PATHOPrelims - 1. Surgical Pathology (Introduction)

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Dr. Roman | June 17, 2015 | PATHOLOGY

SURGICAL PATHOLOGY: An Introduction

Italicized – from the lecture of Dr. Roman th Blue Text – from Surgical Pathology 10 Ed. PATHOLOGY  ANATOMIC PATHOLOGY  Autopsy  Surgical Pathology  Cytology  Forensic Pathology  CLINICAL PATHOLOGY  Hematology  Clinical Chemistry  Blood Banking  Immunology/Serology  Clinical Microscopy  Microbiology

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WHO IS SURGICAL Pathologist? Must be a licensed physician. Must undergo training in an accredited hospital by the Philippine Society of Pathologist Must pass the Specialty Board Examination given by the PSP Board of Pathologists. Either a Diplomate or a Fellow  Pathology Residency = 4 years  Pathologist – “the one who reads the biopsies.”

Surgical Pathologist  Closely affiliated with all surgical specialties, internal medicine, dermatology, neurology, radiology etc.  He needs to understand the clinician‟s needs and respond to them accordingly.  He needs to tell the clinician, not only whether a tumor is benign or malignant but also  Extent of the disease  Grade of malignancy  Adequacy of surgery  Other pertinent information, e.g. Prognosis Subspecialties  Hematopathology  Renal Pathology  Neuropathology  Dermatopathology  Forensic Pathology

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SURGICAL PATHOLOGY REPORT  Should contain all relevant gross and microscopic features of a case  Should be:  Prompt  Accurate  Brief  Avoids unnecessary histologic jargon 5 Major Fields: 1. History  Clinical data known to the pathologist  Name, age, sex, symptoms, surgical findings, type of surgery  Optional; includes the demographics of the patient  Contains the essential clinical data known to the pathologist at the time he dictates a description of the gross specimen(s).  Gives the reader of the report, whether a clinician or another pathologist, an immediate orientation to the nature of the problem that led to that particular operation. 2. Gross  Gross description  Precise and thorough, (with picture is better)  Fresh or fixed specimen, previously opened or intact, any landmark (ex. Suture)  Size, color and location of all lesions should be recorded.  Use metric system – cm used not inches  Exact measurement is better than comparing with objects (e.g. size of an egg – which egg?) – give 3D measurements  It is advisable to give specific dimensions and descriptions rather than to provide comparisons with common objects such as fruits or other vegetables.  Weight is important in many tissues, like thyroid, prostate – normal weight of the prostate = 20g  It is important to be accurate, factual, and noncommittal in the gross description, avoiding subjective interpretations as much as possible.  Use standard color, like red, brown, amber, green rather than blood-stained, pus or milky. – using blood-stained, pus or milky somehow

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imparts the diagnosis rather than describe the specimen  Concluded by stating whether the entire specimen was submitted for tissue processing or sampling was done. 3. Microscopic Description  Optional  Short and straight to the point  Histologic samples are kept by the lab  Usually not written or described 4. Diagnosis  Organ, specific site, operation – morphologic Diagnosis  E.g. Bone, femur, biopsy – Osteosarcoma. 5. Note or Remarks  Differential diagnosis, reasons for interpretation, prognostication and therapeutic consideration  Helps the clinician make his/her diagnosis and plan of care  One of the most important means of clinicopathologic correlation REMEMBER! …no technologic advancement can replace the time-honored practice of two medical specialist discussing how best to treat a patient. SLIDE REVIEW  Slide can be evaluated by several pathologists or by the same pathologist at a different time.  Slides and paraffin blocks are stored indefinitely.  Usual practice is to review outside slides of patient who is referred to his institution prior to therapy  Consultation with other pathologists in unusual, difficult and controversial cases is now a popular practice  Slides sent to different professionals for second opinions. LIMITATIONS OF HISTOLOGIC DIAGNOSIS  Pathologists are physicians and human beings.  They have as great a capacity for error and susceptibility to subjective distractions as other practitioners of the art of medicine.

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 Incomplete communication between clinician and pathologist may make diagnosis difficult or impossible.  If diagnosis is not compatible with clinical diagnosis, send slide to other pathologists for second opinion  Sampling of tissue – need “enough” samples which vary depending on the pathologist  Best biopsy is a cleanly excised, uncrushed wedge that includes a junction between normal and neoplastic tissue.  Adequacy of sample - Adequate volume of tissue permits a choice of fixatives, histochemical studies, bioassay or tissue culture.  Fixation of sample – fix sample at once  Upon removal of the breast tissue, it takes 20 minutes before receptors autolyse secondary to anoxia. If the sample was fixed after > 20 minutes, the test would yield a false negative result. BIOPSIES  Incisional Biopsies  Only a portion of the lesion is sampled.  Strictly of diagnostic nature.  Involves the removal of portion/s of a big mass  Excisional Biopsies  Entire lesion is removed usually with a rim of normal tissue.  Both diagnostic and therapeutic in some cases.  Factors to consider:  Size of the lesion  Depth of lesion  Wide Excision – for suspected malignant lesion Fine Needle Aspiration Biopsy – current trend in tissue sampling. - Accurate results of cytology exam - Usually done with lung and liver biopsy General Rules for Biopsy Procedure:  Larger lesions should be sampled more – variability in pattern may exist and the diagnostic areas may be present only focally.

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 In ulcerated lesion, take from the periphery that includes both normal and diseased tissues biopsy of the central ulcerated area may show only necrosis and inflammation  Biopsy should be deep enough to show relationship between the tumor and stroma Epithelia involved by carcinoma have a tendency to detach from the underlying stroma. This should be avoided whenever possible by careful handling of the tissue.  Deep seated lesion can show peripheral tissue reaction, e.g. inflammation, fibrosis, calcification – If the biopsy is too peripheral, this may only reflect peripheral tissue reaction. Similarly, in a mass of lymph nodes, a deep-seated node may show involvement by a malignant tumor, whereas a superficial node may show only nonspecific hyperplasia.  When several fragments of tissues are obtained, submit everything - Sometimes the smaller or grossly less impressive fragment is the only one that contains the diagnostic elements.

Get at the junction between normal tissue and the ulcer. Obtaining sample in the middle will only yield necrotic tissue.  Crushing or squeezing with forceps should be avoided. – increases artifacts making interpretation difficult  Always place adequate amount of fixative  10% buffered neutral formalin

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Volume of fixative is 10x that of tissue (1:10 ratio)  Consider the need for special studies, e.g. touch preparations, electron microscopy, cytogenetics, molecular pathology, flow cytometry, etc. INTRAOPERATIVE CONSULTATION (FROZEN SECTION)  Required experience, knowledge of clinical medicine and pathology, capacity to make quick decisions under pressure, good judgment and attitude that is conservative but no excessively so.  Cryostat or Freezing microtome.  A simple question:  “Will the result of the frozen section examination influence in any way the surgical procedure?”  If the answer is no, the procedure is not indicated.  E.g. A patient with Hirschsprung’s Disease undergo Frozen Section intraop to determine the area where there are already ganglion cells present before performing a pull-through surgery  Three legitimate purpose:  To establish the presence and nature of a lesion.  To determine adequacy of surgical margins  To establish whether the tissue obtain contains diagnosable material or whether additional sampling is indicated  Diagnosis usually given within 20 minutes – especially with breast specimens  At the time of a frozen section, the diagnosis given verbally to the surgeon should be transcribed verbatim in an appropriate form and a copy of such form incorporated immediately into the chart.  Another copy should remain in the laboratory and be filed with the frozen section slides.  Overall accuracy is pretty good:  CAP sponsored review show a concordance of 98.58%  Causes of discrepancies between frozen sections and paraffin sections:  Misinterpretation of the original frozen section

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Absence of diagnostic tissue in the material frozen but present in tissue not sampled.  Absence of diagnostic tissue in the material frozen but present in permanent section A correlation exists between the accuracy of the procedure and both the tissue type and the nature of the pathologic process. The real aim of the frozen section procedure is to influence the course of the operation. The pathologist should be thoroughly briefed on the patient's clinical history: ideally the surgeon and the pathologist should have discussed the case beforehand. The pathologist should be prepared to advise the surgeon as to the best area to biopsy. Freezing the tissue in isopentane (methylbutane) cooled with liquid nitrogen or with an electronic device saves valuable time and results in fewer artifacts than when the tissue is frozen on the cryostat stage. Parts:  Gross examination – determine if benign or malignant from gross features  Touch imprint – touch the slide on the surface of the tumor to get samples  Samples are examined cytologically  Microscopic examination

 FNAB – breast, thyroid, salivary glands and lung.  It is generally carried out with a „fine‟ needle (OD 0.6–0.9 mm), sometimes under image guidance.  Inexpensive, safe, quick, and – when performed by experienced workers – quite accurate.  It can also induce artifacts of various types in the tissues, which the pathologist should be cognizant of in order to avoid misinterpretation.  Many of the special stains that are routinely used for tissue sections can also be very useful for the evaluation of cytologic material. This includes stains for glycogen, melanin, fat, and mucin.  Cytologic material is also well suited for examination with immunocytochemical, ultrastructural, flow cytometric, cytogenetic, and molecular genetic techniques.  Use 95% alcohol as fixative

DIAGNOSTIC CYTOLOGY  In the hands of a well-trained, experienced individuals offers an extremely high degree of reliability  A positive cytologic diagnosis of malignancy made under these circumstances should be given the same weight as one obtained from a surgical biopsy.  Terminology used should be the same as those used for biopsies e.g. HSIL rather than Moderate Dysplastic cells.  In most organs, the cytology diagnosis should be followed by conventional biopsy before treatment is carried out e.g. cervical cancer.  Pap smear showing severe dysplasia is followed by cervical biopsy to identify layer involvement

LEGAL ASPECT OF SURGICAL PATHOLOGY  Most common reasons for surgical pathologists being brought to trial:  A mistaken diagnosis based on misinterpretation of a slide.  An important lesion or feature present in the specimen was missed  The pathologic diagnosis failed to give the clinician a clear idea about the nature or extent of the lesion or adequacy of the sample because of poor wording or omissions in the report  E.g. Invasion of lymphatics to determine metastasis was not reported  Two essential components of these claims are:

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DIGITAL PATHOLOGY & TELEPATHOLOGY  Images can be transmitted electronically to any part of the globe.  This can be done at various levels, from the email attachment of a few static photographs to sophisticated systems that duplicate almost to perfection the examination of slides under the microscope and are, therefore, accurately referred to as virtual microscopy.

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That the alleged error or omission resulted in physical, emotional, and/or financial damage to the patient That such an error or omission was below the standards for the practice of pathology in that particular community at that particular time.

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 BASIC ELEMENTS OF MALPRACTICE:  DUTY: recognition of an obligation of a physician to treat the patient. (Physician’s responsibility)  BREACH: Neglect to treat within the standard of care. (Responsibility was not done)  PROXIMAL CAUSE: Breach causes injury in a fairly direct manner. (neglect directly affects the patient)  DAMAGE: Injury resulted (negative effect) GROSS TECHNIQUES IN SURGICAL PATHOLOGY  Description of the gross specimen to include:  Measurement  Weight  Color  Consistency  Surface texture  Cut surface texture Remember! Once a lesion is seen intraoperatively, the surgeon must be able to describe the lesion according to its: 5 MedTechs Before Christ - Site – location (e.g. upper outer quadrant of the breast) - Size – in cm - Shape – standard shapes (e.g. oval, irregular) - Surface – smooth, nodular, granular, borders - Symmetry - Mobility - Tenderness - Ballotment – how it feels between the thumb and the index finger - Consistency FIXATION  10% buffered formalin  Relatively inexpensive  Tissue can remain in it for prolonged periods without deterioration

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Compatible with most stains including histochemical stains  As long as the tissue is placed in fixative shortly (24–48 hours) is avoided. “Pure” formalin is 40% solution of gas formaldehyde  Thus a 10% formalin solution represents a 4% solution of the gas, which is 1.3 molar.

Other Fixatives:  ZENKER‟S o Expensive and contains Mercuric chloride o Often used for biopsies of the kidney, bone marrow, lymph node and testes. o Expensive, requires careful disposal of the mercury, and necessitates meticulous attention to fixation times and washing procedures to remove the precipitates of mercury.  BOUIN‟S o Contains picrid acid o Recommended for testicular biopsies o Preservation of nucleic acids is very poor.  CARNOY‟S o Mixture of ethanol, chloroform and glacial acetic acid o Dissolves fats o For identification of lymph nodes in radical resection specimens Considerations:  Volume of fixative is at least 10X that of the tissue.  Container should have a wide mouth - tissue can be removed easily after it has been hardened by the fixation.  Fixative should surround the specimen on all sides.  Large specimens that float on a fixative should be covered by a thick layer of gauze.  In cases of large, flat, heavy specimens that rest on the bottom of the containers, the gauze should be placed between the container bottom and the specimen.  Fixation is usually carried out room temp.  For large specimen, 4°C

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 Speed of penetration of formalin is 1 mm/hr. – fixation time of several hours is needed for most specimens o  Fixation can be hastened by heat (60 C) and by continuous agitation by heater-motor GROSS EXAMINATION  Specimen photography – transparencies or digital images

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 Tissue contamination (“floater”) – some cells retained at the cutting board from the first specimen  Suspect a „floater‟ whenever confronted with any of these situations: 1. A fragment of tissue that looks different from all the others by virtue of the thickness of the section and/or staining intensity 2. A fragment of tissue that is on a slightly different plane from the others, especially if superimposed on them 3. A fragment of tissue showing pathologic changes totally different from the others, and of a type that one would not have expected at all under the clinical circumstances of the case. st o e.g. malignant cells coming from the 1 specimen transferred to the following specimen giving it a false positive result

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It is advantageous to fix the specimen in Carnoy solution, which somewhat clears the specimen by the action of the chloroform at the same time that it fixes it.

 Sampling for histologic evaluation  Must not be more than 3 mm thick  Adipose tissue must be cut even thinner.  Not larger than dimensions of tissue cassette or glass slide  Suture material, metal clips, and other foreign bodies should be removed from the tissues  Discrete areas of calcification or ossification should be dissected out, or else the specimen should be decalcified.  Better preparations will be obtained in organs covered by folded mucosa (e.g., stomach, bowel) if the sections are taken perpendicular rather than parallel to the mucosal folds.  Adequate sampling with proper labeling (lead pencil in folder paper material)  Surgical Margins  A positive margin will likely lead to local recurrence if uncorrected.  Carried out by „painting‟ those margins with India ink or a similar pigment before sectioning.  The smoother the specimen contours and the harder the consistency, the easier to identify the true surgical margins.

 Specimen radiography – for bone lesions, calcified soft tissue masses, breast biopsies and excisions (especially if they had been studied by mammography), cardiac valves, and lymph node groups in which a lymphangiogram had been performed.  Lymph node dissection - one of the most important components in the gross evaluation of a radical operation for cancer.  Consists of dissecting the node-containing fat from the organ in the fresh state, using forceps and sharp scissors.  It is too easy to crush them with the forceps and scissors, especially if they are dissected before fixation.

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SPECIAL TECHNIQUES IN SURGICAL PATHOLOGY The gold standard of Surgical Pathology is still with the use of the H & E stain on a tissue cut 5 micrometer by a microtome. H & E Technique - hematoxylin staining of nuclei is followed by counterstaining of cytoplasms and various extracellular materials by eosin. - In order to function as a nuclear stain, hematoxylin needs to be oxidized („ripened‟) to the purple dye hematein and provided with a net positive charge by combining it („chelating‟) with a metallic salt („mordant‟) - Eosin is an anionic xanthene dye that combines electrostatically with various cytoplasmic components and with tissue such as collagen or muscle, the latter in an amphoteric manner. - It is relatively quick, inexpensive, suitable for most situations, and comparatively easy to master. - It allows an accurate microscopic diagnosis of the large majority of specimens sent to the laboratory. Special Techniques – for difficult slides  Special Stains  Enzyme Histochemistry  Tissue Culture  Quantitative Methods (Histometry)  X-Ray Microanalysis  Electron Microscopy  Immunohistochemistry  Flow Cytometry  Cytogenetics  Molecular Pathology SPECIAL STAINS  PAS (Periodic Acid Schiff) Stain  Demonstrate glycogen and neutral mucosubstances, outlines basement membranes and makes evident most fungi and parasites.  Also useful for the demonstration of the intracytoplasmic crystals in alveolar soft part sarcoma.  Stains for Microorganisms  Gram Stain - allows the separation of bacteria into those that retain the crystal

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violet–iodine complex (gram positive) and those that are decolorized by alcohol or acetone treatment and counterstained by either safranin or fuchsin.  Acid Fast - depends on the high lipid content (mycolic acids and long-chain fatty acids) in the cell walls of mycobacteria, which confer to the cell the ability to complex basic dyes (such as carbolfuchsin) and to retain them following strong decoloration with acid– alcohol. Argentaffin and Argyrophilic stains  Neuroendocrine cells and their tumors, also for reticulin, melanin and calcium  Argentaffin reaction depends on the presence in the tissue of a substance, often of the phenolic group (such as catecholamines or indolamines), that reduces silver (and other metallic) salts  In the argyrophilic reaction, an extraneous reducing agent such as hydroquinone or formalin is added Amyloid stains  Congo red  Regarded as the most reliable and practical technique to detect amyloid. Reticulin Stains  Reticular fibers and basement membranes  Reticular fibers consist of very thin fibers of mainly type III collagen, which are widespread in connective tissue throughout the body.  Basement membranes are largely composed of type IV collagen and laminin.  Main applications of silver-based reticulin stains: (1) Epithelial from nonepithelial neoplasms; (2) Various mesenchymal neoplasms from each other; and (3) In situ from invasive carcinoma. Trichrome Stains  Evaluation of the type and amount of extracellular material  Determine parasitism  The three tissue structures demonstrated by the three component dyes are nuclei, cytoplasm, and extracellular collagen

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 PTAH (Phosphotungstic acid-hematoxylin) stain  To demonstrate intracytoplasmic filaments, e.g. muscle and glial cells  Stains for hemosiderin (Perls), melanin (FontanaMasson) and calcium (von Kossa)  Perls - hydrochloric acid splits off the protein bound to the iron, allowing the potassium ferrocyanide to combine specifically with the ferric iron to form ferric ferrocyanide (Prussian blue).  Fontana-Masson - ammoniacal silver solution is used without a reducing bath. Only substances capable of reducing directly silver salts (i.e., argentaffin) such as melanin are demonstrated.  von Kossa – silver is substituted for calcium in calcium salts; this silver salt is then reduced to black metallic silver by the use of light or a photographic developer.  Stains for neutral lipids  Oil red O is the one most commonly employed.  Inconsequential distinction between fibroma and thecoma in the ovary, support for the diagnosis of renal cell carcinoma and sebaceous gland tumors of skin, and identification of lipid-rich carcinoma in various organs.  Mucin stains  Used to classify gastric incomplete metaplasia into subtypes (sialomucin- and sulfomucin-containing) having supposedly different malignant potentials  Hale colloidal iron stain has become the standard for the identification of renal chromophobe carcinoma  Giemsa stains  For the demonstration of various hematolymphoid elements (including mast cells) and microorganisms.  Elastic fibers  Weigert-type techniques are reasonably specific for elastin and are regarded by many as the method of choice for the demonstration of these extracellular fibers.  The Verhoeff–van Gieson (VVG) stain is more popular because it is quick and outlines the elastic fibers with a strong black color.

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 Myelin stains  Luxol fast blue for the demonstration of myelin.  Formaldehyde-induced Fluorescence - sensitive for the demonstration of catecholamines and indolamines ENZYME HISTOCHEMISTRY  Fell in general disuse because of:  Complex technique  Need for fresh material  Relative non specific  At present time, enzyme histochemical methods use are:  Skeletal muscle-related enzymes (for myopathies)  Acetylcholinesterase (for Hirschprung‟s disease)  Choloroacetate esterase (for myeloid leukemia) ELECTRON MICROSCOPY  Main applications  Renal pathology  Tumor pathology  Limitations  Sampling - only a small proportion of the neoplasm can be studied  Paucity of truly specific ultrastructural features - the number of organelles or other structures that are exclusive of a cell or tissue type is very small  Possible misinterpretation of entrapped nonneoplastic elements as belonging to the tumor – due to the difficulties in evaluating spatial relationships in a small tissue sample.  Diagnostic potential in:  Identification of a tumor as neuroendocrine through the detection of dense-core granules  Assessment of the nature of tumor cells with granular cytoplasm (oncocyte, granular cells etc)  ID of epithelial differentiation  ID of tumors as melanocytic through detection of melanosomes  ID of Langerhan‟s histiocytosis through the detection of Birbeck‟s granules

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ID of tumor as composed of steroidproducing cells from adrenal cortex and gonads through the detection of abundant smooth ER & mitochondria with tubulovesicular cristae  ID of endothelial cells by detection of WeibelPalade bodies  ID of skeletal and smooth muscles by detection of cytoplasmic filaments  ID of Schwann cells by mesoaxons  ID of alveolar soft tissue sarcoma by detection of membrane bound crystals  ID of smooth muscles, neural or other types of differentiation in tumors of GIST family.  Main situations where EM is used:  Differential diagnosis of carcinoma, melanoma and sarcoma.  Differential diagnosis between adenocarcinoma and mesothelioma  Differential diagnosis of anterior mediastinal tumors among thymoma, thymic carcinoid, malignant lymphoma and seminoma.  Differential diagnosis of small round cell tumors of infancy.  Differential diagnosis of spindle cell tumors of soft tissues.  Differential diagnosis between endocrine and non-endocrine tumors. IMMUNOHISTOCHEMISTRY  Application of immunologic principles and techniques to demonstrate molecules in cells and tissues.  Remarkable sensitivity and specificity  Applicability to routine processed material  Feasibility of an accurate correlation with the traditional morphologic parameters  Determine specific or identifying features of the tumors Actin - marker for the identification of smooth muscle cells and myofibroblasts, and for the evaluation of the participation of myoepithelial cells in lesions of breast, salivary glands, and sweat glands. Epithelial Membrane Antigen (EMA) – epithelial cells - Excellent marker for most normal and neoplastic epithelia but is not restricted to them

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Also expressed by mesotheliomas, meningiomas, a variety of mesenchymal neoplasms, and even some malignant lymphomas. - Also been found to be a marker of normal and neoplastic perineurial cells. Glial Fibrillary Acidic protein – nerve cell - Present in normal, reactive, and neoplastic astrocytes; developing, reactive, and neoplastic ependymal cells; and developing and neoplastic oligodendrocytes - Also been documented in peripheral nerve sheath tumors and in mixed tumors of salivary glands and sweat glands. HCG – gestational Trophoblastic Disease HER2/neu (ERBB2) – determines behavior/aggressiveness of breast cancer - When overexpressed, it acts as an oncogene. This overexpression can be seen in breast carcinoma (22%), lung adenocarcinoma (28%), colorectal carcinoma (17%), lung squamous cell carcinoma (11%), and gastric adenocarcinoma (11%). - Predict response to Herceptin (trastuzumab) in carcinoma of breast and other organs HMB-45 – melanin for melanoma - Now known to be expressed by other neural crest-derived tumors, angiomyolipomas of the kidney and other sites, other components of the tuberous sclerosis complex („PEComas‟), and occasional carcinomas as well as several other neoplasms. Keratins – squamous cells - Excellent marker for epithelial differentiation regardless of whether the tumor is of endodermal, neuroectodermal, mesenchymal, or germ cell derivation - Particularly common and prominent in synovial sarcoma, epithelioid sarcoma, and uterine smooth muscle tumors Neuron Specific Enolase - found in the majority of neuroectodermal and neuroendocrine neoplasms, including carcinoid tumors and malignant melanoma. PSA – prostate cancer - Method of choice for the identification of prostatic adenocarcinoma. S-100 – malignant melanoma

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Evaluation of peripheral nerve sheath and melanocytic tumors.

FLOW CYTOMETRY  Measurement of various parameters while a suspension of cells flows through a beam of light past stationary detectors.  Light scattered at various angles by the cells is registered by detectors and converted to electronic signals and analyzed by computers  Cellular features evaluated include cell size, cytoplasmic granularity, cell viability, cell cycle time, DNA content, surface marker phenotype and enzyme content.  Usually used in hematopathology

translocations – has been particularly successful in the fields of leukemias and lymphomas, germ cell tumors, pediatric tumors, and mesenchymal neoplasms  Contributions of CytoGen to Pathology: (1) Defining subsets within putatively histologically homogeneous tumor types (2) Suggesting connections between histologically diverse tumors (3) Highlighting specific changes in histological subtypes (4) Suggesting the site of the primary when a specific cytogenetic change is found in a metastasis (5) Providing clues to tumor classification, causation, and presence of cancerrelated genes.  MOLECULAR PATHOLOGY  Based on the application of recombinant DNA technology.  Can be performed in tissues handled as part of routine work, including formalin-fixed, paraffin-embedded material.  Snap frozen material is superior in regard to the yield and quality of extracted DNA and particularly RNA

Stained cells enter the flow chamber where they pass into the center of a stream of sheath fluid in single file. They are then struck by a focused laser beam and emit scattered and fluorescent light, which is separated according to wavelength by appropriate mirrors and filters. An obscuration bar protects the forward angle light scatter (FALS) detector from exposure to the direct laser beam. Only two fluorescence detectors are shown, for sake of simplicity, but a typical instrument has three or four such detectors, one of which can be used to measure laser light scattered perpendicular to the laser beam by the cells. Signals from the detectors pass to amplifying processors and then to the integral (on-board) computer, which digitizes the signals, stores them, and displays them. NEWER TRENDS  CYTOGENETICS  Karyotypic analysis of human tumors  Detection of nonrandom or specific chromosome abnormalities – such as deletions, amplifications, inversions, and

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