BLOOD SMEAR

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Complete description of Blood Smear technique Revealed

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BLOOD SMEAR {SLIDE DISCUSSION}:

BLOOD SMEAR { SLIDE DISCUSSION}

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Definition “A blood smear is a blood test that gives information about the number and shape of blood cells.”(Textbook of medical physiology- Guyton & Hall) Alternative Names Peripheral blood smear.

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blood smear a blood test used to provide information concerning drugs and diseases that affect the morphology of red and white blood cells and to help diagnose certain congenital and acquired diseases. A properly performed and analyzed blood smear is the most informative of all hematologic tests, allowing examination of erythrocytes, platelets, and leukocytes.

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Blood is typically drawn from a vein, usually from the inside of the elbow or the back of the hand. The site is cleaned. The health care provider wraps an elastic band around the upper arm to apply pressure to the area and make the vein swell with blood. puncture site is covered to stop any bleeding. In infants or young children, a sharp tool called a lancet may be used to puncture the skin and make it bleed. A bandage may be placed over the area if there is any bleeding.

Collection of blood:

Collection of blood capillary Venous

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Capillary blood Apparatus Sites –ball of finger{ring finger left hand}, ear lobe, heel or big toe in infants. Procedure- Bunsen burner or spirit lamp

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Venous blood Apparatus Sites – anticubital fossa , dorsum of hands, femoral vein in children Procedure

Risks of taking blood by i.v. route :

Risks of taking blood by i.v . route Excessive bleeding Fainting or feeling light-headed Hematoma (blood accumulating under the skin) Infection (a slight risk any time the skin is broken) Thrombophlebitis of veins

Precautions to be taken:

Precautions to be taken

BLOOD SMEAR PREPARATION AND STAINING:

BLOOD SMEAR PREPARATION AND STAINING How to Prepare & Interpret Peripheral Blood Smears

PRINCIPLE OF SMEAR PREPARATION :

PRINCIPLE OF SMEAR PREPARATION Smear Preparation A small drop of blood is placed near the frosted end of a clean glass slide. A second slide is used as a spreader. The blood is streaked in a thin film over the slide. The slide is allowed to air-dry and is then stained SPECIMEN EDTA anticoagulated blood is preferred. Blood smears can also be made from fingerstick blood directly onto a slide.

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Other Anticoagulats used for collecting blood Ammonium potassium oxalate Sodium citrate heparin

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REAGENTS, EQUIPMENTS Glass slides, 3 x 1 inch with frosted edge Stopper piercer Lancet Spirit or alcohol

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BLOOD-LANCET Blood-lancet is used for finger needling to sample blood in medical and preventive treatment facilities. According to technical standards maintained by the Euro-Asian Council for Standardization, Metrology and Certification (EASC),

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Dimensions: Weight: 0.4 g Length: 40 mm Width: 5, 5 mm Thickness: 0, 15-0, 2 mm Length of the needle: 4 mm Puncture effort: up to 90 g Advantages: Little puncture effort (90 g at possible 150 g) Increased flexural strength

PROCEDURE(vid) :

PROCEDURE( vid ) Using the stopper piercer, place a drop of blood, about 2 mm in diameter approximately inch from the frosted area of the slide. Place the slide on a flat surface, and hold the narrow side of the nonfrosted edge between your left thumb and forefinger. With your right hand, place the smooth clean edge of a second (spreader) slide on the specimen slide, just in front of the blood drop. Hold the spreader slide at a 30 angle, and draw it back against the drop of blood.

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Allow the blood to spread almost to the edges of the slide. Push the spread forward with one light, smooth, and fluid motion. A thin film of blood in the shape of a bullet with a feathered edge will remain on the slide. Label the frosted edge with patient name, ID# and date. Allow the blood film to air-dry completely before staining. (Do not blow to dry. The moisture from breath will cause RBC artifact.)

POINTS TO BE NOTED:

POINTS TO BE NOTED A good blood film preparation will be thick at the drop end and thin at the opposite end. As soon as the drop of blood is placed on the glass slide, the smear should be made without delay. Any delay results in an abnormal distribution of the white blood cells, with many of the large white cells accumulating at the thin edge of the smear.

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The blood smear should occupy the central portion of the slide and should not touch the edges. The thickness of the spread when pulling the smear is determined by the 1) angle of the spreader slide (the greater the angle, the thicker and shorter the smear), 2) size of the blood drop and 3) speed of spreading.

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=If the hematocrit is increased, the angle of the spreader slide should be decreased. =If the hematocrit is decreased, the angle of the spreader slide should be increased

Common causes of a poor blood smear: :

Common causes of a poor blood smear: Drop of blood too large or too small. Spreader slide pushed across the slide in a jerky manner. Failure to keep the entire edge of the spreader slide against the slide while making the smear. Failure to keep the spreader slide at a 30 angle with the slide. Failure to push the spreader slide completely across the slide.

Biologic causes of a poor smear :

Biologic causes of a poor smear Cold agglutinin - RBCs will clump together. Warm the blood at 37 C for 5 minutes, then remake the smear. Lipemia - holes will appear in the smear. There is nothing you can do to correct this. Rouleaux - RBC’s will form into stacks resembling coins. There is nothing you can do to correct this.

Stains used:

Stains used

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Leishman's stain pH 6.8 phosphate buffer Working stain: Leishman's stain diluted 1:4 with buffer

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Leishman stain uses a methanol solution of staining dyes . 7-10 drops is applied to the slide with the specimen. After 5 minutes, 10-15 drops of a buffer solution is added and mixed with the stain, then the specimen is left staying for 20–30 minutes, then washed off with the buffer solution

Procedure for staining:

Procedure for staining Either spray the wet preparation with Smear Fix, leave for 1-2 mins , wash off the fixative with distilled water and drain.... Or lower the slide gently into a coplin jar of acetic alcohol (3% acetic acid in 95% methanol), fix for 1 minute, wash off the fixative with distilled water and drain. Put slides on a rack and cover with 1ml of Leishman stock - 20 seconds.

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Add 2ml of pH6.8 buffer and tip the rack up and down to mix the solutions, stain for 7 minutes. Rinse quickly in distilled water then treat with pH6.8 buffer - 2 minutes. Rinse quickly in distilled water, shake off the excess and dry on a warm (50 o C) hotplate, or carefully blot dry with fibre -free blotting paper. Clear and mount.

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Low power (10x) scan Determine the overall staining quality of the blood smear. Stain should not be too dark or too pale. There should be no stain precipitate present on smear. RBCs should be appropriate color of reddish pink. Lymphocytes have dark purple nuclei with varying shades of blue cytoplasm

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Some abnormalities may be graded on a 4-point scale: 1+ means 25% of cells are affected 2+ means half of cells are affected 3+ means 75% of cells are affected 4+ means all of the cells are affected

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Neutrophils have dark purple nuclei with reddish, granular cytoplasm. Monocytes have a lighter purple nucleus with a gray-blue cytoplasm. Eosinophils have bright red/orange granules Basophils have dark purple nuclei and granules

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Determine if there is a good distribution of the cells on the smear. Scan the edges and center of the slide to be sure there are no clumps of RBCs, WBCs or platelets. Scan the edges for abnormal cells.

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Red blood cells - red to yellowish red Neutrophils - dark purple nuclei, pale pink cytoplasm, reddish-lilac small granules Eosinophils - blue nuclei, pale pink cytoplasm, red to orange-red large granules Basophils - purple to dark blue nucleus, dark purple, almost black large granules

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Lymphocytes - dark purple to deep bluish purple nuclei, sky blue cytoplasm Platelets - violet to purple granules Parasites ( Leishmania , malaria, etc.) - dark blue-black.

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High power (40x) scan Find an optimal area for the detailed examination and enumerations of cells. The RBCs should not quite touch each other. There should be no area containing large amounts of broken cells or precipitated stain. The RBCs should have a graduated central pallor. Nuclei and cytoplasm of WBCs should be the proper color. Platelets should be clearly visible.

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Smears will be evaluated on the following criteria: Stain quality Length Thickness Shape of feathered edge Proper labeling

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Too blue: Smear too thick Inadequate time in buffer Buffer pH too high Staining time too long Diluted stain overused, requires replenishment Stock solution left exposed to bright daylight

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Too pink: Excessive time in buffer Buffer pH too low Coverslip mounted before film is completely dry

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Too faint: Staining time too short Excessive washing after staining

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Stain deposit: Stain solution left in uncovered jar or tray Stain solution not filtered Dirty slides

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Blue background: Inadequate fixation Prolonged storage before fixation Blood anticoagulated with heparin

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The presence of target cells may be due to: Decreased osmotic fragility Deficiency of an enzyme called lecithin cholesterol acyl transferase Hemoglobin abnormalities ( hemoglobinopathies ) Iron deficiency Liver disease Spleen removal Thalassemia

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Presence of sphere-shaped cells ( spherocytes ) may be due to: Autoimmune hemolytic anemia Hereditary spherocytosis Increased osmotic fragility

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Spherocytes are special red blood cells produced when a red blood cell is not complete removed by the spleen. The spleen cell “bites off” only a portion of the red cell leaving the rest to escape back to the circulation. The presence of spherocytes indicates that red blood cells are being destroyed.

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Presence of elliptocytes may be a sign of hereditary elliptocytosis or hereditary ovalocytosis . Elliptocytes are red blood cells that are oval or cigar shaped. They may be found in various anemias , but are found in large amounts in hereditary elliptocytosis .

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The presence of fragmented cells ( schistocytes ) may be due to: Artificial heart valve Disseminated intravascular coagulation Hemolytic uremic syndrome (HUS) Microangiopathic hemolytic anemia Thrombotic thrombocytopenic purpura " schizein " meaning "cut, cleave, or split

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By mechanical trauma or an intrinsic abnormality of erythrocytes. " schizein " meaning "cut, cleave, or split

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The presence of a type of immature red blood cell called a normoblast may be due to: Cancer that has spread to bone marrow Erythroblastosis fetalis Leukoerythroblastic anemia ( myelophthisis process) Miliary tuberculosis Myelofibrosis Removal of spleen Severe hemolysis Thalassemia

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The presence of burr cells ( echinocytes ) may indicate: Uremia The presence of spur cells ( acanthocytes ) may indicate: Abetalipoproteinemia Severe liver disease

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The presence of teardrop-shaped cells may indicate: Leukoerythroblastic anemia Myelofibrosis Severe iron deficiency Thalassemia major

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The presence of Howell-Jolly bodies may indicate: Myelodysplasia Post- splenectomy Sickle cell anemia The presence of Heinz bodies may indicate: Alpha thalassemia Congenital hemolytic anemia G6PD deficiency Unstable form of hemoglobin

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The presence of slightly immature red blood cells ( reticulocytes ) may indicate: Anemia with bone marrow recovery Hemolytic anemia Hemorrhage The presence of basophilic stippling may indicate: Lead poisoning Myelofibrosis Myelophthisic process The presence of sickle cells may indicate sickle cell anemia.

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Ring stages, Schizont in the lower center, Trophozoite on the left[ P.falciparum ]

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Anemia

references:

references Diagnostic Microbiology - Bailey & Love Textbook of medical physiology- Guyton & Hall Human physiology – A K Jain Practical physiology – Dr. V.D. Joshi. www.bd.com

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