Collection and Transport of Histopathology SpecimensBY LT.COL.FARHAN AKHTAR AFIP

Collection of Biopsy SpecimensFIXATIVE: Surgical specimen should be placed in adequate fixative. 10 % formal saline as soon as possible. For optimal fixation tissue should be immersed ten times of its own volume . CONTAINERS: Jars , Bottles with screw tops should be used Large specimens should not be squeezed .

ContIf specimen is too large to fit easily into the largest size of the container it should be brought as such to the laboratory without delay . Amputated limbs maybe wrapped in rubber sheets . Hollow viceras should be opened at both ends or cut open along their length . The specimen should never be put into water or normal saline because it will hasten autolysis .

ContRAPID FROZEN SECTION: If rapid frozen section is required laboratory staff and pathologist must be notified 1 hour before Arrangement should also be made to notify the laboratory as soon as patient is taken into the theater. Specimen must not be put in fixative . Specimen should be brought with least possible delay .

Contt..REQUEST FORMS: If more than one biopsy is taken from the patient a single request form will suffice for all those specimens . If two or more apparently unrelated biopsies from same patient than separate form should accompany each specimen. Medical officer in charge of the case must complete the forms . In surgical cases the additional information required is as follows.

Cont1- Precise nature of operation performed 2- Whether the entire specimen or only a part of it is being sent to the laboratory. LABELLING OF SPECIMEN: It is responsibility of MO to see that specimens are labeled correctly. Including name of patient , ward , hospital , date and time obtaining the specimen. These particulars should tele with accompanying request form .

RENAL BIOPSYRenal biopsy should be collected in 10 % formal saline . Request form should contain all the relevant clinical information. Specimen for immuno fluorescence should be submitted fresh in normal saline .

LIVER BIOPSYLiver biopsy should be collected in 10% formaline . Request form should mention , reports of LFTs , Hepatitis markers tested , beside the relevant clinical information. Liver biopsy specimen , for the diagnoses of storage disorders, should be collected in absolute alcohol.

BONE SPECIMENShould be collected in 10% formal saline . Information regarding age , gender , site of biopsy, clinical hx, x-ray that includes radiologists opinion.

SPECIMENS FOR IMMUNOHISTOCHEMISTRY AND TUMOR MARKERSSpecimen should be embedded blocks Histopathology tissue collected in 10% formal saline If the case has been reported from AFIP initially then AFIP report is required for immunohistochemistry and tumor markers.

SPECIMEN FOR OESTROGEN AND PROGESTERONE MARKERSThese markers are carried out on parrafin blocks. Parrafin blocks along with previous slides and the report are required if case has been reported from other laboratory.

REVIEW CASESIn these cases a full description of the gross specimen, parrafin blocks slides and clinical information are required. If case reported from some other laboratory then all the slides along with parrafin blocks are to be submitted.

COLLECTION OF CYTOLOGY SPECIMENSFIXATIVE: Two types of smears are used for cytological examination Usually wet-fixed smears are preffered Wet fixed smears are prepared by immediately fixing the slide without allowing it to dry. Fixative recommended are mixture of equal parts of 1- ETHER 2-95% ETHYL ALCOHOL 3-FORMOL ALCOHOL Not less than 15 minutes are required for adequate fixation Coplin Jars are commonly used Papanicolaou and H&E stains are commonly used on wet-fixed smears. Air dried smears are simply prepared by allowing these to dry naturally Smears are stained with Diff Quick/Haemacolor, Giemsa stain, Leishman Stain etc The smear must be labelled regarding the type of fixation besides patient identification so that they can be appropriately stained.

ContinREQUEST FORM: Request forms for cytological specimen must include patients identification data. Type and site of the specimen, clinical details, in case of slides, method of fixation that was used. In cases of cervical smears, date of last menstrual period, use of IUCD, hormone therapy, previous history must be mentioned on request form

RESPIRATORY TRACTSPUTUM: fresh early morning specimen produced by deep cough should be collected Should be immediately brought to laboratory without any fixation. If its not possible to bring unfixed specimen to laboratory the sputum should be pre-fixed by asking the patient to expectorate into wide-mouthed small jar half filled with 70% ethly alcohol. Laboratories where cytology facilities are not available, fresh sputum should be examined. Smears from these areas and other randomly sampled areas should be fixed immediately in 95% ethyl alcohol After 20 mins slides can be dried and transported to a lab for reporting.

ContiBRONCHIAL ASPIRATES, WASHING and BRUSHING: Aspirates collected during bronchoscopy may be centrifuged and smears prepared from cell button. Direct smears should be prepared from bronchial brushing and wet-fixed in 95% alcohol Alternatively they can be processed like aspirates and washings.

ContiBRONCHOALVEOLAR LAVAGE (BAL): Bal involves the infusion and re-aspiration of a sterile saline solution into the air passages. This fluid should be immediately submitted as such to the lab. If a delay in transportation is expected, an equal quantity of 95% ethyl alcohol should be added.

ContiURINARY TRACT: URINE: freshly-voided urine is the specimen of choice in male patients. 1st morning specimen should be avoided as urine has been collected in the bladder overnight and cells have degenerated. In female patients catheterized urine is preffered. Approximately 50-100 ml of urine should be collected in an equal amount of 50% ethyl alcohol. If possible patient should be sent to the laboratory for the collection of fresh sample

Conti.WASHING AND BRUSHINGS OF URETER AND RENAL PELVIS, BLADDER WASHINGS: All washings should be collected in an equal amount of alcohol for fixation. Brushing may also be added to alcohol for fixation. Alternatively, direct smears may be prepared and wet-fixed in an alcoholic fixative.

ContiPLEURAL,PERICARDIAL AND PERITONEAL FLUIDS: Fluids should be collected in clear, dry container. Should be sent to lab as soon as possible. If not possible to send the fluid immediately, it should be stored in a refrigerator at 4 degree c and not allowed to freeze. Can b preserved for several days.

ContiCSF: Cerebrospinal fluid should be collected in clean container Should be transported immediately to the lab. If a delay in transportation more than few hours is expected, an equal amount of 50:50 alcohol ether mixture or 95% ethyl alcohol may be added.

Conti..ALIMENTARY TRACT: Brush and wash samples may be collected from the oesophagus, stomach, lower bowel and rectum. Brush smears should be prepared immediately by rolling on clear glass slide. 5-6 such smears may be wet-fixed in alcohol for Papanicolaou staining. These should be labelled as such and submitted for cytological examination. Wash samples from stomach must be immediately neutralized with N/10 sodium hydroxide and centrifuged rapidly in ice-cold siliconised tubes.

Contin.FEMALE GENITAL TRACT: CERVICAL SMEAR: aim of collecting cervical smear is to obtain a representative specimen from the squamocolumnar junction using Ayres spatula. Having exposed the cervix using a bi-valve speculum, a circumferential sample is obtained by rotating the spatula through 360 degree. If smear does not show endocervical cells, an additional smear may be obtained later form endocervical canal by using a cotton-tipped applicator. Once sample has been obtained it should be placed on glass slide. Slide must be fixed immediately without allowing to dry, by placing 95% alcohol for 15 min. After fixation slide should be sent to the lab.

ContinVAGINAL SMEAR: A specimen for hormonal evaluation is taken from lateral vag wall. If this is not possible a posterior fornix pool specimen may be used. The specimen should be taken by lightly dipping the applicator in the secretions, avoiding forceful scrapping. Smear are prepared and immediately wet-fixed in the same manner as cervical smears. Dried and submitted to the laboratory.

ContiBREAST: NIPPLE DISCHARGE: a few drops should be expressed by pressing the sub-areolar region. Drops are touched onto a clean glass slide and spread with the help of another slide. Smear shold be wet-fixed in an alcoholic fixative and submitted.

A FINE-NEEDLE ASPIRATION CYTOLOGYINTORODUCTION: FNAC is an inexpensive, rapid method of diagnosis. It is safe Rapid Reliable Relatively painless Can be used sample superficial and subcutaneous lesions Can also be used in deep visceral lesions with the help of radiologysts using 1-fluoroscopy 2-tomography 3-angiography 4-ultrasonography To localize the lesions.

ContTECHNIQUE: Proper clinical hx Patient should be explained about the procedure. 21-25 gauge needle can be used. Area should be cleaned with spirit swab. Needle introduced into the mass. -ve pressure is applied by retracting plunger and mass is probed in diff directions. Prior to withdrawl of needle plunger is released allowing equalization of pressure. Cotton swab is applied for few minutes to stop the bleeding.

SLIDE PREPRATIONAfter aspiration syringe is detached from needle. Filled with air Syringe is re-attached to same needle and plunger is pushed to gently express the material on to glass slide. Material is gently spread on slides by using another slide. Slides are allowed air dry. Slides can be stained by Papaicolaou (PAP), Haematoxylin and Eosin, Leishman and Modified Giemsa Stains.

PITFALLSIf slide is too bloody, cells from actual lesion will be diluted in smear. If aspiration time too long then specimen will clot in syringe and needle and wont be spread properly. If slides are incorrectly made nuclear details will be poor and artifacts will be created.

COMPLICATIONSMajor complications of FNAC is bleeding and infection. Incase of deep seated lesions: 1-pneumothorax 2-embolism 3-neurovascular injury 4-major internal bleeding 5-interperitoneal leak of hollow viscus have been reported 6-vasovagul syncope may also occur 7-tumors spread slong needle tract have been reported.

CELL-BLOCK PREPRATIONCan be made from aspirated material. TECHNIQUE: Aspirated material in syringe in rinsed thouroughly in normal saline Centrifuged Supernatant is removed and in sediment. Human plasma and thromboplastin suspension made in normal saline is added in equal quuantity(3 drops each) kept for clot formation. later on this clot is placed in 10% formaline for fixation and dealt like other tisses for histopath examinations. Routine staining (H&E), special stains and even immunohistochemistry can be performed.

POST-MORTEM EXAMINATIONSLT COL FARHAN AKHTAR AFIP RAWALPINDI AFPGMI

REASON FOR PERFORMING POST MORTEM:

Mainly 2 reasons: MEDICO-LEGAL POSTMORTEM: When cause of death not clear MEDICAL POSTMORTEM: Aim to study process of a disease to benefit medical education.

OBJECTIVES OF AN AUTOPSY:To make positive identification. To determine cause of death. To determine mode of death. To determine time of death. To determine extant of internal, external abnormalities. To determine external and internal injuries. To get samples for analysis. To retain tissue for evidence. To obtain photographs for evidential purpose. To provide comprehensive autopsy report. To restore body to best possible cosmetic condition.

COLLECTION OF SPECIMENS FOR BIOCHEMICAL EXAMINATION:All specimen should be collected aseptically. In suitable container. With required preservative. In sufficient quantity. 1.URINE 2.BLOOD. 3.CEREBROSPINAL FLUID.

SPECIMEN TO BE COLLECTED IN AIRCRAFT ACCIDENT:The following specimen should be collected. 1. blood for glucose. 2. blood for lactic acid. 3. blood for alcohol. 4. urine for alcohol. 5. urine for opiates. 6. muscle for lactic acid. 7. blood for carbon monoxide.

SPECIAL PRECAUTIONS IN CASE OF DELAY OF BURIAL:Process of autolysis starts immediately; Due to disease this process is rapid. An early autopsy and on time burrial is recommended. In case of delay following procedure will help: 1. mortury refrigeration. 2. Store in cool, dry place. 3. all wounds should be cleaned. 4. packing the body cavity with farmaline soaked cotton. 5. after closing autopsy incision , the body should be thoroughly washed and wrapped in clean white sheets.

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