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  2. 2. WHAT ISHAEMOCYTOMETRY ? 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 Haemocytometer or by Electronic cell counter. 2
  3. 3. PURPOSE 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 . 3
  4. 4. Broadly , The cell count isdone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
  5. 5. PRINCIPLE OF CELLCOUNTINGThe blood is diluted with appropriate known volume of diluting fluid and then counting is done by using haemocytometer . 5
  6. 6. HAEMOCYTOMETER This is an instrument used for counting the cells in blood or fluid. It consist of a special instrument called counting chamber, cover glass, pipette for diluting the blood, rubber tube with plastic mouth piece for drawing blood or fluid in pipette. 6
  7. 7. COUNTING CHAMBER It is a thick glass slide, 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). 7
  9. 9. There are some diff. type of counting chambers :-1.Old neubauer counting chamber2.Improved neubauer counting chamber3.Burker counting chamber4.Fuch’s rosenthal counting chamber. 9
  10. 10. OLD central platform is set 0.1 mm. below theIn this the NEUBAUER CHAMBERlevel of the two side, which giving the chamber a depthof 0.1 mm. The ruling covers an area of 9 sq.mm.divided into 9 squares of 1 sq.mm. each. The fourcorner squares are subdivided into 16 squares , eachwith an area of 1/16 of a sq.mm. The central ruled areaof 1sq.mm. is divided into 16 larger squares by set oftriple lines. These large squares are further subdividedinto 16 small squares by single lines. 10
  11. 11. IMPROVED NEUBAUERCHAMBER In this the triple lines which dividing the central large square are very much closer to each other. The central ruled area is divided into 25 large squares. These squares are subdivided to form 16 smaller squares each with an area of 1/400 of 1 sq.mm. The depth of improved neubauer chamber is same i.e. 0.1mm. 11
  12. 12. OLD v/s IMPROVED NEUBAUER The space occupied by the triple lines in old neubauer chamber being used to produce extra large squares. In old neubauer chamber the gap b/w triple lines was very wide and the rectangular space b/w them looks as similar as the squares in which cells are to be counted. This makes the count very difficult and chances of error was very high. 12
  13. 13. Cont.  In old neubauer chamber the lines were very dull and some times it was very difficult to recognize them.  BUT, In improved neubauer chamber these all faults are removed.  It’s triple lining are very closer to each other ,these are dark and can easily recognize.  By dividing central square in 25 squares the RBC and Platelet count is become easy to do. 13
  16. 16. BURKER COUNTING CHAMBERLike the neubauer counting chamber , this has a ruledarea of 9 sq.mm. and a depth of 0.1mm. , but it’ssmaller squares are divided by double lining not bysingle one. 16
  18. 18. FUCH’s ROSENTHAL CHAMBERThis chamber was originally designed forcounting cells in CSF ,but as such a relativelylarge area is covered , it is preferred by someworkers for counting blood cells. The depth ofthis chamber is 0.2mm. and the ruled areaconsist of 16mm squares divided by triple lines.These squares are subdivided to form 16 smallersquares , each with an area of 1/16 of 1 sq.mm. 18
  20. 20. COVER GLASSA special cover glass is used which has a very smooth , flattened surface and even thickness.Different Thicknesses are :0.3mm0.4mm (most common)0.5mmTwo sizes are common:-16x22 sq. mm 22x23 sq. mm 20
  21. 21. TOTAL WBC COUNTDiluting fluid – Tuerk fluid Glacial acetic acid – 2ml Gentian violet – about a pinch Distilled water – 97mlSolution is stable at room temp. 21
  22. 22. PROCEDURE Take 950 µl diluting fluid in a clean, dry test tube. Add 50 µl anticoagulated blood sample and mix. Keep for 5 min. at room temp. Mix and fill the chamber with the help of pipette. Count the cells using low power(10x) objective. Cells in 4 large corner squares are to be counted. Count the cells touching the triple lines of the left side and on the top of the square. 22
  23. 23. As shown in the picture 23
  24. 24. CalculationTLC = No. of cell counted x dil. Factor Volume= n x 20/0.4=n x 20 x 10/4= n x 50/cumm.Normal range= 4000 to 11000 per cumm. 24
  25. 25. InterpretationLeucocytopenia ) Decrease (Increase (Leucocytosis)  Bacterial infections(typhoid) Muscular exercise  Viral infection ( Influenza ) High temp.  Protozoal infection(Maleria) Severe pain  Megaloblastic anemia Bacterial or viral infections  Bone marrow depression as in Leukemia aplastic anemia , drugs , Severe hemorrhage radiation etc. 25
  26. 26. NOTEWhile performing RBC count by thistechnique , possibilities of error is very high. So, this technique is now not in use forRBC count. 26
  27. 27. PLATELET COUNTDiluting fluid : 1% Ammonium oxalate.Principle –: 1% Ammonium oxalate is isotonic to platelets and lyses the RBC. WBC remains but they are less in count so they do not interfere in counting the platelets. 27
  28. 28. PROCEDURE Take 950 µl diluting fluid in a clean, dry test tube. Add 50 µl anticoagulated blood sample and mix. Keep for 5 min. at room temp. Mix and fill the chamber with the help of pipette. Keep the charged chamber in moist petridish for 5 to10 min. After that wipe the back of chamber and count the cells at high power (40x) objective . Platelets are also counted in the same squares RBC used to be count. 28
  29. 29. CALCULATIONArea of chamber counted=5x1/25=1/5sq.mm.Depth=0.1mmTotal volume=1/5x0.1=1/50cu.mm.Dilution=1/20Total no. of platelets per cumm==no. of platelet counted/volume x dilutionn/1/50 x 1/20=n x50x20=n x 1000 per cummNormal range=1.5 to 4.0x1000per cumm 29
  30. 30. INTERPRETATION Increase Decrease (Thrombocytosis) (Thrombocytopenia) Polycythemia  Acute leukemia Chronic granulocytic  Pancytopenia as in anemia aplastic anemia After surgery  Liver disease Immediately after  Metal poisoning hemorrhage.  Megaloblastic anemia 30
  31. 31. ABSOLUTE EOSINOPHIL COUNT Diluting fluid = Dunger’s fluid Eosin Yellow = 0.5 g. 95% acetone = 0.5ml. 40%formalin = 0.5 ml. Distilled water = 99 ml. 31
  32. 32. PRINCIPLEBlood is diluted with special diluting fluid whichstains the eosinophilic granules brightly and distinctly.At the same time it lyses the RBC and other WBC. 32
  33. 33. PROCEDURE Take 950 µl diluting fluid in a clean, dry test tube. Add 50 µl anticoagulated blood sample and mix. Keep for 5 min. at room temp. Mix and fill the chamber with the help of pipette. Keep the charged chamber in moist petridish for 5 to10 min. Count the eosinophil under low power(10x)with reduced light. Count in 4 corner squares of the Improved counting chamber . 33
  34. 34. CALCULATIONAEC=Total cell counted x dil. Factor/volume=N x 20/0.4=N x 20 x 10/4=N x 50/cumm 34
  35. 35. INTERPRETATIONIncrease in Allergic conditions Parasitic infection especially in helminths Hyperadrenalism Leukemia Aplastic anemia 35
  36. 36. PRECAUTIONS The preparation of diluting fluids must be proper. Always clean the chamber before and after use. After taking blood in pipette, clean the outer surface of tip before diluting it in the diluting fluid. Always mix the dilution before filling the chamber. 36
  37. 37. Cont.. Avoid bubbles to come in the chamber. Never over flow the chamber with dilution. For decrease the error more cells must be count. Change the cover glass if dirty and scratched. Calculation must be done properly. Clean the microscope before and after every use. 37
  38. 38. THANK YOU 38