HAEMOCYTOMETRY+TLC (2).pptx demonstration

ppt TLC

1. HAEMOCYTOMETRY 1

2. WHAT IS HAEMOCYTOMETRY ? It is a technique used to enumerate the total cell count in the BLOOD or other Biological body fluids. This can be done either by using H aemocytometer or by Electronic cell counter. 2 Hemo : blood Cyto : cell Meter : measurement/counter Thus, it is an instrument used to count the blood cells.

3. 3 Since the No. of blood cells is very high so it is difficult to count them even under microscope. This difficulty is partly overcome by diluting the blood to a known degree with a suitable diluting fluids and then counting them. The sample of blood is diluted in a special pipette and the diluted blood drop is then placed under cover slip placed over special thick glass slide i.e. counting chamber. The drop of diluted blood spread under cover slip in single layer and make the counting of cells easy . Knowing the dilution employed the no. of cells in a undiluted blood can be calculated and the result so obtained is expressed as cells per cubic mm. PRINCIPLE OF CELL COUNTING

4. Broadly , The cell count is done To find out normal and abnormal count of the cells To support and confirm clinical diagnosis of the patient . To find out the response of the patient to the treatment . 4 In certain pathological conditions the value of different type of cells may have the variation. Thus by counting the cells in the blood or body fluids , it can be find out if an individual is normal or not . PURPOSE

5. APPARATUS REQUIRED Hemocytometer, diluting fluids, spirit swabs, pricking needle and compound microscope. HAEMOCYTOMETER It consist of a special instrument called Neubauer’s counting chamber, cover slips, pipette (RBC pipette & WBC Pipette)for diluting the blood with rubber tube and plastic mouth piece for drawing blood or fluid in pipette. The hemocytometer was invented by Louis-Charles Malassez and modified by Neubauer and now a days improved Neubauer counting chamber is used . It is a  special type of  microscope slide consisting of two chambers This is an instrument used for counting the cells in blood or fluid. 5

6. Counting Chamber WBC pipette RBC pipette Cover slip

7. COUNTING CHAMBER The center of which has double ruling area separated by troughs (these four troughs are extending across the slide and set parallel to each other. The fifth one is separating the two ruling areas from each other). It is also named as Improved Neubauer counting chamber. The glass cover slip is held at 0.1 mm above the surface of the counting areas by ground glass ridges on either side of the vertical grooves of the H shape. The depth of the scales is 1/10mm or 0.1mm. 7

8. COUNTING CHAMBER 8 Four vertical troughs One Horizontal trough Counting Grid Area

9. NEUBAUER’S CHAMBER Neubauer’s slide with a cover slip over it, is called a Neubauer’s chamber.

10. 10 Counting Grid Areas on central platform of chamber Counting Grid Each scale is 3mm wide and 3mm long. The whole scale is divided into 9 big squares. Each square is 1mm long and 1mm wide.

11. IMPROVED NEUBAUER CHAMBER COUNTING AREA The ruled area of the hemocytometer consists of 9 large square. With (1x 1mm) 1mm² area of each square, separated by triple lines. 11

12. W W W W The four corner squares are further divided into sixteen smaller squares and are used for WBC counting. Total = 64 small squares

13. 14 The central square is subdivided into twenty five (25) smaller squares and each of these smaller squares is further subdivided into sixteen smallest squares. These are meant for platelet and RBC counting. The platelets are counted in all the small squares of the central square, while the RBCs are counted in five small squares, four of corners and one of center. (total of 80 smallest squares)

14. 15 R R R R R

15. 16

16. 17

17. RBC PIPETTE It has a red bead It has graduations from 0 to mark 101 Size of bulb is larger Size of lumen is smaller Mouth piece is red in color WBC PIPETTE It has a white bead It has graduations from 0 to mark 11 Size of bulb is smaller Size of lumen is larger Mouth piece is white in color DIFFERENCES BETWEEN RBC AND WBC PIPETTE

18. 4X to see the general formation of slide. 10X for WBC counting 40X for RBC counting FOCUSING

19. Counting Rule Count the cells touching the triple lines of the left side and on the top of the square in the same square. Count the cells touching Bottom line, Right line in adjacent square. This is to avoid double counting.

20. 4x magnification 1 mm

21. 10x magnification 0.2 mm

22. 40x magnification

23. PY 2.11 (4) TOTAL LEUCOCYTE COUNT

24. PY2.11(4) (a) Describe the clinical importance of estimation of TLC count PY2.11(4) (b) Identify the WBC pipette and justify PY2.11(4) (c) Demonstrate the dilution of blood for TLC count PY2.11(4) (d) Demonstrate charging of Neubauer’s chamber for TLC count PY2.11(4) (e) Demonstrate the adjustments of microscope for focusing neubauer’s chamber to do TLC count PY2.11(4) (f) Perform total TLC count PY2.11(4) (g) List the precautions for collection of blood, diluting blood sample and charging the chamber PY2.11(4) (h) Enumerate the contents of diluting fluid and mention the function of each constituents PY2.11(4) ( i ) Enumerate the functions of WBCs PY2.11(4) (j) List normal values of WBCs in new born, children and adult male and female and justify the variation in each PY2.11(4) (k) Explain the variations in leucocyte count in different physiological and pathological conditions PY2.11(4) (l) Define leukemia and enumerate causes for it.

25. COUNTING CHAMBER 33 Four vertical troughs One Horizontal trough Counting Grid Area

26. Causes of elevated WBC (Leukocytosis) Infections – most common is bacterial infections It also occur in viral (lymphocytosis) Allergy and drug hypersensitivity Parasitic infections Inflammation: eg . Inflammatory bowel disease, RA, and vacuities Extremely low birth weight Malignancy and myelo-sproliferative disorders: eg . Leukemia's, lymphomas Increased release of WBC from bone marrow:- This occurs in infection, stress, and hypoxia it also occurs due to endotoxin stimulation and steroid administration

27. Causes of low WBC (Leukopenia) Viral infections – eg . HIV, influenza etc. Medications – cytotoxic drugs Chemotherapy/Radiation therapy Malnutrition Non pyogenic bacterial infection- eg . Typhoid fever. Aplastic anemia