Artefaccts in hemat part 1

1. 1 Presented by Dr. Shrikant Sonune Guided by Dr Ashok Patil, Dr Shilpa Kandalgaonkar, Dr Suyog Tupsakhare, Dr Mahesh Gabhane. Dr. Gaurav Agarwal Investigations & Artifacts in Hematology

2. The study of blood components and coagulation. It includes, 1) Analysis of the concentration, structure and functions of the cells and their precursors in the bone marrow. 2) Analysis of chemical constituents of plasma or serum intimately, linked with blood cell structure and functions. 3) Study of functions of the platelets and proteins involved in blood coagulation.

3. A. Complete Blood Count B. Complete Hemogram C. Tests of Hemostatic Function

4. 1. Hemoglobin (Hb) concentration. 2. Total erythrocyte count (RBC). 3. Total leukocyte count (WBC). 4. Examination of blood film (smear) for, - Assessment of red cell morphology. - Differential white cell count.

5. 1. Packed cell volume (PCV). 2. Mean corpuscular volume (MCV). 3. Mean corpuscular hemoglobin concentration (MCHC). 4. Platelet count. 5. Erythrocyte sedimentation rate (ESR).

6. 1. Bleeding Time. 2. Clotting Time. 3. Prothrombin time. 4. Partial Thromboplastin Time. 5. Determination of Thrombin Time. 6. Determination Fibrinogen.

7. Sound knowledge Advance planning Collection of adequate and appropriate specimens Sufficient documentation Biosafety and decontamination Correct packaging Timely assessment of results

8. The procedure in which an operator bleeds a specific amount of blood of the subject for a particular investigation can be termed as Collection of blood. Samples can be collected from: 1. Veins : Most commonly used. 2.Capillaries. 3.Arteries: in case of arterial blood gas analysis and sometime in pediatric patients.

9. Pricking the skin with needle or lancet. Site usually ring finger, prick should be 2-4mm deep to ensure free flow of blood. Only few drops are collected by this method. Recommended for the Bleeding & clotting time.

10. Blood is obtained by Venepuncture. More reliable results are obtained. With single prick- multiple tests can be done Test can be repeated, as the sample is available. Recommended for all hematological investigation except few e.g. BT, CT, BLOOD GAS LEVEL.

11. 1. Anatomy and physiology 2. The criteria for choosing a vein 3. The device to use 4. Skin preparation 5. Personal safety infection control policy

12. The superficial veins of the upper limbs are most commonly used for Venepuncture.

13. Sterile gloves Syringes and needles Tourniquet Specimen containers (Bulbs) 70 % alcohol or 0.5% chlorhexidine Sterile gauze swabs Adhesive dressings Rack to hold the specimen containers.

14. A vacutainer blood collection tube is a sterile glass or plastic tube with a closure that is evacuated to create a vacuum inside the tube facilitating the draw of a predetermined volume of liquid. Most commonly used to draw a blood sample directly from the vein. Vacutainer tubes may contain additives designed to stabilize and preserve the specimen prior to analytical testing.

15. Ash fluoride

16. Gauge of needle (22 gauge) Squeezing of finger. Initial drops - Discarded. Ring finger is preferred. No liquid anticoagulant.

17. 1. Whole blood for glucose, urea, pH estimation 2. Serum Total protein, Albumin, Globulin, Bilirubin, cholesterol, 3. Plasma- chloride, fibrinogen, ascorbic acid.

18. Clotted specimen. Improperly labeled or unlabeled specimen. Specimen too old. Failure to meet volume criteria. Improperly collected (diluted) capillary specimen. Leaking tube. Delay in transport. Collection of specimen in wrong tube. Hemolyzed smear

19. Granular fluid Colt consume platelets & other cells hence reduces the count. Basically arise from inadequate quantity of anticoagulant. Not mixing the anticoagulant properly.

20. Specimen Collection: An improper choice in the Venepuncture site, such as drawing from a distal site to the antecubital region of the arm rather than drawing from an antecubital site, has been shown to result in more hemolysis.

21. Prolonged tourniquet time causes the interstitial fluid to leak into the tissue and cause hemolysis. Cleansing the Venepuncture site with alcohol and not allowing the site to dry may cause hemolysis.

22. An improper Venepuncture, indicated by a slow blood flow, may indicate occlusion due to the lumen of the needle being too close to the inner wall of the vein, causing hemolysis. The use of a small-bore needle, resulting in a large vacuum force applied to the blood, may cause shear stress on the red blood cells, causing them to rupture.

23. The use of a large bore needle may result in a much faster and more forceful flow of blood through the needle, resulting in hemolysis. Syringe Draws Pulling the plunger of a syringe back too far while using a large bore needle, may cause enough pressure for hemolysis to result during collection. The pressure may be greater than a standardized evacuated tube.

24. Transferring into a tube by pushing down on the syringe plunger in order to force blood into a tube may cause hemolysis. Vigorous mixing or shaking of a specimen may cause hemolysis. Prolonged contact of serum or plasma with cells may result in hemolysis.

25. Exposure to excessive heat or cold can cause RBC rupture and hemolysis. Specimen Transport: Mechanical trauma during transport may occur.

26. Redraw the specimen. The most common sites to draw from are the median cubital, basalic, and cephalic veins from the antecubital region of the arm. Avoid using a needle that is too small or too large. The tourniquet should be released after no more than one minute, and excessive fist clenching should be avoided.

27. Without touching, allow the Venepuncture site to air dry before collecting blood. Avoid drawing the syringe plunger back too forcefully when collecting blood with a needle and syringe.

28. Avoid pushing the plunger too forcefully when transferring to a tube. Ensure all blood collection assemblies are fitted securely. Gently invert the blood collection tube and mix additive specimens thoroughly according to manufacturers recommendations.

29. Almost 1hr not much changes takes place regarding cell morphology. By 3hr changes may be discernible. By 12-18 hrs striking changes that includes Neutrophils nuclei stain homogenously Nuclear lobes may become separated Vacuole appear in cytoplasm Ragged cytoplasmic membranes RBCs show progressive crenation & sphering.

30. Neutrophils nuclei stain homogenously Nuclear lobes may become separated RBCs show progressive crenation & sphering Homogeneous staining

31. Cell counts are affected by The state of the blood circulation, Nutrition Time & condition of pt If strictly comparable values are required, there should also be half an hour of bed rest before the sample is drawn, Color Atlas of Hematology Practical Microscopic and Clinical Diagnosis Harald Theml,M.D.

32. Agents which prevent the coagulation of blood are called as anticoagulants.

33. EDTA (Ethylene di amine tetra Acetic acid) Tri-sodium Citrate Heparin Double oxalate Sodium fluoride

34. It is the most commonly used anticoagulant in routine practice. The potassium & sodium salts of EDTA are powerful anticoagulants. Mechanism Of Action: It acts by chelating the calcium molecules in the blood. 1.2 mg of EDTA is required for each ml of blood to get the desired results. EDTA is used for mainly Blood counts.

35. COMPOSITION : EDTA Dipotassium salt -44.5 g EDTA Disodium salt 41.0 g 1mmol NaOH-75 ml Water 1 liter.

36. ADVANTAGES : It is the anticoagulant of choice in routine hematological work. Best for platelet counts. DISADVANTAGES: RBC morphology is hampered if the concentration is more than the required. Not good for coagulation studies.

37. It is used for coagulation studies. Mechanism of action : It also works on the principle of calcium chelation. For ESR estimation, 4 volumes of blood are added to 1 volume of sodium citrate solution & well mixed.

38. Having pH 6.9. Trisodium citrate, dihydrate( 102 mmol / l) - 30 g Sodium dihydrogen phosphate, monohydrate ( 1.08 mmol / l ) - 0.15 g Dextrose ( 11 mmol / l ) - 2 g Water - 1 litre

39. ADVANTAGES : Tri-sodium citrate is used for coagulation studies Used in blood banking and blood transfusion purposes.(if citrate phosphate dextrose is not available )

40. It is used for chemistry , blood gas analysis & emergency tests. It is the best anticoagulant for osmotic fragility tests & immunophenotyping. The lithium or sodium salt of heparin at a concentration of 10- 20 IU / ml blood is used generally. It is not suitable for blood counts as it induces platelet & leucocytes clumping & gives faint blue color on Peripheral Smear.

41. It acts by forming complex with calcium i. e. calcium fluoride. It prevents glycolysis by blocking phosphorylase enzyme in red cells. It is the ideal additive used for blood sugar estimation. Precaution should not delay the investigation.

42. 1. Loss of CO2- it diffuse from plasma to air. 2. Conversion of glucose into lactic acid by glycolysis. 3. Formation of NH3 from nitrogenous substance like urea. 4. Passage of substances through the red cell membrane. 5. Conversion of pyruvate to lactate.

43. RBC morphology is hampered if the concentration is more than the required in case of EDTA. Heparin is not suitable for blood counts as it induces platelet & leucocytes clumping & gives faint blue color on Peripheral Smear. Incorporation of dust particles may takes place if the anticoagulants are not stored properly.

44. Clinical Significance: Hemoglobin concentration is always as a part of routine blood test. Part of complete blood test Increase in amount of Hb is Polycythemia Decrease in amount of Hb is Anemia

45. 1. Acid- Hematin (Sahli) method 2. Colorimetric or Spectrophotometric method 3. Oxyhaemoglobin (HbO2) Method 4. Talliqvist method. 5. Chemical method.

46. One of the most commonly used technique. Simple, cost effective.

47. Why so Hemoglobinmeter is used ??? 1. Ability of Hb to combine with oxygen. 2. Presence of known amount of iron in each gram of Hb. 3. Ability of a solution of a derivative of Hb to refract specific wavelengths of light , thus giving typical absorption bands

48. The Hb present in a measured amount of blood is converted by dilute HCL into acid hematin. Which in dilution is golden brown in color . The intensity of color depends on the concentration of acid hematin. Which in turn depends on concentration of Hb. The color of solution is matched against golden brown tinted glass rods by direct vision. The readings are obtained against in gm%.

49. Hb Acid Hematin Acid Hematin brown color Standard HCl

50. Capillary blood Thoroughly mixed anticoagulated (EDTA or Double oxalate) venous blood. Amount 0.02ml.

51. Pour 1/10 HCl into Hb tube. Specimen collected by Hb pipette. Pour into Hb tube. Wait for 10 min Add distilled water drop by drop Till it mach with standard. The reading on Hb tube is considered as concentration of Hb.

52. Normal Values : Hb, g/dl - MEN 13-18 - WOMEN 12-16.5 - CHILDREN (up to 1 year) 11.0-13.0 - CHILDREN (10-12 years) 11.5-14.5

53. Simple fairly accurate. Less time consuming Cost effective

54. Acid hematin is not a true solution, some turbidity may occur. Measures only oxy Hb & reduced Hb. Other forms such as carboxy Hb & met Hb are not established.

55. 1. Faulty instruments Hb pipette- blockage, wet pipette. Hb tube dirty tube. Hemoglobinmeter- Defective or improper comparator.

56. 1. Not taking exact amount of blood 2. Not cleaning the outer surface of Hb-tube. 3. Not giving the enough time or giving more than required time to react with HCl. 4. Visual error 5. Adding more amount of distilled water. 6. During reading keeping stirrer in the Hb tube.

57. Cyanmethaemoglobin Method Tallqvist Method Oxyhaemoglobin method Alkaline Haematin method Chemical method

58. This is most accurate & most wildly used method of estimation of Hb. It is internationally recommended method for best quality control.

59. BLOOD + POTASSIUM CYANIDE, ferricynide = Methaemoglobin & carboxyhaemoglobin Then , cyanmethaemoglobin Absorbance of solution is measured in photoelectric calorimeter at wavelength of 540 nm.

60. Reagent 1. Drabkins Cyanide- ferricynide solution. Composition Pot. ferricyanide 200mg Pot. Cyanide -- 50 mg Sod bicarbonate 1gm Distilled water -- 1000ml

61. Cyanide is poisonous Highly recommended to use the rubber teat . Turbidity may cause faulty results.

62. Take 0.02 ml of blood Mix with 4ml of 0.04% liquor ammonia The formed solution is matched in a colorimeter at 540nm

63. Common sources of error in measuring hemoglobin include anything that will cause turbidity and interfere with a Spectrophotometric method. Examples are a very high WBC or platelet count and hemoglobin's that are resistant to lysis.

64. Iron content of Hb is 0.347 % Total iron content of blood is estimated chemically & Hb content is found out by dividing result by 3.47.

65. A drop of blood collected finger is absorbed on a piece of blotting paper Dried for a few seconds Compared with the references standard of color paper made by manufacturer Used in survey purpose.

66. Visual error Subjective variability Results are not reliable If figure is squeezed then false results

67. Can be measured by Two methods which includes Counting RBCs by manual method Electronic cell counter

68. Male 5.0 +/- 0.5 x 1012/l 4.5 to 5.5 million / cb. mm Female 4.3+/- 0.5 x 1012/l 3.8 to 4.8 million / cb. mm

69. Principle The blood is diluted 200 times in the red pipette and the cell are counted in the counting chamber, knowing the dilution employed, their number in undiluted blood can easily be calculated.

70. RBC pipette Neubauer chamber

71. 1. NaCl - 0.05gm- Maintain tonicity 2. Na2SO4- 2.05gm -Anticoagulant 3. HgCl2- 0.25g antifungal 4. Distilled water 100ml- medium for solution.

72. Position the Neubauer chamber over microscope. Finger prick 0.5ml of blood sample Hayems fluid up to 101mark Shake the RBC pipette Discard 2-3 drops Load on Neubauer chamber. Wait for 2-3min Counting of RBCs That is multiplied by the 10,000

73. 1mm 1mm Area= 1X1=1mm, Total nos of squre 400. area of each squre= 1/400mm.

74. Area= 1X1=1mm, Total nos of squre 400. Area of each squre= 1/400mm.

75. Area of smallest square= 1/400 sq mm Depth= 0.1mm Vol of smallest square= 1/4000cb mm Nos of RBCs counted are in 80 square Hence 80 X 1/4000= 1/50. Hence nos of RBCs in 1cb mm vol is = 50 X 200 X nos conted. i.e. 10,000X nos counted.

76. Very high WBC High concentration of very large platelets Agglutinated RBCs, Roulex formation Cell fragments or other debris

77. Inaccurate amount of blood taken. Inaccurate amount of fluid Mannual error during taking fluid Improper mixing Improper loading improper distribution Counting errors Calculation error Improper instruments

78. Clinical Significance: 1. Increase in Total leukocyte count of more than 10,000/cu mm is known as leukocytosis . 2. Decrease or less than 4,000/cu mm as leucopenia.

79. - Adults : 4,000-10,000/cu mm - At Birth : 10,000-25,000/cu mm - 1 to 3 years : 6,000-18,000/cu mm - 4 to 7 years : 6,000-15,000/cu mm - 8 to 12 years : 4,500-13,500/cu mm

80. The Glacial acetic acid lyses the red cells The gentian violet slightly stains the nuclei of the leukocytes. The blood specimen is diluted 1:20 in a W.B.C pipette with the diluting fluid. The cells are counted under low power of the microscope by using a counting chamber. The number of cells in a undiluted blood are reported per cu mm of whole blood

81. 1) Double oxalated or EDTA blood 2) Capillary blood.

82. 1. Glacial acitic acid (hemolysis of RBCs without affecting WBCs) 2. Gention violet (stains the nuclei of leucocytes) 3. Distilled water act as medium.

83. Position the Neubauer chamber over microscope. Finger prick 0.5ml of blood sample Turks (WBC) fluid up to 11mark Shake the WBC pipette Discard 2-3 drops Load on Neubauer chamber. Wait for 2-3min Counting of WBC in (X) That is multiplied by the 50

84. Inaccurate amount of blood taken. Inaccurate amount of fluid Improper mixing Improper loading improper distribution Counting errors Calculation error Improper instruments

85. Unusual RBC abnormalities that resist lysis Nucleated RBC Fragmented WBCs Unlysed particle Specimen containing fibrin, cell fragmnets or other debris

Artefaccts in hemat part 1

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