Hematology - 06 Laboratory Evaluation of RBC

HEMATOLOGY 5. LABORATORY EVALUATION OF RBC OUTLINE • Red Blood Cell Indices • Quality Assurance • Hematology Laboratory o Mean Cell Volume (MCV) • Complete Blood Count o Mean Cell Hemoglobin (MCH) o Hemacytometry o Mean Cell Hemoglobin o Limitations of Manual Concentration (MCHC) Counting o RBC Distribution Width • RBC Count, Hemoglobin, and (RDW) Hematocrit o Red Blood Cell (RBC) Count o Hemoglobin Determination o Hematocrit Determination o Rule of Three • QUALITY ASSURANCE • “It is important in HEMATOLOGY LABORATORY to ensure that the right test is being carried out on the right specimen and that reliable results are delivered with accuracy and precision without delay to the appropriate receiver” o Receiver - attending physician QUALITY ASSURANCE • • • • Aka Quality Assurance System Concerned with the total process happening inside the laboratory. Monitors quality of the entire laboratory performance. o Pre-analytical phase o Analytical phase o Post-analytical phase. Used to represent practices that are generally recommended for ensuring that desired quality goals are achieved. o Recommended practices are in a quality manual External Quality Assessment ▪ Proficiency testing ▪ Hematology - performed once a year ▪ Unknown sample are from National Reference Lab (National Kidney and Transplant Institute) Post-analytical Phase • o Includes factors after testing (turnaround time, clerical works, etc.) 1. Accuracy in transcription and filling of results 2. Content and Laboratory report 3. Narrative report 4. Reference interval and therapeutic range 5. Timeliness in communicating critical values 6. Patients and physician satisfaction 7. Turnaround time 8. Cost analysis HEMATOLOGY LABORATORY • Frequently performed tests in Hematology Laboratory: o COMPLETE BLOOD COUNT (CBC) o Reticulocyte Count COMPLETE BLOOD COUNT • • • • • • aka Full Blood Count, Full Blood Exam Most frequent blood test o Most common test for newly admitted patients Provides a detailed information about the cells in the blood Screening test for most diseases Sample: EDTA blood Includes measurement of: o Red Blood Cell Count o Hemoglobin o Hematocrit o RBC Indices o White Blood Cell Count o WBC Differential Count o Platelet Count HEMACYTOMETRY • • • Pre-analytical Phase o Includes factors in acquiring, handling, transporting, and processing a patient specimen prior to actual analysis 1. Selection of assay relative to the patient need 2. Implementation of assay selection 3. Patient identification and preparation 4. Specimen collection technique 5. Specimen collection apparatus used 6. Specimen transport, preparation and storage 7. Monitoring of specimen condition Analytical Phase o Includes factors related to the analysis (methodology procedures, etc.) 1. Laboratory personnel competence 2. Assay and instrument selection 3. Assay validation – includes linearity, accuracy, precision, analytical limits and specificity 4. Internal Quality Control ▪ Short-term quality control • Manual quantitative evaluation of formed elements of the blood (RBC, WBC, Platelets) Materials: o Counting Chamber o Thoma pipet (diluting pipet) o Suction device o Thick coverslip o Cell counter o Diluting Fluids COUNTING CHAMBER • • • • • Aka Hemacytometer “Heart” of manual blood cell counting Improved Neubauer Counting Chamber – the most commonly used hemacytometer/counting chamber Depth: 0.1 mm o Distance between counting chamber & coverslip Has 2 counting area, each counting area (primary square): o 3 mm x 3mm (9 mm2) o 9 secondary squares (1 mm2 each) ▪ 4 corner large square - used for manual WBC ct • Subdivided into 16 tertiary squares ▪ 1 central large square – used for manual RBC ct • Subdivided into 25 tertiary squares • Each secondary squares are subdivided into 16 quaternary squares RBC COUNT, HEMOGLOBIN, AND HEMATOCRIT RED BLOOD CELL (RBC) COUNT • • • • • • • • General Formula: # of cell counted x dilution factor Depth x Area Unit: cells/mm3 (conventional) Other types of counting chambers: o Neubauer ▪ Different from improved Neubauer o Fuchs-Rosenthal ▪ Used for absolute eosinophil count ▪ Depth: 0.2 mm o Speirs-Levy o Tuerk o Bass-Jones • • THOMA PIPET • • • • Stem - shaft below the bulb; 1ml volume Two types: RBC Thoma Pipet (RBC Pipet) o Markings: 0.5, 1, and 101 o Color of bead: RED o Volume in the bulb: 100 WBC Thoma Pipet (WBC Pipet) o Markings: 0.5, 1, and 11 o Color of bead: White o Volume in the bulb: 10 • • • DILUTING FLUID • Used mainly to disperse blood cells to facilitate cell counting o RBC Diluting Fluid: ideal is isotonic o WBC Diluting Fluid: ideal is hypotonic o Platelet Diluting Fluid: must preserve platelet integrity while inhibiting their aggregation LIMITATIONS OF MANUAL COUNTING • • • • • Experience is needed to make technically adequate smears consistently Non-uniform distribution of WBC and RBC over the smear o Result of inadequate blood smear Subjective, labor-intensive, and statistically unreliable. Imprecise Cell identification errors in manual counting aka Red Cell Count, Erythrocyte Count Total number of red blood cells per liter of whole blood. Specimen: EDTA Whole Blood Red Blood Cell – transports oxygen and remove waste products from the peripheral tissues o ↑ morning, ↓ evening o ↑ in polycythemia o ↓ anemia Reference Values (x 1012/L): o Female: 3.6 – 5.6 o Male: 4.2 – 6.0 o Newborn: 5.0 – 6.5 Decreased RBC Count: o Blood loss o Hemorrhage o Bone Marrow Failure o Iron, Folate, Vitamin B12** deficiencies o Hemolysis o Certain cancers o Anemia Increased RBC Count: o High altitude o Congenital Heart Disease o Polycythemia vera o Dehydration Diluting Fluids: o Isotonic ▪ When hypotonic, RBC will swell o Has a high specific gravity o Easy to prepare o Good preservative o Has a buffering action o Does not initiate growth of molds RBC Diluting Fluids o Dacie’s Fluid/Formol citrate – best RBC diluting fluid o NSS – used in case of excessive rouleaux formation (coin-like stacking of RBC) and autoagglutination o 3.8% Sodium citrate o Hayem’s o Gower’s o Toissons’s o Bethel’s Manual RBC Count Procedure: 1. Mix specimen thoroughly (EDTA whole blood) 2. Aspirate EDTA whole blood to 0.5 marks of RBC pipet. Wipe excess blood not touching the tip of the pipet 3. Aspirate RBC diluting fluid to 101 mark (0.5:101 dilution) 4. Shake RBC pipet in figure of 8 motion for 5 minutes 5. Discard few drops (the stem of RBC pipet may contain pure diluting fluids) (0.5:100 or 1:200 dilution) 6. Change the counting chamber (ensure that it will not spill on the sides of the counting chamber 7. Stand for 3 minutes 8. Count RBC in 5 tertiary squares (central and four corner) of the central secondary square (A1, A2, A3, A4, A5 in the picture) HEMOGLOBIN DETERMINATION • • • • NOTE: o Count RBC on both counting site of the hemacytometer and get the average. o Allowable difference of RBC count between 2 chamber is 15-16 counts o Repeat procedure when above allowable diff. General Formula: • • o o o o o • Unit: cells/mm3 SI unit: x 1012/L (cells/mm3 ÷ 1,000,000) Dilution Factor: 200 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): 0.0025 mm2 Simplified Formula: • CYANMETHEMOGLOBIN METHOD • • • • • RBC Count = # of cells counted x 10,000 o Note: When dividing fractions, take the reciprocal of each fractions and change the operation to multiplication. o Used when the dilution is 200 and 5 RBC 3° squares Sample Problem 1: You are performing a manual RBC count on an EDTA sample. You used a dilution of 1:200 using RBC thoma pipet and counted an average of 590 RBC in the 5 tertiary squares (4 corner and one center) of the central secondary squares of both counting sites of the hemacytometer. What is the total RBC count? o # of cells counted: 590 o Dilution: 1:201 (1/200) o Area: 0.04 mm x 5 = 0.2 mm (1/5 mm) o Depth: 0.1 mm (1/10 mm2) Screening test for anemia and may detect RBC breakdown (hemolytic anemia) Hemoglobin o Gives red color to the blood o Carries oxygen from the lungs to the tissues and takes carbon dioxide ▪ ↑ morning, ↓ evening ▪ ↑ birth due to active red bone marrow ▪ ↑ in high altitudes due to hypoxia ▪ ↑ in strenuous muscular activity due to androgen influence ▪ ↑ in smokers due to formation of carboxyhemoglobin ▪ ↓ when in lying position due to the dilution of interstitial fluid Reference values: g/dL g/L Female 12 - 15 120 - 150 Male 14 - 18 140 - 180 Newborn 16.5 - 21.5 165 - 215 Increased Hemoglobin o Sickle cell anemia o Thalassemia o Transfusion reaction o Hemolysis o Dehydration o Polycythemia vera o High altitude Decreased Hemoglobin o Anemia o Blood loss o Iron, folate, and vitamin B6, B12 deficiencies • • aka “Hemoglobincyanide” Reference method for hemoglobin determination Standard approved by Clinical and Laboratory Standard Institute (CLSI) Can measure all forms of hemoglobin except sulfhemoglobin Reagent - Drabkin’s Reagent (contains): o Potassium ferricyanide o Potassium cyanide o Non-ionic detergent – improves RBC lysis to release Hgb o Dihydrogen potassium phosphate Principle: o • Absorbance of cyanmethemoglobin at 540 nm is directly proportional to the hemoglobin concentration Procedure: 1. In 5mL of Drabkin’s reagent in a test tube, add 0.02cc/20uL of blood using sahli pipette. Rinse the pipette with reagent several times 2. Mix well and stand for 10 minutes. Ensures lysing and conversion. 3. Read absorbance using spectrophotometer at 540 nm • • Sources of Error: o Cyanmethemoglobin is sensitive to light ▪ Use amber-colored bottle/container ▪ Protect from light o Turbid specimen can cause false increase: ▪ Lipemia ▪ Extremely elevated WBC count (>20 x 109/L) ▪ Extremely elevated platelet count (>700 x 109/L) ▪ Presence of HbS and HbC ▪ Easily precipitated globulins (e.g. Waldenstrom’s macroglobulinemia, multiple myeloma) Other Developments: o Azide Methemoglobin ▪ Avoids dilution and interference from turbidity ▪ Read at 570 nm and 880 nm o SLS – methemoglobin ▪ Avoids the use of toxic material ▪ Uses sodium lauryl sulfate (SLS) • o OTHER HEMOGLOBIN METHOD • • • • Sahli’s Acid Hematin Method 1. Place N/10 HCl into Hb tube up to 2 grams. 2. Blood sample in Sahli’s Hb piptette up to 20 uL. 3. Add blood sample to acid solution. 4. Mix with a stirrer. 5. Allow to stand for 10 minutes. 6. Add distilled water drop by drop till the color of the solution matches to brown glass standard. 7. Take the reading of the lower meniscus from the graduated tube in grams. o Uses comparator block to compare color of blood o Empty tube in the middle Copper Sulfate Method o aka Specific Gravity Method o Used for screening blood donors o Copper sulfate: S.G 1.053 = 12.5 g/dL Hgb o >12.5 g/dL Hgb – drop of blood sinks o <12.5 g/dL Hgb – drop of blood floats Gasometric method (Oxygen Capacity Method) o Principle: Hgb will combine and liberate a fixed quantity of Oxygen. The blood is hemolyzed with saponin and the gas is collected and measured in a van slyke apparatus o Oxygen combining capacity of blood. 1.34ml O2 /g of hgb o Formula: Fetal Hemoglobin (HbF) Determination Alkali Denaturation Test Principle: HbF resists alkali denaturation while HbA does not Normal Value Adults: 0.5 – 8% 1 year old: 1% Acid Elution Test (Kleihauer-Betke Test) Resists acid elution, thus appear as isolated darkly – staining cell among a background of pale staining ghost cells 1-5% of rbc contain residual HbF Hemoglobin S (HbS) Determination o Sodium Metabisulfite Test: ▪ Principle: Sodium metabisulfite is a reducing agent that will deoxygenate hemoglobin causing polymerization of RBC forming sickle cells ▪ Presence of sickle cell = presence of HbS Dithionite Tube Test: ▪ Principle: Sodium hydrosulfite (dithionite) converts Fe2+ to Fe3+ in hemoglobin resulting to deoxygenation of hemoglobin. • Polymerization of RBC ▪ Solubility: • Normal Hemoglobins: Soluble • HbS: Insoluble (turbid solution) HEMOGLOBIN ELECTROPHORESIS • Alkaline Hemoglobin Electrophoresis o Principle: Hemoglobin molecules at alkaline Ph assumes negative charge and migrate towards anode ▪ Hemoglobin contain proteins composed of amino acids that have isoelectric point (pH is at net 0) ▪ Anode - positively charged electrode o Medium: Cellulose Acetate/Agarose Gel (pH 8.4-8.6) o Migration: ▪ Fastest: HbA/HbA1 ▪ Slowest: HbC, HbA2, HbE, HbO-Arab ▪ Same Migration: • HbS, HbD, HbG • HbC, HbE, HbO Alkaline Acid • Acid Hemoglobin Electrophoresis o Principle: Some hemoglobins assume a negative charge (migrate towards the anode), whereas others assume positive charge (migrate towards the cathode) ▪ Cathode - negatively charged electrode ▪ Separates the Hgbs that migrate towards the same area at an alkaline pH o Medium: Citrate agar (pH 6.0-6.2) o Migration: ▪ Separates HbS from HbD and HbG ▪ Separates HbC from HbE and HbO HEMATOCRIT DETERMINATION • • aka “Packed Cell Volume” (PCV) Hematocrit o Volume of packed RBC that occupies a given volume of whole blood. o ↑ in polycythemia, macrocytic anemia, hypochromic anemia, sickle cell anemia, spherocytosis, thalassemia o ↓ anemia, age (>50 y.o.) o Reference values: % Female 35 - 49 Male 40 - 54 Newborn 48 - 68 • Other Macrohematocrit Methods: o Haden’s Modification Method: ▪ Anticoagulant: 1.1% Sodium oxalate o Van Allen’s Method ▪ Anticoagulant: 1.6% Sodium oxalate o Sanford-Magath Method ▪ Anticoagulant: 1.3% Sodium oxalate o Bray’s Method ▪ Anticoagulant: Hepain MICROHEMATOCRIT METHOD • • Uses capillary tubes/microhematocrit tube: o Length: 75 mm o Inner Bore: 1.2 mm o Volume: 0.05 mL Two types of capillary tubes: Heparinized Plain Color of band Red Blue Additive Heparin None Uses Used when blood Used when blood is directly collected is from a prefrom dermal collected EDTA puncture blood MACROHEMATOCRIT METHOD • • • Uses Wintrobe tube o Length: 115 mm o Diameter (bore): 3.0 mm o Hematocrit marking (White): 10cm-0 (from top) o ESR marking (Red): 0-10cm (from top) Specimen: Whole Blood (using Double Oxalate anticoagulant) Procedure: 1. Fill the Wintrobe tube with blood up to 10 mark (white) 2. Place it inside a test tube with cottons in between to make it stable 3. Centrifuge at 2000-3000 RPM for 30 mins 4. Remove the tubes and carefully place it in Wintrobe tube stand 5. Measure the height of RBC layer 6. It is expressed in percentage (%) • Materials: o Capillary tubes (red/blue) o Clay sealant o Microhematocrit centrifuge o Microhematocrit reader • Procedure: 1. Perform skin puncture and fill 2 heparinized capillary tubes (3/4) 2. Seal the end with the colored ring of the capillary tube with clay and wax (4-6mm) 3. Centrifuge for 5 minutes at 10,000-15,000 g 4. Read using microhematocrit reader (buffy coat should not be included) ▪ Inclusion of buffy coat may cause false increase • • Layers of Microhematocrit Tube: o Descending Layer: ▪ First Layer: Plasma ▪ Second Layer: Buffy coat (WBC, plts) ▪ Third layer: Packed red blood cell ▪ Fourth layer: Seal/plug o Ascending Layer: ▪ First Layer: Seal/Plug ▪ Second Layer: Packed red blood cell ▪ Third layer: Buffy coat (WBC, platelets) ▪ Fourth layer: Plasma Sources of Error: o False Increase: ▪ Insufficient centrifugation ▪ Inclusion of buffy coat ▪ Hemoconcentration ▪ Dehydration ▪ Disorders such as Macrocytic anemia, Hypochromic anemia, and sickle-cell anemia ▪ Trapped plasma in packed RBC layer (~0.2 L/L) o False Decrease: ▪ Improper sealing of capillary tubes ▪ Increased concentration of anticoagulant ▪ Prolonged centrifugation ▪ Acute blood loss ▪ Hemolysis ▪ Inclusion of interstitial fluid (dilute whole blood) MEAN CELL VOLUME (MCV) • • • • • • • Used for validating test results of Hemoglobin & Hematocrit Works only on normocytic, normochromic specimens. o Normal MCV & MCHC Formula: o Hemoglobin (g/dL) = RBC count (x 1012/L) x 3 o Hematocrit ± 3% = Hemoglobin (g/dL) x 3 Example: Reference value: 80-100 fL (normocytic) o Microcytic: <80 fL o Macrocytic: >100 fL (megalocytes = >120 fL) MEAN CELL HEMOGLOBIN (MCH) • • • Average weight of hemoglobin in an individual RBC in picograms (pg) Directly proportional to the size and hemoglobin concentration of an RBC Not used in classification of anemia Formula: • Reference range: 26 – 32 pg • RULE OF THREE • • Average size or volume of an individual RBC in femtoliter (fL) Increased: Megaloblastic anemia, Hemolytic anemia with reticulocytosis, liver disease and normal newborn, autoagglutination, severe hyperglycemia (<600 mg/dL), Leukocytosis Decreased: Iron deficiency anemia, sideroblastic anemia, thalassemia, and Lead poisoning Formula: MEAN CELL HEMOGLOBIN CONCENTRATION (MCHC) • • • • • • Average conc. of hemoglobin in each individual RBC Ratio of hemoglobin weight to RBC volume Increased: Spherocytic cells, errors in RBC (hemolysis), presence of cold agglutinins. Decreased: Thalassemia, Iron Deficiency Anemia Formula Reference value: 32 – 36 g/dL or % (normochromic) o <32 g/dL: hypochromic cells (increased central pallor) o >36 g/dL: Hyperchromic (spherocyte - new term) RBC DISTRIBUTION WIDTH (RDW) • When wrong result in rule of three, rerun the sample o After rerun, Hct w/in acceptable range = random error o Hct outside rule of three, check peripheral smear to assess RBC morphology RED BLOOD CELL INDICES • • • • aka Erythrocyte indices Used to mathematically define cell size and hemoglobin content within cells Used to morphologically differentiate anemias Includes: o Mean Cell/Corpuscular Volume (MCV) o Mean Cell/Corpuscular Hemoglobin (MCH) o Mean Cell/Corpuscular Hemoglobin Concentration (MCHC) o Red Blood Cell Distribution Width (RDW) • • • • • Coefficient of variation of RBC volume Index of anisocytosis Determined from RBC histogram Reference value (RDW-CV): 11.5-14.5% o Refers to the width of the curve Interpretation: o Normal RBC Histogram: Single peak between 80100 fL (normal MCV) o Anisocytosis: Increased curve width (<80 to >100 fL) o Microcytosis: Shift to the left (<80 fL) o Macrocytosis: Shift to the right (>100 fL)