PREPARATION AND EXAMINATION OF BLOOD SMEAR

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1 UNIT 3 Structure PREPARATION AND EXAMINATION OF BLOOD SMEAR 3.0 Objectives 3.1 Introduction 3.2 Bood Fim Preparation Thick Bood Fim Thin Bood Fim Fixing Bood Fims 3.3 Staining of Bood Fims Leishman s Stain Giemsa s Stain Wright s Stain 3.4 Microscopic Identification of Parasites in Bood Maaria Leishmaniasis (Kaa azar) Fiariasis 3.5 Let Us Sum Up 3.6 Answers to Check Your Progress 3.0 OBJECTIVES After competing this unit, you shoud be abe to: demonstrate your ski in obtaining bood by finger pricking for making a fresh bood smear; demonstrate correct way of making thin and thick bood fims; use appropriate stains for bood smear for detection of specific parasites; and identify various parasites in the stained thick or thin bood fims. 3.1 INTRODUCTION In the preceding Unit, you have gained the ski for examining iving microorganisms under the microscope using various objects commony avaiabe. They were a fresh specimens. You aso gained experience in simpe and differentia staining techniques whereby we can preserve those microorganisms for future study. In this unit, you wi earn to take out bood from the finger to make a bood fim. Bood fims need to be made thin or thick according to the specific diagnosis to be confirmed. For specific parasites circuating in bood, specific staining techniques have to be foowed. Remember, normay bood aso contains various ces other than the parasites causing the infection. Examination of a stained bood fim under a microscope is a very important diagnostic technique. 3.2 BLOOD FILM PREPARATION In order to prepare a bood fim, aways take a new side which is first ceaned and freed from grease by washing in 70% acoho, poished and dried with a fine inen coth. This is important because previousy used cean sides, or uncean new sides, may produce artefacts which are difficut to interpret on microscopic examination. For making a bood fim, bood is most convenienty obtained, by pricking the tip of the 27

Practica Manua Microbioogy third finger with a ancet. Two types of bood fims are made for the microscopic examination of bood smear, i.e. thick bood fim and thin bood fim.

2 Practica Manua Microbioogy third finger with a ancet. Two types of bood fims are made for the microscopic examination of bood smear, i.e. thick bood fim and thin bood fim. Let us discuss these in the subsequent two sub-sections Thick Bood Fim Thick bood fim is used for examination of a arger quantity of bood quicky, so as to discover ow eves of parasitaemia, e.g. maaria parasites and microfiariae. It is ess suitabe for examination of individua parasites in order to make a species-specific diagnosis. A drop of bood, about 3 mm in diameter, is deposited in the center of a side and is spread with the head of a pin, or with the corner of another side to cover an area of 10 mm in diameter. The density of the fim shoud be such that the hands of a watch can just be seen through it. The fim is thoroughy dried at 37 o C in an incubator for 30 min. The thick fim is dried and the haemogobin is aked out before staining by immersing in distied water for 5 min Thin Bood Fim Thin bood fim is used to demonstrate the morphoogica characteristics of the organisms and of the ces that contain them, most ceary. Thin fims aid in diagnosis of species and pasmodium. Thin fims are ideay one ce thick, i.e. a ces are ceary dispayed for examination. A sma drop of bood, about 1.5 mm in diameter, is paced near one end of the side and immediatey, the end of a second side is appied to the first side at an ange of about 45 o. The second side is then drawn back to contact the drop, which is then aowed a second or two to spread aong the junction of the two sides. The drop is a to d, thin fim: a, drop of bood at one end of the side; b spreader (another gass side) hed at an ange of 45 o and pushed in the direction of the arrow; c making a smear; d thin bood fim competed; e and f thick fim four drops of bood at the corners of a haf inch square; g and h thick and thin fims on the same side 28 Fig. 3.1: Preparation of thin and thick fims of bood

3 then spread aong the first side by a continuous forward movement of the side at 45 o. Ideay the fim shoud cover two-thirds of the ength of the side, shoud have a tai, and shoud not extend to the edges of the first side. The fims are aowed to dry and then fixed with absoute methano and then stained. For anaemic bood a rapid smearing is required whereas for thick concentrated bood smearing shoud be done sowy. A we spread fim shows no ines extending across or downwards through the fim and the smear shoud be tongue shaped Fixing Bood Fims Ony thin bood fims are fixed. Absoute methano is used as fixative. The side bearing the thin bood fim is fooded with absoute methano. The reaction is aowed for three to five minutes. The methano is then poured off and the side is air dried. Staining of the bood fim can now be carried out. 3.3 STAINING OF BLOOD FILMS Bood ces have structures that are acidophiic and some that are basophiic, so they vary in their reactions (ph). The nucei are basophiic and stain bue. The highy basophiic (acidic) basophi granues aso stain bue. Haemogobin (being basic) stains acidophiic or red. Stains that are made up of combinations of acid and basic dyes are caed Romanowsky Stains and its various modifications are avaiabe such as Wright s, Leishman s, Giemsa s and Jenner s stains. Most use Methyene Bue as the basic stain, though Toudine Bue is used by some. Most use Eosin as the acid stain though Azure I and Azure II are aso used. We sha now study various modifications of Romanowsky stain. First et us take Leishman s Stain Leishman s Stain Leishman s Stain is used for visuaizing protozoa in bood fims. Dried but unfixed bood fims are used. When first undiuted stain is used, the methano fixes the fim. Later on diution with distied water the proper staining is carried out. Steps for Staining with Leishman s Stain 1) Pour undiuted stock stain soution on the unfixed fim and aow to react for 1 min. 2) Then add doube the voume of distied water with a pasture pipette and mix the fuids by aternatey aspirating and expeing them. Aow the diuted stain to react for 12 min. 3) Then food the side genty with distied water, aowing the preparation to differentiate in the distied water ti the fim appears bright pink in coour. This usuay takes 30 sec. 4) Remove the excess of water with botting paper and dry in the air. Then observe under microscope. 5) Record your observation. Observe a teacher-prepared side for your guidance. 29

4 Practica Manua Microbioogy Giemsa s Stain This stain is used for demonstrating protozoa and spirochaetes in bood. Steps for Staining with Giemsa s Stain 1) Fix the fim with methano for 3-5 min and then dry it. 2) Then food the side with diute stain and keep for 15 min or onger. 3) Then wash off with neutra distied water and air dry. Observe under high power and oi immersion ens of microscope. 4) Record your observation. Observe a teacher-prepared side for your guidance Wright s Stain This stain is used to demonstrate protozoa and heminthic parasites in bood. Steps for Staining with Wright s Stain 1) Both thick and thin fims of bood may be prepared. Sides with thick fims shoud be immersed in distied water for 5 min to remove the haemogobin and then dried. 2) Cover the fim with 1 m of Wright s stain. Aow to react for 3 min. 3) Food the side with 0.1 M Phosphate buffer ph 7. Leave for 6 min. 4) Wash sides in a gente stream of distied water. 5) Observe the thick fim with 5x objective and the thin fim with oi immersion 100x objective under the microscope. 6) Record your observation. Observe a teacher-prepared side for your guidance. 3.4 MICROSCOPIC IDENTIFICATION OF PARASITES IN BLOOD 30 The diagnosis of most parasitic infections is dependent on the aboratory. For intestina and bood parasites, morphoogic demonstration of diagnostic stages is the principa means of diagnosis.

5 Bood and tissue parasites whose diagnostic forms circuate in the periphera bood are generay diagnosed by the demonstration of parasites in the stained thick or thin fims of bood. For various protozoan and heminthic infections, such as maaria, fiaria, etc., this is a routine procedure. Let us now start with the microscopic identification of maaria parasites in bood Maaria Maaria is caused by 4 species in humans P. faciparum, P. vivax, P. maariae, and P. ovae. P. faciparum causes the most serious form of the disease. Fims shoud, if possibe, be taken during the pyrexia and no antimaaria drugs shoud have been administered beforehand. Bood fims are best made from fresh bood to which anticoaguant has not been added, such as that obtained from finger prick. Anticoaguants may interfere with parasite morphoogy and staining. It is essentia that the fim be we stained, a good guide is the staining of eukocytes in the fim; if this is satisfactory then any maaria parasites present woud be detected. In thin fims, examination of edges of fim is advisabe since the parasites may be more numerous there than in the centre. In the diagnosis of cinica maaria, thin bood fims are usuay sufficient except perhaps in some P. vivax and P. maariae infections. The absence of parasites during an apyrexia interva, however, by no means excudes maaria and repeated examination of fims, incuding thick fims, may be required before the diagnosis can be estabished. Smears shoud be viewed under oi immersion ens. Standard high power magnification is inadequate to distinguish maaria parasites from pateets precipitated stain and nonspecific debris. Essentiay, a maaria parasite is recognized by its red-staining nucear materia and bue-staining cytopasm, and by its occurrence within the erythrocyte. To inexperienced workers, artefacts may sometimes simuate maaria parasites. Various species of pasmodium are differentiated and by their own morphoogica characteristics by the changes which they induce in the ces which they parasitize. Remember that bood side shoud be made when the person is having high temperature. You may have difficuty in identifying maaria parasite in the beginning. With your teacher s hep you may succeed. Let us now demonstrate the presence of microfiariae in the bood Leishnamiasis (Kaa azar) Leishmaniasis is a disease caused by the fageated protozoans, caed Leishmania donovani, a hemofageate, that is transmitted to humans by the bite of bood sucking arthropods (sand fy). The disease is seen in various states of India, especiay Bihar. The parasites can be demonstrated as bone marrow/bood smears stained with Wright s or Giemsa s stain Fiariasis Fiariasis is a chronic heminthic infection of humans and animas which is caused by parasites beonging to the super famiy Fiarioidea. The parasites causing Fiaria in India are Wuchereria bancrofti, Brugia maayi, etc. Preiminary diagnosis of fiaria infection invoves demonstration of microfiariae in the bood of the patient. The microfiariae are simpe in structure. They are vermiform in shape and, in stained preparations, appear to be composed of a coumn of nucei which is interrupted by spaces or specia ces which are precursors of the body organs. Some species of the microfiariae are enveoped in a sheath, whereas in others, the sheath is absent. Bood is coected from the patient between 10:00 p.m. and 01:30 a.m., at night. The eve of microfiariae in the bood is higher during this period. Thick and thin 31

6 Practica Manua Microbioogy bood fims are prepared. The fims may be stained by Wright s stain for visuaization of the microfiariae. You may have difficuty in identifying microfiariae in the beginning. With your teacher s hep you may succeed in identifying it. Check Your Progress 1) Why are thick bood fims not fixed but dried ony? 2) Name the parasite that causes the most-severe infection of Maaria in India. 3) Expain the basis of the staining of ces in bood. 4) Expain the function of Methano in Romanowsky stains. 5) Why shoud no antimaaria drugs be administered before a bood fim for maaria is to be made? Points to Remember 1) Care shoud be taken whie pricking the finger. The acoho disinfectant shoud be aowed to dry before the area is punctured, or there may be fixation of erythrocytes, which wi interfere with the preparation and staining of thick fims. 2) Dried bood fim can stay for a coupe of days in hot dry weather but they get spoied if they are not fixed, in hot and humid cimate that exists in India. 3) Thick and thin fims are best made on separate sides, so as to avoid the accident of inadvertent fixation of thick fim. 4) It is important that the ph of distied water used in the staining procedure be neither acid nor akaine. 32

7 3.5 LET US SUM UP Bood and tissue parasites where diagnostic forms circuate in the periphera bood are generay diagnosed by the demonstration of parasites in the stained thick or thin fims. You know that bood contains various types of ces. For diagnostic confirmation of certain diseases a microscopic examination of bood is done by preparing a thick and thin fims on a gass side and then staining it with specific stains. Staining procedures are based on acid base reaction of the ces or on some enzymatic reaction. Stained bood fims may be used for diagnosis of infections such as maaria, fiaria and Leishmaniasis. Sef Activity Draw the abeed diagram(s) of the bood parasites you have observed/studied in your hospita. 3.6 ANSWER TO CHECK YOUR PROGRESS 1) Thick bood fims must be treated to remove hemogobin. Otherwise whie fixing hemogobin wi come out of the ces and interfere in staining reaction. 2) P. faciparum. 3) Bood ces have structures that are acidophiic and some basophiic, so they vary in their reaction (ph). The nucei are basophiic and stain bue. The highy basophiic (acidic) granues in basophis stain bue. Haemogobin, being basic, stains acidophiic or red. 4) Methano acts as Fixative. 5) The antimaaria drug wi reduce the number of parasites in the bood, making detection difficut and often ending up in fauty diagnosis. 33

PREPARATION OF BLOOD FILMS FOR MALARIA DETECTION

PREPARATION OF BLOOD FILMS FOR MALARIA DETECTION Materials for Preparation of Malaria Smears: Clean and wrapped slides Sterile lancets 70% ethanol and water Absorbent cotton wool Surgical gloves Lint-free