Some additional guidelines should be followed to create the. Examination of blood films microscopic examination progressing from low power to highpower under low magnification to. The best place to examine blood cell morphology is the feathered edge of the blood smear where red cells lie in a single layer, side by side, just barely touching one another but not overlapping. B staining of blood smear leave it for 12 minutes for fixation of the smear. Making and staining a blood smear a wellmade blood smear is a beauty to behold, and likely to yield interesting and significant information for a research project. Some additional guidelines should be followed to create the best blood smear.
Preparation of peripheral blood smear and staining. Many areas of the smear will be too thick for evaluation, however, there are usually multiple cell monolayer regions present on the smear that should be acceptable for cytological assessment. The smear is covered with stain for approximately ten minutes, then diluted with water and allowed an additional ten minutes for the cells to properly stain. Here, the methods for making and staining smears are given, as well as a list of sources for high quality slides, stain, and chemicals. Blood smears and the use of wrights stain iowa state university.
Wrights stain is named for james homer wright, who devised the stain in 1902 based on a modification of romanowsky stain. It is not appropriate to use the same edge of a slide for more than a single film. Learn vocabulary, terms, and more with flashcards, games, and other study tools. A blood film or peripheral blood smear is a thin layer of blood smeared on a microscope slide and then stained in such a way to allow the various blood cells to be examined microscopically. The slides should be dried as quickly as possible to prevent. The purpose of preparation of blood smear blood film is to study the morphology of rbcs, differential leukocyte count and reticulocyte count. Let it stand for 2 minutes, the methanol present in the stain fixes the smear onto the glass slide. Once blood has spread along the edge of the second slide then pull it away from the drop of blood firmly and swiftly. The role of the peripheral blood smear in the modern haematology laboratory automated haematology cell counting the laboratory practice of haematology has evolved tremendously over the past few decades with automated analyser generated complete blood counts cbc having fully replaced the original manual individual parameter assay methods. A well prepared blood smear is necessary for microscopic examination of blood. It works by precipitating proteins and carbohydrates. Papanicolaou described three chief objectives for staining of cytological smears.
The blood smear should occupy the central portion of the slide and should not touch the edges. Papanicolaou stain pap stain protocol ihc world, llc. Giemsa stain is a buffered thiazineeosinate solution designed to provide coloration of blood cells similar to the original product described by giemsa. Bain, in dacie and lewis practical haematology twelfth edition, 2017. For staining the thin blood smear the giemsa stain is used in 1. Cell fixationis done by placing the slide in methanol ch3oh. Choose from 500 different sets of preparing staining a blood smear flashcards on quizlet. Giemsa stain is a type of romanowsky stain, named after gustav giemsa, a german chemist who created a dye solution.
Apart from staining malarial parasites, giemsa stain has a variety. Blood films with excessive tails or gritty feathered ends indicate a spreader edge that is rough or dirty, or an accumulation of leukocytes due to either slow spreading or a very high leukocyte count. Blood smear technique aspirated material may contain enough blood andor liquid to allow. After staining with giemsa, the slide can be examined under the microscope. It is also used for staining bone marrow aspirates, urine samples and to demonstrate malarial parasites in blood smears. Do not make the smear too thick or it will fall off the slide.
It is used to diagnose diseases, such as malaria, which are caused by parasitic infections the collected blood sample is smeared onto a glass slide and stained with giemsa stain. An unstained blood smear is clearly seen by eye on the microscope slide, however, the blood cells are barely visible under the microscope figure 1. A properly prepared blood smear is essential to accurate assessment of cellular morphology. The video shows the procedure for preparation of a peripheral blood smear and its staining prior to observation under a microscope. Megan brashear, cvt, vts ecc, demonstrates technique in staining a blood smear for microscopic evaluation. Staining blood smears with dilutions of 10x or 20x in either water, saline, dilute sodium bicarbonate or vitamin c only gave faint blue staining of white blood cells after 3. Blood edtablood as is, purpletop tubes or edtablood preserved in sample buffer preferred. Start a free trial of quizlet plus by thanksgiving. These dyes have the property of making subtle distinction in shades of staining. The remarkable property of the romanowsky dyes of making subtle distinctions in shades of staining, and of staining granules differentially, depends on two components. To illustrate the simplicity of the method of making and staining slide smears, let me say that the physicians in the receiving ward of the cook county hospital, working two at a time, and diagnosing from 100 to 200 cases daily, find time to make, stain and examine bloodsmears by this method to help in differentiating typhoid, malaria, and. Stain only one set of smears, and leave the duplicates unstained. Wewilluseadropletofbloodtomakeathin%smear,dryit,fixandstainitand observe under a microscope.
The most dependable stain for blood parasites, particularly in thick films, is giemsa stain containing azure b. Blood smears are used to determine leukocyte differentials, to evaluate erythrocyte, platelet and leukocyte morphology, and, if necessary, to estimate platelet and leukocyte counts. The stain must be diluted for use with water buffered to ph 6. The extra time and care taken during the field season will be rewarded later when the smears must be scanned, and parasites identified and counted. Prepare a second smear by repeating steps 35, using the edge of the first slide to spread a drop of blood on the second slide. Leishman staining technique principle, preparation. Pap staining is used to differentiate cells in smear preparations of various bodily secretions. Video is prepared by the faculty and students of the department. Making and staining a blood smear university of vermont. A phlebotomist, a person specifically trained to draw blood, first cleans and sterilizes the injection site with an antiseptic. They have a very darkly staining, dense, round nucleus. It is hoped that this paper will provide an aid to those who wish to employ the blood smear as a diagnostic tool. Procedure for staining pour leishmans stain dropwise counting the drops on the slide and wait for 2 minutes.
Using a capillary tube, place a 23mm drop of blood about 1cm from the frosted end of a clean slide that is on a flat surface. The thin blood films are prepared from whole blood with clean, dry and wrapped microscope slides cfr. Histology laboratory microscope orientation and blood. Bloodfilmpreparationand stainingprocedures berendhouwen,md,phd. It was primarily designed for the demonstration of malarial parasites in blood smears, but it is also employed in histology for routine examination of blood smear. It consists of a mixture of eosin an acidic stain, and methylene blue a basic stain in methyl alcohol and is usually diluted and buffered during the staining procedure. Wait until the thin and thick films are completely dry before staining. However, the red blood cells took on more stain too, appearing darker. Either should be tested for proper staining reaction before use. Introduction to peripheral blood smear examination. If using a needle and syringe, first remove the needle and then touch the end of the syringe to the slide. Used in hematology, this stain is not optimal for blood parasites. Making the blood smear before any stained smear can be used for a diagnosis.
Allow the blood to spread along the edge of the slide. Now, cover the well dried, thin blood smear with undiluted leishman stain solution by counting the drops of leishman stain. Histology staining of lymph node a, gastric mucosa infected with h. This was the case even after 30 minutes staining figure 4. Blood films should be fixed in methanol within 4 hours preferably within 1 hour of preparation. A blood filmor peripheral blood smearis a thin layer of blood smeared on a glass microscope slide and then stained in such a way as to allow the various. Using the corner of a clean slide, spread the drop of blood in a circle the size of a dime diameter 12 cm. The giemsa blood smear test is a test to detect blood parasites. Seed haematology the role of the peripheral blood smear. Take a second slide and lie the edge flat on the smear slide. The mechanism of action of wrights stain is also discussed. The procedure of giemsa staining varies as per the purpose of staining that means whether the staining is done for the examination of blood cells or to find the parasites in the blood smear and accordingly the blood smears are prepared as thin blood films or thick blood films.
The latter will prove useful if a problem occurs during the staining andor if you wish later to send the smears to a reference laboratory. Learn preparing staining a blood smear with free interactive flashcards. The narrow cytoplasmic rim in a wellstained smear appears clear, sky. A simple single differential stain of blood smears using. A variety of methods are available for preparing and staining blood smears, the most common of which are discussed in this atlas. Romanowsky stains are used universally for routine staining of blood films. Because of the widespread muclear abnormalities of cancer cells and their diagnostic significance, good staining of the nucleus is of primary importance transparency of cytoplasm. Right blood as seen at 400x under light microscope. Prior to staining, cells must be fixed to the glass slide with acetone free methanol, either alone or in solution with. 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. High ph, prolonged staining, or insufficient washing can result in excessively bluestaining blood films.
Neel, dvm, dacvp clinical associate professor, clinical pathology nc state college of veterinary medicine raleigh, nc, 27607 introduction although tremendous advances have been made in the field of pointofcare hematology analyzers, examination of a well prepared, well stained blood smear remains the. Wrights stain is a type of romanowsky stain, which is commonly used in hematology laboratory for the routine staining of peripheral blood smears. After 2 minutes, add twice the amount of distilled water or phosphate buffer solution and mix the content by swirling or by blowing gently. The authors approach is to look at the smallest cellular elements, the platelets, first and work.
Papanicolaou stain also papanicolaous stain and pap stain is a multichromatic staining histological technique developed by george papanikolaou, the father of cytopathology. Cytology sample collection and preparation for veterinary. Preparation of blood smear with different staining method dr. Module staining of pbf and interpretation of normal and abnormal red cell morphology hematology and blood bank technique 72 hematology and blood bank technique notes smears are routinely stained with romanowsky stains with very good results. Add leismans stain drop by drop till it covers whole of the smear. Push the second slide back until it contacts the drop of blood. Low ph, inadequate staining time, degraded stains, or excessive washing can result in excessively pinkstaining blood films. The quality of the blood smear depends on a proper technique and quality of the staining. Leishman stain is a neutral stain for blood smears which was devised by the british surgeon w. This occurs when there is inadequate contact with the stain solutions.