IntroductionClinical laboratory in the 21st generation has evolved from simple observation and description of blood and its components to a highly automated and extremely technical science. However, some of the more basic tests have not changed dramatically over the years. This research presents the significance of manual and semi automated methods that can be used in lieu of automated instrumentation.Although most routine cell-counting procedures in the laboratory are automated, it may be necessary to use manual methods when counts exceed the linearity of an instrument, when an instrument is nonfunctional and there is no backup, in remote laboratories, or in a disaster situation when testing is done in the field.The used of automated cell-counting analyzers has directly affected the availability, accuracy, and clinical usefulness of the CBC and WBC differential count. Some parameters that are available on laboratory instrumentation, but cannot be derived manually, have provided further insight into various clinical conditions.This research emphasizes the procedures for manual counting of leukocytes. Manual cell counts and differentiation were originally the only means of enumerating and classifying cellular elements in blood and body fluids. To a large extent, the laboratory evaluation of leukocytes, erythrocytes, and platelets has now been automated. If automated leukocyte is available, why must laboratory scientist learn manual techniques for evaluating them? There are several reasons.Thesis statementManual computation is relevant in counting white blood cell in patient.

Objectives1. Emphasize how and why manual and semi-automated method would be convenient in a laboratory in counting white blood cells in patients blood.2. Ensure that the use of manual and semi-automated method in WBC count is accurate.

Overview of the white blood cellsImportance of white blood cellsThe primary function of leukocytes as a group is defense against any invader into the body that may be recognized as foreign. Each cell type has a specific function and behaves as a separate but related system. Five principal types of leukocytes normally circulate in peripheral blood: neutrophils, eosinophils, basophils, monocytes and lymphocytes. Several different criteria may be used to classify leukocytes. According to granularity leukocytes may be classified as granulocytes (neutrophils, eosinophils, basophils) and nongranulocytes (monocytes and lymphocytes). The granulocytes contain distinct granules in their cytoplasm when stained and examine on a routine smear, whereas nongranulocytes have no cytoplasmic granules and have as small, rounded nuclei.Neutrophils are the most common PMN and are produced in 7 to 14 days and remain in the circulation for 6 hours. The primary function of the neutrophil is phagocytosis, killing and digestion of bacterial microorganisms. Acute bacterial infections and trauma stimulate neutrophil production, resulting in an increased WBC count. Basophils also called as mast cells and especially eosinophils are involved in the allergic reaction. They are capable of phagocytosis of antigen-antibody complexes. As the allergic response diminishes the eosinophil counts decreases. Lymphocytes are divided into two types: T cells which mature in thymus and B cells which mature in bone marrow. T cells involved primarily with cellular-type immune reactions, whereas B cells participates in humoral immunity. The primary function of lymphocytes is to fight chronic bacterial infection and acute viral infections.Interfering factors that may lead to increase and decrease of white blood cell in patient

Factors that may lead to increase and decrease of white blood cellsEating, physical activity and stress may cause an increased WBC count and alter the different values. Final months in pregnancy and labor may be associated with increased WBC levels. Patients who have had a splenectomy have a persistent wild to moderate elevation of WBC counts. The WBC count tends to be lower in the morning and higher in the late afternoon. The WBC count tends to be age-related. Normal newborns and infants tend to have higher WBC count than adult. Drugs that may cause increased WBC levels include adrenaline, allopurinol, aspirin, chloroform, epinephrine, heparin, quinine, steroids and triamterene. Drugs that may decrease WBC levels include antibiotics, anticonvulsants, antihistamines, antimetabolites, antithyroid drugs, arsenicals, barbiturates, chemotherapeutic agents, diuretics, and sulfonamides.

Procedures that may take place in drawing white blood cell count in patientsBefore the test is taken, explain the procedure to the patient and tell the patient that no fasting is required. During the actual test is taken, Collect approximately 5 to 7 ml of venous blood in a lavender-top tube. After the test is taken, apply pressure to the venipuncture site.

Meaning of an increased WBC countInfection: WBCs are integral initiating and maintaining the bodys defense mechanism against infection.Leukemia neoplasia or other myeloproliferative disorders: These neoplastic cells are produced by the marrow and are released into blood stream.Other malignancy: Advanced non-marrow cancers (e.g. lung) are associated with leukocytosis.Trauma, stress, or hemorrhage: the WBC count is probably under hormonal influence (e.g. epinephrine).Inflammation: the pathophysiology of this observation is complex, including recognition of neurotic or normal tissue as foreign so that a WBC response is instituted. Dehydration: not only is dehydration a stress that, itself, increases the WBC count, but also by virtue of hemoconcentration, the WBC count increases.Thyroid storm: the WBC is probably influence by thyroid hormones.Steroid use: glucocorticosteriods stimulate WBC production.

Meaning of a decreased WBC countDrug toxicityBone marrow failureOverwhelming infectionsDietary deficiencyCongenital marrow aplasiaBone marrow infiltrationAutoimmune diseaseHypersplenism

Manual method of white cell countPrinciple Whole blood is mixed with a weak acid solution to dilute the blood and hemolyze the red blood cells. Then loaded into a Hemacytometer and counted.Specimen Whole blood may be obtained from a venous EDTA sample or a free flowing capillary puncture and diluted 1:100 with a Unopette.ReagentsA prepared kit is available that uses the Unopette system for white blood cellcounts. The reservoir contains 3% glacial acetic acid. A 25 ul capillary pipette is used to aspirate the blood sample and make a 1:20 dilution in the reservoir. (Alternatively, the Unopette containing ammonium oxalate and result in a 1:100 dilution may be used).

Procedure(1) Dilute the specimen - let stand for 10 minutes to allow red cells to hemolyzed.(2) Expel first 3 to 4 drops of diluted specimen to clean capillary bore.(3) Charge the Hemacytometer (both sides) with the diluted specimen.(4) Cells must settle for a minimum 3 to 5 minutes after placing Hemacytometer in moist chamber.(5) Count white blood cells. Use low power objective and low light. Viewed under low power, leukocyte nuclei appear slightly iridescent but not retractile; cells should have a visible cell wall and nucleus; use fine focus to differentiate them from artifacts. Count all WBCs within the 9 large squares (1:100) and those WBCs touching upper and right-hand perimeter lines. Count second side of Hemacytometer in the same manner. Validation that each side of the chamber was charged equally - Total number of cells counted on each side of the counting chamber should agree within 10 percent of each other - calculate acceptable range using lower count.CalculationThe depth of the counting chamber is 0.1 mm and the area counted is 4 sq mm (4 squares are counted, each with an area of 1.0 sq mm therefore, 4 X 1.0 sq mm = a total of 4 sq mm). The volume counted is: area X depth = volume. 4 mm2X 0.1 mm = 0.4 mm3(cubic millimeters).Here is the formula:

Sources of ErrorImproper collection of blood specimens causes variable results.Wet or dirty pipets.Not allowing cells to settle for an adequate amount of time.Poor pipetting technique causes high or low counts. Poor pipetting technique includes:Undershooting Unopette with blood.Overfilling Unopette with blood.Air bubbles in the shaft.Not mixing the blood specimen thoroughly.Failure to expel 3 or 4 drops in the pipet tips before charging the Hemacytometer.Overfilling the chamber of the hemacytometer, which causes erroneously high counts.Not mixing the diluted specimen prior to filling the Hemacytometer.Uneven distribution of cells in the counting chamber causes erroneous results.Counting artifacts.Dirty or scratched Hemacytometer.Failure to mix anticoagulated blood thoroughly before use.

Evolution of automated cell countersTraditionally, the blood counts were performed manually using the hemocytometers and the differential leukocyte counts by studying the peripheral blood smears (also referred to as the 100-cell slide differential, eye count leukocyte differential or manual counts). The Coulter principle led to the availability of the Coulter counters and thereafter, the development of sophisticated automated blood-cell analyzers. The level of sophistication has been rising ever since. Technological advancements have made it possible to incorporate increasingly more analysis parameters as possible into single instrument platforms, in order to minimize the need to run a single sample on multiple instruments. The modern versions of analyzers are capable of measuring white blood cells (WBC), WBC differentials (5 part differentials), red blood cells (RBC), hemoglobin (HGB), platelets, mean corpuscular volume (MCV),and mean platelet volume, and automatically calculating hematocrit (HCT), mean corpuscular hemoglobin (MCH), MCH concentration (MCHC), RBC distribution width, plateletcrit, and platelet (PLT) distribution width. The other crucial considerations in automatic analyzers are the speed with which they perform and the number of specimens they can process per batch (reduction in turnaround time due to high throughput).

Limitations in manual method of white blood cell countExperience is needed to make technically adequate smears consistently.Non-uniform distribution of WBCs over the smear, with larger leukocytes concentrated near the edges and lymphocytes scattered throughout.There is a non-uniform distribution of red blood cells as well, with small crowded red blood cells at the thick edge and large flat red blood cells without central pallor at the feathered edge of the smear.It is subjective, labor-intensive,and statistically unreliable (only 100-200 cells are counted).It is imprecise with reported Coefficients of Variation (CV) ranging from 30 110 %.Cell identification errors in manual counting: This is mostly associated with distinguishing lymphocytes from monocytes, bands from segmented forms and abnormal cells (variant lymphocytes from blasts). The monocytes tend to be underestimated and the Lymphocytes tend to be overestimated.

Advantages of manual method in WBC countNot all samples can be evaluated by automated methods for one reason or another. For example, when a leukocyte count is extremely low or high, it may be necessary to perform manual count because of loss of instrument linearity (capability to count cells accurately) at the extreme ends of the spectrum.Other samples may require manual cell counting including those with abnormal proteins, clumped platelets, or antibody elements in the plasma that interfere with an instruments ability to count leukocytes.For some laboratories automated method may be evaluated by manual methods, although automated leukocyte and erythrocyte counts should be evaluated with single-channel automated cell counter as a backup rather than a manual method. Extremely abnormal leukocytes, such as those seen in leukemia, may not, in some cases, be accurately differentiated by automated methods.

ConclusionUsing manual method would be definitely more time consuming but to consider the linearity of the computation of the specimens white blood cell count, it is needed to use the manual method for back up purpose and variety of any laboratory use. Creating these automated analyzers we may lessen the number of laboratory technologists but considering the extent of how far can these analyzers give an exact result and if ever there would be an emergency cases and there is no supply of electricity, would question that we need to learn the manual techniques in many laboratories.

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