Blood cell counters Blood cell counter cell count) eure" ATE Used For haematotogieal me 8 has proved to be much ee el meen, Semitic le than microseopie eel! cow mated Tbecanise a investigator is sill most valuable and in mang Neamees the ned cha A number of ditterent priciples are used in cell * Blectrical/aperture impedance : Lights ‘attering(Optical method) oa pcemigaton and quantitative butty-coat analysis, 1g Kechiniques all major classes of blood cells can be identified and even subclasses can be measured, Electrical method/Aperture impedance change Impedance measuremer rabesdanes meastarement js most commonly used for cell counting. This prints of ent takes advantage of the fact that blood cells are less conductive than the diluent electrolyte. The principle is explained below 1. when an electrical potential is is applied to two electrodes dipped into an electrolyte solution, an electric current can be measured due to the transport of ions from one electrode to the other. If the electrodes are seperated by an insulator, the flow of electric current will drop to zero, ‘The current will reappear if a small hole is introduced into the insulator, but 3. the magnitude of the current will be small because the insulator is still partially effective. 4. A small particle such as blood cell with conductivity lower than than that of the electrolyte solution passing through the aparture from one chamber to another will temporaraly decrease the current because a smaller volume of electrolyte is able to pass through the aparture at the same time, The current will regain its original value when the particle has passed through the aparture When a cell containing fluid is sucked through the aparture of an insulator separating wo electrode chambers, each change in current registered as a pulse indicates the passange of 8 us allowing the cells to be counted. The magnitude of cell particle through the aparture, th each pulse is propotional to the size of the cell particle passing through the aparture. The of measurement implies that each measured pulse is attributed to the passage of a single cell particle through the aparture of the separating membrane, ‘Therefore, a blood specimen 5 million cells per microlitre must be diluted having a cell concentration of aboutappropriately to ensure that individual cells are counted. Two electrodes from the ohmmeter are placed each in one chamber. Resistance of the path through the hole is measured When blood cell is in the aperture as shown in the figure below. The resistance is high when a cell particle is in the aparture and low when there is no Particle in the aparture. Constant current source (CCS) and voltage amplifier replace the ohmmeter. The resistance of the aperture and will be either high or low, depending on whether or not the blood cell is inside the aperture. Amplifier convert the current pulse to voltage pulse.El A lectrode Perture Exiemar Electrode ° U=RxI Blood sample is aspirated, measured to a predetermined volume, diluted at the specified ratio, then fed into each transducer. The transducer chamber has a minute hole called the aperture. On both side of the aperture, there are the electrodes between which flows direct current. Blood cells suspended in the diluted sample pass through the aperture, causing direct current resistance to change between the electrodes. As direct current resistance changes, the blood cell size is detected as electric pulses. Blood cell count is calculated by counting the pulses, and a histogram of blood cell sizes is plotted by determining the pulse sizes, Also, analyzing a histogram makes it possible to obtain various analysis data = Joc Supey Transcucer Chavber =] Resa Extema! Electrode + z eaamne Blood Cet Suspennor ‘ é Aperture Internal Electroge Figure 9-2-1: DC Detection MethodCOULTER COUNTER (Electrical Impedance) Blood cell counter usually consists of three parts: 1- Electr El trical part which includes power supply, WBC and RBC preamplifier and amplifier boards, microprocessor boards, interface boards and other electrical ‘components 2- Pneumatic supply that provides compressed and vacuum air which is needed for different parts of counter to work 3-Diluents part which includes all the components necessary to aspirate the whole blood sample, dilute it and measure the cells of the blood depending on electrical conductivity [eam] ruse Staxac Bo -| Seine monzonras | weer counter FIG. At-1 SCHEMATIC DIAGRAM OF COULTER PRINCIPLE Ina COULTER COUNTER instrument, a tube with a small aperture on the wall is immersed into a beaker that contains particles suspended in a low-concentration electrolyte. Two. electrodes, one inside the aperture tube and one outside the tube but inside the beaker, are placed and a current path is provided by the electrolyte when an electric field is appliedCigu le " gure 1). Th >. The impedance ber ween the el the electrodes is then measured. The aperture ereates what is called a», Heda "sensing Zone" them Particles in rolyte, ean be counted by passin ow throug concentra igh the a ation, suspended c apertur idea in the ele equivale AS a particle Caivatent to the immarea PACE Mses though the aperture, volume oF electrolyte HSCS a short-term chamae fe nue oF the particle is displaced from the sensing zone, This #¢ in the impedance aeross the aperture Operation The COULTER COUNTE! sence LTER COUNTER instrument determines the number and size of particles through a ani Cleettically conductive liquid. This is done by forcing the suspension to flow ‘gh a small aperture having an immersed electrode on cither side. As a particle passes through the ape rough the aperture, it changes the resistance between the electrodes. ion having a magnitude proportional to particle This produces a voltage pulse of short dure size. The series of pulses is then electronically scaled and counted. When the stopcock is opened, a controlled external vacuum initiates flow from the beaker through the aperture and unbalances the mercury siphon, Closing the stopcock then isolates the system from the external vacuum and the siphoning action of the mercury continues the sample flow. The advancing mercury column contacts the start and stop probes to activate the electronics counter. The probes are placed precisely O.5ml apart, providing a constant sample volume for all counts. Other fixed sample volumes are available. The voltage pulses are amplified and fed to a threshold circuit having an adjustable threshold level. If this level is reached or exceeded by a pulse, the pulse is counted. The threshold level is indicated on an oscilloscope screen by a brightening of the pulse segments above the threshold, facilitating the selection of appropriate counting levels. By taking a series of counts at selected threshold levels, data is directly obtained for plotting cumulative frequency (larger than stated size) versus particle size. Integration of all or part of the resultant curve provides a measure of the particle content of the suspension. Before plotting, the counts are first corrected for coincident particle passages (doublets, triplets, etc.). These corrections are quite precise and if kept to a moderate level (say, 15%), an overall accuracy of measurement of better than 1% is readily achieved. Fig. 20-2 is a block diagram of the electrical functions of an early Coulter Counter.Counter wan Fig. 20-2 Hectronie circuitry of the Coulter Counter (courtesy Coulter Flectronies tne ). Erros that affect cell counting I affect measurement of cell concentrations are: General errors- General errors that v |. Unsatisfactory blood sampling and stor 2. Inadequate dilution of the specimen 3. Fibrin prespitates in the specimen 4. Inadequate lysis of red blood cells when counting whit blood cells 5. Lack of homogeneity in the distribution of blood cells in the dilution Technical errors- These may be as a result of the following: 1, Fluctuations in the electric current 2. Incorrect setting of the size threshold of the instrument 3. Dust particles in the diluent 4. Leakage in the suction system of the instrument 5. Partial or total obstruction of the aparture 6. Multiple cell passage at high cell counts(coincidence error) 7. Carry- over from one measurement to subsequent measurement General errors can be avoided by: 1. Ensuring satisfactoryblood sampling ang storage 2. Ensure adequate dilution of the specimen 3. Ensure measures are in place to prevent fibrin precipitation 4. Ensure adequate lysing of red blooe cells when measuring white blood cells Ensure homogeneity of the distribution of blood cells in the dilution by proper shaking 3. Technical errors can be avoided by:3 Hise comet sclatog COMESCd oan © tle of diluent should ren Of the size stabilizer 4 The fittings of movable ald be checked dof ts fae te 5, Tite Suction system | P'S should be coated waste solution dis 5. \The ster a oan e aparture between case (92 «5, Messurements and clement Ct™Ol¥!e chambers should be checked afte each 6. Ensure thatthe specineen ti @ small sof brush itnecemary WBC/count . Colorimeter — hemoglobirY Optical flow sensor > RBC/count. a) Topic SummarySummary Automated cell counting provides greater accuracy and precision compared to nmanual celle counting methods, The primary principles of operation, electronic impedance and optical scatter, are used by most automated hematology analyzers. Radiofrequency (RE) is sometimes weed in conjunetion with electroni . : pedance. changes in electrical resistance The measurable voltaze aperture, ‘The electronic impedance method detects and measur Is pass through a sensi between two electrode: s are plotted on frequency distribution graphs, or histograms, that allow the ev d on cell volume. e electromagnetic current. Measurable changes in the RF signal of cell populations bi RF resistance uses high-voltag are proportional to cell interior density, or conductivity. Impedance and conductivity ean be nal distribution cytogram or scatterplot, which plotted against each other on a two-dimensi allows the evaluation of cell populations using cluster analysis. Optical scatter systems (flow cytometers) use detection of interference in a laser beam or light, source to differentiate and enumerate cell types. Reticulocyte analysis has been incorporated into the primary cell-counting instruments of all major manufacturers. All use either fluorescent or other dyes that stain nucleic acid in reticulocytes before the cells are counted using fluorescence or absorbance and light scatter ng ch instrument has limitations related to methodology that may result in instrument flagg ion of automated data. Likewise, inherent specimen problems Ea of specific results or suppressi may result in instrument flagging that indicates possible rejection of automated results ‘Automated hematology analyzers have had a significant impact on laboratory work flow, automation of the WBC differential. In addition, newer parameters that can now the immature reticulocyte fraction (IRF) and the reticulocyte particularly be measured, such as F th hemoglobin concentration (RET-He and CHr), have documented clinical utility.