Principles of specimen collection in pathology Jumphol Mitchai MD Department of pathology KKU

Pathology services Surgical pathology Cytopathology Cytogenetics 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.

การเตรียมสิ่งส่งตรวจ 1 การติดสลากบนภาชนะใส่ข้อมูลผู้ป่วยให้ครบถ้วน ชื่อสกุล HN เพศ อายุ แพทย์เจ้าของไข้ หอผู้ป่วย มีการติดสลากบนทุกภาชนะ 2 การเขียนใบนำส่ง ใส่ข้อมูลผู้ป่วยในทุกช่องรายการข้อมูล 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

วิธีการเตรียมสิ่งส่งตรวจทาง cytopathology 1 สิ่งส่งตรวจที่ป้ายบนสไลด์ให้แช่ใน95%Alc. ทันที 2 Fluid specimen ให้นำส่งห้องlabทันที หรือเก็บในตู้เย็นได้นาน12-24ชม. 3 เขียนใบนำส่งที่มีรายละเอียดของผู้ป่วย และประวัติการเจ็บป่วย 4 ติดสลากระบุข้อมูลผู้ป่วยบนภาชนะหรือสไลด์ 5 ส่งห้องปฏิบัติการทันที

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

เกณฑ์การปฏิเสธสิ่งส่งตรวจ ชื่อสกุล และHN ไม่ชัดเจน ชื่อสกุลในใบนำส่งและบนภาชนะไม่ตรงกัน HNไม่ตรงกับข้อมูลในเวชระเบียน ไม่ระบุหน่วยงานนำส่ง, แพทย์เจ้าของไข้ ไม่มีประวัติผู้ป่วยและผลการวินิจฉัยทางคลินิก ชนิดของสิ่งส่งตรวจไม่ตรงกับใบนำส่ง สภาพสิ่งตรวจไม่เหมาะสม

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