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Principles of specimen collection in pathology Jumphol Mitchai MD Department of pathology KKU Pathology services • Surgical pathology • Cytopathology – Exfoliative cytology – Body fluids – Fine needle aspiration (FNA, FNAC, FNAB) • Cytogenetics • Autopsy Surgical pathology • Examined all tissues and foreign objects removed from living patients • Specimens not to be submitted must be written as hospital policy • Practice of pathologists • Pathological diagnosis : “ Gold standard in medicine ” Specimen collection in surgical pathology • Incisional biopsy : for diagnostic purpose • Excisional biopsy : for diagnostic and therapeutic purposes • Tissue collection by instruments : needle biopsy, core biopsy, punch biopsy, curettage, shave biopsy, Bard biopsy, endoscopic biopsy • Necropsy = biopsy from dead body • Organs from operation rooms General rules for the biopsy • Larger the lesion - more numerous biopsies • Ulcerated tumor – recommend peripheral biopsy • Avoid necrosis and hemorrhage areas • All fragments must be sent to the pathology department • Not crushing, squeezing, cautery • Immediately fix in fixatives • Orientating specimen e.g. deep margin, superior and inferior margins by using sutured threads (long, short) Containers • • • • • • Transparency Lid Leak-proof Wide mouth, flat bottom Size corresponding to specimen volume Label Fixation of the tissue • Hard tissues take more time to thoroughly fixed than soft tissue. • Penetration rate, around 1 mm./hr. in general (variable, depends on consistency of the tissue). • 10 times volume of fixative over the specimen volume is needed. Fixatives Neutral buffered formalin - fixation time 12-24 hours. • Formalin (40% aqueous solution of formaldehyde) - 100ml • Sodium dihydrogen orthophosphate (monohydrate) - 4g • Disodium hydrogen orthophosphate (anhydrous) - 6.5g • Distilled water - 900ml This fixative is suitable for most histological purposes. It is to be preferred to formol-saline (a single 10% solution of formalin in 9% aqueous NaCl) as formalin pigment is avoided. Specimens may be stored in this fluid. The solution is isotonic. การเตรียมสิ่ งส่ งตรวจ 1 การติดสลากบนภาชนะใส่ ขอ้ มูลผูป้ ่ วยให้ครบถ้วน ชื่อสกุล HN เพศ อายุ แพทย์เจ้าของไข้ หอผูป้ ่ วย มีการติดสลากบนทุกภาชนะ 2 การเขียนใบนาส่ ง ใส่ ขอ้ มูลผูป้ ่ วยในทุกช่องรายการข้อมูล specimen(organ), method of specimen collection, characteristics, clinical informations) Filling the request forms (1) 1. identified of the patient 2. identified specimen ( gross appearance, cyst, vascular, inking for margin) type of surgery ( biopsy, wide excision) surgical finding 3. adequate clinical history (necessary) prior diagnosis, immune status (HIV), prior treatment : radiation, chemotherapy bone lesion : X-ray finding Filling the request forms (2) bone lesion : X-ray finding hematolgic disease : CBC, bone marrow female reproductive lesion : LMP, hormone hepatitis : LFT 4. Clinical diagnosis or differential diagnosis Immunohistochemistry and HER2 study • Use formalin-fixed paraffin embedded tissue (paraffin block) • Must have request forms Frozen section • Intraoperative consultation • Fresh tissue was immediately freezed in liquid nitrogen and make an ultrathin section in cryostat • Frozen section is not a permanent section Cryostat • Cryostat = microtome in the freezing cabinet. Frozen sectioning Indications for frozen section • Benign or malignant lesions • Adequacy of surgical margins • Metastatic lymph node : sentinel nodes in breast carcinoma • Presence or absence of ganglion cells in large intestinal wall of Hirschsprung disease Diagnosis of frozen section • Benign VS malignant : not specific diagnosis • In case of doubt – wait for permanent section Specimens for frozen section • Fresh tissue – < 1cm. in size is recommended. • Put in plastic bag, tightly closed with rubber bands • Label on the plastic bag • Place the plastic bag in ice • Send it to pathology lab immediately Inappropriate specimens for frozen section • Large tumor or specimens • Highly infectious specimens : TB, HIV • Follicular lesion of thyroid (can not diffentiate between benign and malignant) Cytopathology • The study of individual cells or small clusters of cells for diagnosis and screening of diseases, including cancers • practice of screeners, cytotechnicians, cytopathologists Specimen collection in cytopathology • Specimens – Smear slide • Cervix (Pap smear) • Fine needle aspiration (FNA) • Bronchial brush – Fluid • Sputum • Bronchial wash • Body fluids, urine, CSF วิธีการเตรี ยมสิ่ งส่ งตรวจทาง cytopathology 1 สิ่ งส่ งตรวจที่ป้ายบนสไลด์ให้แช่ใน95%Alc. ทันที 2 Fluid specimen ให้นาส่ งห้องlabทันที หรื อเก็บในตูเ้ ย็นได้ นาน12-24ชม. 3 เขียนใบนาส่ งที่มีรายละเอียดของผูป้ ่ วย และประวัติการเจ็บป่ วย 4 ติดสลากระบุขอ้ มูลผูป้ ่ วยบนภาชนะหรื อสไลด์ 5 ส่ งห้องปฏิบตั ิการทันที Cytogenetics • Study structure of chromosome from cell culture • FISH in chromosome of leukemia • Specimens – Whole blood – Bone marrow – Amniotic fluid Specimen collection for cytogenetics • Sterile techniques • 2-5 ml. of blood or bone marrow in heparinized tube or sterile heparinized syringe • 20 ml. of amniotic fluid in sterile disposable syringe • Send to lab immidiately (with request form) or keep in refrigerator not more than 12 hours Service hours for cytogenetics • • • • For blood specimen : Mon, Tue, Fri 9.00-16.00 For Bone marrow : Mon, Thur 9.00-16.00 For amniotic fluid : Tue 9.00-15.00 Blood and bone marrow for FISH : Mon-Fri 9.00-16.00 เกณฑ์ การปฏิเสธสิ่ งส่ งตรวจ • • • • • • • ชื่อสกุล และHN ไม่ชดั เจน ชื่อสกุลในใบนาส่ งและบนภาชนะไม่ตรงกัน HNไม่ตรงกับข้อมูลในเวชระเบียน ไม่ระบุหน่วยงานนาส่ ง, แพทย์เจ้าของไข้ ไม่มีประวัติผปู ้ ่ วยและผลการวินิจฉัยทางคลินิก ชนิดของสิ่ งส่ งตรวจไม่ตรงกับใบนาส่ ง สภาพสิ่ งตรวจไม่เหมาะสม Thank you