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ESR, PCV and Buffy coat- Significance.pptx
- 1. ESR, PCV andBuffy coat
- 2. • The erythrocytesedimentation rate (ESR) measures the rate of settling (sedimentation) of erythrocytes in anticoagulated whole blood • Anticoagulated blood is allowed to stand in a glass tube for 1 hour and the length of column of plasma above the red cells is measured in millimeters; this corresponds to ESR • ESR and CRP are two commonly used tests to screen for inflammation
- 3. STAGES OF ERYTHROCYTESEDIMENTATION RATE • There are three stages of erythrocyte sedimentation: • Stage 1: Formation of rouleaux or lag phase (10 minutes): Red cells stack together like a pack of coins. The ESR depends mainly on this stage • Stage 2: Sinking of rouleaux or decantation phase (40 minutes): Rapid and constant sedimentation. The longer the tube, the longer is this stage and higher the value of ESR • Stage 3: Packing of rouleaux (10 minutes): Slow sedimentation
- 4. FACTORS AFFECTING ERYTHROCYTE SEDIMENTATIONRATE • Red cells, composition of plasma, and technical factors affect ESR • Red cells: Alteration of ratio of red cells to plasma affects ESR. Decreased red cell mass in anemia increases ESR • Conversely, increased red cell mass in polycythemia decreases ESR. • Macrocytes tend to sediment rapidly than microcytes • Sickle cells and spherocytes are unable to form rouleaux and therefore ESR is low in sickle cell disease and hereditary spherocytosis. In these conditions, ESR is not reliable as an indicator of illness.
- 5. • Plasma: Themost important factor affecting ESR is the composition of plasma • Increase in fibrinogen, other acute phase proteins (C-reactive protein, haptoglobin, ceruloplasmin, α1-antitrypsin, etc.) and immunoglobulins increase ESR • Increased proteins in plasma reduce negative charge on the surface of red cells and reduce the zeta potential (the electrical repulsion between red cells); this brings red cells closer together and facilitates rouleaux formation • Removal of fibrinogen by defibrination and increase of albumin retard ESR
- 6. • Technical factors: •ESR increases with room temperature • Tilting of the tube • Length and bore of the tube affect ESR
- 8. SIGNIFICANCE OF ERYTHROCYTE SEDIMENTATIONRATE • ESR is elevated in a wide range of organic diseases • ESR is not a specific and diagnostic test for any disease • It is helpful in differentiating functional from organic disease • Raised ESR signifies presence of some organic disease, which needs evaluation • Most of the inflammatory and neoplastic diseases are associated with an increase in ESR
- 9. • ESR correlateswith disease activity and therefore it is helpful in monitoring disease activity and response to therapy in acute rheumatic fever, bacterial endocarditis, tuberculosis, rheumatoid arthritis, temporal arteritis, polymyalgia rheumatica, and Hodgkin’s disease • ESR is an important criterion in establishing the diagnosis of temporal arteritis and polymyalgia rheumatica
- 10. • ESR isnot significantly raised in typhoid fever, malaria, infectious mononucleosis, angina pectoris, osteoarthritis, acute appendicitis, peptic ulcer, acute allergy, and unruptured ectopic pregnancy • In emergencies ESR is not helpful in diagnosis
- 11. • Very highor extreme elevation of ESR (≥100 mm at 1 hour) is seen in • Infections • Paraproteinemias • Metastatic malignancy • Polymyalgia rheumatica • Temporal arteritis • Rheumatoid arthritis
- 12. • ESR isdecreased in • Polycythemia • Congestive cardiac failure • Dehydration • Sickle cell anemia • Hereditary spherocytosis • Hypofibrinogenemia
- 14. INDICATIONS FOR MEASUREMENTOF ERYTHROCYTE SEDIMENTATION RATE • It is recommended to measure ESR • (i) when infectious, inflammatory, or a neoplastic disease is suspected in symptomatic individuals in whom a specific diagnosis has not been established, • (ii) to monitor disease activity in tuberculosis, inflammatory arthritis, rheumatic fever, Hodgkin’s disease, giant cell arteritis, and polymyalgia rheumatica • (iii) as a diagnostic criterion for temporal arteritis and polymyalgia rheumatica
- 15. METHODS FOR ESTIMATIONOF ERYTHROCYTE SEDIMENTATION RATE • Westergren method • Wintrobe method • Automated method – Examples are commercially available SEDIMAT, Ves-matic, ESR STAT PLUS, and Zeta sedimentation ratio
- 16. Westergren method • InternationalCouncil for Standardization in Hematology recommends Westergren method for estimation of ESR • Equipment and Reagent • Westergren ESR tube: This is a straight glass pipette measuring 300 mm in length and calibrated in mm from 0-200 (top to bottom) • The markings are only over the lower 2/3rds of the tube. The tube is open ended • Internal diameter should not be less than 2.55 mm. The tube should be clean and dry.
- 17. Westergren and Wintrobetubes for measurement of ESR
- 18. • Westergren stand:This holds the tube in a motionless, vertical position • Anticoagulant-diluent solution: Trisodium citrate dihydrate is the anticoagulant of choice. Its composition is as follows: • Trisodium citrate dihydrate 32.08 gm • Distilled water upto 1000 ml
- 19. Westergren ESR standwith ESR tubes
- 20. • It isfiltered through a sterile membrane (0.22 μm) into a sterile container and stored in a refrigerator at 4°C • It keeps for several months, but if it becomes turbid (due to the growth of moulds), it should be discarded • Alternate anticoagulant is EDTA (1.5 mg/ml); however, such anticoagulated blood sample should be diluted with trisodium citrate just before the test (1 volume of trisodium citrate to 4 volumes of blood)
- 21. • Specimen: • Venousblood is collected in trisodium citrate solution in 4:1 (blood: citrate) proportion • If specimen is kept at room temperature, test should be carried out within 4 hours of blood collection; if stored at 4°C, a delay of up to 6 hours is permissible • Blood anticoagulated with EDTA can be tested within 24 hours if stored at 4°C (provided it is diluted with trisodium citrate before testing)
- 22. Method 1. Mix anticoagulatedblood sample thoroughly. The Westergren tube is filled with the blood sample up to the “0” mark. A rubber bulb or a mechanical device should be used for filling. There should be no air bubbles in the blood. 2. The tube is placed in a strictly vertical position in the ESR stand and left undisturbed for 1 hour. It should not be kept in direct sunlight and should not be subjected to vibrations
- 23. 3. After exactly1 hour, read the height of the column of plasma above the red cell column in mm 4. Express the result as: Erythrocyte sedimentation rate = ——— mm in 1 hour
- 24. Reference range byWestergren method • Males <50 years : 0-15mm in 1 hr • Males > 50 years : 0-20 mm in 1 hr • Females < 50 yrs: 0-20 mm in 1 hr • Females > 50 years : 0-30 mm in 1 hr • New born : 0-2 mm in 1 hr • Children to puberty : 0-13 mm in 1 hr
- 25. Other methods • WintrobeMethod • Wintrobe tube can be used for estimation of both ESR and packed cell volume (PCV) • After obtaining ESR in the first hour, the tube can be spun in a centrifuge to get PCV • Wintrobe’s method is more reliable when ESR is low, while Westergren’s method is more sensitive for high ESR • EDTA or double oxalate is used as an anticoagulant • Length of Wintrobe tube is shorter (110 mm) and internal diameter is about 3 mm
- 26. • Reference rangeby wintrobes method • Males : 0-9mm in 1 hr • Females : 0-20mm in 1hr • Children : 0-13 mm in 1 hr
- 27. • Automated methods •These require special analyzers or centrifuge • They save technician time, require small sample volume and generate more rapid results
- 28.
- 29. • Packed cellvolume (PCV) is the volume occupied by the red cells when a sample of anticoagulated blood is centrifuged • It indicates relative proportion of red cells to plasma • PCV is also called as hematocrit or erythrocyte volume fraction • It is expressed either as a percentage of original volume of blood or as a decimal fraction
- 30. USES OF PCV •Detection of presence or absence of anemia or polycythemia • Estimation of red cell indices (mean cell volume and mean corpuscular hemoglobin concentration) • Checking accuracy of hemoglobin value (Hemoglobin in grams/dl × 3 = PCV)
- 31. • There aretwo methods for estimation of PCV: macro method (Wintrobe method) and micro method (microhematocrit method). • Micro method is preferred because it is rapid, convenient, requires only a small amount of blood, capillary blood from skin puncture can be used, and a large number of samples can be tested at one time • This method is also more accurate as plasma trapping in red cell column is less
- 32. MACRO METHOD (WINTROBEMETHOD) • Principle • Anticoagulated whole blood is centrifuged in a Wintrobe tube to completely pack the red cells • The volume of packed red cells is read directly from the tube • An advantage with this method is that before performing PCV, test for erythrocyte sedimentation rate can be set up
- 33. Equipment • 1. Wintrobetube: This tube is about 110 mm in length and has 100 markings, each at the interval of 1 mm. Internal diameter is 3 mm. It can hold about 3 ml of blood. • 2. Pasteur pipette with a rubber bulb and a sufficient length of capillary to reach the bottom of the Wintrobe tube. • 3. Centrifuge with a speed of 2300 g.
- 34. • Specimen • Venousblood collected in EDTA (1.5 mg EDTA for 1 ml of blood) or in double oxalate • Test should be performed within 6 hours of collection.
- 35. Method 1. Mix theanticoagulated blood sample thoroughly. 2. Draw the blood sample in a Pasteur pipette and introduce the pipette up to the bottom of the Wintrobe tube. Fill the tube from the bottom exactly up to the 100 mark. During filling, tip of the pipette is raised, but should remain under the rising meniscus to avoid foaming.
- 36. 3. Centrifuge thesample at 2300 g for 30 min (To counterbalance a second Wintrobe tube filled with blood from another patient or water should be placed in the centrifuge). 4. Take the reading of the length of the column of red cells. Hematocrit can be expressed either as a percentage or as a fraction of the total volume of blood sample.
- 37. Significance • In anemia,PCV is below the lower level of normal range. PCV is raised in dehydration, shock, burns, and polycythemia. • After centrifugation of anticoagulated whole blood, three zones can be distinguished in the Wintrobe tube from above downwards- plasma, buffy coat layer (a small greyish layer of white cells and platelets, about 1 mm thick), and packed red cells
- 38. Anticoagulated blood-filled Wintrobehematocrit tubes after centrifugation, showing normal PCV, low PCV (anemia), and thick buffy coat layer
- 39. • Normal plasmais straw-colored • It is colorless in iron deficiency anemia, pink in the presence of hemolysis or hemoglobinemia, and yellow if serum bilirubin is raised (jaundice) • In hypertriglyceridemia, plasma appears milky
- 40. • Increased thicknessof buffy coat layer occurs if white cells or platelets are increased in number (e.g. in leukocytosis, thrombocytosis, or leukemia) • Smears can be made from the buffy coat layer for demonstration of lupus erythematosus (LE) cells , malaria parasites, or immature cells
- 41. Preparation of smearfrom buffy coat
- 42. MICRO METHOD • Principle •Anticoagulated whole blood is centrifuged in a capillary tube of uniform bore to pack the red cells • Centrifugation is done in a special microhematocrit centrifuge till packing of red cells is as complete as possible • The reading (length of packed red cells and total length of the column) is taken using a microhematocrit reader, a ruler, or arithmetic graph paper
- 43. Microhematocrit capillary tubesfollowing centrifugation in a microhematocrit centrifuge
- 44. Equipment 1. Microhematocrit centrifuge:It should provide relative centrifugal force of 12000 g for 5 minutes 2. Capillary hematocrit tubes: These are disposable glass tubes 75 mm in length and 1 mm in internal diameter They are of two types: plain (containing no anticoagulant) and heparinised (coated with a dried film of 2 units of heparin). For plain tubes, anticoagulated venous blood is needed. Heparinised tubes are used for blood obtained from skin puncture 3. Tube sealant like plastic sealant or modeling clay; if not available, a spirit lamp for heat sealing 4. Microhematocrit reader; if not available, a ruler or arithmetic graph paper.
- 45. • Specimen • Venousblood collected in EDTA (dipotassium salt) for plain tubes or blood from skin puncture collected directly in heparinised tubes • Venous blood should be collected with minimal stasis to avoid hemoconcentration and false rise in PCV
- 46. Method 1. Fill thecapillary tube by applying its tip to the blood (either from skin puncture or anticoagulated venous blood, depending on the type of tube used). About 2/3rds to 3/4ths length of the capillary tube should be filled with blood. 2. Seal the other end of the capillary tube (which was not in contact with blood) with a plastic sealant. If it is not available, heat-seal the tube using a spirit lamp
- 47. 3. The filledtubes are placed in the radial grooves of the centrifuge with the sealed ends toward the outer rim gasket. Counterbalance by placing the tubes in the grooves opposite to each other 4. Centrifuge at relative centrifugal force 12000 g for 5 minutes to completely pack the red cells 5. Immediately remove the tubes from the centrifuge and stand them upright. The tube will show three layers from top to bottom: column of plasma, thin layer of buffy coat, and column of red cells
- 48. • 6. Withthe microhematocrit reader, hematocrit is directly read from the scale. If hematocrit reader is not available, the tube is held against a ruler and the hematocrit is obtained by the following formula : • To obtain PCV, the above result is multiplied by 100
- 49. Reference range • Adultmales: 40-50% • Adult females (nonpregnant): 38-45% • Adult females (pregnant): 36-42% • Children 6 to 12 years: 37-46% • Children 6 months to 6 years: 36-42% • Infants 2 to 6 months: 32-42% • Newborns: 44-60%
- 50.
- 51. • A buffycoat suspension is a concentrated suspension of leukocytes and platelets that make up a part of the anticoagulated blood sample obtained by the process of density gradient centrifugation • In a test tube with whole blood, a thin grey-white layer of buffy coat is formed between the plasma and the hematocrit, consisting of leukocytes and platelets, both less dense than erythrocytes. • Buffy coat accounts for about less than 1% of the total blood volume taken in a tube.
- 52. • The colorof the buffy coat usually remains between yellow to light brown, but there might be some variations in the color as the color depends on the concentration of neutrophils.
- 53. Uses of buffycoat • Buffy coats are important for DNA isolation from blood samples. Especially in the case of the mammalian blood sample with non-nucleated RBCs, DNA extraction is performed from white blood cells as leukocytes are about ten times more concentrated source of nucleated cells. • Buffy coat preparation also allows the differentiation of white blood cells as they are more concentrated in the buddy coat than in the whole blood sample.
- 54. • The platelet-richbuffy coats have become an alternative source for the platelet concentration method as the buffy coat preparation causes less platelet activation and damage. • A quantitative buffy coat is a standard laboratory test to detect infection with malaria or other blood parasites like trypanosomes, Leishmania, and Histoplasma. • Buffy coat preparation is a cheaper method of blood cell separation, and it is also the ideal method to meet the emergency requirement for platelets.
- 55. Buffy coat preparation 1.Asmall narrow test tube or a bore plastic is taken and filed with an EDTA-treated anticoagulated blood sample with a Pasteur pipette. 2.The blood sample is then centrifuged for a total of 15 minutes at the RCF of 1000g. The time might differ with laboratories as the time might range between 10 to 25 minutes. 3.Following centrifugation, the supernatant containing the plasma above the buffy coat layer is removed onto a new tube.
- 56. 4. The buffycoat layer, along with some red cells present immediately below the buffy coat is transferred to one end of a slide. Buffy coat and the red cells are mixed with the tip of the pipette. 5. A thin preparation is made on the slide with a smooth- edged spreader, and the slide is allowed to air dry. 6. When dry, the preparation is fixed with absolute methanol or ethanol for 2 minutes. 7. The slide is then stained using either the Field’s thin-film staining technique or with the Giemsa staining method