RBC COUNT

DR.ANINDITA SAHARBC COUNT

RED BLOOD CELLSRed blood cells, orerythrocytes, are the most abundant cells in the bloodstream and contains hemoglobin, the compound that carries oxygen through the body.. Any disruption of thered blood cells, its quantity, shape, size, structure or life cycle can therefore affect the oxygen-carrying capacity of the blood.

Shape and size of RBCsAred blood cellis a biconcave disc. Simply it is a round ball that is squeezed from two opposite ends to appear, widest at the sides and narrowest in the middle.

Ared blood cellmeasures about 6 to 8 micrometers in diameter (average = 7.8 um) with an average thickness of 2 micrometers (2.5 um at the thickest point and less than 1um at the center). Although ared blood cellis wider than some capillaries, its flexibility allows it to become distorted as it squeezes through narrow passages and then restores to its original shape.

METHODS OF RBC COUNT: Visual haemocytometer( manual method )

Automated method

MANUAL METHOD:

EQUIPMENTS REQUIRED: 1)Haemocytometer or improved Neubauers chamber 2)RBC pipette

FLUIDS REQUIRED :

RBC diluting fluid

Blood sample (capillary blood or EDTA anticoagulated blood can be used )

RBC DILUTING FLUIDRBCs are around only 5 millions/cumm of blood.Counting this much number is highly imposible.Therefore the blood sample is diluted with the help of RBC diluting fluid.It fixes and preserves RBCs.It is isotonic to RBCs.

NEUBAUER CHAMBER:

The improved neubauers chamber has a metalized surface and improved neubauer rulings.

The chamber has two ruled areas.

Each ruled area:

dimension = 3mm x 3mm

consists of 9 large squares each measuring 1mm x 1mm

Depth 0.1 mm

The central large square is divided into 25 squares.Each of this 25 square is further divided into 16 small squares.Each group of 16 small squares is separated by closely ruled triple lines.The 4 corner squares are further divided into 16 small squares.

Coverslips used in neubauers chamber:The coverslips used for the chamber are special.They are thick and optically flat so that the volume occupied by the diluted blood in each large square is 0.1 ml when covered by the coverslip.

RBC PIPETTE:The pippete has a bulb for dilution and mixing and is caliberated to deliver 20ul or 0.02 ml of blood.The bulb contains a red bead which facilitates the mixing of blood with the diluting fluid.Freely mobile bead indicates dry pipette.Markings on pipette are 0.5 , 1 , 101.

DILUTING FLUIDTwo types of diluting fluids are used for RBC counting.

1) Hayems fluid 2) Dacies fluid

Composition of Hayems fluid:

a. Sodium chloride -0.5 gm b. Sodium sulfate -2.5 gm c. Mercuric chloride -0.25 gm d. Distilled water 100 ml.

Composition of Dacies fluid:

i. Trisodium citrate 3 gm. ii. Formalin 1 ml. iii. Distill water 99 ml.

This diluting fluid is cheap and commonly used.

PROCEDUREAssemble all the equipment; draw the blood directly from finger or collected sample into RBC pipette upto 0.5 mark. Wipe off the tip of pipette to remove extra blood, if present. Then immediately draw up the diluting fluid upto 101 mark.Now rotate the pipette gently for 2 3 mins so that the diluting fluid gets mixed properly. This will give dilution 1: 200. Place the cover slip in position over the ruled area of chamber. Once again mix the solution thoroughly by rotating the pipette.

Discard first 1 2 drops of blood from pipette.Now apply slight pressure on the rubber tube of pipette, so that the third drop of fluid is in hanging position. Touch the tip of pipette (hanging drop) against the edge of cover slip. The angle between pipette and cover slip is 45. With this process, chamber gets filled with the fluid. This is known as charging of the chamber.The care should be taken that, no air bubble is present inside the chamber and there is no over filling beyond the ruled area.

Leave the counting chamber as it, without disturbing for about 3 min. This will allow the settling down of RBCs. Place the chamber on stage of microscope. Adjust the light and ruled area. Make sure that, the distribution of RBC over the chamber is uniform.

Now count the RBC using 40x in Central Square. The central square is subdivided into 25 squares. Out of 25 squares, count four at each corner and one at center. Each of these five squares is subdivided into 16 small squares. Thus, RBCs are counted in 16 x 5 = 80 small squares.In case of marginal cells, count the cells on L line, i.e. either right and lower or left and upper margin. Make the total of cells counted in 5 squares.

Let the no. of RBC counted in 80 small squares = NVolume of area in which RBCs counted = ( 1/5 x 1/5 x 0.1 ) x 5=1/50No of RBCs in 1/50 mm3= N No of RBCs in 1 mm3= Nx50Dilution factor = 200 RBC count per mm3 = Nx50x200=N x 10000

No. of cells in neubeaur chamber= N x dilution factorVolume of chamber in which cells countedFor RBC N x 10000

ERRORS OF RBC COUNTTECHNICAL ERRORS: defective apparatus/defective technique Technical errors can be minimised by the use of accurately caliberated apparatus and by careful technique.INHERENT ERROR: The distribution of RBCs is of an irregular pattern in the counting chamber.This affects the accuracy of visual count which can be minimised by counting a large no. of RBCs.

AUTOMATED METHODElectronic counter is based on the principle of aperture impendence method . Anticoagulated blood is diluted with particle free diluting fluid such as physiological saline or phosphate buffer saline.Particles passing through a chamber in single file scatter the light and convert by a detector into pulses proportional to size of the cells which are then counted electronically.

Advantages:1) Easy and rapid method2) Many thousand of cells are counted compared to fewer cells counted in manual method.Disadvantages:1)Costly equipment2) Caliberation error3) Altered composition of diluent causes erroneous results.4)Gaint platelets are counted as RBCs.5)High WBC count alters results.

Reference range:

Adult male 4.5 to 5.5 million/cummAdult female 3.8 to 5.2 million /cummAt birth 6 to 8 million/cumm

Conditions of decreased RBC count:1)Anaemia2)PregnancyConditions of increased RBC count:Physiological: 1)new borns and infantsPathological : 1) polycythemia(polycythemivera,kidney tumours,high altitude) 2) dehydration 3 ) hypoxia(COPD, congenital heart disease)

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