British Journal of Medical & Health Sciences (BJMHS)

Vol. 2 Issue 4, April - 2020 Comparison Of Count By Automated And Manual Methods, A Study And Review Of Literature In A Medical College Hospital In Kashmir 1Dr Jangbhadur Singh Associate Professor, ,2Sawaira Parvaiz Msc haematology Lab. Tec.3Dr Afiya Shafi, 4Dr Nadia Jeelani,5Dr Mabruka Jeelani SKIMS Medical College . Srinagar, Kashmir, India.

Abstract to 8 cuµ in volume. Normally, are of several shapes viz. Spherical or rod shaped and become oval Introduction: Estimation of platelet counts is of or disc shaped when inactivated, arising from the prime importance in clinical practice as well as in fragmentation of megakaryocytes. Megakaryocytes certain diseases.Automated analysers commonly are giant cells in the bone marrow, form platelets by used for platelet analysis may produce erroneous pinching off and extruding pieces of cytoplasm. results in presence of particles or light scatter like fragmented red blood cells and giant platelets and platelet clumps, so alternative methods like using chamber counting and peripheral blood smear examination can be used for validation. Aims and Objectives: Comparison of platelet counts by three methods: 1. Automatic analyser. 2. Manual platelet count using counting chamber and 3. Traditional method , counting average platelets per ten high power fields[100X] and multiplying the same by 15000. Methods: Four hundred Ethylene Diamine tetra-acetic Acid (ED TA) samples were analysed over a period of six months .Statistical analysis was done by using microsoft software. Descriptive statistics like mean and percentage were used for data interpretation. Sensitivity and specificity of the manual methods was compared with that of automated analyzesr. Results: The mean platelet count by Hemostasis, the process of stopping bleeding, is automated method was 1.70/µl. The mean platelet the primary function of platelets. To accomplish this, count by Neubauer chamber is 1.77/µl and mean platelets contain lysosomes (chemicals capable of platelet count by PBF was 1.81/µl. The P value for breaking down other substances), clotting factors, and automated versus Neubauer chamber was 0.57 and a growth factor that stimulates healing. Traveling in for automated method versus PBF was 0.87 the circulation, platelets join with other blood Suggesting that there was no significant variation in components to limit blood loss. Platelets may also the platelet count between the manual methods when help maintain the integrity of the vascular lining and compared with automated analyzer. Conclusion stimulate proliferation of vascular smooth muscle. :Platelet count by manual method using chamber for counting as well as by traditional method using Blood is a complex process. Activated peripheral blood smears for platelet counting are by factors at the site of an injured blood vessel, validated as alternative and reliable methods of platelets aggregate (collect) to form a plug, change platelet counting. shape, discharge their granules, and initiate the generation of thrombin, an enzyme that converts Keywords—Platelet count; automated analyser; fibrinogen to fibrin. Thrombin causes them to become peripheral blood smear; thrombocytes, sticky and adhere irreversibly to each other, as well as I. INTRODUCTION to the break in the vessel wall. The granules attract more platelets, and thrombin begins formation of a Platelets or thrombocytes are the formed elements true clot with a net of fibrin. of blood. Platelets are small colorless, non nucleated and moderately refractive bodies, these formed Platelets, besides being acute phase reactants, are also influenced by the patient`s health and elements of blood are considered to be the fragments [1, 2]. of cytoplasm. Platelets are 2 to 4 µ in diameter and 7 nutritional status Sometimes the platelets have

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Vol. 2 Issue 4, April - 2020 dumbbell shape, comma shape, cigar shape or any normal or prolonged bleeding time but defective clot other unusual shape. Inactivated platelets are without retraction. processes or filopodia and the activated platelets Giulio Bizzozero, an Italian pathologist is credited develop processes or filopodia. Platelet is constituted with identifying platelets as an important component of by cell membrane, microtubules, and cytoplasm. blood and their essential involvement in the Platelets are inactive and execute their actions mechanism of blood clotting at the end of the only when activated. Activated platelets immediately nineteenth century [3]. It is a well known fact that an release many substances. This process is known as accurate and reproducible platelet counts are of platelet release reaction. Platelets are responsible for utmost importance in day-to-day clinical practice formation of intrinsic prothrombin activator. This aiding not only in diagnosis but also in the treatment substance is responsible for the onset of blood and prognostication of a wide variety of diseases [4]. clotting. In the blood clot, blood cells including Severe thrombocytopenia can present as internal and platelets are entrapped in between the fibrin threads. external bleeding which maybe fatal and require Cytoplasm of platelets contains the contractile emergency platelet transfusion. At the other extreme, proteins, namely actin, myosin and thrombosthenin, thrombocytosis can cause thrombotic events [5]· In which are responsible for clot retraction. Platelets infections like malaria and dengue-with accelerate the heamostasis. Platelets secrete 5-HT thrombocytopenic patients facing the potential risk of which causes the constriction of blood vessels. Due to bleeding-an accurate count is imperative. Platelet adhesive property, the platelets seal the damage in count is also one of the parameters to assess the blood vessel like capillaries. By formation of severity of dengue [6]. It is also important to temporary plug, the platelets seal the damage in blood prognosticate patients undergoing cytotoxic therapy vessels. Platelet-derived growth factor formed in for hematological malignancies. Studies have shown cytoplasm of platelets is useful for the repair of the that platelet count is often reduced than normal in endothelium and other structures of the ruptured patients of Nonalcoholic fatty liver disease (NAFLD), blood vessels. thereby providing a supportive indicator in addition to liver biopsy in these patients. At present, platelet Platelet disorders occur because of pathological count is also used for prognostication of fibrosis and variation in platelet count and dysfunction of platelets. [7]. cirrhosis of the liver Various principles of doing Platelet disorders are: Thrombocytopenia, automated platelet counts are impedance, optical thrombocytosis, thrombocythemia, Glanzmann’s scattering, optical fluorescence, and immunologic flow thrombasthenia. cytometry. The manual phase contrast microscopy Thrombocytopenia is the decrease in platelet method, although considered the gold standard was count. It leads to thrombocytopenic purpura ; it is a abandoned as it was time-consuming and not precise disorder characterized by prolonged bleeding time. at low counts. However, clotting time is normal. Characteristic Immunological flow reference method also known feature of this disease is spontaneous bleeding under as the RBC platelet ratio method replaced the manual the skin from ruptured capillaries. It causes small tiny phase contrast microscopy method as the hemorrhagic spots in many areas of the body. The international decade, which has led to greater hemorrhagic spots under the skin are called purpuric precision in platelet counting and has resulted in its spots (purple colour patch like appearance). That is [8]. extensive use in laboratories worldwide. A few why this disease is called purpura. Thrombocytopenia limitations of estimating platelet counts on Leishman is caused due to many conditions like acute infections, stain smears are improper staining and stain artifacts. acute leukemia, aplastic and pernicious , [9] To overcome this, study by Uma Shankar et al has chickenpox, smallpox, splenomegaly, scarlet fever, shown that even the unstained peripheral smears typhoid, tuberculosis, purpura, Gaucher’s disease. prepared from fresh blood can be used to evaluate a Thrombocytosis is the increase in platelet count. platelet counts. However micro platelets are difficult to Thrombocytosis can occur due to conditions like appreciate and large platelets resemble WBCs on allergic conditions, asphyxia, hemorrhage, bone unstained smears which must be considered [10]. A fractures, surgical operations, splenectomy, rheumatic major factor contributing to the accuracy of platelet fever, and trauma. counts is the duration of sample storage post collection. The standard guidelines state that Thrombocythemia is the condition with persistent processing of hematological samples should be done and abnormal increase in platelet count. within six to eight hours of collection, to avoid Thrombocythemia occurs due to many conditions like variations and errors in reporting the different carcinoma, chronic leukemia, Hodgkin’s disease. hematological parameters [9, 11]. A study by Jain et al Glanzmann’s thrombasthenia is an inherited has shown that platelet counts done on automated hemorrhagic disorder caused by structural or analyzer s showed an unacceptable bias even within functional abnormality of platelets. It leads to less than four hours of sample storage at 33 degree thrombasthenic purpura. However the platelet count is centigrade for platelet counts [12]. In the present study, normal. It is characterized by normal clotting time, all the samples were run within a span of five hours from the time of collection.

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Vol. 2 Issue 4, April - 2020 Although hematology analyzers provide reliable Platelet count is important diagnostic tool so it is full blood counts based on their linearity limits, they necessary to count the platelets accurately. Before the are known to be inaccurate and poorly reproducible at widespread use of automated counters manual enumerating platelets in severe thrombocytopenia platelet count was done. The manual count is the which maybe secondary to interference from cells or oldest means of counting platelets and remarkably, is materials of a similar size to platelets or due to light still used as the gold standard international reference scatter like in the case of fragmented RBCs, method (18). Recently, a new immunoplatelet counting microcytic RBCs, lipemic samples, debris in patients procedure has been proposed as the new taking cytotoxic drugs or in the presence of giant international reference method. Although modern platelets or platelet clumps. According to a study done impedance counters are rapid, precise and by Barbara et al [8]. It was found that majority of the reproducible, they tend to overestimate the platelet automated analyzers overestimated the platelet count count in samples that contain cellular debris, e.g. especially when the levels were less than 200×103/µL. thalassaemia, thrombocytopenic purpura (TTP). It is Overestimation of thrombocytopenic platelet counts necessary to count as the transfusion threshold for may result in the substantial under transfusion of platelets is now set at 10×10⁹/l. [19,20]. Recent platelets in high risk patients in need of platelet analyzers work on optical counting methods. [21, 22]. transfusion resulting in significant risk of under New methods increase the accuracy of the count as transfusion [8] · both normal and large- sized platelets are easily discriminated from other cell population. Therefore manual platelet estimation using various methods has been commonly used in settings of low Automated hematology analyzer: platelet count for microscopically corroborating the In most of the laboratories, the hematology auto analyzer counts. The evaluation of the peripheral analyzer is utilized to count platelets in patient blood blood smear till date still constitutes an indispensable samples. Platelets are difficult to count because of tool in the evaluation of patients with hematologic [13]. their small size, marked variation in size, tendency to disorders Manual evaluation offers aggregate and overlapping of size with microcytic red added assessment of platelet size, shape, cells, cellular fragments, and other debris. In granulation, and can also be used to analyze platelet hematology analyzers, this difficulty is addressed by aggregation or satellitism. It is also essential for any mathematical analysis of platelet volume distribution standard of care test to be consistent, and a study [4] so that it corresponds to log normal distribution. carried out by Al-Hosni et al demonstrated that Platelets are counted by electrical impedence method manual platelet count estimation is reproducible in in the RBC aperture, and a histogram is generated trained competent hands when a standardized with platelet volume on X-axis and relative cell methodology is used. In our study the manual frequency on Y-axis. Particles greater than 2fl and methods were done independently by two observers less than 20fl are classified as platelets by the and average values obtained were used so as to keep analyzer. interobserver bias to the minimum. An average of interobserver CVs in the range of 10-25% has been However, varying platelet count results may be reported in previous studies as against CVs of less observed with different hematology analyzer for the than 3% for automated blood counts [14]. A few studies same blood sample, which make the comparison very have suggested the standardization of the manual difficult. The other methods for estimation of platelet platelet count methods in relation to the microscopes count are manual counting by counting chamber, PBS used for doing the counts, the region of the smears examination, immunoplatelet counting and examined on the slides and the laboratory personnel radioisotope labeling technique. [23]. Automated involved. Also proposals that determination of a hematology analyzers are frequently used in clinical correction factor be performed in each laboratory in laboratories to assess and monitor health condition of order to improve the accuracy and reliability of platelet patients. estimates [15] · In today’s general screenings, CBC tests are Average life span of platelets is 10 days. It varies performed using automated hematology analyzers. In between 8 and 11 days. Platelets are destroyed by addition to reporting RBC, PLT, and WCB counts, an tissue macrophage system in spleen. So, analyzer also measures the oxygen-containing splenomegaly decreases platelet count and hemoglobin (HGB) and determines a range of other splenectomy increases platelet count. parameters such as the mean cell volume (MCV), PLT width distribution, and (HCT), that is, the The accurate platelet count estimation has an -to-plasma ratio. Hence, an automated important role in diagnosis and treatment of analyzer can provide much more information than a thrombocytopenia cases. The reliability of platelet manual count. count is highly desired where the platelet transfusion is necessary. Thrombocytopenia is commonly Automated analyzer is adventitious in speed with associated with various conditions like bacterial efficient handling of a large number of samples, sepsis, terminal liver disease renal failure, leukemia, accuracy and precision in quantitative blood tests, malignancy, after chemotherapy etc [16, 17]. ability to perform multiple tests on a single platform, significant reduction of labor requirements and it is www.jmhsci.org BJMHS450083 179 British Journal of Medical & Health Sciences (BJMHS)

Vol. 2 Issue 4, April - 2020 invaluable for accurate determination of red cell Technical Errors indices. Errors in bood collection. Automated analyzer have some disadvantages like:  Prolonged, tight application of a tourniquet leads to venous stasis and false elevation of cell  Comments on red cell morphology cannot be count. generated. Abnormal red blood cell shapes (such as  Excessive squeezing of finger puncture fragmented cells) cannot be recognized. results in dilution of blood with tissue fluid.  Flags: Flagging of a laboratory test result  Inadequate mixing of blood with anticoagulant demands labor-intensive manual examination of a leads to formation of clots in blood sample and falsely blood smear. low count.  Erroneously increased or decreased results  Non-mixing of anticoagulated venous blood due to interfering factors imme-diately before testing will cause falsely low  Expensive with high running costs. count. Haemocytometer: Errors in pipetting A haemocytometer consists of a counting chamber, a cover glass for the counting chamber and  Use of wet, chipped, or dirty pipettes the diluting pipettes. The haemocytometer or  Use of improperly calibrated pipettes Neubauer chamber consists of a thick rectangular  Not wiping blood adherent to outside of glass slide with an`H’ shaped trough, forming two pipette counting areas. Beyond the two vertical arms of the  Using bulb pipettes that are difficult to trough are two raised shoulders which support the calibrate and which break off easily. specially made thick, optically flat cover glass. Space Errors in filling of chamber between the cover glass and surface of the counting area [depth] is exactly 0.1mm. Each counting area is  Incorrect filling of chamber, spillage into 3×3 mm. This produces a total counting volume of moats 0.9cu.mm. When the ruled area is viewed under the  Not allowing 2 minutes for cells to settle. 10x objective of the microscope it shows 5 squares.  Drying of edges of preparation Each having 1mm² area. This 1mm² sections in the  Not using specified coverglass four corners are divided into 16 equal squares. The  Chamber containing dirt or air bubble. square in the centre of the ruled area is divided into 25 equal squares. In turn each of these 1/25mm² squares are further divided into 16 portions. Peripheral blood smear:

Manual methods are time consuming, subjective and tedious with high levels of imprecision [10]. Automated hematology analyzers though rapid in giving results, at times give erroneous values in the presence of giant platelets, platelet clumps, fragmented and microcytic red blood cells. [24, 25, 26]

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Vol. 2 Issue 4, April - 2020 It is a standard protocol in most hematology machine. Ten and 25 high-power fields were laboratories, that abnormal platelet values generated microscopically averaged and then multiplied by by cell counter be followed by a manual examination 15,000 and 20,000 to arrive at a platelet count in of Leishman staind peripheral blood smear [27, 28, 29] 1,000 per micro liter. Multiplying by 15,000 gives a platelet count reasonably close to automated machine PBS or peripheral blood smear is highly counts in 1000 per microliter. informative hematological tool for RBC`S, WBC`S, platelets morphology and counting. PBS screening is Anitha K,et al., (2013) conducted the study to commonly used for disease diagnosis and its progress compare the platelet estimation by peripheral smear and therapeutic response. A peripheral blood smear method and automated method and the result of this has three parts: head, body and tail. A blood smear study suggest that platelet estimation by peripheral should be examined in an orderly manner. The best smear method is a reliable, rapid, easy and economic, morphological details of PBS are seen in the area it can be done even in rural setup for early diagnosis where red cells are just touching one another. The of thrombocytopenia in pregnancy, as it is equivalent platelet count results obtained by this method of PBS to the automated method. examination had no statistically significant difference Parvaiz. A. Shah, et al., (2013) studied the role of when compared with hematology analyzer platelet [30, 31]. mean platelet volume in the pathogenisis, severity and count results for same blood sample outcome of isochemic stroke. The prospective study Microscopic examination of PBS is performed by comprised of 100 patients each of isochemic stroke preparing, staining and examine a thin film of blood. It with equal number of age and sex matched control provides valuable information on the status of blood group. High mean platelet volume and low platelet cells and presence of parasitic elements. The blood count was found in the study group with in comparison film is the only permanent record of hematological to the control group was statistically significant. investigations. Therefore a well made well stained Observations showed that, mean platelet volume smear is essential. The film must be prepared in a bears an inverse relationship to immediate outcome technically correct manner so that the cells are evenly from ischemic stroke independent of stroke subtype. distributed throughout the smear. An ideal blood film should be smooth and not interrupted by ridges and Carlo L. Balduini, et al., (2014) put together all holes. It should cover ½ to ¾ of the area of the slide. It data of subjects enrolled in the three population based should be thickest at the origin or the head and studies concluding the age related changes in platelet gradually thin out forming a tail without defined border count were actually very large: platelet count in old at the end. The thickness and the length of the film will age was reduced by 35% in men and by 25% in depend on the angle of the spreader, the size of the women with respect to early infancy. Most of the drop of blood and speed at which the spreader is reduction occurred in childhood and in old age, with pushed forward. It is essential to use a clean slide. A only minor changes in adulthood. Thus, there is no glass slide cleaned with alcohol and wiped dry gives longer any doubt that age is a major determinant of the best result. A spreader e.g. margin free slides with platelet count in healthy people. ground glass edges, should be used to make the film. After preparation the blood smear should be stained Zainab Mohammedi Golwala, et al., (2016) as soon as possible. Studied forty cases and controls. Platelet count, PCT and the ratios of MPV/platelet count, MPV/PCT, In the present study, we analysed the platelet PDW/platelet count, PDW/PCT and MPV × count results obtained by hematology cell analyser, PDW/platelet count × PCT were significantly different Neubauer counting chamber and Peripheral Blood among children who survived compared to those who Smear, examination in randomly selected blood died. On multiple regression analysis the ratio of samples. MPV/PCT, PDW/ platelet count and MPV/platelet count were risk factors for mortality with an odd ratio Review Of Literature of 4.31, 3.86, 3.45 respectively which is 95% of CI. In Wallace H Coulter discovered the Coulter 67% of patients who died MPV/PCT ratio was above Principle in the late 1940s (though a patent was not 41.8 and PDW/platelet count was above 3.86. In 65% awarded until October 20, 1953). Coulter was of patients who died MPV/platelet count was above influenced by the atomic bombs dropped on 3.45. Hiroshima and Nagasaki. These events motivated Sajad Geelani, et al., (2017) studied the variation Coulter to simplify and improve blood cell analysis so in platelet count between automated analyzer and that large populations could be screened rapidly, as manual platelet counting. It was found that platelet would be necessary in the event of a nuclear war. count by manual method is higher as compared to the Partial funding of the project came from a grant award automated method in the laboratory. Higher platelet from the Office of Naval Research. [32, 33] count by manual method is because of large platelet Webb Dl, et al., (2004) studied the platelet count size which analyzers are not able to count. visually in a peripheral blood smear and compared it Balakrishnan A, et al., (2018) received 250 with an automated machine platelet count. 35 samples in EDTA tubes for platelet counts during the peripheral blood smears were made from blood study period. 121 were female and 129 were males. specimens counted on an automated blood cell www.jmhsci.org BJMHS450083 181 British Journal of Medical & Health Sciences (BJMHS)

Vol. 2 Issue 4, April - 2020 Mean age of the patients was 45.8 years. Study has 2. Processing of blood samples for platelet count shown that manual methods are reliable to validate estimation with automated analyzer, PBS and the automated counts in routine practice under Neubauer chamber. standard conditions. In addition essential that 3. Statistical analysis. pathology laboratory personals and clinicians who rely 1. Collection: on platelet counts for various scenarios in day to day practice understand the limitation of instrumentation in A total of 400 blood samples from random use and the measurement uncertainty of automated Kashmiri population were used to conduct the study. platelet counters. All the venous blood samples were collected in EDTA vials. Each blood sample was properly mixed and the Vyankatesh T. Anchinmane, et al., studied 100 vials were properly labeled. randomly collected blood samples in EDTA anticoagulant vacutainer tubes. Each blood sample 2. Processing: was processed for platelet count estimation with automated hematology analyzer and Leishman’s 2.1. Hematology automated analyzes; stained PBS examination. The statistical analysis was The platelet count was obtained by processing done by using Pearson’s correlation test to access the blood samples in fully automatic hematology analyzer agreement between both the methods. Study (COULTER LH 750). It is a quantitative, automated concluded that platelet count estimation by PBS hematology analyzer and leukocytes differential method is reliable and statistically significant when counter for invitro diagnostic use in clinical compared to hematology analyzer method. laboratories. The coulter H750 hematology analyzer provides automated reticulocyte analysis and Aims and objectives: enumeration of nucleated red blood cells (NRBC’S) as Platelet count is one of the critical parameters in well as an automated method for enumeration of patient care, at times being a decisive factor for RBC’S and WBC’S in body fluid. diagnosis and treatment. There are several methods The purpose of the LH700 series is to separate of platelet count used in hematology laboratory. These methods are: the normal patient, with all normal system generated parameters, from the patient who needs additional  Manual counting method. studies of any of these parameters. These studies  Automated hematology analyzer counting might include further measurements of cell size and method. platelet distribution, manual WBC differential or any  Platelet count estimation by PBS method etc. other definitive test that helps diagnose the patient’s condition. The present study was undertaken to estimate platelet counts by automated hematology analyzer and correlate them with results obtained from PBS and Neubauer chamber. Aims and objectives:Platelet count is one of the critical parameters in patient care, at times being a decisive factor for diagnosis and treatment. There are several methods of platelet count used in hematology laboratory. These methods are:  Manual counting method.  Automated hematology analyzer counting method.  Platelet count estimation by PBS method etc. The present study was undertaken to estimate platelet counts by automated hematology analyzer and correlate them with results obtained from PBS and Neubauer chamber Materials and methods; PARAMETERS: The investigation on blood platelet count on the WBC or leukocyte count random Kashmiri population was carried out in pathology and hematology department of SKIMS- RBC Red blood cell or erythrocyte count Medical college Hospital (Bemina). The study involved Hgb Hemoglobin concentration following steps for meeting various objectives of study: Hct Hematocrit (relative volume of erythrocytes) 1. Collection of blood sample in EDTA coated MCV Mean corpusculas (erythrocyte) volume vials. MCH Mean corpuscular (erythrocyte) heamoglobin

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Vol. 2 Issue 4, April - 2020 MCHC Mean corpuscular(erythrocyte) numbers of cells pass through the aperture at one heamoglobin concentration point of time. There are two electrodes on either side of the aperture as the solution in which the cells are RDW red cell (erythrocyte volume) distribution suspended is an electrolyte solution; an electric width current is generated between the two electrodes. Plt platelet or thrombocyte count When cell passes through this narrow aperture across which a current is flowing, change in electrical MPV Mean platelet (thrombocytes) volume resistance (I. e. Momentary interruption of electrical LY% Lymphocyte percent current between the two electrodes) occurs. A small pulse is generated due to a temporary increase in MO% Monocyte percent impedance. This pulse is amplified, measured, and NE% Neutrophil percent counted. The height of the pulse is proportional to cell volume. The width of the pulse corresponds with the EO% Eosinophil percent time required for the cell to traverse the aperture. BA% Basophil percent Cells that do not pass through the center of the aperture generate a distorted pulse that is not LY# Lymphocyte number representative of the cell volume. Some analyzers use MO# Monocyte number hydrodynamic focusing to force the cells through the central path so that all cells take the same path for NE# Neutrophil number volume measurement. An anticoagulated whole blood EO# Eeosinophil number sample is aspirated into the system, divided into two portions, and mixed with a diluent. One dilution is BA# Basophil number passed to the red cell aperture bath (for red cell and NRBC% Nucleated red blood percent platelet counting), and the other is delivered to the WBC aperture bath (where a reagent is added for NRBC# Nucleated red blood cell number lysis of red cells and release hemoglobin this portion RET% Reticulocyte percent is used for leukocyte counting followed by estimation of hemoglobin). Particles between 2-20 Fl are counted RET# Reticulocyte number as platelets, while those between 36-360 fl are *HLR% High light scatter reticulocytes % counted as red cells. Hemoglobin is estimated by light transmission at 535 nm. *HLR# High light scatter reticulocyte% 2.1.2 Light scatter IRF Immature reticulocyte fraction Each cell flows in a single line through a flow cell. MRV Mean reticulocyte volume A laser device is focused on the flow cell as the laser light beam strikes a cell it is scattered in various *MSCV Mean sphered cell volume directions. One detector captures the forward scatter *PCT Plateletcrit light (forward angle light scatter or FALS) that is proportional to cell size and a second detector *PDWS Platelet distribution width captures side scatter (SS) light (90 °) that corresponds Automated hematology analyzers work on different to the nuclear complexity and granularity of principles ; cytoplasm. This simultaneous measurement of light scattered in two directions is used for distinguishing  Electrical impedance between granulocytes, lymphocytes, and monocytes.  Light scatter  Fluorescence 2.1.3. Fluorescence  Light absorption Cellular fluorescence is used to measure RNA  Electrical conductivity. (reticulocyte), DNA (nucleated red cells), and cell Most analyzers are based on a combination of surface antigens. different principles. 2.1.4. Light absorption 2.1.1 Electrical impedance Concentration of hemoglobin is measured by This is the classic and time-tested technology for absorption spectrophotometry, after conversion of counting cellular elements of blood. As this method of hemoglobin to cyanmethemoglobin or some other cell counting was first developed by Coulter compound. In some analyzers, peroxidase Electronics, it is also called as Coulter principle. Two cytochemistry is used to classify leukocytes; the electrodes placed in isotonic solutions are separated peroxidase activity is determined by absorbance. by a glass tube having a small aperture. A vacuum is 2.1.5. Electrical conductivity applied and as a cell passes through the aperture, flow of current is impeded and a voltage pulse is Some analyzers use conductivity of high generated. The requisite condition for cell counting by frequency current to determine physical and chemical this method is high dilution of sample so that minimal composition of leucocytes for their classification.

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Vol. 2 Issue 4, April - 2020 2.2 Peripheral blood smear  Add equal number of the drops of buffer  Principle solution. Mix the reaction mixture adequately by blowing on it through a pipette. Wait for 10 minute. The polychromic staining solution (wright, Leishman, Giemsa) contain methylene blue and  Watch the smear by using tap water. eosin. These basic and acidic dyes induce multiple  Stand the slide in a draining rack or on the colors when applied to cells. Methanol acts as fixative laboratory counter to dry. and also as a solvent. The fixative does not allow any 2.2.5 Examination of film further change in the cells and make them adhere to the glass slide.  First examined the stained smear under the low power. Three zones will appear Head, body and  Procedure tail. A thin smear is prepared by spreading a small  The portion slightly before the tail-end where drop of blood evenly on a slide. the red cells begin to overlap is chosen.  Then a drop of immersion oil is placed on the 2.2.1 Making a film smear and examines the film by moving from one field  Take a clean, dry slide. to next.  Transfer a small drop of blood near the edge  Count the platelets in at least 10 oil immersion of slide. fields. The average number of platelet is multiplied by 15×104 and the platelet count is expressed as  Place the spreader slide at an angle of 30 3 degree.Pull back the spreader until it touches the drop lacs/mm . of blood. Let the blood run along the edge of the spreader.  Push the spreader forward to the end of this slide with a smooth movement .Dry the blood smear at room temperature. Adequate drying is essential to preserve the quality of the film.

2.3 Neubauer chamber 2.3.1 Principle Whole blood sample is mixed with a diluent (1% Ammounium oxlate) in which red cells are lysed while the leukocyte, platelets and reticulocytes remain intact. The dilution for platelet count is 1:200. The sample was incubated for some time and mounted on haemocytometer. The cells were allowed to settle and

then counted in a specific area of the haemocytometer 2.2.2 Identification marking chamber under the microscope. The number of  By using glass marker or lead pencil, write the platelet was calculated per microlitre. identification number on the slide. 2.3.2Material 2.2.3 Fixing the smear . Blood sample (EDTA) . Neubauer counting chamber  The blood smear is fixed with methanol for 2- . Adjustable pipettes 3 minutes to prevent distoration of cells . 1% Ammonium oxalate solution for dialution 2.2.4 Staining the film . Test tubes . rack  Covered the smear with the staining solution . Glass capillary tubes (Leshman stain) by adding 10-15 drops on the smear. . Petridish with wet filter paper Wait for 1 minute. . Microscope

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Vol. 2 Issue 4, April - 2020

Number of platelets×200 2.3.3 Method Platelets/mm3 = 0.1  Test tubes were labeled and 3.98ml of 1% = Number of platelets ×2000 ammonium oxalate is taken in each test tube.  To this add 20µl of well mixed anticoagulated 3 Statistical analysis venous blood and mix thoroughly. Dilution of blood is Statistical analysis was done by using Microsoft 1:200. office software. Descriptive statistics like mean and  Diluted samples were mixed by inversion. percentage were used for data interpretation.  Mirrored surface of the counting chamber and Sensitivity and specificity of the manual methods was glass cover slip is cleaned gently. compared with the autoanalyzer.  Place coverslip over the heamocytometer.  Capilary tube full of well mixed diluted sample Result and discussion was filled.  Capillary tube was touched to the edge of the loading groove on one side of the counting chamber In present study, 400 samples of blood were and we allowed the diluted sample to fill the chamber collected from the random Kashmiri population and  The counting chamber was placed in a were processed by automated analyzer (Backman petridish in a moist filter paper for 15 minutes to allow coulter LH 750) and manual platelet counting the platelets to settle (Neubauer chamber) and Peripheral blood  Bottom the chamber was wiped carefully to examination . remove excess moisture from the moist petriplate and A total of 400 samples were received in EDTA place chamber on the microscopic stage tubes for platelet counts during the study period, 167  Using the 10x objective, we focused on the were males and 233 were females. engraved counting area to look for the central 1mm square The result demonstrated variation in platelet count between automated analyzer and manual platelet  Then we change to 40x objective counting.  The platelets will appear like highly refractile particles Age wise variation of platelet count using  Count platelets in all 25 small squares the automated platelet count analyzer, Neubauer and area covered by 25 squares is equivalent to 1 sq.mm PBF is given in table 1.

Plt. count Plt Age Plt. count Frequency by .count group Male Female by of patients Neubauer by Years Analayzer chamber PBF 0-10 9 4 5 2.85 2.71 2.78 11-20 53 27 26 1.71 1.84 1.86 21-30 135 36 99 1.39 1.54 1.58 31-40 88 34 54 1.47 1.57 1.59 41-50 60 33 27 1.56 1.64 1.67 51-60 38 23 15 1.47 1.44 1.48 61-70 15 9 6 1.39 1.56 1.58 71-80 2 1 1 1.80 1.93 1.97 TOTAL 400 167 233 13.64 14.37 14.51

Table 1: Age wise variation of platelet count.

2.3.4Calculation The formula used for calculating the cell count was

Platelets/mm3 = number of platelet counted × dilution area counted×depth of fluid Where; Dilution = 200 Area =1mm2 Depth = 0.1mm

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Vol. 2 Issue 4, April - 2020 colorless, discoid or elliptical refractile bodies. In Leishman`s stained PBS, platelets appears as light 3 blue colored, round, oval or rod shaped structures. 2.5 plt.count by Platelet are multifunctional and plays key role in 2 Analayzer hemostasis, thrombosis and wound repair. The 1.5 normal range of platelets count in healthy human being is 15×104 to 40×104 platelets perµl. The 1 plt.count by thrombocytopenia is one of the critical conditions neubauer 0.5 where patients platelet count decreases below the chamber (34,35) 0 normal range. plt.count by The platelet count is commonly done in PBF laboratories by evaluation of PBS, Neubauers

chamber counting or by automated hematology

0-10 years 0-10

61-70 years 61-70 21-30 years 21-30 years 31-40 years 41-50 years 51-60 years 71-80 11-20 years 11-20 analyzer. The platelet counting is more difficult as compared to RBCS count and WBC count. Until Total Std. Method Mean P value recently, the Brecher and Cronkite platelet counting cases deviation method described in 1950, was consider as reference Automated 400 1.705 0.485 0.57 method while comparing with platelet counting by method semi-automated and automated method. These Neubauer 400 1.776 0.403 manual phase contrast microscopic method was chamber considered as gold standard which had significant limitation in view of imprecision. This method results are highly variable and depends upon individual Table 2: Comparison between automated analyzer training. Few researchers mentioned that the risk of and Neubauer chamber. error estimation is upto 10% to 20% for this manual platelet counting method. (35) Total Std. Method Mean P value cases deviation In the year2001, a joint task force of ISLH and Automated ICSH recommended a new immunological based 400 1.705 0.485 0.87 method reference method for platelet counting. This method PBF 400 1.813 0.422 utilizes monoclonal antibodies to platelet surface antigens conjugated to suitable flurophore. This method permits the possible implementation of new Table 3: Comparison between automated analyzer reference method for calibration of hematology (36) and PBF. analyzer. The mean platelet count by Automated method The platelet count with hematology analyzer is was 1.70/µl. The mean platelet count by Neubauer usually precise. In hematology analyzers, the principle chamber is 1.77/µl and mean platelet count by PBF of particle impedence is utilized which was first was 1.81/µl. The P value for automated versus described by Wallace H. Coulter in 1954. The Neubauer chamber was 0.57 and for automated hematology analyzer platelet count accuracy is method versus PBF was 0.87 Suggesting that there compromised while processing blood samples with was no significant variation in the platelet count low platelet counts or with blood samples with between the manual methods when compared with abnormal platelets morphology like giant platelets or automated analyzer. blood samples having presence of non platelet particle like RBC, WBC fragments. The accuracy of Discussion: platelet count is also compromised in hematology The precise, accurate and reliable assessment of analyzer due to inadequate calibrations and lack of .[37, 38] platelet count is required to avoid unnecessary adequate quality control material Despite of platelet transfusion in the treatment of severe advances in hematology automation, manual counting thrombocytopenia patients. The accuracy is also methods has its own importance in hematology needed after platelet transfusion treatment to check laboratories for validating results of other methods for the therapeutic response. platelet counting. Till date, even the accurate and best quality hematology analyzer also cannot replace the The platelets circulate in the blood as small disc manual counting evaluation. (39) and are derived from megakaryocytes in the bone marrow. Megakaryocyte constitutes <1% of myeloid Web et al and Bajpai et al had reported slightly 4 cells in the bone marrow. One megakaryocyte can better results with 15.0×10 platelets per µl as platelet 4 give rise to one thousand to three thousand platelets. multiplication factor instead of 20 ×10 platelets per µl (40, 30). The platelets are about 3µm in diameter and are non- The PBS platelet count estimation method gives nucleated. The life span of normal platelet is about approximate platelet count and not the exact on and is seven to twelve days and is destroyed by splenic the drawback of this method. However PBS method macrophages. In wet preparation, platelet appears as precisely comments on adequacy of platelet count in

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Vol. 2 Issue 4, April - 2020 patient blood samples and hence this method has 4) A1-Hosni ZS, Al-Khabori M, Al-Mamari S, et al. upper hand over other methods in diagnosing Reproducibility of Manual Platelet Estimation thrombocytopenia cases and avoiding unnecessary Following Automated Low Platelet Counts. Oman platelet transfusion. Medical Journal. 2016 ; 31 (6) : 409-413. 10. 5001/omj. 2016. 83 Neubauer chamber inspite of being the best manual counting technique, an error still occurs due to 5) Umashankar T, Thomas BM, Sahana P random distribution of the cells in the chamber. This Estimation of platelet count in unstained peripheral results in variation in cell number in different areas of blood smears in comparison with stained smears and the same size. This distribution follows Poisson`s law. evaluation of its efficacy. Estimation of platelet count More number of cells should be counted to reduce this in unstained peripheral blood smears in comparisori error. with'stained smears'and evaluation of its efficacy. Malays J Patho1. 2014 Dec ; 36 (3) The present study has shown that manual methods are reliable to validate the automated counts in routine 6) Phakhounthong K, Chaovalit P, Jittamala p, practice under standard conditions and can be used BlacksellsD, Carter MJ, Turner P et al. Predicting the as alternative methods for platelet estimation as well severity of dengue fever in children on admission as the standard methods in rural clinical set ups based on clinical features and laboratory indicators: where automatic analysers are a remote possibility. application of classification, tree analysis. BMC Pediatrics. 2018 ; 18 : 109. Doi : 10. 1186/5 12887- In addition, it is essential that pathology laboratory 018-1078-y. personals and clinicians who rely on platelet counts for various scenarios in day to day practice 7) Milovanovic Alempijevic T, Stojkovic Lalosevic understand the limitations of the instrumentation in M, Dumic I, et al. Diagnostic Accuracy of Platelet use and the measurement uncertainty of automated Count and Platelet Indices in Noninvasive platelet counters. Assessment of Fibrosis in Nonalcoholic Fatty Liver Disease PatienSts. Canadian Can J Gastroenterol However, limitation of the study are a small Hepatol. 2017; 2017 : 6070135. Doi: 10. 1155/2017/ sample size and that reproducibility of the manual 6070135. methods in case of thrombocytopenia, thrombocytosis and normal ranges have not been studied 8) De la Salle BJ, McTaggart PN, Briggs C, independently. Harrison P, Dore CJ, Longair I, et al. The accuracy of platelet counting in thrombocytopenic blood samples Conclusion: distributed by the UK National External Quality The study highlights the differences in platelet Assessment Scheme for General Haematology. Am J count in the population by using automated and Clin Pathol. 2012 jan; l37(1) : 65-74. doi. org/10. manual counting methods. During analysis it was 1309/AJCP86JMB FUCFCXA found that platelet count by manual methods is a little 9) Buttarello M. quality specification in hematology higher as compared to the automated method, the the automated blood cell count. Clin Chim Acta. 2004 possible reason for this could be the large sized ; 346 : 45-54. Doi : 10. 1 0 1 6/j. cccn. 2004. 02. 03 platelets (Giant platelets with size ranging from 10- 20µm) which the analyzers are not able to count. 10) Briggs C, Harrison P, Machin SJ. Continuing developments with the automated platelet count. Int The study tries to validate the manual methods for LabHem 2007;29 :77-91. 10. 5958/2394-6792. 2016. use in circumstances where in a fully automated 00066.1. hematology analyzer is not available as is usually seen in in primary healthcare centers or urban health 11) National Accreditation Board for Testing and centre. This also reaffirms that manual methods may Calibration Laboratories (IN). NABL 112 Specific also be used to validate the results shown by Criteria for Accreditation of Medical Laboratories. analyzer. Gurgaon: National Accreditation Board for Testing and Calibration Laboratories; 2016. p.36

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