CYTOLOGY

MAY-GRÜNWALD GIEMSA STAIN:- This is one of the common Romanwsky stains used in cytology. It is useful for studying cell morphology in air-dried smears. It is superior to Papanicolaou to study the cytoplasm, granules, vacuoles, basement membrane material etc. For nuclear staining Papanicolaou is superior. Contents of the staining reagents:- May-Grünwald solution                                               0.2%  Methanol                                                                       99 %May-Grünwald´s eosin-methylene blue                    0.2 % Contains:- Eosin G, Methylene blue Giemsa solution:- Methanol                                                                       73 % Glycerol                                                                         26 %Giemsa´s Azur-Eosin-Methylene blue                      0.6 % Contains: Azur I, Eosin G, Methylene blue Phosphate buffer:- Potassium dihydrogen phosphate/ disodium hydrogen phosphate x 2H2O  (67.0 mmol/l) Storage:- Giemsa solution, May-Grünwald solution: protected from light at 2-25°C.Unopened reagents may be used until the expiry date on the label.Phosphate buffer: at 2-8°C. Unopened reagents may be used until the expiry dateon the label. Preparation of working solutions:- 1. Buffered water: Dilute phosphate buffer with deionised or distilled water1:20, e.g. 30 ml phosphate buffer + 570 ml deionised or distilled water.2. Giemsa working solution : Mix 84 ml of Giemsa solution into 516 ml ofbuffered water.3. May-Grünwald working solution: Mix 360 ml of May-Grünwald solutioninto 240 ml of buffered water. Staining method:- 1. Fix the air-dried smear specimen in methanol for 10 -20 minutes2. Stain with May-Grünwald working solution for 5 minutes3. Stain with Giemsa working solution for 12 minutes4. Wash with clean buffered water for 2, 5 and 2 minutes5. Dry the slides in upright position at room temperature6. Mount the slides with a coverslip using DPXAny modifications to the staining procedure/working solutions may affect thestaining result, and are subject to precise method validation Sources of errors:- Irregular distribution of the blood smear on a glass slide may result in an erroneous cell counts. Alcohols used for wiping the skin may cause hemolysis and artifacts. Do not let the specimens dry at any stage of the staining procedure. Wash properly to avoid dye artifacts. Buffered water is strongly recommended for washing. Staining result is dependent on pH. Alkaline pH increases blue and acidic pH pink or reddish tinge in the stained specimen. Ziehl-Neelsen stain:- Reagents:-   (1) Carbol Soft FuchsinBasic Fuchsin 1 gmAbsolute alcohol 10 mlAdd the basic fuchsin to the alcohol in a 100 ml flask and mix, on amagnetic stirrer for 30 minutes. Add 100ml of 5% aqueous phenol. Mixwell. Filter and store in a brown glass bottle. (2) Acidified Methylene Blue0.25% methylene blue in 1% acetic alcohol (3) 0.5% Acid AlcoholDistiller water 700 mlAbsolute alcohol 300 mlHydrochloric acid 5 ml (6) 5% Sulphuric AcidDistilled water 475 mlSulphuric acid 25 ml Staining Method:- Place fixed slides on the staining rack in serial order, smeared side up. Slides should be separated by a 1 cm gap, and should never touch one another. Cover slides individually with filtered Ziehl’s carbol fuchsin working solution. Heatslides from underneath with the flame of a Bunsen burner, an alcohol lamp or an alcohol soaked cotton swab until vapour starts to rise. Staining solution should never be allowed to boil. Do not allow the stain to dry. Keep slidescovered with hot, steaming carbolfuchsin for 5 minutes by re-flaming as needed. Rinse slides gently with water to remove excess carbolfuchsin. Drain off excess rinsing water from slides. Sputum smears appear red in colour.   Decolourising: Cover slides with 25% sulfuric acid or acid-alcohol solution and allow to stand for 3 minutes, after which the red colour should have almost completely disappeared. If needed, repeat sequence until the red colour disappears, but do not overdecolourise. Gently wash away the sulfuric acid or acid alcohol and the excess stain with water. Drain off excess rinsing water from slides.   Counterstaining: Cover slides individually with 0.3% methylene blue counterstaining solution and allow to stand for 1 minute. Rinse slides individually with water. Drain water off the slides, which are then allowed to air dry.   A properly stained smear should show a light blue colour due to methylene blue. Results:- Tubercle bacilli, hair shafts, Actinomyces, some fungal elements- red. Background: pale blue.

CYTOLOGY STAINING METHODS:- INTRODUCTION:- Consistency and reliability are most important in cytological interpretation. Cytologists rely heavily on the quality and appearance of the stain. The Papanicolaou stain is recommended for the staining of alcohol fixed cytology slides. Romanowsky stains may also be used for wet fixed slides, but are primarily applied to air-dried smears.Special stains are used as per requirements: Modified Ziehl Neelson (for acid fast bacilli), Gram staining (Bacteria), Mucicarmine (mucins), PAS (for glycogen, fungal wall, lipofuscin, etc), Oil red O (lipids), Perl’s Prussian blue (iron), modified Fouchet’s test (bilirubin), etc. Recently, immunocytochemistry is also being increasingly used in cytology specimens. These special stains and immunocytochemistry will be discussed along with respective sections inhistopathology as the principles and methods remain the same.  STAINING IN CYTOLOGY:- The universal stain for cytological preparations is the Papanicolaou stain. Harris’hematoxylin is the optimum nuclear stain and the combination of OG6 and EA50 give the subtle range of green, blue and pink hues to the cell cytoplasm. Papanicolaou stain:- Papanicolaou formula:- 1. Harris’ hematoxylin:- Hematoxylin                               5g Ethanol                                        50mlPotassium alum                        100gDistilled water (50°C)               1000 mlMercuric oxide                           2-5gGlacial acetic acid                     40ml 2. Orange G 6:- Orange G (10% aqueous)                50ml Alcohol                                               950mlPhosphotungstic acid                     0-15g 3. EA 50:- 0.04 M light green SF                      10ml 0.3M eosin Y                                     20ml Phosphotungstic acid                     2g Alcohol                                              750mlMethanol                                           250mlGlacial acetic acid                           20mlFilter all stains before use Original Papanicolaou staining method:-  1. 96% ethyl alcohol 15 seconds                                                                                                                                                     2. 70% ethyl alcohol 15 seconds                                                                                                                                                     3. 50% ethyl alcohol 15 seconds                                                                                                                                                     4. Distilled water 15 seconds                                                                                                                                                             5. Harris hematoxylin 6 minutes                                                                                                                                                       6. Distilled water 10 dips                                                                                                                                                                   7. Hydrochloric acid 0.5% solution, 1-2 quick dips                                                                                                                       8. Distilled water 15 seconds                                               

INTRODUCTION:- The established approach to the evaluation of ovarian function and endocrine disorders in the woman is based on serial biochemical analyses of hormones, such as estrogen, progesterone, luteinizing hormones and their metabolites. In women who suffer from menstrual disorders and abnormalities of the ovarian cycle, the biochemical analyses can be effectively supplemented by the old fashioned endometrial biopsies, or studies of endocervical mucus. In addition, the cervicovaginal smear may sometimes provide useful information and has the advantage of being easy to obtain, rapidly evaluated, and inexpensive. The cytologic approach is particularly valuable if laboratories specializing in endocrine analysis are not readily available. The principle of the cytologic hormonal analysis is simple. The degree of maturation of the squamous epithelium of the female genital tract depends on steroid hormones, mainly estrogen.  HORMONAL ASSESSMENT:- Naturally occurring estrogen, or the parenteral administration of estrogen or its natural or synthetic substitutes in adequate amounts, produces a rapid and complete maturation of the normal squamous epithelium of the female genitaltract with a resulting preponderance of mature superficial squamous cells in smears. The effect takes place regardless of the prior hormonal status, exceptduring pregnancy. Conversely, complete atrophy of the squamous epithelium of the vagina and cervix may be equated with complete absence of estrogenic activity. However, there are no reliable data linking intermediate degrees of maturation of the squamous epithelium with the action of a specific hormone or hormones. Evaluation of the endocrine status of a menstruating woman during the childbearing age belongs among the most difficult tasks in diagnostic cytology. There is considerable variation in the smear patterns from one patient to another,even if matched for age and menstrual history. Several conditions must be fulfilled before a successful hormonal evaluation of the squamous epithelium may be undertaken. There must be absence of inflammation or cytolysis. There must be no recent medication, either topical or systemic, especially with compounds known to affect the squamous epithelium of the lower genital tract. There must be no history of radiotherapy or recent surgery to the vagina or cervix.  An adequate baseline investigation must have been performed in menstruating women. This should include daily smears during at least one and preferably two complete cycles, or their chronologic equivalent. In nonmenstruatingpatients, two or three smears may suffice.The smears should be obtained from the proximal portion of the lateral wall of the vagina, care being taken to avoid contamination with material from the adjacent cervix. The Karyopyknotic Index (KI):- The karyopyknotic index expresses the percentile relationship of superficial squamous cells with pyknotic nuclei to all mature squamous cells. Usually, 200 to 400 consecutive cells in three or four different fields on the smear are evaluated. The peak of KI usually coincides with the time of ovulation and was estimated at 50% to 85% of total cells. The Eosinophilic Index (EI):- The eosinophilic index expresses the percentile relationship of mature squamous cells with eosinophilic cytoplasm to all mature squamous cells, regardless of the status of the nucleus. In a normal menstruating woman, the peak of EI coincideswith the peak of KI and may reach 50% to 75% at the time of ovulation. The Maturation Index (MI):- The maturation index expresses the maturation of the squamous epithelium as a percentile relationship of parabasal cells to intermediate cells to superficial cells. The count should be performed on single cells. For example, in a normal menstruating woman at the time of ovulation, an MI of 0:35:65 would indicate that the smear contained no parabasal cells, 35% of intermediate cells, and 65% of superficial cells. Other Indices:- The folded-cell index represents the relationship of mature superficial or intermediate squamous cells with folded cytoplasm to all mature squamous cells. The crowded-cell index represents the relationship of mature squamous cells lying in clusters of four or more cells to all mature squamous cells. Alternative Ways of Reporting Hormonal Status:- It has been a common practice to base the evaluation of the maturation of the squamous epithelium on an overall visual impression gained during the routine screening of smears. This simplest of methods has not failed in revealing majorabnormalities of smear patterns. By comparing the current smear pattern with original baseline smears, a good appreciation of changes in smear pattern may be gained. Small variations in smear pattern have no diagnostic meaning but maystrongly influence the indices and thus give a false impression of hormonal “effects.” The reporting of smears based on this overall visual impression is always given in reference to age, menstrual history, and possible clinicalsignificance. Some examples follow: Patient age 35: “Midcycle smear pattern—consistent with functioning ovaries.”Patient age 52: “Absence of maturation of squamous cells consistent with menopause.” Patient age 25: “Absence of maturation of squamous cells—abnormal for age.” Patient age 60: “High level of maturation of squamous cells not consistent with clinical menopause. It is assumed that this patient is not receiving estrogens or other drugs that may account for this smear pattern. DETERMINATION OF THE TIME OF OVULATION FROM CERVICOVAGINAL SMEARS:- A precise determination of the time of the ovulation is important in artificial insemination and in in-vitro fertilization. The use of the cervicovaginal smears to establish the time of ovulation or the status of the endometrium has been of llimited reliability.  It is recommended that cytologic methods for estimation of ovulation or status of the endometrium be supplemented by other procedures, such as temperature curves and endometrial biopsies. The examination of endocervical mucus may also be of assistance. Cyclic changes in the physicochemical properties of the cervical mucus have been known for a greatmany years. Prior to ovulation, the mucus tends to be viscous and when placed on a glass slide, form crystalline, fern-like structures, whereas at the time of and after ovulation, the mucus is more liquid and does not crystallize. Cytologic evaluation for menstrual abnormalities:- 1. Cytologic hormonal evaluation may be of assistance in the evaluation of amenorrhea (cessation of menses), in women who have never menstruated (primary amenorrhea) or who stopped menstruating at a young age after a period of

INTRODUCTION:- The use of fine-needle aspiration (FNA), a method of aspiration biopsy cytology, continues to grow throughout the World. Improvements in imaging, computed tomography scan (CT), and ultrasound (USG) have fueled the growth of FNA among both radiologists and clinicians. The dominant clinical sites for FNA still remain breast, thyroid, and lymph nodes among superficial tissues. CLINICAL SKILLS REQUIRED:- Aspiration biopsy may be indicated whenever there is a palpable tumor mass or a lesion visualized within any organ. For the physician or more specifically for the pathologist performing FNA, some familiarity with general anatomy is essential. For the physician or more specifically for the pathologist performing FNA, some familiarity with general anatomy is essential. For the pathologist performing this biopsy some sharpening of clinical skills, both obtaining a focused clinical history and performing a physical examination are required. Clinicians performing aspiration biopsy obviously lack this essential ingredient of experience and knowledge of morphology. Despite the recognized participation and value of cytotechnologists to an aspiration biopsy service, the pathologist must be actively involved in the aspiration biopsy, making both the initial and final evaluation of the smears. The Thin-needle Aspiration Method:- Thin needle generally 22, 23, 25, and 27 gauge, are used for the performance of aspiration biopsy, most often 1.5 in. in length. Special situations may dictate shorter needles and even higher gauge. For example, the very small cutaneous metastasis of breast carcinoma on the chest wall may be sampled more easily with a 27-gauge, 1-in.or even ½-in. needle and with a small, 3.0- to 5.0-mL syringe, approaching the nodule in a plane perpendicular to the skin surface, in the manner of performing a tuberculin skin test. Radiologists most often use the Chiba needle of 21 and 22 gauge for transthoracic and transabdominal aspirations. If one employs only the thin-needle technique, there are virtually nocomplications, the exceptions being FNA of the thorax (pneumothorax) or some cases of excessive bleeding with transabdominal aspiration biopsy. Basic Equipment:- The basic equipment used for rapid and efficient performance of thin-needle aspiration biopsy are as follows. 1. Cameco Syringe Pistol, Aspir-Gun, or other type aspiration handle; 2. 10 or 20-mL disposable plastic syringe with LuerLok or straight tip, depending on aspiration gun handle size; 3. 22 to 27-gauge, 0.6- to 1.0-mm external diameter disposable needles, 3.8 and 8.8 cm, 15 and 20 cm long, with or without stylus; the needle hub should be clear; 4. Alcohol skin preparation sponges; betadine skin sponges for deeper aspirations, transabdominal, transthoracic, bone (where the cortex is not intact or the periosteum is elevated), or deep soft tissue; 5. Sterile gauze pads;6. Microscopic glass slides with frosted ends;  7. Small vial of balanced salt solution and/or RPMI tissue culture transportmedia;  8. Suitable alcohol spray fixatives for immediate fixation of wet smears. 9. 10 or 20 mL capped tube with 10% neutral buffered formalin for cell-block.10. Optional vial of local anesthesia, 1-2% lidocaine; topical spray anesthesia for aspirates in children or intraoral aspirates; vials of lidocaine that dentists use for local anesthesia and the dispensing equipment may be useful.  A small plastic tray easily holds all the equipment. Majority of the smears are to be air-dried and later stained with a Romanowsky method, the Diff-Quik stain being preferred. Some smears are usually wet-fixed in 95% ethyl alcohol. Aspiration Technique:- To be successful with an aspiration biopsy, it is important to follow the preliminary steps listed here: 1. Review the history of the patient. Determine the clinical problem and its relevance to the lesion to be biopsied.2. Determine whether the biopsy is justified. 3. Palpate the mass, attempting to determine its location in relation to surrounding structures. Estimate its depth. Decide on the optimal direction of the needle to accomplish the aspiration biopsy. A mass located deeply in tissue in usually best approached perpendicularly to the skin surface. Small and superficially lying tumors are best approached by penetrating the skin at or very close to horizontal plane, then feeling for the mass with the needle tip. The patient should be placed in a comfortable position for the aspiration biopsy, but the mass must be easily palpable and immobilized during the biopsy. Step 4 is very important for head and neck lesions. The prominence of an enlarged lymph node, or lump, may sometimes depend on whether thepatient is supine or erect. The sternocleidomastoid muscle bulk and its close proximity to the cervical lymph nodes require positioning the patient such that the biopsy needle passes through only a minimum of soft tissue and muscle before reaching the target. Avoid aspirating a mass by traversing the sternocleidomastoid muscle. For the aspiration of thyroid lesions, it is usually helpful to place a small pillow under the patient’s upper back, extending the neck with the head tilted back. 5. Take time to examine the patient thoroughly. Discuss your preliminary assessment of the patient’s lesion. This is an opportunity to describe whatwill take place during the aspiration and what is to be accomplished withit.  6. Obtain informed consent. This consent should indicate that name of the patient who is having the aspiration, the name of the doctor performing the aspiration and a listing of discussed complications. PERFORMING THE FNAC:- It is essential that the FNAC be performed by a doctor who has knowledge ofanatomical structures and pathological lesions expected in the particular region. Smear Preparation:- 1. Immediately after completing the aspiration biopsy, the needle should be quickly removed from the syringe; pulled back on the syringe pistol to fill the syringe with air. 2.The needle should be reattached and placed near the center and touching the surface of a plain glass slide. 3. Advancing the plunger of the syringe, will express a small drop of the sample, approximately 2–3 mm in diameter, onto the sl  4. This procedure should be quickly continued over a series of five to six slides. 5. Invert another plain glass slide over the drop; as it spreads from just the weight of the slide, pull the two slides apart horizontally in a single gentle motion. 6. As an alternative, when the drop spreads in a circular fashion, again

INTRODUCTION:- Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of similar cells from the same origin that together carry out a specific function. Organs are then formed by the functional grouping together of multiple tissues. TYPES OF TISSUES :- Following types of tissues make up all organs of the body:- A. Epithelium B. Connective tissue-supporting tissue C. Muscle-striated, smooth and cardiac D. Nervous tissue E. Blood-It is found in blood vessles, which are part of connective tissue. A. Epithelium:-  Epithelial tissue covers the whole surface of the body. It is made up of cells closely packed and ranged in one or more layers. This tissue is specialized to form the covering or lining of all internal and external body surfaces. * Epithelial tissue that occurs on surfaces on the interior of the body is known as endothelium.               * Epithelial tissue, is usually separated from the underlying tissue by a thin sheet of connective tissue called as basement membrane. *The basement membrane provides structural support for the epithelium and also binds it to  neighboring structures. Types of Epithelial Tissues:- It can be divided into two types according to location. *Covers the external surface and line all the body cavities and tubes. *Secretory; found in glands. 1.Simple epithelium:-Simple epithelium can be subdivided according to the shape and function of its cells. *Squamous (pavement) epithelium:- Squamous (pavement) epithelium is a type of epithelial tissue made of thin, flat, scale-like cells that fit closely together, like paving stones.It forms a thin lining that allows easy diffusion and filtration and is found in places such as the air sacs of the lungs, blood vessels, and kidney capsules.They form the lining of cavities such as the mouth, esophagous, anus, uterine cervix and make up the outer layers of the skin. Simple Cuboidal Epithelium:- Cuboidal cells are roughly square or cuboidal in shape. Each cell has a spherical nucleus in the centre. Cuboidal epithelium is found in glands and in the lining of the kidney tubules as well as in the ducts of the glands. Simple Columnar Epithelium:-  Columnar epithelial cells occur in one or more layers. The cells are elongated and column-shaped.  Columnar epithelium forms the lining of the stomach and intestines. Goblet cells (unicellular glands) are found between the columnar epithelial cells of the colon. They secrete mucus, a lubricating substance which keeps the surface smooth. Glandular Epithelium:- Columnar epithelium with goblet cells is called glandular epithelium. Columnar and cuboidal epithelial cells often become specialized as gland cells which are capable of synthesizing and secreting certain substances such as enzymes, hormones, milk, mucus, sweat and saliva. Stratified Epithelium:-  Where body linings have to withstand wear and tear, the epithelia are composed of several layers of cells and are then called compound or stratified epithelium. The top cells are flat and scaly and it may or may not be keratinised (i.e. containing a tough, resistant protein called keratin). Human skin is an example of, keratinised, stratified epithelium. The lining of the mouth cavity is nonkeratinising, stratified epithelium. B. Connective Tissue:- Connective tissue is a type of tissue in the body that supports, binds, protects, and connects other tissues and organs. It is characterized by having cells scattered within an extracellular matrix (a non-living material made of fibers and ground substance).This is the most widespread tissue in the human body. *The most common cell type is fibroblast, which produces fibres and other intercellular materials.The two most common types of fibres are: collagen (collagenous) and elastic. *Collagen fibres are for strength while elastic fibres provide elasticity to the tissue.Both the cells and the fibres are embedded in the intercellular substance.The consistency of this substance is highly variable from gelatin-like to a much more rigid material. Classification of Connective Tissue:- I.Connective Tissue Proper – encompasses all organs and body cavities, connecting one part with another and, equally important, separating one group of cells from another.This includes adipose tissue (fat), areolar (loose) tissue, and dense regular tissue. II. Specialized Connective Tissues — this group includes cartilage, bone, and blood. Cartilage and bone form the skeletal framework of the body.Blood is circulated in the the vessles,made of connective tissues. Muscles:- Muscles are specialized body tissues made of elongated cells called muscle fibers that acts like an engine. Its primary job is  contract (shorten) and relax (lengthen) to create movement, maintain posture, and circulate blood throughout your body. There are three types of muscles in the body. Smooth muscle:-  Muscle tissue that contracts without conscious control, having the form of thin layers or sheets made up of spindle-shaped, unstriated cells with single nuclei.It is present in the walls of the internal organs, such as the stomach, intestine, bladder, and blood vessels. Cardiac muscle:-  This type of muscle occurs only in heart. Its cells are joined end to end. The resulting fibers are branched and interconnected in complex networks. Each cell has a single nucleus. At its end, where it touches another cell, there is a specialized intercellular junction called an intercalated disc, which occurs only in cardiac tissue. Cardiac muscles work involuntarily and can continue to function without being stimulated by nerve impulses. Skeletal muscle:- It is also called voluntary muscle, striped muscle, or skeletal muscle.It is the most common of the three types of muscle in the body. Striated muscles are attached to bones and produce all the movements of body parts in relation to each other. Striated muscle is under voluntary control. Its multinucleated fibers are long and thin and are crossed with a regular pattern of fine red and white lines, giving the muscle its distinctive appearance and its name. These cross striations are better seen with phosphotungstic acid hematoxylin stain. BONE:-  Bone is the basic unit of the human skeletal system and provides the framework for and bears the weight of the body, protects the vital organs, supports mechanical movement, hosts hematopoietic cells. Cartilage and bone are specialized connective tissues and consist of cells and extracellular matrix. The matrix of all connective tissues consists of

INTRODUCTION:- Laboratory sample processing includes steps from the receipt of the specimen in the laboratory to the delivery of a stained slide ready for microscopic examination. Throughout processing, the identity and integrity of the specimen must be maintained, and the principles of universal precautions followed. SPECIMEN PROCESSING:- The laboratory should confirm the identity and integrity of the specimenreceived. Specimens are accepted only when ordered by physicians or otherpersons authorized by law. Each sample must have a request completed by theauthorized provider prior to processing. 1. Specimen Preparation :- (a) Smears:- The preparation objective of direct smears is a slide with an evenly andthinly applied cellular specimen that is free of mechanical distortion and free of drying artifact when the slide is fixed in alcohol. Smears fixed inalcohol (wet fixation) are usually stained by the Papanicolaou method; airdried smears are usually stained with a Romanowsky stain. Smearspreserved with spray fixatives should be soaked in 95% alcohol. (b) Liquid Specimens:- *Liquid specimens should be processed according to the manner in whichthey are submitted. *Liquid specimens may be received fresh, with heparin, with preservative (alcohol or other fixative), or with physiologic solution or tissue culture medium. *Additional processing should be considered for grossly bloody specimens prior to slide preparation. *Blood clots should be removed and processed as a cell block. *Specimens of low cellularity and low volume may be cytocentrifugeddirectly. * High volume specimens are usually concentrated prior to preparation.Centrifugation is frequently used with the re-suspended pellet used fordirect smears. 2. Specimen staining:- The Papanicolaou stain is recommended for the staining of alcohol fixedcytology slides. Romanowsky stains may also be used for wet fixed slides,but are primarily applied to air-dried smears. (a) Papanicolaou Stain:- *The Papanicolaou stain uses a standard nuclear stain, hematoxylin, and twocytoplasmic counterstains, OG-6 and EA. *The outcome of this method is crisp nuclear detail and transparency of the cytoplasm, which allows the examiner to clearly visualize cellular morphology. *Either a progressive or regressive technique may be used for nuclear staining. Several automatic programmable stainers are available.  (b) Romanowsky Stain :- *A Romanowsky stain is recommended for air-dried smears. *Romanowsky stains, mixtures of eosin and methylene blue, are a family of polychromestains that produce their effect by the production of azure dyes as a result of demethylation of thiazines and the acidic component eosin. *Unlike the Papanicolaou stain they are metachromatic. *Most Romanowsky stains used in cytology are aqueous stains as opposed to the methyl alcohol based stains of hematology. *Many commercial stains are available, and most consist of a methanol-based fixative, and two dyes which result in differentiation of cytoplasmic and nuclear components.  *Most Romanowsky stains are rapid and are useful in enhancing pleomorphism, and distinguishing extracellular from intracytoplasmic material. 3. Dehydration, Clearing and Coverslipping:- (a) Dehydration and Clearing:- *After staining, the sample is dehydrated by a series of increasing concentrations of alcohol followed by rinsing in clearing solutions. *The last clearing solution should be colorless and its refractive index should be closeto that of the coverslip, slides and mounting medium. *Xylene is the most commonly used clearing agent. Xylene clearing must be performed in a well ventilated area or fume hood to limit exposure to xylene fumes.  *Slides should remain in the clearing agent until coverslipping is performed. (b) Coverslipping:- *Mounting medium used to bond the slide and the coverslip should be compatible with the clearing agent, transparent, and have a refractive index similar to the glass slide and the stained specimen. *Adequate mounting medium should be applied to protect the cellular material from air-drying and shrinkage, and to prevent fading of the cell sample. *The cellular material should be covered by a suitably sized coverslip or covering material of appropriate quality *Different methods used to coverslip include placing the mounting medium on the coverslip, then inverting the coverslip onto the slide surface, or lowering the slide onto a coverslip containing adequate mounting medium. Glass coverslips, coverfilm and automated coverslippers are available.  *deally, the mounting medium should be allowed to dry before the slides are reviewed to reduce movement of cellular material during the slide examination. *Chemical waste collected throughout the staining, dehydration, clearing and coverslipping processes must be disposed of or recycled. *The stained and labeled slide(s) should be matched with its requisition or other laboratory document that displays the same information.  *The information on the slide must correspond to the information on the requisition or laboratory document.

INTRODUCTION:- Cytology is the field of diagnostic medicine which deals with study of individual cells and/or tissue fragments spread on glass slides and stained. The final quality of cytodiagnosis depends to a large extent on quality of preparation of the material. It has an advantage of providing a rapid diagnosis. Cytological study can be done on various discharges from body (urine, nipple, sputum, vaginal, sinus, etc), scrapings obtained (buccal mucosa, gastric, respiratory), tap done from fluid collected in body (pleural, peritoneal, pericardial) or aspiration from palpable lumps.  HEALTH AND SAFETY :- There are potential hazards in handling fluid specimens like unfixed sputum,urine and other body fluids. All employees should be aware of all health andsafety aspects of laboratory, including:-  *   fire drills                                                                                                                                               *   storage                                                                                                                                               *   disposal of chemicals                                                                                                                       *   use of electrical equipment                                                                                                             *   storage and disposal of biological*    infectious material. Modern approach is to use ‘Universal Precautions’ to treat all unfixed specimens with care and to handle them in biological safety cabinets. Centrifuges should use sealed buckets. Main aspects of safety in cytology laboratory:- 1. Specimen reception- suitable container (disinfectant, autoclave proof);availability of suitable disinfectant (hypochlorite); protocol for leakage/spillage.  2.Specimen preparation– Protective clothes, coats, gloves, eye protection etc;Safety cabinet, Disposal pots with disinfectant, Refrigerator for specimenstorage 3. Specimen disposal– Disposal protocol, Autoclave, clinical waste collection. 4. Fire hazards and evacuation(Rescue,exit) procedure. 5. Storage of chemicals- Inflammables, Poisons, Toxic substances  6. First aid. SPECIMEN COLLECTION:- Most specimens are received in the laboratory either as direct cell spreads onslides or as cell suspensions (fluids). Most of Hospitals have FNA (fine needleaspiration) clinic where FNA is done by cytopathologist. Medical technologistis required in the clinic to spread the sample on glass slides and fix the slidesappropriately. Some clinics also perform rapid staining to check adequacy ofmaterial.  TRANSPORT AND INFORMATION :- *Specimens should be sent to the laboratory as early as possible in suitable containers. *Lids should be properly secured to prevent any leakage and specimens should be sealed in plastic bags. *Glass slides should be kept in suitable slide boxes. *All specimens and slides should be properly labeled with patient’s name and number. *Fixed smears should be submitted in containers that protect against breakage. *Slide containers are available in a variety of shapes, volumes and material. *Optimal design features include easily opened containers which stay closed during transport, shock resistant material, and enough room to prevent slides from adhering to one another or the container. *The slides should be marked clearly with the patient’s name, as well as other identifiers if possible. *If more than one site is sampled, the slides must be clearly marked as to their site. Slides in fixative should be submitted in leak proof containers that protect against breakage and are clearly labeled with the patient’s name and specimen site(s). *Each fluid specimen should be placed in a clearly labeled container that is leak proof. *Enclosure in a transport bag indicating biohazardous contents is careful if a courier system or manual delivery is planned. *Paper requisitions that accompany slides in fixative should be placed in an outside pocket to avoid exposure to any leakage of fixative. *Needles should never be transported with fluid specimens.  *Large glass collection containers should be avoided. *Specimen should be accompanied by properly filled requisition form. The requisition must also provide space for the date the sample was collected, the test to be performed, the source of the material, and the name and address or other suitable identifiers of the authorized person requesting the test. *The request form should contain essential patient identification data- name, age, sex,ward/OPD, hospital number, any previous sample number. Precise information should be given regarding type of specimen, any fixative used, relevant clinical information and any previous treatment.  *A written procedure must be in place to handle specimens that are received without adequate information on the request form. *High risk specimens should be clearly marked with biohazard stickers or labels. *The specimen after checking all labels should be given a lab identification number. The laboratory identifier may be generated manually or electronically and may be numeric or alphanumeric and may also be bar coded. Criteria for the rejection of specimens :- *Unlabeled slides, slides labeled with nonpermanent writing utensils or paper  labels and broken slides.*Mismatched specimens and requisition forms.*Specimen without accompanying requisition form. STORAGE OF SPECIMENS:- *Samples should be immediately prepared from the specimen. *Record the date of preparation on the specimen container and refrigerate any remainingspecimen. Specimen can be stored for one week before disposal. *Ideally, samples need to