HAEMOCYTOMETRY

HAEMOCYTOMETRY1WHAT 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 Haemocytometer or by Electronic cell counter. 2PURPOSEIn 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 . 3Broadly , The cell count is doneTo 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 .

4PRINCIPLE OF CELL COUNTINGThe blood is diluted with appropriate known volume of diluting fluid and then counting is done by using haemocytometer .5 HAEMOCYTOMETERThis 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.

6COUNTING CHAMBERIt 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). 7COUNTING CHAMBER

8 There are some diff. type of counting chambers :-

1.Old neubauer counting chamber2.Improved neubauer counting chamber3.Burker counting chamber4.Fuchs rosenthal counting chamber.9OLD NEUBAUER CHAMBER In this the central platform is set 0.1 mm. below the level of the two side, which giving the chamber a depth of 0.1 mm. The ruling covers an area of 9 sq.mm. divided into 9 squares of 1 sq.mm. each. The four corner squares are subdivided into 16 squares , each with an area of 1/16 of a sq.mm. The central ruled area of 1sq.mm. is divided into 16 larger squares by set of triple lines. These large squares are further subdivided into 16 small squares by single lines. 10 IMPROVED NEUBAUER CHAMBER 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. 11OLD v/s IMPROVED NEUBAUERThe 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.12Cont.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 OLD NEUBAUER CHAMBER

14IMPROVED NEUBAUER CHAMBER 15

BURKER COUNTING CHAMBER Like the neubauer counting chamber , this has a ruled area of 9 sq.mm. and a depth of 0.1mm. , but its smaller squares are divided by double lining not by single one. 16 BURKER COUNTING CHAMBER

17 FUCHs ROSENTHAL CHAMBER This chamber was originally designed for counting cells in CSF ,but as such a relatively large area is covered , it is preferred by some workers for counting blood cells. The depth of this chamber is 0.2mm. and the ruled area consist of 16mm squares divided by triple lines. These squares are subdivided to form 16 smaller squares , each with an area of 1/16 of 1 sq.mm. 18FUCHs ROSENTHAL CHAMBER

19COVER GLASSA special cover glass is used which has a very smooth , flattened surface and even thickness.Different Thicknesses are :0.3mm0.4mm (most common)0.5mm Two sizes are common:-16x22 sq. mm 22x23 sq. mm

20 TOTAL WBC COUNT Diluting fluid Tuerk fluid

Glacial acetic acid 2ml Gentian violet about a pinch

Distilled water 97ml

Solution is stable at room temp.

21PROCEDURETake 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 side and on the top of the square.

22As shown in the picture

23 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.24InterpretationIncrease (Leucocytosis)Muscular exercise High temp.Severe painBacterial or viral infectionsLeukemiaSevere hemorrhageDecrease ( Leucocytopenia )Bacterial infections(typhoid)Viral infection ( Influenza )Protozoal infection(Maleria)Megaloblastic anemiaBone marrow depression as in aplastic anemia , drugs , radiation etc.25NOTE While performing RBC count by this technique , possibilities of error is very high . So, this technique is now not in use for RBC count.26PLATELET 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.

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PROCEDURETake 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 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.

28CALCULATIONArea 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 cumm29INTERPRETATION Increase (Thrombocytosis)PolycythemiaChronic granulocytic anemiaAfter surgeryImmediately after hemorrhage. Decrease (Thrombocytopenia)Acute leukemiaPancytopenia as in aplastic anemiaLiver diseaseMetal poisoningMegaloblastic anemia

30 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 PRINCIPLE Blood is diluted with special diluting fluid which stains the eosinophilic granules brightly and distinctly. At the same time it lyses the RBC and other WBC.32PROCEDURETake 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 to10 min.Count the eosinophil under low power(10x)with reduced light. Count in 4 corner squares of the Improved counting chamber .

33CALCULATIONAEC=Total cell counted x dil. Factor/volume=N x 20/0.4=N x 20 x 10/4=N x 50/cumm34INTERPRETATIONIncrease inAllergic conditionsParasitic infection especially in helminthsHyperadrenalismLeukemiaAplastic anemia

35PRECAUTIONSThe 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.

36Cont..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.37THANK YOU38