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How to submit biopsy specimens for histopathology

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How to submit biopsy specimens for histopathology

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How to submit biopsy specimens for histopathology

  1. 1. Dr. MD. SAIDUZZAMAN SAYID MBBS, BCS (Health) Lecturer Department of Pathology Dinajpur Medical College, Bangladesh
  2. 2. Overview  Introduction  Submission Form  Tissue Fixation  Packaging  Submitting multiple sites  Endoscopic biopsies  Denoting margins  Things to avoid  Other things to know  Contacting us
  3. 3. Introduction Cell Tissue Organ Structure Biochemical Function Anatomy Biochemistry Physiology Any change from normal Pathology Disease
  4. 4. Introduction Pathology is the study of the structural, biochemical and functional changes in cells, tissues and organs that underlie disease.
  5. 5. Introduction  It serves as the bridge between the basic sciences and clinical medicine, and is the scientific foundation for all medicine.
  6. 6. Introduction  Clinical correlation is important for optimal diagnosis.
  7. 7. Introduction Histopathological examination is used to provide diagnostic information that is important for timely diagnosis of disease to determine treatment plan.  Fresh tissue is extremely fragile & subject to autolysis.
  8. 8. Introduction Loss of specimen is a tragic result both for patient & pathologist Handle all specimens with care & respect. Handle quickly & correctly.
  9. 9. Tissue for study obtained from Biopsies Autopsies
  10. 10. Requirement to send specimen Requisition form/submission form Container Fixative
  11. 11. Submission form
  12. 12. Submission form One slip for one patient Fill properly including clinical history, pre-operative, operative, post-operative diagnosis, organ or tissue. More than one specimen for same patient. Label specimen name with letter A,B,C,D etc. Label specimen container similarly. e.g. A- tissue from right cheek B- tissue from left cheek.
  13. 13. Submission Form Help Us Help You ** Please provide anatomical site, lesion description, and pertinent clinical information on the submission form**  Anatomical location, as well as critical clinical information may allow your pathologist to provide you with the best possible diagnosis and/or differentials
  14. 14. Submission Form  If you have a list of differentials you’d like to rule out, please mention such. Again, please make every effort to provide necessary information in the designated areas on biopsy submission form. It will help us help you help your patients.
  15. 15. Submission Form  First of all & most importantly surgeon should take adequate care to avoid contamination of tissue with tissue from other patient.  This may happen in operation room, clinic, or in pathology lab. Things to be taken care for during & after biopsy surgery
  16. 16. Specimen container  Plastic or glass jar  Label matching requisition slip  Reg no.  Full name  Age, Sex  Word no, Bed no  Site & side  More specimen mark as A, B, C, D etc.  Signature of doctor with date
  17. 17. Tissue Fixation 1. Should prevent autolysis & putrefaction of the cell 2. Should penetrate evenly and rapidly 3. Should harden the tissues 4. Increase the optical density 5. Should not cause shrinkage or swelling of the cells 6. Must not react with the receptor sites & thus must not interfere with the staining procedure 7. Must be cheap and easily available Aims
  18. 18. Fixation Small intestine well preserved Autolyzed Small intestine Good fixative is most important in the production of satisfactory results in histopathology
  19. 19. Simple Fixatives  Formalin
  20. 20. ADVANTAGES & DISADVANTAGES ADVANTAGES 1. Rapid penetration 2. Easy availability & cheap 3. Does not over harden the tissue 4. Fixes lipids for frozen sections 5. Ideal for mailing
  21. 21. ADVANTAGES & DISADVANTAGES DISADVANTAGES 1. Irritant to the nose, eyes and mucous membranes 2. Formation of precipitate of paraformaldehyde which can be prevented by adding 11- 16 % methanol 3. Formation of black formalin pigment, Acid formaldehyde hematin
  22. 22. Formulae 10% Formalin (Unbuffered)  40% Formaldehyde 100 ml  Tap Water 900 ml
  23. 23. Formulae 10% Neutral Buffered Formalin (NBF)  40% Formaldehyde 10 ml  Tap Water 90 ml  Sodium Hydrogen phosphate 0.4gm  Disodium Hydrogen phosphate 0.65gm  pH 7.2-7.4 Buffered formalin prevents formation of pigment acid formaldehyde hematin formed from hemoglobin at acidic pH.
  24. 24. Tissue Fixation Specimen submit in 10% Formalin Formalin tissue ratio 10:1 No other fixative should be used Specimen should be in a container that can be sealed & will not leak
  25. 25. Tissue Fixation  Frozen section  Cultures  Renal & skin tissues for immunoflurescence  Flow cytometry  Chromosome studies  Electron microscopy Submit fresh tissue Not in Formalin
  26. 26. Tissue Fixation This is an example of an 20 cm diameter mass lesion which was fixed at the clinic and subsequently sent to the lab in a plastic, labeled, zip lock bag devoid of any formalin.
  27. 27. Tissue Fixation Incomplete parallel cuts minimum of 2 cm apart (bread loafing) can be utilized to assist with appropriate tissue fixation for solid organ. Be sure to avoid complete transection or too many cuts which can both result in loss of tissue orientation! Large solid specimens
  28. 28. Tissue Fixation  Hollow specimen like cystic cavities: Hollow specimen cavity either opened or filled with formalin by syringe or catheter or packed with gauge or cotton soaked in formalin. Cystic lesions are injected with formalin after removal of original fluid.
  29. 29. Tissue Fixation When tissue float Large specimen that floats on fixative should be covered by a thick layer of gauze.
  30. 30. Tissue Fixation Large flat tissue Large, flat, heavy specimen that rest on bottom of the containers, the gauze should be placed between the container bottom and specimen.
  31. 31. Tissue Fixation Large samples can be held to fix (at least 24 hrs) at your clinic prior to submitting to the lab to help avoid shipping large volumes of formalin which may be costly and hazardous
  32. 32. Submitting specimens  The container should be large enough to accommodate the specimen and filled with enough formalin to completely cover & surround the specimen.  The specimen should be float freely in the container for adequate fixation.
  33. 33. Submitting specimens  Submit whole specimen in a single laboratory  Don’t divide specimen to submit in different laboratories Material on which diagnosis is made (slides, blocks) can be stored for long time & can be evaluated by different observers or by the same observer at different time. Lobular carcinoma in fibroadenoma
  34. 34. Submitting specimens Don’t discard any tissue removed from the body Submit for histopathology Apparently innocent looking tissue may contain ugly behavior (malignancy)
  35. 35. Packaging • Container should have large enough opening • Fresh tissue is malleable and can manipulate to fit into container • Upon fixation tissue becomes rigid and can not remove easily without cutting or breaking the container. No
  36. 36. Packaging Formalin filled jars containing specimens should be placed in a plastic bag, box, or other container with absorbent material to absorb any leakage YES The container should be couriered or brought to the laboratory in a biohazard bag with a completed requisition
  37. 37. Packaging  Paperwork should be placed in a separate plastic bag to avoid contact with formalin if leaking does occur. Such contact can result in altered and illegible paperwork. NO
  38. 38. Submitting Multiple Sites  Submit multiple specimen of same patient in multiple separate appropriately labeled jar. YES
  39. 39. Submitting Multiple Sites  If multiple specimens are submitted in a single container (which is less ideal) there needs to be some method of tissue identification (i.e. suture) to denote respective anatomical sites. YES
  40. 40. Endoscopic Biopsies All fragments submit in a same container YES
  41. 41. Endoscopic Biopsies screen cassette  The optimal method to submit endoscopic biopsy is to place it in a screen cassette after which the cassette should be placed in an appropriately labeled formalin filled jar. If individual cassettes are labeled properly (sharpie or no. 2 pencil), multiple cassettes can be place in one jar. YES
  42. 42. Endoscopic Biopsies  Do not submit endoscopic biopsies wrapped in gauze. Specimens may become lost or may be crushed during the attempted retrieval process. NO
  43. 43. Denoting Margins - Ink the area of interest - Ink prior to bread loafing (if needed) - Allow ink to begin drying before placing the specimen in formalin  Surgical Ink
  44. 44. Denoting Margins - Used to indicate margins or for orientation - Use variable numbers and/or colors of suture - Provide a clear description on the submission form denoting what the sutures indicate (i.e. one suture = cranial margin)  Tagging
  45. 45. Denoting Margins * Submission of samples from the post-surgical bed * Any tumor / neoplastic cells in these specimens is evidence of remaining microscopic disease * Similar to “submitting multiple sites” clearly label and submit each region individually  Tumor Bed Samples
  46. 46. Mapping on a larger scale Cystoprostatectomy Prostate Posterior bladder Anterior bladder Ureter Red = Right Green = Left
  47. 47. Things to Avoid  Please help keep our technician’s fingers safe and DO NOT submit specimens with needles for any reason! NO
  48. 48. Things to Avoid  Please do no staple or suture tissue to cardboard. It can damage tissue and prevent appropriate margin assessment NO
  49. 49. Other Things to Know It is important for you to realize that after all is said and done the pathologist typically evaluates 1 to 4, 5µm thick sections from the entire specimen which is submitted. Images depicting a mass from which a section is taken, embedded in paraffin, and subsequently sectioned to a thickness of 5µm for microscopic evaluation.
  50. 50. Specimen identification and labeling Tissue specimen received in the laboratory have a request form that lists the patient information, history & description of the site of origin.  The specimen are labeled by giving ID number in the laboratory.
  51. 51. Specimen identification and labeling  Any discrepancies of specimen identification noted by pathology assistant should contact with pathologists and/or clinician if there are any questions.
  52. 52. Causes of rejection of specimen  Specimen not in formalin  Unlabeled or improperly labeled container  Without requisition slip or incomplete requisition slip.
  53. 53. Other Things to Know Our Staffs is here working hard for you!  Our staffs and pathologists are here to assist you
  54. 54. Contacting Us If you have any questions about how to best submit your sample or have questions regarding any other issues, please contact the laboratory Mobile No - 0171-3337793 0171-1361198

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