Tlc dlc

Useful for MBBS students

1. Total leucocyte count Total differential count Dr Neha Mahajan MD Pathology

2. Haematopoeisis

3. G R A N U L O P O E I S I S

4. Type of WBC’s Granulocytes—have large granules in their cytoplasm Neutrophils ( 40 to 75%) Eosinophils (1 to 6%) Basophils (0 to 1%)

5. Types of WBC’s Agranulocytes —do not have granules in their cytoplasm Lymphocytes(20 to 40%) Monocytes ( 2 to 10%)

6. Granuloctyes Neutrophils Stain light purple with neutral dyes Granules are small and numerous—course appearance Several lobes in nucleus 65% of WBC count Diapedesis,inflammation

7. Granulocytes Eosinophils or Acidophils : Large, numerous granules Nuclei with two lobes 2-5% of WBC count Found in lining of respiratory and digestive tracts Protections against infections caused by parasitic worms and involvement in allergic reactions Secrete anti-inflammatory substances in allergic reactions

8. Basophils Least found- 0.5 to 1% Contain histamine,serotonin,heparin —inflammatory chemical Granulocytes

9. Agranulocytes Lymphocytes Smallest WBC Large nuclei/small amount of cytoplasm Account for 25% of WBC count Two types—T lymphocytes—attack an infect or cancerous cell, B lymphocytes—produce antibodies against specific antigens (foreign body)

10. Agranulocytes Monocytes Largest of WBCs Dark kidney bean shaped nuclei Highly phagocytic

11. Total leucocyte count

12. White cell count (WBC) White cell count (WBC) is the total number of leukocytes in a volume of blood, expressed as thousands/µl. WBC can be done by manual methods or by automated cell counters. Normal Values: Newborn 9.0-30.0 x 103/ μl 1 week 5.0-21.0 x 103/ μl 1 month 5.0-19.5 x 103/ μl 6-12 months 6.0-17.5 x 103/ μl 2 years 6.2-17.0 x 103/ μl Child/adult 4.8-10.8 x 103/ μl

13. Principle of WBCs count test Free-flowing capillary or well-mixed anticoagulated venous blood is added to a diluent ) at a specific volume in the thoma pipette. The diluent lyses the erythrocytes but preserves leukocytes and stains the nuceli . The diluted blood is added to the hemacytometer chamber.

14. Specimen: EDTA- anticoagulated blood or capillary blood is preferred . Reagents, supplies and equipment: White blood cells count diluting fluid Turks' solution which is formed of: Glacial acetic acid 3 ml Crystal violet 1 ml 100 ml distilled water.

15. Equipment White blood cells count diluting fluid Thoma white pipette Hemacytometer and coverslip Microscope Lint-free wipe Alcohol pads

16. haemocytometer chamber Thoma white pipette Rubber sucking tube

17. Hemacytometer The hemacytometer counting chamber is used for cell counting. It is constructed so that the distance between the bottom of the coverslip and the surface of the counting area of the chamber is 0.1 mm. The surface of the chamber contains two square ruled areas separated by an H-shaped moat.

18. Hemacytometer

19. Procedure Draw the blood up to 0.5 mark in the thoma pipette. Wipe the outside of the capillary pipette to remove excess blood that would interfere with the dilution factor. Holding the pipette almost vertical place into the fluid. Draw the diluting fluid into the pipette slowly until the mixture reaches the 11 mark, while gently rotating the pipette to ensure a proper amount of mixing. Place the pipette in a horizontal position and firmly hold the index finger of either hand over the opening in the tip of the pipette, detach the aspirator from the other end of the pipette now the dilution of the blood is completed

20. Procedure Mix the sample for at least 3 minutes to facilitate hemolysis of RBCs. Clean the hemacytometer and its coverslip with an alcohol pad and then dry with a wipe. Before filling the chamber, discard the first four to five drops of the mixture on apiece of gauze to expel the diluent from the stem.

21. Procedure Carefully charge hemacytometer with diluted blood by gently squeezing sides of reservoir to expel contents until chamber is properly filled.

22. Procedure for counting WBC’s Under 10 x magnifications, scan to ensure even distribution. Leukocytes are counted in all 4 large squares of counting chamber. Count cells starting in the upper left large corner square. Move to the upper right corner square, bottom right corner square, bottom left corner square and end in the middle square. Count all cells that touch any of the upper and left lines, do not count any cell that touches a lower or right line.

23. Calculations Dilution factor 20 Volume= Area x depth TLC/ uL = No of WBCx Correction for Volume X dilution No of large squares (4) = N x 20 x10 4 = N x 50

24. Corrected TLC Diluting fluid does not lyse nucleated RBC`s/erythroblasts Falsely counted as WBC Corrected TLC/ uL = TLC x 100 NRbc /100 Wbc + 100

25. White Blood Cell Differential Count

26. Definition The relative percentage of each type of white blood cells in peripheral blood. This experiment is a part of blood routine test.

27. PERIPHERAL BLOOD SMEAR A properly prepared blood smear is essential to accurate assessment of cellular morphology The wedge smear is the most convenient and commonly used technique for making PBS

28. PERIPHERAL BLOOD SMEAR Wedge technique of making PBS A. Correct angle to hold spreader slide B. Blood spread across width of slide C. Completed wedge smear

29. PERIPHERAL BLOOD SMEAR Characteristics: It is smooth without irregularities, holes, or streaks When the slide is held up to light, the featheredge of the smear should have a “rainbow” appearance The whole drop is picked up and spread Well-made PBS

30. tail body head

31. PERIPHERAL BLOOD SMEAR Examples of unacceptable smears

32. PERIPHERAL BLOOD SMEAR Examples of unacceptable smears

33. Observing direction: Observe one field and record the number of WBC according to the different type then turn to another field in the snake-liked direction * avoid repeat or miss some cells

34. Romanowsky staining Leishman's stain : a polychromatic stain Methanol : fixes cells to slide methylene blue stains RNA,DNA blue-grey color Eosin stains hemoglobin, eosin granules orange-red color pH value of phosphate buffer is very important 42

35. Procedure Thin smear are air dried. Flood the smear with stain. Stain for 1-5 min. Experience will indicate the optimum time. Add an equal amount of buffer solution and mix the stain by blowing an eddy in the fluid. Leave the mixture on the slide for 10-15 min. Wash off by running water directly to the centre of the slide to prevent a residue of precipitated stain. Stand slide on end, and let dry in air. 43

36. Features of a well-stained PBS Macroscopically: color should be pink to purple Microscopically: RCS: orange to salmon pink WBC: nuclei is purple to blue cytoplasm is pink to tan granules is lilac to violet Eosinophil: granules orange Basophil: granules dark blue to black 44

37. Optimal Assessment Area: RBCs are uniformly and singly distributed Few RBC are touching or overlapping Normal biconcave appearance 45

38. Principle White Blood Cells Check for even distribution and estimate the number present (also, look for any gross abnormalities present on the smear). Perform the differential count. Examine for morphologic abnormalities. 46 Manual differential

39. Red Blood Cells, Examine for : Size and shape ( Anisocytosis,Poikilocytosis Relative hemoglobin content. Polychromatophilia . Inclusions.   Rouleaux formation or agglutination 47

40. WBC estimation Under 40X Using the × 40 high dry with no oil. Choose a portion of the peripheral smear where there is only slight overlapping of the RBCs. To do a WBC estimate by taking the average number of white cells and multiplying by 2000. 48

41. Platelet estimation Under 100X Use the oil immersion lens estimate the number of platelets per field. Look at 5-6 fields and take an average. Multiply the average by 20,000. Note any macroplatelets. 49

42. Platelets per oil immersion field (OIF) <8 platelets/OIF = decreased 8 to 20 platelets/OIF = adequate >20 platelets/OIF = increased 50

43. Manual differential counts These counts are done in the same area as WBC and platelet estimates with the red cells barely touching. This takes place under × 100 (oil) using the zigzag method. Count 100 WBCs including all cell lines from immature to mature. Reporting results Absolute number of cells/µl = % of cell type in differential x white cell count 51

44. Microscopic exam 10× (low fold): overall smear quality, rouleaux , agglutination or parasites 100× (oil Len): WBC Diff, RBC morphology RBC Morphology WBC- Total count Differential count Platelets Abnormal cells Parasites Peripheral smear reporting

45. Normal blood smear 53

46. normal Neutrophil count (40 to 75%) Neutrophilia 54 Absolute neutrophil count greater than 7500/ uL Causes Acute bacterial infections- abscesses,pneumonia,meningitis,UTI Tissue necrosis- burns,injury,MI Acute blood loss Acute haemorrhage Myeloproliferative disorders Posisoning

47. Neutropenia Absolute netrophil count less than 2000/ uL Mild- 2000 to 1000/ uL Moderate-1000 to 500/ uL Severe- < 500/ uL CAUSES I )Decreased or ineffective production in bone marrow Infections- bacterial,protozoal,viral Haematologic disorders- megaloblastic anemia,aplastic anemia,aleukemic leukemia Drugs Ionising radiation Congenital disorders

48. II) Increased destruction in peripheral blood Neonatal isoimmune neutropenia Systemic lupus erythematosus Felty`s syndrome III) Increased sequestration in spleen Hypersplenism

49. Eosinophila Absolute eosinophil count greater than 600/ uL CAUSES 1.Allergic diseases- Asthma,rhinitis,urticaria 2.Skin diseases- Eczema,pemphigus,Dermatitis herpetiformis 3.Parasitic infections with tissue invasion- filariasis,trichinosis,echinoccocoosis 4.Hematologic disorders- MPD,Hodgkins disease,Peripheral T cell lymphoma

50. 5.Carcinoma with necrosis 6.Radiation therapy 7.Lung diseases- loefflors syndrome,tropical eosinophilia 8.Hypereosinophilia syndrome

51. Monocytosis Increase in absolute monocyte count > 1000/ uL Causes Infections- TB,SABE,Malaria,Kala Azar Recovery from neutropenia Autoimmmune disorders Hematologic diseases- MPD,Monocytic leulemia,hodgkins disease Others-ulcerative colitis,chron`s disease,sarcoidosis

52. LYMPHOCYTOSIS Absolute lymphocyte count more than upper limit of normal for age( 4000/ uL in adults,>7200/ uL in adolescents,>9000/ uL in children and infants) Causes Infections- Viral- acute infectious lymphocytosis,neoatitis,CMV,rubella,mumps,varicella Bacterial- pertussis,TB Protozoal - toxoplasmosis

53. Hematological disorders- ALL, CLL, Multiple myeloma, Lymphoma Others- Serum sickness, post vaccination, drug reactions

54. Platelets Small,1 to 3 um in diameter,purple structures with tiny irregular projections on surface Occur in clumps Pseudothrombocytopenia Platelet sateletism

55. Summary Normal haematopoeisis Total leucocyte Count Manual differential count Peripheral smear Interpretation

56. Thank you