytosis Thalassemia (characterized by numerous target cells) Sickle Cell Anemia Initial Hemolysis (%NaCl) Complete Hemolysis (%NaCl) 0.45 0.35-0.30 0.65 0.45 0.35 0.20 0.35 0.20 SUCROSE HEMOLYSIS TEST • • • • OSMOTIC FRAGILITY CURVE • • • Normal Curve indicates RBC starts to lyse at 0.45% NaCl Solution and are completely lysed at 0.35-0.30% NaCl Solution Shift to the left – increased osmotic fragility Shift to the right – decreased osmotic fragility; high resistance to osmotic stress Aka Sugar Water Test Indication: Paroxysmal Nocturnal Hemoglobinuria (PNH) or the Marchiafava Micheli Syndrome o PNH - RBC are susceptible to lysis by complement due to cell membrane lacking complement regulatory proteins resulting to intravascular hemolysis causing hemoglobinuria during sleep or after waking Procedure: 1. Washed RBC (patient’s RBC) are incubated at isotonic sucrose solution with normal ABO-compatible serum 2. At Low ionic concentration, red blood cells absorbed complement components from the serum o Normal Cells – resistance to hemolysis at such condition; contains complement regulatory proteins o PNH red cells – more sensitive and will lyse under this condition. ▪ More sensitive to complement components found in serum causing hemolysis AUTOMATION: AUTOMATED HEMATOLOGY ANALYZERS • • • INCUBATED OSMOTIC FRAGILITY TEST • • Useful for detection of Mild Hereditary Spherocytosis Blood is incubated at 37C for 24 hours Automation in widely utilized today Provides the Standard Hematology Parameters: o RBC count and Indices, Hgb, Hct o WBC count o Platelet count o WBC Differential Count: ▪ 3-part (segmenters, lymphocytes, monocytes) • Segmenters - neu, eos, baso ▪ 5-part (Neu, Eos, Baso, Lympho, Mono) ▪ 6-part (Neu, Eos, Baso, Lympho, Mono, nRBC) General Principles: 1. Electronic Impedance 2. Radiofrequency 3. Optical Scatter COMMON BASIC COMPONENTS • • • Hydraulics o Includes aspirating unit, dispensers, dilutors, mixing chambers, aperture baths and/or flow cells and hemoglobinometer o Aspirating unit - probe; absorbs blood Pneumatics o Generates vacuum and pressure for operative valves and moving the sample through the hydraulic system Electrical System o Controls operational sequences of the total system o Includes electronic analyzers and computing circuity for processing data generated. • • • ELECTRONIC IMPEDANCE (EI) • • • • • • Aka low-voltage direct current or coulter principle o Based on the development of Coulter in 1950s Most common methodology used Developed by Coulter in the 1950’s Examples: o Beckman Coulter (LH series, GEN-S, HmX, A-T) o Sysmex (XE and XT series) o Abbott (Cell-Dyn, 3700, Ruby, Sapphire,.) o ABX (Micros 60, Pentra series) Principle: o Detection and measurement of changes in electrical resistance (impedance) produced by cells as they traverse a small aperture. Electrical resistance between the two electrodes, or impedance in the current, occurs as the cells pass through the sensing aperture, causing voltage pulses that are measurable o Each cell passes through aperture causing electrical resistance/change producing electrical pulse ▪ Cells are poor conductors of electricity o Pulses are measured which are significant RADIOFREQUENCY • • • • • • • • • • Sample is diluted in an isotonic solution o Isotonic - preserve cell shape and characteristic Dilution is instrument-dependent Has 2 aperture o RBC aperture - simultaneously counts RBC & plts ▪ Small enough to only allow RBC and plts to enter o WBC aperture RBC and Platelets are simultaneously counted WBC counting – sample is mixed with lytic reagent that lyse RBCs making WBC enter WBC aperture Hemoglobin and WBC are simultaneously assessed o Lysed RBC release Hgb o Most commercially available lytic solution convert Hgb to Cyanmethemoglobin Oscilloscope o Displays the pulses generated by the cells as they interrupt the current. o Number of impulses = the number of cells o Amplitude of impulses = size/volume of cells o With threshold circuits – allows discrimination of cells of specific volume Volume Distribution Histogram o Frequency distribution graph o Depicts the volume distribution of the cells counted o X-axis – volume (fL) o Y-axis – relative number of cells • Aka RF resistance, high-voltage electromagnetic current Used in conjunction with low-voltage DC impedance Measures conductivity (relative to cellular content) o Conductivity is relative to nuclear cytoplasmic ratio, nuclear density, cytoplasmic granulation It’s used to detect cell size, based on the cellular density Example: o Coulter (LH series, GENfi-S, HmX, A-T) o Sysmex (XE-2100, XT 2000i, HST-N) Measures two different cell properties: o DC impedance – proportional to cell volume (amplitude) o RF pulse/Conductivity – proportional to intracellular density • • Plotted using cytogram or scatterplot o No. of dots represent concentration of specific cell type This allows 5-part WBC differential • Different angles proportional to cell characteristics o Forward-angle light scatter (0°) – cell vol./cell size o Forward low-angle light scatter (2 to 3°) & Forward high-angle (5 to 15°) - can relate to size or volume and allows for description of the refractive index of cellular components o Orthogonal light scatter (90°) aka side scatter correlates w/ degree of internal complexity (granularity and lobularity) ▪ Separates granular cells ERRORS WHICH MAY BE ENCOUNTERED IN RF & EI • • Instrumental Errors 1. Aperture plugs - positive error (false ↑) 2. Bubbles in the specimen - positive error (false ↑) 3. Extraneous electrical impulses - pos error (false ↑) 4. Excessive lysing of RBCs - negative error (false ↓) 5. Improper setting of aperture current - pos/neg error Nature of Specimen 1. Giant platelets ▪ Counted as RBC or WBC ▪ False↑ - RBC, WBC; False↓ - plt ct 2. Fragments of WBC cytoplasm - counted as plt or RBC ▪ False↑ - RBC, plts 3. Some abnormal RBCs resists lysis (False↑ - WBC ct) OPTICAL SCATTER • • • • Optical scatter system = flow cytometer Uses LASER (helium-neon) or non-LASER light (tungstenhalogen lamp) o LASER - monochromatic light emitted at single wavelength Used to measure light scattering properties of cells o Scattering properties are measured by optical scatter Principle: o • A diluted blood specimen passes in a steady stream through which a beam of laser light is focused o As each cell passes through the sensing zone of the flow cell, it scatters the focused light o Scattered light is detected by a photodetector and converted into an electrical pulse (directly proportional to the number of cells passing through flow cell) Light Scattering results from the interaction between: o Absorption – wavelength of light that is not transmitted o Diffraction - bending of light around corners with the use of small angles o Refraction - bending of light because of a change in speed with the use of intermediate angles o Reflection - light rays turned back by the surface or an obstruction with the use of large angles o Light are scattered at an angle AUTOMATION: HISTOGRAMS RBC HISTOGRAM • • • • • • Count cells as RBC if volume is 36-360 fL o Volume is correlated with MCV value Normal – a single peak between 70-110 fL Shift to the left – microcytosis Shift to the right – macrocytosis Bimodal curve – two population of RBCs o Seen in: 1. Post-blood transfusion 2. Cold agglutinin disease 3. Hemolytic anemia with schistocytes 4. Anemia with different cell sizes Can measure cells with volume of 24 fL o Not counted as RBC o 24 - 36 fL are not counted as RBC WBC HISTOGRAM • • Count & plot of cells in WBC aperture bath larger than 45 fL Normal – has three distribution peaks o First Peak (35-90 fL) – small lymphocytes o Second Peak (90-160 fL) – mononuclear cells (monocytes, reactive lymphocytes, immature WBC) ▪ Immature WBC - abnormal ▪ Band cells - only immature WBC normally seen o Third Peak (160-450 fL) – granulocytes ABNORMAL WBC HISTOGRAM PATTERNS • • Region Code (R) flags: o Signal irregularities in WBC distribution o R1 – increased interference in the area left of the lymphocyte peak (approx. 35fL) (left of 1st peak) ▪ Caused by sickled RBC, nRBC, clumped and giant platelets counted in WBC aperture. o R2 – excessive overlap of cell populations at the lymphocyte/mononuclear cell boundary (approx. 90fL) (2nd peak) ▪ Atypical lymphocytes, blast, or plasma cells o R3 – caused by excessive overlap of cell population at the lymphocyte/mononuclear cell boundary (approx. 160 fL) (2nd peak) ▪ Increased presence of immature granulocytes (bands, metamyelocytes) o R4 – caused by extension of cell distribution past the upper end of WBC threshold (approx. 450fL) (3rd peak) ▪ Very high granulocyte population o RM – error code for multiple region overlap Other Flags o H – parameter values is higher than set normal limits o L – parameter values is lower than set normal limits PLATELET HISTOGRAM • • Cells in RBC aperture bath with size range of 2-20 fL Atypical platelet histogram can result in some disorder when large platelets are present o Platelets are not counted as plateless CONDITIONS CAUSING INTERFERENCE IN HEMATOLOGY ANALYZES