5.6. BLOOD CELL COUNTER Changes in the normal functioning of an organism are often accompanied by changes in the blood cell count. Therefore, the determination of the number and size of blood cells per unit volume often provides valuable information for accurate diagnosis. | The blood constitutes 5-10% of the total body weight and in an average adult, it amounts to 5-6 litres. Blood consists of corpuscles suspended in a fluid called plasma in the proportion of 45 parts of corpuscles (cells) to 55 parts of plasma.ie Cicerone 5s, ‘The-percentage of cells jn the blood is called the haematocrit value or packed| ‘The majority of the corpuscles in blood are Red blood cells (Erythrocytes)| others being white blood cells (Leucocytes) and platelets (Thrombocyytes). Blood cells are divided into groups according to their form and function as} shown in'the table. | Number of Cell | Mean Cell Volume Blood Cell Typ in mm (MCV) in pm? © 1, Erythrocytes . | (4.8-5.5)Ix 1x 104 “90 2. Leucocytes 5000-10,000 - (@ Neittrophills | 2000-7500 450 () Lymphocytes | 1500-4000 250 (c) Eosinophils | 40-400 450 (@Basophills | 10-100 450 (e)Monocytes | 200-800 600 3. Thrombocytes | 1.5 x 105-4 x 105 8 () Erythrocytes (Red Blood Cells) Red blood cells have the form of a bi- concave disc with a mean diameter of about 7.5} and thickness of about 1.7 jt. The mean surface area of the cell is about 134 jum? There are about 5.5 million of them in every cubic millimeter of blood in men and nearly 5 million in women. In the whole body, there are about 25 billion erythrocytes and they are constantly being destroyed and replaced at a rate of about 9000 million per hour. The normal red cell lasts approximately 120 days befor iti destroyed.Basics of Biomedical Instrumentation The exythrocytes have no nucleus. They are responsible for carrying oxygen fom the lungs tothe tissues and carbon dioxide from the tissues to the lungs. i) Leticocytes (White Blood Cells) Leucocytes are spherical cells having 4 nucleus. There are normally 500-10,000 white cells per cubic mm of blood but their number varies during the day. ‘They live for seven to fourteen days and there is a rapid turn over, with constant destruction and replacement. Responsibility Leveocytes are responsible to form the defence mechanism of the body against infection. (in, ) Thrombocytes (Platelets) Platelets are usually tiny, round, oblong or irregularly shaped cells of the blood with an average diameter of approximately 2p. There are usually 2,50,000-7,50,000 platelets in every cubic mm of blood. Responsibility They play an important role in the blood coagulation process. 5.6.1. CALCULATION OF SIZE OF CELLS. (/) Mean Celt Volume (MCV) Itis calculated from the PCV (Packed Cell Volume) and the number of red cells present per litre of blood. For example, if PCV is 0.45, i.e., 1 litre of blood contains 0.45 litres of red cells and if there ure 5 x 10!2 red cells per litre, then 0.45 Mean volume of one cell = =90f0 “mM Sx 102 Femolitres‘Normal mean red cell volume is 86 + 10 //1. (i) Mean Cell Haemoglobin (MCH) Itis calculated from the Hb and red cell count. For example, Ifthere are 15 g of Hb per decilitre (dl) of blood, there will be 150 gram Hb per picogram (pg) Normal meat cell haemoglobin is 29.5 + 2.5 pg. iif) Mean Cell Haemoglobin Concentration (MCHC) It can be calculated if PCV and Hb per dl are known. For example, | IEPCV is 0.45 and Hb is 15 pg per dl 15 thea, MCHC = 945 g/dl = 333 g/dl (iv) Mean Platelet Volume (Pv) It is the ratio of the integrated platelet volume to the platelet count and is expressed in femolitres. (v) Plateletcrit (PCT) Itis the percentage of the total specimen volume occupied by the platelets. pers = MPV Ux PLL x(10%/1) (vi) Red Cell Distribution Width (ROW) It is a numerical expression of the width of the size distribution of red cells. tis derived by analog computation, s : 20" - 80") percentile volume. joy, K RDW index = (99575 g0 percentile volume[542] Basits of Biomedical Instrumentation KK - constant, the calibration factor to produce a result of 10 for a riormal population. (vif Platelet Distriwtion Width (PDW) This index is refated fo the size range covered by those platelets lying between the sixteenth and eighty fourth percentile. ETHODS OF CELL COUNTING [bp sii ‘scopic MetRtod . » The most common and routinely applied method of counting blood cells ven today, particularly in small laboratories isthe microscopic method in which the diluted sample is visually examined and the cells-counted. > ee it suffers from ‘Several Comimion drawbacks. ‘> Apart from the inherent error of the system, which may be about 10%, there is an additional subjective error of + 10% entailing poor reproducibility of the results. (ii) Automatic Optical Method | This method is based on collecting scattered light from the blood cells and convertin, , it into electrical pulses for counting. A sample of dilute blood is taken in a glass container. ° It is drawn through a counting chamber in which the blood stream is reduced in cross-section by a concentric high velocity liquid sheath. A sample optical system provides a dark field illuminated zone on the stream and the light scattered in the forward direction is collected on the cathode of a photomultiplier tube. % Pulses are produced in the photomultiplier tube corresponding to each cell.Bio-Chemical Measurement Ba] > Secs a ee at eae to an adjustable amplitude discriminator. * The disrimintor provides ples of equal ample, which ae wed 1 __ drive a digital display. + Instruments based on this technique take about 30S for completing the’ = count. %& Anaccuracy of 2% is attainable. The instruments require about 1 ml of| blood sample. Read out |} Dsatiinator “Amplifier Photo multiplier =—— Sheath fow Volume Cl es EE TNT aea = ° Basics of Biomedical Instrumentation] (iif Coulter (Gaonductivity Method) i i A wide mage of particle counting instruments based on the coulter | ri Ihave been designed to meet a wide variety of needs in the 1 haem Baboratory. eee +> Theselsamesents range from the small counters used primarily for red “1 viljeeeell counts in very small hospitals and clinics, to the mult- cramer: raicroprocessor controlled, instrument featuring fully cndaatng of samples and printing of results. ‘Volume: finite Ampliie Counter Digital % In above figure , a glass measuring tube °c” provided with an aperture ‘*isimmersed into the suspension. a i | Fig. 5.16. Block diagram of blood cell Coulter counter| Bio-Chemical Measurement > The pressure difference created betweeh the two sides of the aperture A constant current is normally passed between the'electrodes E, and E,- The electric resistance of the liquid measured. between these two ‘electrodes changes rapidly when a particle having electric conductance _ differing from the condictanee of the electrolyte passes through the 4 This results in the generation of a voltage pulse, ‘hich is amplified in a preamplifier of high pain and low noise level. 4 The output signal, of this stage goes to a discriminator, which compares the amplitude of the pulse arriving at its input with the preset triggering level, ‘> If the input signal exceeds the triggering level, the discriminator gives out a pulse of constant shape and amplitude. 4 These pulses go to a counting circuit for the display of the measured | parameter. iv) Laser based Cell Counting 4 This technique is used to determine the number of RBC’s, WBC's and platelets. % The cell volume of the red blood cells and the haemoglobin concentration can also be obtained by this method. Principle 4 The principle used in this lascr based blood cell counting is “the angle of scattered light is different for different sized blood cells”. “The blood is diluted and passed through the capillary tube.& The laser light is passed through the glass tube and the blood cells {n the tube scatter the light. The scattering angles of platelets and RBC are different. ‘They are detected by two different photo detectors. : % The detectors are given the digital voltmeter which gives the density of blood cells and platelets. : Lysing agent is used to destroy the RBC’s and the WBC number can be determined. ‘& The haemoglobin concentration in the RBC’s also can be measured by this method. +o o, ‘Sample blood Detector 2 Laser beam Capillary tube Fig. 5.17. laser based cell counting Difference between RBC and WBC Red Blood Cells (RBC) % Red blood cells also known as erythrocytes, % RBCs are the most abundant type of cells in the human organism> In biology, RBCs are known as anucleated cells. (without a nucleus). ot % The space that would have been occupied by the mucléus allows for more _ pigment known as hemoglobin to be present inside each cell. = >, This pigment is responsible for spteading oxygen around the human % Hemoglobin predominantly consists of iron, which when bound with oxygen, gives blood its red edlor. White Blood Cells (WBC) + White blood cells also known as Leukocytes. 4 WBC’s are responsible for immune response of the human organism. RBC's Vs WBC’s % The main difference between red blood cells and white blood cells is in their respective Functions: aa 4 While RBC’s are responsible for transport of respiratory gases, white blood cells provide defense mechanisms for fighting foreign micro organisms entering the human organism. _ 4 The two types have different structures while red blood cells do not have a nucleus, white blood cells do. This is what human blood looks like through a 1000 X microscope. Red blood cells one of a red color, hence their name. The color is made possible due to the presence of hemoglobin. White blood cells, on the other hand are colorless. * Hemoglobin works essentially as a storer of oxygen which can be further transported to various parts of the body and is responsible for the overall amount of energy an individual has. ‘© When people do not have enough red blood cells, they often show symptoms of anemia, the feeling of tiredness. A rendering of the white and the red cells, side by side,5.43] Basics of Biomedical Inscrumentation |_—_——__—______ i > Red bloott cells are-more Populous and have a longer lifespan than white blood cefis.