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Band Granulocyte Stab Cell The juvenile cell or the band cell are the youngest granulocytes normally found in the peripheral blood. It is not segmented but may be slightly indented at one two points. The chromatin is continuous thick and coarse, and parachromatin is scanty. Cytoplasm: contains specific and a few non-specific granules and is pink or colorless. Nucleus: eccentric with heavy, thick chromatin masses. The ratio of segmented to band forms is of clinical significance and is normally about Cytoplasm: abundant and slightly eosinophilic pinkish or colorless and contains specific granules.

The neutrophilic granules are very fine in texture and do not overlay the nucleus. Eosinophilic Granulocyte and Precursors Eosinophils mature in the same manner as neutrophils. The eosinophlic myeloblast is not recognizable as such. In the eosinophilic promyelocyte in the Wright-Giemsa stained preparation the granule are at first bluish and later mature into orange granules, which are larger than neutrophilic granules are round or ovoid and are prominent in the eosinophilic myelocyte.

Eosinophils with more than two nuclear lobes are seen in vitamin B12 and folic acid deficiency and in allergic disorders. Cytoplasm: densely filled with orange-pink granules so that its pale blue color can be appreciated only if the granules escape. The granules are uniform in size, large and do not cover the nucleus.

Basophilic Granulocyte and Precursors The early maturation of the basophilic granulocyte is similar to that of the neutrophlic granulocyte. Nucleus: Indented giving rise to an S pattern. It is difficult to see the nucleus because it contains less chromatin and is masked by the cytoplasmic granules. Cytoplasm: Pale blue to pale pink and contains granules that often overlie the nucleus but do not fill the cytoplasm as completely as the eosinophilis granules do.

Nucleus: Round or oval and at times notched and indented. The chromatin is delicate blue to purple stippling with small, regular, pink, pale or blue parachromatin areas. The nucleoli in number are pale blue, large and round. Cytoplasm: Relatively large in amount, contains a few azurophile granules, and stains pale blue or gray.

The cytoplasm filling the nucleus indentation is lighter in color than the surrounding cytoplasm. The surrounding cytoplasm may contain Auer bodies. Promonocyte The earliest monocytic cell recognizable as belonging to the monocytic series is the promonocyte, which is capable of mitotic division. Nucleus: Large, ovoid to round, convoluted, grooved, and indented.

The chromatin forms a loose open network containing a few larger clumps. There may be two or more nucleoli. Cytoplasm: sparse, gray-blue, contains fine azurophilic granules. Nucleus: Eccentric or central, is kidney shaped and often lobulated.

The chromatin network consists of fine, pale, loose, linear threads producing small areas of thickening at their junctions. No nucleolus is seen. The overall impression is that of a pale nucleus quite variable in shape. Cytoplasm: Abundant, opaque, gray-blue, and unevenly stained and may be vacuolated. Lymphoid precursor cells travel to specific sites, where they differentiate into cells capable of either expressing cell- mediated immune responses or secreting immunoglobulins.

The influence for the former type of differentiation in humans is the thymus gland; the resulting cells are defined as thymus-dependent lymphocytes, or T cells. The site of the formation of lymphocytes with the potential to differentiate into antibody-producing cells has not been identified in humans, although it may be the tonsils or bone marrow.

In chickens it is the bursa of Fabricius, and for this reason these bursa-dependent lymphocytes are called B cells. Nucleus: Central, round or oval and the chromatin has a stippled pattern. Cytoplasm: Non-granular and sky blue and may have a darker blue border.

It forms a thin perinuclear ring. Nucleus: Oval but slightly indented and may show a faint nucleolus. The chromatin is slightly condensed into a mosaic pattern. Cytoplasm: there is a thin rim of basophlic, homogeneous cytoplasm that may show a few azurophilic granules and vacuoles.

Lymphocytes There are two varieties and the morphologic difference lies mainly in the amount of cytoplasm, but functionally most small lymphocytes are T cells and most large lymphocytes are B cells.

The chromatin is dense and clumped. A poorly defined nucleolus may be seen. Cytoplasm: It is basophilic and forms a narrow rim around the nucleus or at times a thin blue line only. Its chromatin is dense and clumped. Cytoplasm: abundant, gray to pale blue, unevenly stained, and streaked at times.

These are large granular lymphocytes LGLs. Formation of platelets Thrombopoiesis Platelets are produced in the bone marrow by fragmentation of the cytoplasm of megakaryocytes. The precursor of the megakaryocyte-the megakaryoblast- arises by a process of differentiation for the hemopoietic stem cell. Megakaryocyte development takes place in a unique manner.

The nuclear DNA of megakaryoblasts and early megakaryocytes reduplicates without cell division, a process known as endomitosis.

As a result, a mature megakaryocytes has a polyploidy nucleus, that is, multiple nuclei each containing a full complement of DNA and originating from the same locust within the cell. Mature megakaryocytes are 8 n to 36 n. The final stage of platelet production occurs when the mature megakaryocyte sends cytoplasmic projections into the marrow sinusoids and sheds platelets into the circulation. It takes approximately 5 days from a megakaryoblast to become a mature megakaryocyte.

Each megakaryocyte produces from to platelets. The platelet normally survives form 7 to 10 days in the peripheral blood. The cell is smaller than its mature forms but larger than all other blast cells. Multi-lobulated nuclei also occur representing a polyploid stage. Several pale blue nucleoli are difficult to see. The parachromatin is pink. Cytoplasm: the cytoplasm forms a scanty, bluish, patchy, irregular ring around the nucleus.

The periphery shows cytoplasmic projections and pseudopodia like structures. The immediate perinuclear zone is lighter than the periphery. It is larger than the megakaryoblast and in the process of maturation it reaches the size of the stage III cell.

Nucleus: large, indented and poly-lobulated. The chromatin appears to have coarse heavily stained strands and may show clumping. The total number of nucleoli is decreased and they are more difficult to see than in the blast cell. The chromatin is thin and fine. Granular Megakaryocyte The majority of the megakaryocytes of a bone marrow aspirate are in stage III which is characterized by progressive nuclear condensation and indentation and the beginning of platelet formation within the cytoplasm.

Cytoplasm: a large amount of polychromatic cytoplasm produces blunt, smooth, pseudopodia-like projections that contain aggregates of azurophilic granules surrounded by pale halos. These structures give rise to platelets at the periphery of the megakaryocytes. Nucleus: no nucleus is present. What is hemopoiesis and how is the process regulated? What are the hemopoietic tissues during fetal life, in infancy, in childhood and in adulthood?

What are the effects of the hormone erythropoietin on red cell development and maturation 4. Explain what megaloblastic erythropoiesis is. State the main functions of blood. Blood must be collected with care and adequate safety precautions to ensure test results are reliable, contamination of the sample is avoided and infection from blood transmissible pathogens is prevented. Unless an appropriately designed procedure is observed and strictly followed, reliability can not be placed on subsequent laboratory results even if the test itself is performed carefully.

All material of human origin should be regarded as capable of transmitting infection. Specimens from patients suffering from, or at risk of, hepatitis or human immunodeficiency virus HIV infection require particular care.

When collecting blood sample, the operator should wear disposable rubber gloves. The operator is also strongly advised to cover any cuts, abrasions or skin breaks on the hand with adhesive tape and wear gloves. Care must be taken when handling especially, syringes and needles as needle-stick injuries are the most commonly encountered accidents.

Do not recap used needles by hand. Should a needle-stick injury occur, immediately remove gloves and vigorously squeeze the wound while flushing the bleeding with running tap water and then thoroughly scrub the wound with cotton balls soaked in 0. Three general procedures for obtaining blood are 1 Skin puncture, 2 venipuncture, and 3 arterial puncture.

The technique used to obtain the blood specimen is critical in order to maintain its integrity. Even so, arterial and venous blood differs in important respects.

Arterial blood is essentially uniform in composition throughout the body. The composition of venous blood varies and is dependent on metabolic activity of the perfused organ or tissue. Site of collection can affect the venous composition. Venous blood is oxygen deficient relative to arterial blood, but also differs in pH, carbon dioxide concentration, and packed cell volume.

Blood obtained by skin puncture is an admixture of blood from arterioles, venules, and capillaries. Increased pressure in the arterioles yields a specimen enriched in arterial blood. Skin puncture blood also contains interstitial and intracellular fluids.

It is also used when venipuncture is impractical, e. Note: Edematous, congested and cyanotic sites should not be punctured. Cold sites should not be punctured as samples collected from cold sites give falsely high results of hemoglobin and cell counts. Site should be massaged until it is warm and pink. If the heel is to be punctured, it should first be warmed by immersion in a warm water or applying a hot towel compress. Otherwise values significantly higher than those in venous blood may be obtained.

After the skin has dried, make a puncture mm deep with a sterile lancet. A rapid and firm puncture should be made with control of the depth. A deep puncture is no more painful than a superficial one and makes repeated punctures unnecessary. The first drop of blood which contains tissue juices should be wiped away. The site should not be squeeze or pressed to get blood since this dilutes it with fluid from the tissues.

Rather, a freely flowing blood should be taken or a moderate pressure some distance above the puncture site is allowable. The veins in the antecubital fossa of the arm are the preferred sites for venipuncture. They are larger than those in the wrist or ankle regions and hence are easily located and palpated in most people.

Puncture of the external jugular vein in the neck region and the femoral vein in the inguinal area is the procedure of choice for obtaining blood. Method 1. Assemble the necessary materials and equipment. Attach the needle so that the bevel faces in the same direction as the graduation mark on the syringe. The gauge and the length of the needle used depend on the size and depth of the vein to be punctured. The gauge number varies inversely with the diameter of the needle.

The needle should not be too fine or too long; those of 19 or 21G are suitable for most adults, and 23G for children, the latter especially with a short shaft about 15mm.

The point of the needle will thus be embedded in the stopper without puncturing it and loosing the vacuum in the tube. Allow it to dry in the air or use a dry pad or cotton. The area should not be touched once cleaned. Apply a tourniquet at a point about cm above the bend of the elbow making a loop in such a way that a gentle tug on the protruding ends will release it. Alternatively, the veins can be visualized by gently tapping the antecubital fossa or applying a warm towel compress.

Using the assembled syringe and needle, enter the skin first and then the vein. The needle should be pointing in the same direction as the vein. If the needle is properly in the vein, blood will begin to enter the syringe spontaneously. If not, the piston is gently withdrawn at a rate equal to the flow of blood. Apply a ball of cotton to the puncture site and gently withdraw the needle.

Instruct the patient to press on the cotton. The sample should never be shaked. With the vacutainer system, remove the tube from the vacutainer holder and if the tube is with added anticoagulant, gently invert several times. Reinspect the venipuncture site to ascertain that the bleeding has stopped. It also frequently allows the performance of additional tests that may be suggested by the results of those already ordered or that may occur to the clinician as afterthoughts.

Difference between peripheral and venous Blood Venous blood and peripheral blood are not quite the same, even if the latter is free flowing, and it is likely that free flowing blood obtained by skin puncture is more arteriolar in origin. The PCV, red cell count and hemoglobin content of peripheral blood are slightly greater than in venous blood. This may be due to adhesion of platelets to the site of the skin puncture. The multiple sample needle used in the vacutainer method has a special adaptation that prevents blood from leaking out during exchange of tubes.

These blood gas measurements are critical in assessment of oxygenation problems encountered in patients with pneumonia, pneumonitis, and pulmonary embolism. Arterial punctures are technically more difficult to perform than venous punctures. Increased pressure in the arteries makes it more difficulty to stop bleeding with the undesired development of a hematoma.

Arterial selection includes radial, brachial, and femoral arteries in order of choice. Sites not to be selected are irritated, edematous, near a wound, or in an area of an arteriovenous AV shunt or fistula. Avoid rough handling of blood at any stage.

Do not eject the blood from the syringe through the needle as this may cause mechanical destruction of the cells. Transfer the blood from the syringe by gently ejecting down the side of the tube. Stopper and store in a refrigerator at 4OC.

Blood should not be stored in a freezer because the red cells will hemolyse on thawing. Hypotonic solutions will lead to hemolysis. The blood should be allowed to escape freely. What are the sources of blood sample for hematological investigations?

What are the anatomical sites of collection in these sources in the different age groups? How do you minimize or avoid the occurrence of hemolysis in blood samples for hematological investigations?

What is the difference between samples collected from these two sources in terms of hematological parameters? In other words, certain steps are involved in blood coagulation, but if one of the factors is removed or inactivated, the coagulation reaction will not take place.

The substances responsible for this removal or inactivation are called anticoagulants. While clotted blood is desirable for certain laboratory investigations, most hematology procedures require an anticoagulated whole blood.

EDTA and sodium citrate remove calcium which is essential for coagulation. Calcium is either precipitated as insoluble oxalate crystals of which may be seen in oxalated blood or bound in a non-ionized form.

Heparin works in a different way; it neutralizes thrombin by inhibiting the interaction of several clotting factors in the presence of a plasma cofactor, antithrombin III. Sodium citrate or heparin can be used to render blood incoagulable before transfusion. Ethylenediamine tetraacetic acid Ethylenediamine tetraacetic acid EDTA has become the standard hematology anticoagulant because of its very efficient and complete anticoagulation and its lack of effect on the size morphology or number of blood cells in the specimen.

Its disodium or tripotassium salt are used. The anticoagulant recommended by the ICSH is the dipotassium salt. It is the preferred anticoagulant for cell counts and morphological studies. It exerts its effect by tightly binding chelating ionic calcium thus effectively blocking coagulation. The dilithium salt of EDTA is equally effective as an anticoagulant, and its use has the advantage that the same sample of blood can be used for chemical investigation.

The amount of EDTA necessary for the complete chelation of Calcium is balanced with the desire to minimize cellular damage so that standardizing bodies have recommended a concentration of 1. This concentration does not appear to adversely affect any of the erythrocyte or leucocyte parameters. Nine volumes of blood are added to 1 volume of the sodium citrate solution and immediately well mixed with it. Sodium citrate is also the anticoagulant for the erythrocyte sedimentation rate ESR ; for this, 4 volumes of venous blood are diluted with 1 volume of the sodium citrate solution.

Balanced or double oxalate Salts of oxalic acid by virtue of their ability to bind and precipitate calcium as calcium oxalate serve as suitable anticoagulants for many hematologic investigations. Three parts of ammonium oxalate is balanced with two parts of potassium oxalate neither salt is suitable by itself, i. Heparin Heparin is an excellent natural anticoagulant extracted from mammalian liver or pancreas.

Heparin prevents clotting by inactivating thrombin, thus preventing conversion of fibrinogen to fibrin. It is the best anticoagulant when absolute minimal hemolysis is required e.

It is unsatisfactory for leucocyte and platelet and leucocyte counts as it causes cell clumping and also for blood film preparation since it causes a troublesome diffuse blue background in Wright-stained smears.

It is used in the proportion of 0. Define anticoagulant. List the anticoagulants that are commonly used in hematology. How does each of these anticoagulants exert their functions? Write the proportion of the volume of blood to the volume of each if these anticoagulants. A great deal of information can be obtained from the examination of a blood film.

With the use of automatic counting devices that determine hemoglobin, hematocrit, red cell, white cell, and platelet counts together with MCV, MCH, MCHC, and RDW, white cell differential, and histograms, there is a tendency to place less emphasis on the routine examination of the peripheral blood film.

Of course, in a laboratory without access to such automated information, the microscopic examination of the peripheral blood film is invaluable. Examination of the blood film is an important part of the hematologic evaluation and the validity or reliability of the information obtained from blood film evaluation, the differential leucocyte count in particular depends heavily on well-made and well- stained films.

While blood film preparation is a disarmingly simple straight - forward procedure, there is abundant and continuing evidence that the quality of blood films in routine hematology practice leaves much room for improvement. If not made from skin puncture, films should be prepared within 1 hour of blood collection into EDTA.

Adequate mixing is necessary prior to film preparation if the blood has been standing for any appreciable period of time. Method I. Another slide, the spreading slide placed in front of the drop of blood at an angle of to the slide and then is moved back to make contact with the drop. The drop will spread out quickly along the line of contact of the spreader with the slide.

The drop should be of such size that the film is cm in length approx. It is essential that the slide used as a spreader have a smooth edge and should be narrower in breadth than the slide on which the film is prepared so that the edges of the film can be readily examined. If the edges of the spreader are rough, films with ragged tails will result and gross qualitative irregularity in the distribution of cells will be the rule.

The bigger leucocytes neutrophils and monocytes will accumulate in the margins and tail while lymphocytes will predominate in the body of the film. The faster the film is spread the thicker and shorter it will be. The bigger the angle of spreading the thicker will be the film. If these are not available, writing can be done by scratching with the edge of a slide. A paper label should be affixed to the slide after staining.

Fig 4. If the drop is not too large and if the cover glasses are perfectly clean, the blood will spread out evenly and quickly in a thin layer between the two surfaces.

After they are stained they are mounted film side down with permount film side down on glass slides. The spinner slide produces a uniform blood film, in which all cells are separated a monolayer and randomly distributed. White cells can be easily identified at any spot in the film On a wedge smear there is a disproportion of monocytes at the tip of the feather edge, of neutrophils just in from the feather edge, and of both at the later edges of the film.

This is of little practical significance, but it does result in slightly lower monocyte counts in wedge films. Preparation of thick blood smears Thick blood smears are widely used in the diagnosis of blood parasites particularly malaria. It gives a higher percentage of positive diagnosis in much less time since it has ten times the thickness of normal smears. Five minutes spent in examining a thick blood film is equivalent to one hour spent in traversing the whole length of a thin blood film.

Method Place a small drop of blood on a clean slide and spread it with an applicator stick or the corner of another slide until small prints are just visible through the blood smear. This corresponds to a circle of approximately 2cm diameter.

What is a thin blood film? Which technique of blood film preparation is commonly employed and how is the method of preparation? What are the desirable qualities of a thin blood film? What are the possible effects of using a blood sample that has been standing at room temperature for some time on blood cell morphology?

Principle of staining Acidic dyes such as eosin unites with the basic components of the cell cytoplasm and hence the cytoplasm is said to be eosinophilic acidic.

Conversely, basic stains like methylene blue are attracted to and combine with the acidic parts of the cell nucleic acid and nucleoproteins of the nucleus and hence these structures are called basophilic. Other structures stained by combination of the two are neutrophilic 5. Wright stain In its preparation, the methylene blue is polychromed by heating with sodium carbonate. It is purchased as a solution ready to use or as a powder.

Staining Method 1. Place the air-dried smear film side up on a staining rack two parallel glass rods kept 5cm apart. Cover the smear with undiluted stain and leave for 1 minute. The methyl alcohol in the satin fixes the smear. When it is planned to use an aqueous or diluted stain, the air dried smear must first be fixed by flooding for minutes with absolute methanol.

Dilute with distilled water approximately equal volume until a metallic scum appears. Allow this diluted stain to act for minutes. Without disturbing the slide, flood with distilled water and wash until the thinner parts of the film are pinkish red.

Giemsa stain Instead of empirically polychromed dyes, this stain employs various azure compounds thionine and its methyl derivative with eosin and methylene blue. This is an alcohol-based Romanowsky stain that required dilution in pH 7. It gives the best staining of malaria parasites in thick films.

Staining of thick smears The stains used employ the principle of destroying the red cells and staining leucocytes and parasites. The method using Giemsa stain is satisfactory. Cover the air-dried smear with a diluted Giemsa using buffered distilled water at pH 6. Do not fix the films before staining. Leave the stain to act for minutes. Wash with distilled water and air dry. Panoptic staining Panoptic staining consists of a combination of a Romanowsky stain with another stain, e.

This improves the staining of cytoplasmic granules and other bodies like nucleoli of blast cells. Jenner-Giemsa method 1. Dry the films in the air then fix by immersing in a jar containing methanol for minutes. For bone marrow films leave for minutes. Transfer the films to a staining jar containing Jenner's stain freshly diluted with 4 volumes of buffered water and leave for 4 minutes. Transfer the slides without washing to a jar containing Giemsa stain freshly diluted with 9 volumes of buffered water pH 6.

Allow to stain for minutes. Transfer the slides to a jar containing buffered water, pH 6. Place the slides on end to dry.

Transfer the slides without washing to a jar containing Giemsa's stain freshly diluted with 9 volumes of buffered water pH 6. It is buffered to the correct pH and neither solution requires dilution when staining thick films.

They stain fresh blood films, well, particularly thick films. The rapid technique is ideally suited for staining blood films from waiting outpatients and when reports are required urgently. Place the slide on a staining rack and cover the methanol-fixed thin film with approximately 0.

Leave to stain for 1 minute. The stain can be easily applied and mixed on the slide by using 1ml graduated plastic bulb pipettes.

Wash off the stain with clean water. Wipe the back of the slide clean and place it in a draining rack for the film to air-dry. Drain off the excess stain by touching a corner of the slide against the side of the container.

Wash gently for about 5 seconds in clean water. Drain off the excess water. Drain off the excess stain. Wash gently in clean water. Wipe the back of the slide clean and place it upright in a draining rack for the film to air-dry. What is the general principle of staining blood films with Romanowsky dyes? What are the various Romanowsky stains used for staining of blood films?

Describe the appearance of cells and cell components in Romanowsky- stained thin blood films. What are the staining problems that give rise to unsatisfactory results? How do you correct these problems? What is panoptic staining? What is the advantage of panoptic stains over simple Romanowsky dyes? What is the principle of thick film staining? List two dyes that are commonly used in thick blood film staining?

It is not recommended for routine red cell counts because the number of cells which can be counted within a reasonable time in the routine laboratory will be too few to ensure a precise result.

Yet it is still necessary for the technologist to be able to use this method effectively and to know its limitations. Any cell counting procedure includes three steps: dilution of the blood, sampling the diluted suspension into a measured volume, and counting the cells in that volume.

Counting Chambers The hemocytometer is a thick glass slide with inscribed platforms of known area and precisely controlled depth under the coverslip. The ruled portion may be in the center of the chamber single chamber or there may be an upper and lower ruled portion double chamber. The double chamber is to be recommended since it enables duplicate counts to be made rapidly. When an optically plane cover glass is rested on the raised bars there is a predetermined gap or chamber formed between its lower surface and the ruled area fig.

This is called the depth of the chamber and it varies with the type of the chamber. The ruled area itself is divided by microscopic lines into a pattern that varies again with the type of the chamber. The counting chamber recommended for cell counts is a metallized surface Bright-line double cell Improved N e u b a u e r r u l e d c h a m b e r.

N o n - m e t a l l i z e d hemocytometer are less expensive, but they are not recommended. It is more difficult to count WBCs reliable using this type of chamber because the background rulings and cells are not as easily seen. Not-metallized chambers are also more difficult to fill. Ordinary Neubauer counting chamber The central platform is set 0. The engraving covers an area of 9mm2 divided into 9 squares of 1mm2 each.

The central ruled area of 1mm2 is divided into 16 large squares by sets of triple lines. These large squares are further subdivided into 16 small squares by single lines. The width of the triple lines dividing the large squares is the same as the width of a small square. Two adjacent sides of the ruled area are bounded by triple lines, the other two by single lines.

Each side is, therefore, divided into 20 equal divisions the width of 16 small squares and 4 sets of triple lines. The Improved Neubauer Counting Chamber The depth between the lower surface of the cover glass which is on the raised bars and the ruled area is 0. The central square of these nine is divided by engraved lines into tiny squares of arranged in 25 groups of 16 by triple boundary lines.

Each large square is 1mm2, each of the 25 medium squares is of 0. Fuchs-Rosenthal counting chamber This chamber was originally designed for counting cells in cerebrospinal fluid, but as such a relatively large area is covered, it is preferred by some workers for counting leucocytes. The depth is 0.

Burker ruled counting chamber Like the Neubauer counting chamber, this has a ruled area of 9mm2 and a depth of 0.

To count white cells using Burker Chamber, the four large corner squares are used 4mm2 and the same calculation as describe for the Improved Neubauer ruled chamber is used. Dilution of the Sample Dilution of sample is accomplished by using either a thomma pipette or the tube dilution method. With tubes larger volumes of blood and diluting fluid are used and the greater will be the accuracy as compared with the smaller volumes used in the thomma pipette techniques.

Thomma pipettes are small calibrated diluting pipettes designed for either white cell or red cell count.

Counting and Calculation The diluted cells are introduced into the counting chamber and allowed to settle. Cells lying on or touching the upper or left boundary lines are included in the count while those on the lower and right boundary lines are disregarded.

Fig 6. Calculation No. EDTA anticoagulated blood or capillary blood can be used for counting white cells. Heparin or sodium citrate anticoagulated blood must not be used. Principle Whole blood is diluted 1 in 20 an acid reagent which hemolyzes the red cells not the nucleus of nucleated red cells , leaving the whit cells to be counted. White cells are counted microscopically suing an Improved Neubauer ruled counting chamber hemocytometer and the number of WBCs per liter of blood calculated.

The glacial acetic acid causes erythrocyte lysis while the gentian violet lightly stains the leucocytes permitting easier enumeration. When blood is drawn up to the 0.

Once the pipette accurately filled to the mark, the rubber suction or mouth piece is carefully removed, with the pipette held horizontally and only one finger sealing the tip. Both ends of the pipette may then be sealed with special small rubber sealing caps or with the middle finger on the tip and the thumb on the other end.

The pipette is shaken mechanically or manually for 2 minutes. A bead contained in the bulb of the pipette aids in the mixing. If shaking is done manually, the shaking motions should be varied and alternated. Once the diluted blood in the pipette has been thoroughly mixed, a few drops are expelled to discard the cell-free diluting fluid in the long stem of the pipette.

With the index finger forming a controlled seal over the end of the pipette, which is held at an angle of , the tip of the pipette is brought up to the edge of the cover glass and by gentle release of index finger pressure, fluid is allowed to run out slowly until the counting platform is covered. The fluid is drawn into the chamber by capillary attraction.

Care must be taken not to overfill the chamber which will result in overflow into the channels. Charging is accomplished by using disposable capillary tubes or long stem Pasteur pipettes. The chamber is placed in position on the microscope stage and is allowed to stand for 2 or 3 minutes so that the cells will settle.

All apparatus should be cleaned thoroughly after each use. Pipettes should be periodically cleaned with potassium dichromate cleaning solution or hydrogen peroxide. Hemocytometers should be washed in distilled water immediately after use and dried with gauze or tissue paper. They should be stored in such a way as to avoid breakage and scratching of the counting surface. Performance of the Count The counting chamber is surveyed with the low power objective to ascertain whether the cells are evenly distributed.

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