Specimen Accessioning And Processing - Histology And Cytology - PDF

SPECIMEN ACCESSIONING AND PROCESSING Wong Chun Keung 28 Feb 2024 1 SPECIMEN RECEPTION Function of anatomical pathology: Diagnosis of disease based on the following examinations of organs and tissues: Macroscopic examination Microscopic examination Immunologic examination Chemical examination Molecul...

SPECIMEN ACCESSIONING AND PROCESSING Wong Chun Keung 28 Feb 2024 1 SPECIMEN RECEPTION Function of anatomical pathology: Diagnosis of disease based on the following examinations of organs and tissues: Macroscopic examination Microscopic examination Immunologic examination Chemical examination Molecular examination https://commons.wikimedia.org/wiki/File:Lab_worker_with_blood_samples.jpg 2 BASIC PROCEDURES OF SPECIMEN RECEPTION AND ACCESSIONING IN HISTOPATHOLOGY AND CYTOLOGY The Medical Laboratory Technologist (MLT) should be proficient in: Handling urgent requests with first priority Ensuring specimens and laboratory request forms fulfilling the acceptance requirements Segregating specimens into histopathology and cytology requests Segregating surgical specimens into all-embedded cases (handled by MLT) and cut-up cases (handled by doctors) 3 LABORATORY INFORMATION SYSTEM (LIS) REGISTRATION The Medical Laboratory Technologist should be proficient in registering and accessioning all specimens received in the LIS correctly The functions of LIS: Recording of laboratory results Tracing of laboratory results Reporting of laboratory results Statistics and data processing https://en.wikiversity.org/wiki/Information_Systems/Internet 4 USE LABORATORY REQUEST FORM FOR LIS REGISTRATION AND SPECIMEN ACCESSIONING The Medical Laboratory Technologist should check that the laboratory request form should provide the following information: (For LIS registration) Patient demographic data Requesting ward & unit Requesting hospital/clinic Specimen nature & site Specimen collection date & time Date of specimen received Examination requested Doctor’s name, code & signature Clinical History Clinical Diagnosis https://www.slideshare.net/sayid881/how-to-submit-biopsy-specimens-forhistopathology 5 USE LABORATORY REQUEST FORM FOR LIS REGISTRATION AND SPECIMEN ACCESSIONING    Specimen should be labeled properly with patient information Make sure the information shown on laboratory request form match with those on specimen label Attach one of a pair of labels with identical laboratory number to the specimen and the request form respectively https://www.slideshare.net/mieime/specimen-collection-dr-manarnursing Input the Laboratory Number and Patient Demographics on the Laboratory Request Form to the LIS to Complete the Specimen Registration 6 THE MEDIC AL LABORATORY TECHNOLOGISTS SHOULD UNDERSTAND The following contact points for tracing missing information on laboratory form or specimen: Check the doctor code from the laboratory form Check the unit / ward from the laboratory form Check the operating theater (OT) list Contact the information counter of the hospital 7 ALL TECHNIC AL STAFFS SHOULD BE PROFICIENT IN THE BASIC STEPS OF OPERATING THE LIS SYSTEM To log in the system To register a request for laboratory examination To amend a request (for changing data) To print the day book (Summary of registration) To log off the system https://www.limsforum.com/astrix-on-demand-webinar-planning-and-executing-alabvantage-lims-validation/91205/ 8 USE GENERIC CLINIC AL REQUEST SYSTEM (GCRS) LABEL ATTACHED TO THE SPECIMEN FOR LIS REGISTRATION (NO LAB. FORM) Read by barcode reader for specimen registration This is an example of test request for Chemical Pathology examination https://manual.cpy.cuhk.edu.hk/wardmanual/Infor/L_Mex/IGF1.pdf 9 USE GENERIC CLINIC AL REQUEST SYSTEM (GCRS) LABEL ATTACHED TO THE SPECIMEN FOR SPECIMEN ACCESSIONING Specimen received labeled with a unique specimen number with a barcode Read the barcode with barcode reader for specimen registration Stick another barcode label with laboratory number to the specimen container, read this barcode with the barcode reader to complete the registration https://www.flickr.com/photos/6 1202488@N00/2551240388 A worksheet is generated automatically by the computer system Check and make sure the information of worksheet generated match with those printed on the specimen label https://pngimg.com/image/7733 10 GCRS – ANATOMICAL & CELLULAR PATHOLOGY (ACP) The GCRS Label usually divides ACP specimens into 5 types of test codes in Hospital Authority: Cytology Examination AN Cytology (non-gynaecological) AG Cytology (gynaecological) AF Cytology (Fine Needle Aspiration) Histology Examination A Histopathology AS Frozen Sectioning https://en.wikipedia.org/wiki/Stool_test 11 GCRS – ANATOMICAL & CELLULAR PATHOLOGY (ACP) If there is a “U” present in the label just before the test code: It implies that the GCRS request for the patient is urgent Example UA: Urgent Histopathology 12 PROCEDURES OF SPECIMEN GROSSING BY PATHOLOGIST Printing of specimen cassettes Matching of cassette and specimen number Selection of tissue blocks by pathologist Specimen Cassette Placing of tissue blocks into the cassettes Wrapping of tiny tissues with wrapping paper Closing of cassettes Making remarks to cassettes if necessary https://www.flickr.com/photos/40 412379@N02/35853278236 https://www.fishersci.es/shop/products/cassette-iislotted-tissue-cassettes-tube-packs-12/16344056 13 EXAMPLES OF SURGICAL SPECIMENS TO BE GROSSED BY PATHOLOGIST Skin Lung Liver Stomach Colon Ovary Pancreas Bone Tongue Breast Esophagus Thyroid 14 PREREQUISITES FOR HANDLING OF ALL EMBEDDED SPECIMENS BY MEDIC AL LABORATORY TECHNOLOGISTS For small in sample size or volume Specimens not require grossing or blocktaking by pathologists before processing Suitable for very few blocks to be taken Orientation for specimen sectioning is not critical Grossing or block-taking by trained technical staffs (AMT, MT, etc) https://en.wikipedia.org/wiki/Stool_test 15 EXAMPLES OF ALL-EMBEDDED SPECIMENS Nasal biopsy Fallopian tubes Esophagus biopsy Gastric biopsy Cervical biopsy Bladder biopsy Pleural biopsy Uterine curettage, etc https://link.springer.com/article/10.1186/1475-925X-9-48 16 SMALL BIOPSY WRAPPING USING WRAPPING PAPER https://www.cancerdiagnostics.com/BB0023-Bio-Paper-Biopsy-Wraps-Blue-2-x-3-BX-500 17 SMALL BIOPSY WRAPPING PROCEDURES USING WRAPPING PAPER Perform the procedures in the fume hoods Make sure the specimens are adequately fixed by buffered formalin Match patient label on specimen container with request form: For same specimen number and patient particulars before wrapping Match also the specimen number and patient initials printed on the cassettes with those of specimen containers before wrapping Use both manual matching and computer matching if available Only one container should be handled at one time Use clean wrapping paper labelled with specimen number and separate clean forceps for each specimen container 18 PROCEDURES OF USING THE FUME HOOD Ensure the date on the maintenance sticker of the hood is within an allowable period Make sure a proper function of the alarms and monitors for flow rate of the fume hood Observe air movement for proper operation Set the sash height as indicated by the arrow or label If the alarm sounds or air flows indicate low flow, turn off the fume hood and lower the sash immediately Stick a warning label stopping others using the fume hood Notify the maintenance team for follow up checking https://2020.igem.org/Team:HK_CPU-WFNWYY/Safety 19 WORKFLOW FROM TISSUE SPECIMEN TO PRODUCE A PARAFFIN TISSUE BLOCK 20 https://mmegias.webs.uvigo.es/02-english/6-tecnicas/3-parafina.php PROCEDURES OF TISSUE PROCESSING BY AUTOMATIC TISSUE PROCESSOR Using automatic tissue processor for tissue processing Ensuring adequate fixation of tissue block in buffered formalin Placing cassettes of tissue blocks into cassette baskets Selecting an appropriate processing program Selecting a suitable level / volume of reagent immersion according to the number of cassette baskets present (if required) Starting the automatic tissue processor Recording the number of tissue cassettes to be processed Replacing the reagents and performing maintenance procedures after the processing cycle (if required) https://epredia.com/solutions/tissueprocessing-embedding-solutions/revos/ 21 EMBEDDING CENTER Components of embedding center include: Cold plate Heated workspace Paraffin wax reservoir Paraffin dispense paddle Touchscreen controls Built-in lighting, etc https://www.de-di.gr/en/products/instruments/embedding-instruments/ 22 TEMPERATURE OF MELTED WAX IN EMBEDDING CENTER Kept 2-4 degrees Celsius above melting point of paraffin wax Common melting point of paraffin wax is around 58°C Higher temperatures may results in Separation of the paraffin components Difficulty in ribboning of microtomy Temperature of embedding center should be measured by the machine digitally and by calibrated thermometer 23 PROCEDURES OF HANDLING MERCURY THERMOMETER BREAKS Evacuate the spillage area Ventilate the place Wear latex or neoprene glove Try to contain the spill Do not vacuum mercury to prevent the harmful vaporization Prevent the mercury from spreading such as to cracks Use scotch tape or other tapes to pick up beads of mercury Use flashlight to detect remaining beads of mercury if required https://www.hazchem.com/mercury-thermometers-what-do-do-when-they-break/ 24 TISSUE EMBEDDING – PARAFFIN TISSUE BLOCK WITH BASE MOLD https://www.flickr.com/photos/euthman/275484520 / 25 TISSUE EMBEDDED IN PARAFFIN WAX https://www.istockphoto.com/hk/%E7%85%A7%E7%89%87/arranged-histology-metal-mold-with-tissue-block-gm500479719-43369904 26 TISSUE EMBEDDING – TISSUE PARAFFIN BLOCK SEPARATED FROM BASE MOLD https://flickr.com/photos/euthman/275487829 27 PARAFFIN MICROTOMY BY ROTARY MICROTOME https://www.pinterest.com/pin/the-technique-of-getting-fixed-tissue-into-paraffin-is-calledtissue-processing-tissue-processing-is-designed-to-remove-all-e--723883340085398491/ 28 PARAFFIN TISSUE SECTIONS FLOATING ONTO WATER BATH  Optimum temperature for water bath is 40°C–50°C, which is 5°C –10°C below that of paraffin’s melting point  Shift the ribbon of paraffin sections toward the water bath  Gently lay the paraffin sections out on the bath in a "dragging" motion  Allow the paraffin sections to sit on the water bath for a few moments  Heated water help to expand any compression and remove the folding and wrinkling in the section for approximately 20-45 seconds https://www.protocols.io/view/hubmap-tissue-sectioning-for-ffpe-specimens-bahdib26.html 29 PROCEDURES OF PARAFFIN SECTION(S) PICK UP FROM WATER BATH Use the forceps to separate and select the section(s) required Use the correct pre-labeled microscope slide that matches the paraffin block under sectioning https://www.youtube.com/watch?v=HTVrs47Z4FQ Dip the slide into water bath, and place it adjacent to and slightly under the chosen section(s) Adjust the section(s) to the center of clear glass area of the slide with forceps, and lift upwards out of the water bath Put the slide into the slide staining rack for air dry overnight or dry by a 50°C oven or hot plate for 1 hour https://www.protocols.io/view/hubmap-tissue-sectioning-for-ffpe-specimens-bahdib26.html https://skrooll.com.ph/hi1210-floatation-bath-leica/ 30 MAJOR STEPS IN HAEMATOXYLIN AND EOSIN (H&E) STAIN Remove the paraffin wax of unstained tissue section with xylene Hydrate the section with ethanol solutions of decreasing concentrations and then to water Stain the nuclei with hematoxylin stain Complete the nuclear stain with bluing solution Remove the excess background stain in the step of differentiation Perform the counterstain with eosin Bring the section from water through low grade ethanol; high grade ethanol; to xylene, before mounting with the mounting medium 31 MOUNTING OF STAINED TISSUE SECTION WITH MOUNTING MEDIUM The mounting medium is used to adhere the coverslip to the slide Apply a small amount of mounting media onto the tissue section Carefully place one end of a coverslip onto the slide Slowly lower the coverslip using a dissecting needle or forceps Allow the slide to air dry at room temperature in a horizontal position Make sure that there are no air bubbles, dirts or artefacts during mounting https://www.nationaldiagnostics.com/2011/09/30/mounting-tissue-sections/ Make sure that the coverslip is located at the center of clear glass area of the slide 32 PURPOSE OF THE H&E STAIN Haematoxylin stains cell nuclei blue Eosin stains in varying shades of pink for: The Cytoplasm The extracellular matrix, Other structures Facilitates the microscopic examination of Cells, structure and morphological changes https://www.leicabiosystems.com/en-gb/knowledgepathway/he-staining-overview-a-guide-to-best-practices/ 33 STAINED TISSUE SECTIONS MOUNTED ON MICROSCOPIC GLASS SLIDES https://www.flickr.com/photos/euthman/275496597 34 MOLECULAR BIOLOGY STUDY Molecular and biochemical processes in cells Such as DNA replication, RNA transcription as well as protein translation Seek for additional diagnostic information Presence of certain infections (viral or bacterial) Presence of various diseases not detected or differentiated by traditional laboratory methods Better prediction of patients outcomes https://courses.lumenlearning.com/waymaker-psychology/chapter/outcomegene-environment-interaction/ 35 EXAMPLES OF SAMPLE PREPARATION FOR MOLECULAR BIOLOGY STUDY Use some solvents to dissolve the samples Use some chemicals to extract the analytes of interest from the matrix of the sample Separate the target analytes from the interfering substances Addition of chemicals and steps to enhance the detection of the target analytes Addition of some reagent to change the target analytes to some measurable derivatives https://www.flickr.com/photos/snre/6946913449 36 EXAMPLES OF CLINICAL SPECIMENS NEED STORAGE AND RETENTION Surgical tissues, all-embedded, fixed in formalin Surgical tissues, cut-up cases, fixed in formalin Fresh surgical tissue Fresh surgical tissue sent for frozen sectioning Post-mortem specimens Cytology specimens such as body fluids Fine needle aspirates Paraffin blocks Stained and unstained tissue in glass slides https://freebie.photography/home/slides/tallboy.htm 37 CONSIDERATIONS TAKEN FOR STORAGE AND RETENTION OF SPECIMENS Condition of storage – Suitable environment and prevent damage, deterioration and loss Proper labeling – Adequately labeled and easily accessible Minimal retention time – Minimum period of time to be elapsed for the items to be destroyed or disposed of Maximal retention time – Maximum period of time beyond the minimal retention time for further keeping of the items, for special reasons of better laboratory service 38 EXAMPLES OF DOCUMENTS NEED STORAGE AND RETENTION Laboratory request forms Worksheet records (Electronic and hard copies) Records of fax and telephone reports Laboratory reports (Electronic and hard copies) Internal audits reports and risk assessment records Equipment maintenance records Internal quality control records, External quality assurance records Standard operation procedures https://www.flickr.com/photos/kulturarvsprojektet/6498637005/ 39 Tissue Types and Structures 40 4 BASIC TYPES OF TISSUE Epithelial tissue Connective tissue Muscle tissue Nervous tissue https://www.flickr.com/photos/146824358@N03/4106 6513194 41 CHARACTERISTICS OF DIFFERENT TYPES OF TISSUE Epithelial tissue Provides the covering Linings of the various passages inside the body and the skin Connective tissue Supports different tissues as well as binds them together Examples are bone, lymph tissues and blood Muscle tissue Such as the smooth and striated muscle Nerve tissue Transmit "messages" from and to various parts of the body 42 I N TRODUCTION OF THE EP I THELIAL TI S S UES Line and cover the body surface, tubes and cavities Important in the activities such as physical protection, absorption, secretion, etc Surface epithelia – One or more layers of cells All epithelia – Support by a basement membrane Basement membrane – Separate the epithelia from the supporting tissue underneath https://humanbiology.pressbooks.tru.c a/chapter/7-3-tissues/ 43 TYPES OF EPITHELIA https://en.wikipedia.org/wiki/Epithelium 44 FUNCTION OF DIFFERENT EPITHELIA Simple squamous – Flat and sheet like appearance, one single layer Simple cuboidal – Cube like appearance, one single layer Simple columnar – Column like appearance, one single layer Stratified squamous – Flat and sheet like appearance, 2 or more layers Stratified cuboidal – Cube like appearance, 2 or more layers Pseudostratified columnar - Column like appearance, one single layer, with appearance of more than one layer Transitional – Multiple layers and varying cell shapes 45 https://philschatz.com/anatomy-book/contents/m46048.html Loose Connective Tissue – e.g. under epithelia of body Connective tissue proper Connective tissue Dense Connective Tissue – e.g. tendons Adipose Connective Tissue – e.g. in breasts Specialized connective tissues Reticular Connective Tissue – e.g. in lymph nodes Elastic Connective Tissue –e.g. walls of large arteries Embryonic Connective Tissue Mesenchymal Connective Tissue–e.g. around adult blood vessel Mucus Connective tissue – e.g. umbilical cord CLASSIFICATION OF CONNECTIVE TISSUE 46 GENERAL FUNCTIONS OF CONNECTIVE TISSUES Supporting and binding other tissues such as the epithelia, nerves and blood vessels. Defending and protecting the body against infection (by means of white blood cells). Providing metabolic support to cells as the medium for diffusion of nutrients and waste products. Healing of wound. Storing fat by the adipose tissue to act as reserve and insulation. https://www.ncbi.nlm.nih.gov/books/NBK5385 34/figure/article-36687.image.f2/ 47 TYPES OF MUSCLE TISSUE https://theory.labster.com/muscle_tissue/ 48 SKELETAL MUSCLE CHARACTERISTICS https://www.virtualhomeschoolgroup.org/vhsgfiles/03_Science/03_Biology/02_Anatomy_and_Physiology/M05_Mus cle_Histology_and_Movement/L1/Published/Study%20Notes/Study_Notes.html 49 STRUCTURE OF MUSCLE FIBER https://courses.lumenlearning.com/wm-biology2/chapter/types-of-muscle-tissue-and-fibers / 50 COMPONENTS OF NERVOUS SYSTEM https://courses.lumenlearning.com/suny-wmopen-biology2/chapter/components-of-the-nervoussystem/ 51 STRUCTURE OF THE NEURON 52 https://open.oregonstate.education/aandp/chapter/12-2-nervous-tissue/ SI G NAL S PASS T H ROUG H NE URONS https://organismalbio.biosci.gatech.edu/chemical-and-electrical-signals/neurons/ 53 TRAVEL OF MESSAGES IN NERVOUS SYSTEM https://chrisganatomy.weebly.com/nervous-system.html 54 NETWORK OF NEURONS IN THE NERVOUS SYSTEM https://www.sciencephoto.com/media/307893/view/nerve-cells 55 TYPES OF NEUROGLIA https://www.virtualhomeschoolgroup.org/vhsgfiles/03_Science/03_Biology/02_Anatomy_and_Physiology/M07_The_Ner vous_System/L1/Published/Study_Notes/Study_Notes.html 56 THYROID - LINED WITH SIMPLE CUBOIDAL EPITHELIUM https://ohiostate.pressbooks.pub/vethisto/chapter/thyroid-gland/ 57 GALL BLADDER - LINED WITH SIMPLE COLUMNAR EPITHELIUM https://mmegias.webs.uvigo.es/02-english/a-imagenes-grandes/epitelio_simple_prism.php 58 LARGE INTESTINE - LINED WITH SIMPLE COLUMNAR EPITHELIUM https://mmegias.webs.uvigo.es/02-english/2-organos-a/imagenes-grandes/digestivo-grueso.php 59 PARANASAL SINUSES - LINED WITH PSEUDOSTRATIFIED CILIATED COLUMNAR EPITHELIUM https://qut.pressbooks.pub/anatomyandphysiology/chapter/chapter-22-the-respiratory-system/ 60 URETHRA - LINED WITH STRATIFIED COLUMNAR EPITHELIUM TRANSITIONAL EPITHELIUM OF URINARY BLADDER https://www.noelways.com/courses/Anatomy%20and%20Physiology%20I/Lab s/Histology/Tissue_Types/Transitional_Epi/Tran_Epi_6b.html https://mmegias.webs.uvigo.es/02-english/a-imagenes-grandes/epitelio_estrat_prismatico.php 61 ECTOCERVIX - LINED WITH STRATIFIED SQUAMOUS EPITHELIUM https://flickr.com/photos/euthman/311128962 62 GYNECOLOGICAL CYTOLOGY Gynecological cytology is always called PAP test A cervical sample is observed under the microscope A screen for a lesion or cancer that can develop into cervical cancer https://pngimg.com/image/25887 63 DIFFERENT TYPES OF TERMINOLOGY IN HISTOLOGY AND CYTOLOGY Lesion – It is defined as a change in the structure of a organ or part of an organ in the body or due to disease or injury Neoplasia – Abnormal and uncontrolled growth of cells or tissues with the abnormal growth itself named neoplasm. It can be benign or malignant Dysplasia – Presence of cells of an abnormal type within a tissue, which may signify a stage preceding the development of cancer Tumours – Groups of abnormal cells forming growths, lumps or mass. They grow and behave differently, to be benign or cancerous 64 DIFFERENT TYPES OF TERMINOLOGY IN HISTOLOGY AND CYTOLOGY Benign – A condition, growth or tumor that is not cancerous. Not invading to nearby tissue. No spreading to other parts of the body. Malignant – Presence of cancerous cells. Able to invade nearby tissues. and spread to other sites of body Cancer – It is defined as a disease that is caused by the uncontrolled division of abnormal cells spread into surrounding tissues in a the body 65 CERVICAL INTRAEPITHELIAL NEOPLASIA (CIN) https://www.alamy.com/stock-photo/pap-test-pap-smear.html?sortBy=relevant 66 HISTOLOGY OF CIN1, CIN 2, CIN3 Proportion of cervical epithelium exhibiting dysplastic cells determines the grade of the dysplasia CIN-1 (low-grade) - Lower 1/3 or less of the epithelium CIN 2 (high grade) – Up to two thirds of the thickness of the epithelium covering the cervix has abnormal cells. CIN 3 (high grade) – The entire thickness of the epithelium covering the cervix has abnormal cells. 67 COMMON CYTOLOGY DESCRIPTIONS Nuclear to cytoplasmic (N/C) ratio Nuclear enlargement Nuclear hyperchromasia Coarse chromatin in nucleus Nuclear membrane irregularities Description of nucleoli 68 WHAT ARE THE SYMPTOMS OF CERVICAL INTRAEPITHELIAL NEOPLASIA? Usually no symptoms Need cervical diagnostic methods using screening and examinations to find out Docter use screening and examinations to diagnose whether the patients should be treated and/or monitored 69 COMMON DIAGNOSTIC METHODS OF CERVICAL INTRAEPITHELIAL NEOPLASIA (CIN)? HPV screening Cytologic examination (Papanicolaou smear) Cervical biopsy and histologic examination Colposcopy, etc The selection, sequence and combinations of diagnostic methods used depend on the most updated international guidelines and the advice provided by doctors 70 TUTORIAL Histopathology Laboratory Tour https://www.youtube.com/watch?v=hNdCZ_ka Dek Journey of a Specimen: Specimen Receiving https://www.youtube.com/watch?v=TJLj6SMVp8 https://eslbrazil.blogspot.com/p/videos-aprenda-ingles-com-videos.html 71 SPECIMEN ACCESSIONING AND PROCESSING TISSUE TYPES AND STRUCTURES 72