1. HAEMOCYTOMETRY 1

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. 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. Broadly , The cell count isdoneTo 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. PRINCIPLE OF CELLCOUNTINGThe blood is diluted with appropriate known volume of diluting fluid and then counting is done by using haemocytometer . 5 6. HAEMOCYTOMETER This is an instrument used for counting the cells inblood or fluid. It consist of a special instrument called countingchamber, cover glass, pipette for diluting the blood,rubber tube with plastic mouth piece for drawingblood or fluid in pipette.6 7. COUNTING CHAMBER It is a thick glass slide, center of which has doubleruling area separated by troughs (these four troughsare extending across the slide and set parallel to eachother. The fifth one is separating the two ruling areasfrom each other).7 8. COUNTING CHAMBER 8 9. There are some diff. type of counting chambers :-1.Old neubauer counting chamber2.Improved neubauer counting chamber3.Burker counting chamber4.Fuchs rosenthal counting chamber. 9 10. OLD central platform is set 0.1 mm. below theIn this theNEUBAUER 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. IMPROVED NEUBAUERCHAMBERIn 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. OLD v/s IMPROVED NEUBAUER The space occupied by the triple lines in oldneubauer chamber being used to produce extralarge squares. In old neubauer chamber the gap b/w triple lineswas very wide and the rectangular space b/wthem looks as similar as the squares in whichcells are to be counted. This makes the count verydifficult and chances of error was very high. 12 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. Its 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 14. OLD NEUBAUER CHAMBER 14 15. IMPROVED NEUBAUERCHAMBER15 16. BURKER COUNTING CHAMBERLike the neubauer counting chamber , this has a ruledarea of 9 sq.mm. and a depth of 0.1mm. , but itssmaller squares are divided by double lining not bysingle one.16 17. BURKER COUNTING CHAMBER17 18. FUCHs 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 19. FUCHs ROSENTHAL CHAMBER 19 20. COVER GLASSA special cover glass isused which has a verysmooth , flattenedsurface and eventhickness.Different Thicknesses are :0.3mm0.4mm (most common)0.5mmTwo sizes are common:-16x22 sq. mm 22x23 sq. mm20 21. TOTAL WBC COUNTDiluting fluid Tuerk fluidGlacial acetic acid 2mlGentian violet about a pinchDistilled water 97mlSolution is stable at room temp. 21 22. PROCEDURE Take 950 l diluting fluid in a clean, dry test tube. Add 50 l 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 sideand on the top of the square. 22 23. As shown in the picture23 24. CalculationTLC = No. of cell counted x dil. FactorVolume= n x 20/0.4=n x 20 x 10/4= n x 50/cumm.Normal range= 4000 to 11000 per cumm.24 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 Leukemiaaplastic anemia , drugs , Severe hemorrhage radiation etc.25 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. PLATELET COUNTDiluting fluid : 1%Ammonium oxalate.Principle : 1% Ammoniumoxalate is isotonic toplatelets and lyses the RBC.WBC remains but they areless in count so they do notinterfere in counting theplatelets. 27 28. PROCEDURE Take 950 l diluting fluid in a clean, dry test tube. Add 50 l 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 to10min. After that wipe the back of chamber and count the cellsat high power (40x) objective . Platelets are also counted in the same squares RBC usedto be count.28 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. INTERPRETATIONIncreaseDecrease(Thrombocytosis) (Thrombocytopenia) Polycythemia Acute leukemia Chronic granulocytic Pancytopenia as inanemia aplastic anemia After surgery Liver disease Immediately after Metal poisoninghemorrhage. Megaloblastic anemia30 31. ABSOLUTE EOSINOPHIL COUNT Diluting fluid = Dungers fluid Eosin Yellow = 0.5 g. 95% acetone = 0.5ml. 40%formalin = 0.5 ml. Distilled water = 99 ml. 31 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. PROCEDURE Take 950 l diluting fluid in a clean, dry test tube. Add 50 l 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 to10min. Count the eosinophil under low power(10x)with reducedlight. Count in 4 corner squares of the Improved countingchamber . 33 34. CALCULATIONAEC=Total cell counted x dil. Factor/volume=N x 20/0.4=N x 20 x 10/4=N x 50/cumm34 35. INTERPRETATIONIncrease in Allergic conditions Parasitic infection especially in helminths Hyperadrenalism Leukemia Aplastic anemia35 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. 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. THANK YOU38