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METACHROMATIC STAINING

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INTRODUCTION:- There are certain basic dyes belonging to aniline group that will differentiate particular tissue components by giving them a different color to that of original dye. The phenomenon is known as metachromasia. METACHROMASIA:-Metachromasia takes place when certain negatively charged groups on the tissue react with cationic dyes. On polymerization the original colour of the dye changes to another colour (eg mast cell stain pink with toluidine blue). Thionin and toluidine blue dyes are commonly used for quick staining of frozen selection using their metachromatic property to stain nucleus and cytoplasm differently.Metachromasia is enhanced when intermolecular distances are reduced. Factors which enhance metachromasia are:- 1. Increasing concentration of dye. 2. Decreasing temperature. 3. pH 4. Water a polar solvent, contributes to the efficiency of van der Waal’s forces by which the molecules are held together. In tissues, where there is a high concentration of anions e.g. in sulphated mucopolysaccharides, the cationic dye molecules may be held in such close proximity to one another that van der Waal’s forces can exert their influence and cause the dye to polymerize. Consequently the colour changes from blue to red. Tissue components often demonstrated by metachromatic stains:- Amyloid material, Mast cell granules Mucin Cartilage Amyloid Stain -Various stains are used to demonstrate amyloid CRYSTAL VIOLET STAIN FOR AMYLOID:- Aim: To demonstrate amyloid in tissue sections. Principle: Amyloid (a glycoprotein) exhibits metachromasia in tissue sections when stained with crystal violet and other cationic dyes. Control: positive control. Reagents:- Crystal violet solution Stock solution Crystal violet                                          14 gm 95% alcohol                                           100 ml Working solution Stock solution                                      10 ml                                                                                                                                                    Distilled water                                      300 ml                                                                                                                                                Concentrated hydrochloric acid        1 ml Procedure:- *Deparaffinize and bring the sections to water. *Put working crystal violet solution for 1 to 2 minutes and check under microscope. *Rinse in tap water. *Mount in water or in water soluble media. *Put on the coverslip seal the edges with nail polish (Do not let it dry.) Result:- Amyloid                                               purple violet Other tissues                                      blue CONGO-RED STAIN FOR AMYLOID:- Aim:- To demontrate amyloid in tissues. Principle:- Diazo dye attaches itself to amyloid fibrils. The union is affected by H bonds between the OH groups of amyloid and amino side groups of the dye. Congo red dye forms non-polar hydrogen bonds with amyloid. The green birefringence of congo red stained amyloid by polarized light is considered diagnostic of amyloid. Control:- Known positive tissue. Reagents Congo red solution Congo red                            1.0gm Distilled water                     100ml Saturated solution of Lithium Carbonate Lithium carbonate 1.3gm Distilled water 100ml Procedure:- *Bring section to water. *Pour congo red solution for 20 minutes. *Pour off the solution and cover the slide with lithium carbonate for 1.5 minutes to differentiate. *Wash with water. *Counter-stain with hematoxyline for 5 minutes. *Differentiate with 1% acid alcohol. *Wash in running tap water. *Dehydrate, clear in xylene and mount in DPX. Result:- Amyloid                                              bright red which gives apple green birefringence in polarized light. Nuclei                                                 blue Other structures                               unstained to yellow Notes:- 1. Sections must be cut at 8 to 10 microns for birefringence 2. Solution must be filtered through glass wool, not paper filters for birefringence to occur 3. Tissue fixed in solutions other than formalin may display false positive birefringence.

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Microtome Techniques and Applications

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INTRODUCTION:- A microtome (from the Greek mikros, meaning “small”, and temnein, meaning “to cut”) is a tool used to cut extremely thin slices of material, known as sections. MICROTOME:- Various types of microtomes are available. Most commonly used microtome for routine histopathology is rotary microtome. The most common applications of microtomes are:- Traditional Histology Technique:- Tissues are hardened by replacing water with paraffin. The tissue is then cut in the microtome at thicknesses varying from 2 to 50 µm. From there the tissue can be mounted on a microscope slide, stained with appropriate aqueous dye(s) after prior removal of the paraffin, and examined using a light microscope. Cryosectioning Technique:- Water-rich tissues are hardened by freezing and cut in the frozen state with a freezing microtome or microtome-cryostat; sections are stained and examined with a light microscope. This technique is much faster than traditional histology (15 minutes vs 16 hours) and is used in conjunction with medical procedures to achieve a quick diagnosis. Cryosections can also be used in immuno histochemistry as freezing tissue stops degradation of tissue faster than using a fixative and does not alter or mask its chemical composition as much. Electron Microscopy Technique:- After embedding tissues in epoxy resin, a microtome equipped with a glass or gem grade diamond knife is used to cut very thin sections (typically 60 to 100 nanometer). Sections are stained with an aqueous solution of an appropriate heavy metal salt and examined with a transmission electron microscope (TEM). This instrument is often called an ultramicrotome. The ultramicrotome is also used with its glass knife or an industrial grade diamond knife to cut survey sections prior to thin sectioning. These sections are of 0.5 to 1 µm thickness and are mounted on a glass slide and stained to locate areas of interest under a light microscope prior to thin sectioning for the TEM. Thin sectioning for the TEM is often done with a gem quality diamond knife. Botanical Microtomy Technique:- Hard materials like wood, bone and leather require a sledge microtome. These microtomes have heavier blades and cannot cut as thin as a regular microtome. Rotary Mictrotome:-  It is most commonly used microtome. This device operates with a staged rotary action such that the actual cutting is part of the rotary motion. In a rotary microtome, the knife is typically fixed in a horizontal position A rotary action of the hand wheel actuate the cutting movement. Here the advantage over the rocking type is that it is heavier and there by more stable. Hard tissues can be cut without vibration. Serial sections or ribbons of sections can easily be obtained. The block holder or block (depends upon the type of cassette) is mounted on the steel carriage that moves up and down and is advanced by a micrometer screw. Auto-cut microtome has built in motor drive with foot and hand control. With suitable accessories the machine can cut thin sections of paraffin wax blocks and 0.5 to 2.0 micrometer thin resin sections. Advantages:- 1. The machine is heavy, so it is stable and does not vibrate during cutting.2. Serial sections can be obtained.3. Cutting angle and knife angle can be adjusted.4. It may also be used for cutting celloidin embedded sections with the helpof special holder to set the knife. In the figure to the left, the principle of the cut is explained. Through the motion of the sample holder, the sample is cut by the knife position 1 to position 2), at which point the fresh section remains on the knife. At the highest point of the rotary motion, the sample holder is advanced by the same thickness as the section that is to be made, allowing for the next section to be made. The flywheel in microtomes can be operated by hand. This has the advantage that a clean cut can be made, as the relatively large mass of the flywheel prevents the sample from being stopped during the sample cut. The flywheel in newer models is often integrated inside the microtome casing. The typical cut thickness for a rotary microtome is between 1 and 60 µm. For hard materials, such as a sample embedded in a synthetic resin, this design of microtome can allow for good “Semi-thin” sections with a thickness of as low as 0.5 µm. Sledge Microtome:- it is a device where the sample is placed into a fixed holder (shuttle), the sledge placed upon a linear bearing, a design that allows for the microtome to readily cut many coarse sections. Applications for this design of microtome are of the preparation of large samples, such as those embedded in paraffin for biological preparations. Typical cut thickness achievable on a sledge microtome is between is 10 and 60 micron. Cryomicrotome:- For the cutting of frozen samples, many rotary microtomes can be adapted to cut in a liquid nitrogen chamber, in a so-called cryomicrotome setup. Thereduced temperature allows for the hardness of the sample to be increased, such as by undergoing a glass transition, which allows for the preparation of semithin samples. However the sample temperature and the knife temperature must be controlled in order to optimise the resultant sample thickness. Ultramicrotome:- A ribbon of ultrathin sections prepared by room temperature ultramicrotomy, floating on water in the boat of a diamond knife used to cut the sections. Theknife blade is the edge at the upper end of the trough of water. An ultramicrotome is a main tool of ultramicrotomy. It can allow for the preparation of extremely thin sections, with the device functioning in the same manner as a rotational microtome, but with very tight tolerances on the mechanical construction. As a result of the careful mechanical construction, the linear thermal expansion of the mounting is used to provide very fine control of the thickness. These extremely thin cuts are important for use with transmission electron microscope (TEM) and Serial Block-Face Scanning Electron Microscopy(SBFSEM), and are sometimes also important for light-optical microscopy. The typical thickness of these cuts is between 40 and 100 nm for transmissionelectron microscopy

CYTOLOGY

Hormone Wellness Assessment

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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

CYTOLOGY

FINE NEEDLE ASPIRATION CYTOLOGY

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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

CYTOLOGY

MORPHOLOGY OF ORGANS

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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

CYTOLOGY

Quality Assurance and Quality Control in Cytology Laboratories

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INTRODUCTION:- Cytopathologists are concerned about and committed to quality assurance and quality control in their laboratories. These practices include, among others, the use of intralaboratory and extradepartmental consultations, case reviews, correlation of cytologic and histopathologic specimens and review of completed diagnostic reports.  QUALITY ASSURANCE MEASURES:- Cytopathology is a practice of medicine and represents a medical consultation, in both gynecologic and nongynecologic anatomic sites. The basic principles of quality assurance apply to all types of cytologic specimens. The following represents several minimum quality assurance measures. 1. Laboratory Directors:- *The laboratory should be directed by a legally qualified physician with a specialist qualification in pathology, including special training and expertise in cytopathology.  *The director or designated medical professional is responsible for proper performance and reporting of all tests done in the cytopathology laboratory.  *The director or designated cytopathologist should be physically present in the laboratory to direct the staff, be available for consultations, review all reactive and abnormal gynecologic cytology samples, review fine needle aspiration samples, and review all nongynecologic samples. 2. Cytotechnologists:- A suitably qualified person should be recruited for this position. 3. Physical Laboratory Facilities:- *The laboratory should be clean, well lighted, adequately ventilated, and functionally arranged so as to minimize problems in specimen handling, evaluation, and reporting.  *The area for specimen preparation and handling should be separate from the area where specimens are evaluated and reported.  *Formaldehyde and xylene (if in use) should be carefully monitored due to the possible presence of hazardous vapor concentrations. 4. Safety Precautions:- *Laboratory personnel must be protected against hazards (chemical, electric, fire, infections, or others) by using well-ventilated hoods and biologic safety hoods for handling potentially infectious material. * Fire precautions should be posted and tested. 5. Equipment:- *An adequate number of binocular microscopes of good quality and proper working order must be available. * Laboratory instruments and equipment should be under periodic maintenance to monitor and ensure malfunctions do not adversely affect analytical results. 6. Specimen Collection:- *Cytologic specimens should be accepted and examined only if requested by a licensed medical practitioner and collected in accordance with instructions regarding recommended collection techniques. *The cytopathology laboratory should inform the originator of the sample if the specimens are “unsatisfactory” and detail adequacy qualifiers such as presence or absence of a transformation zone component or obscuring factors in “satisfactory samples”. 7. Preparation, Fixation, and Staining Procedures:- *The specimens must be identified with the patient’s name and/ or a unique identifier and must be accompanied by a requisition form with the requesting physician’s name, address, date of specimen collection, specimen source, and appropriate clinical information about the patient.  *When the specimen arrives in the laboratory the laboratory staff affix an accession number or bar code label on each slide for further identification.  *The laboratory should have written criteria for rejecting specimens.  *Fixation while the specimen is still wet is recommended for conventional cell samples.  *The Papanicolaou staining procedure is strongly suggested for most cytologic samples, unless additional staining procedures are warranted. *Staining solutions and chemicals used in the cytopathology laboratory should be labeled with the time of preparation, purchase, or both. Staining solutions should be filtered regularly to avoid contamination and should be covered when not in use. *Effective measures to prevent cross-contamination between gynecologic and nongynecologic specimens during the staining process must be used. 8. Slide Evaluation Workload:- *Regulations as to the number of specimens a cytotechnologist may evaluate in a 24-hour period are currently set at 100 slides per an 8-hour day. * This regulation may not do justice to the various conditions that influence thequality of the slide evaluation performance. * The percentage of atypical cases evaluated versus the percentage of negative cases in varying populations as well as screening of nongynecologic specimens should be considered when workloads are established. *This regulation ensures that the number and type of cytologic samples evaluated do not, through fatigue, adversely affect the cytotechnologist’s performance. 9. Cytologic Terminology:- *The vaginal/ectocervical/endocervical cytology sample should be interpreted preferably by using the Bethesda System.  *The nongynecologic material should be interpreted in medical terms. 10. Laboratory Records, Logs, and Files:- *Each specimen should be recorded and a sequential accession number assigned together with the name of the patient and the originator of the sample.  *Test records must be retained for at least 5-10 years.  *The negative gynecologic cell samples should be retained on file for a minimum of 5 years and negative fine needle aspirates for 10 years or indefinitely if they exhibit abnormal features.  *The modern cytopathology laboratory should use a computerized file system.

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CYTOLOGY : DISPOSAL OF HUMAN WASTE

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INTRODUCTION:- Hospital waste is “Any waste which is generated in the diagnosis, treatment or immunization of human beings or animals or in research” in a hospital. Hospital Waste Management means the management of waste produced by hospitals using such techniques that will help to check the spread of diseases through. DISPOSAL OF HUMAN WASTE:-The laboratory should conform to the local practices and guidelines for safedisposal of human and chemical waste generated in the laboratory. WHO MEDICAL WASTE CATEGORIES:- Infectious:- Materials containing pathogens if exposed can cause disease:- Human anatomical waste: waste from surgery and autopsies on patients withinfectious diseases; Sharps: disposable needles, syringes, saws, blades, broken glasses, nails orany other item that could cause a cut; Pathological: tissues, organs, body parts, human flesh, fetuses, blood andbody fluids; Non Infectious (Hazardous):- Pharmaceuticals: drugs and chemicals that are returned from wards, spilled,outdated, contaminated, or are no longer required;  Radioactive: solids, liquids and gaseous waste contaminated with radioactivesubstances used in diagnosis and treatment of diseases like toxic goiter. Non Infectious (Non Hazardous):- Domestic waste: from the offices, kitchens, rooms, including bed linen,utensils, paper, etc Care needs to be taken to dispose off the Infectious and non-infectious hazardouswaste. The non Infectious (Non Hazardous) waste can be disposed off withregular garbage disposal. Cytology laboratory generates waste in the form of remnants of fluids (peritoneal, pleural, cysts, etc), sputum, and left over specimen of liquid cytology. The specimens need to be discarded only after chemical decontamination using at least 1% sodium hypochlorite solution; and then discharged into drains/sewers where it is taken care of by the principle of dilution and dispersal. Any solid waste needs to be disposed off according to hospital waste management. Before disposal the specimen need to be segregated after proper identification. Segregation by color coding system:- Three categories :-Infectious waste – Red bagsDomestic waste – Green BagsSharps – Needle cutters / Puncture proof containers. Transportation:-Containers: puncture proof, leak proof,Bags: sturdy, properly tiedTransport trolleys: designated & timelyStaff protection: provided with protective clothing and other itemsNever put hands in a bag puncture proof, leak proof, The infectious material in red bags will go for incineration:- The sharps can either go to incinerator or following autoclaving/chemicaldisinfection can be mutilated. They should never be thrown in regular garbage. Chemical waste collected throughout the staining, dehydration, clearing andcoverslipping processes must be disposed of or recycled according to state andlocal regulations.

CYTOLOGY

SPECIMEN PROCESSING & STAINING

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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.

CYTOLOGY

SPECIMEN COLLECTION & STORAGE

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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

Uncategorized

EXFOLIATIVE CYTOLOGY

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INTRODUCTION:- Exfoliative cytology, which is a quick and simple procedure, is an important alternative to biopsy in certain situations. In exfoliative cytology, cells shed from body surfaces, such as the inside of the mouth, are collected and examined. This technique is useful only for the examination of surface cells and often requires additional cytological analysis to confirm the results. Exfoliative cytology differs from the more precise sampling of known lesions, like needle biopsy. It categorizes collected samples only by analyzing the presence of abnormal or atypical cells, or by showing the presence of malignant cells. When a woman has a pap smear, she may have a result that show atypical cells. If this is the first exfoliative cytology test that shows atypical cells, then usually, the Pap smear is repeated in six to twelve months. If however, repeated showings of atypical cells are present in exfoliative cytology results, further tests may be undertaken to determine if cancerous cells are present. Doctors or dentists may also use exfoliative cytology to check for the presence of cancer in the mouth or throat. The test takes a few skin scrapings and can show the presence of either malignant or atypical cells. Malignant and atypical cells will probably require a person to undergo a biopsy or closer examination of the area in question to rule out cancer. Cytologic examination of a serous effusion is of paramount importance because the finding of cancer cells in such a specimen denotes that the patient has cancer that is not only advanced but also almost always incurable. Apart from the finding of cancer cells, cytologic examination of pleural, peritoneal, and pericardial effusions may also reveal information about inflammatory conditions of the serous membranes, parasitic infestations, and infection with bacteria, fungi, or viruses. COLLECTION METHOD:- In this method, cells are collected after they have been either spontaneously shed by the body (“spontaneous exfoliation”) or manually scraped/brushed off of a surface in the body (“mechanical exfoliation”). An example of spontaneous exfoliation is when cells of the pleural cavity or peritoneal cavity are shed into the pleural or peritoneal fluid. This fluid can be collected via various methods for examination. Examples of mechanical exfoliation include Pap smears, where cells are scraped from the cervix with a cervical spatula, or bronchial brushings, where a bronchoscope is inserted into the trachea and used to evaluate a visible lesion by brushing cells from its surface and subjecting them to cytopathologic analysis. Spontaneous exfoliation: Peritoneal fluid, pleural fluid, pericardial fluid, urine, cysts, washings (peritoneal, bladder) The fluid is collected into a clean, dry container, which need not be sterile, and sent to the laboratory as soon as possible. If the fluid cannot be sent immediately, it should be stored in a refrigerator at 40C and not allowed to freeze. We do not require anticoagulant or fixative to be added to the fluid. The appearance to the naked eye of a serous effusion sometimes reveals clues about the cause of the effusion and the nature of its cellular contents. Therefore, for every serous effusion received by the laboratory, note should be made of its volume, color, clarity, and any unusual physical features, such as malodor, opalescence, or high viscosity Mechanical exfoliation: Cervical pap smear, brushings (Bronchial, gastric, biliary, oral, etc). Cervical smear is a reliable method for diagnosis of cervical cancer. The smears are usually taken in Gynecology ward or OPD, but sometimes patients are sent to laboratory for smear purposes. Patients should be advised NOT to douche, use vaginal medications, or have intercourse 24 hours prior to the pap smear preparation. Patients should NOT schedule pap smear exams during menses. These situations may obscure cellular details or remove diagnostic material from the cervix or vagina. The smear is obtained under direct vision after introduction of speculum. A wooden tongue depressor cut with scissors to fit the contour of cervix may be used. Commercially prepared plastic or wooden scrapers are widely available for this purpose. The scraper is rotated under pressure to 360° for 4-5 full rotations. The material is spread on a pre labelled slide and fixed immediately. Several types of brushes have also been introduced to overcome the disadvantages of scrapers alone (not being able to reach endocervical canal and transformation zone where the carcinomas originate). Brushes are also used to scrape cells in respiratory tract, oral mucosa, esophagus, stomach, duodenum, colon and biliary tract. It is preferable to obtain the brush sample before the biopsy because the latter results in bleeding, which both obscures the lesion, and detracts from the quality of a subsequently collected cytologic sample, whereas interpretation of the biopsy is not affected by the reverse order of collection. Collection of a good brush sample usually requires an experienced assistant, because the operator may well be engaged in maneuvering the end of the scope and holding the lesion in focus while the assistant manipulates the brush. Therefore, it is ideal that a Cytology staff member be present for immediate slide preparation of the specimen. They are all taken under vision- direct or through fibreoptic endoscopy. In all these cases a lot of care needs to be taken to make smear immediately from the brush by gently rotating the brush on slides and fixing them immediately. The material should not be crushed. If liquid-based cytology is used, the head of the broom is detached and dropped into the preservative vial. Once received in laboratory, the usual precautions need to be taken as discussed in specimen receiving, handling and storage. When only one slide is received, it should be preferable to stain it with Papanicolaou stain. When multiple slides are received- some are air dried (stain with MGG, special stains, etc) and some are wet fixed (stain with Papanicolaou). The liquid specimens need to be commented upon the volume, color and turbidity. Specimens need to be centrifuged and cytocentrifuged depending on cellularity. After concentrating they can be used for both air dried smears and wet fixation.

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