Methods

HISTOPATHOLOGICAL DIAGNOSIS •

Definition: is the diagnosis established on the basis of microscopic examination of the histological sections. • Is carried out on products/tissue fragments harvested by/gathering by/obtained by: 3. BIOPSY • (collection of fragments of tissues / organs obtained during life); Methods of harvesting/collecting: e. Microbiopsy: • biopsy puncture (for deep organs: the liver, spleen, kidneys, bone marrow); • curettage (for mucous membranes or superficial lesions: endometrium, uterine cervix, mouth, skin); • endobiopsy (gastric, rectal, laryngeal, bronchial mucosa). b. Surgical biopsy : • Incisional (for diagnostic purpose) • Excisional (for diagnostic and therapeutic purpose). 2. SURGICAL EXCISION SPECIMENS 3. COLLECTION OF FRAGMENTS OF TISSUES / ORGANS DURING NECROPSY -Necropsisc specimens

Ticket send to Anatomo Pathological Laboratory Medical service that serve for collecting the specimen ............ Nr. Registration / Date ...........

To Anatomo Pathological Laboratory, Name and surname of the patient .............................................. ..... Age…….Profession ......... ........ Residence .............................. .................. Clinical diagnosis ................................................ ............................... Physiological and pathological History......... Treatments carried out ……….. Biopsy specimen............................. Fixative .................

The physician/ doctor signature and seal who performed the specimen collection

HISTOPATHOLOGICAL DIAGNOSIS Time of tissue processing of the specimen: • Specimen collection (indications): large injuries => multiple biopsies, ulcerated lesions=> specimen collection at the level of the edge injury • Fixation: 10% buffered formalin, Carnoy fluid, Bouin • Specimen inclusion in paraffin, and sectioning to the microtome (3-5 microns) • Staining of the histological sections

HISTOPATHOLOGICAL DIAGNOSIS Staining methods: Usual techniques: Hematoxilin-Eosin staining, trycromic staining (van Gieson, Simionescu, Masson);

Histochemical techniques: PAS (neutral MPZ, glicogen, basal membranes, fungi); Fontna/Grimelius (neurosecretor granules); Gordon-Sweets, Gomori (reticuline fibres; basal membranes); Perls (hemosiderin-trivalent iron); Fontana (melanin); von Kossa (calcium); Sudan Black, Sudan 3, Scharlach (neutral lipids); Congo Red (amyloid); Alcian Blue (acid mucine).

HISTOPATHOLOGICAL DIAGNOSIS Medical service / Anatomo Pathological Lab …… Registration Nr …………… Date of report...........

Anatomo Pathological Report Name and surname of the patient ……………………….Age……. Hospital where the patient is admitted ……………… Clinical diagnosis.............................................. ..... Histological details of the lesions observd at microscopical examination….. Final conclusion (histopathological diagnosis).............................. .................. The pathologist signature and seal

CYTODIAGNOSIS • Definition: is the diagnosis established on the basis of microscopic examination of the morphological changes of cells (isolated) contained in different pathological samples displayed on the slides (smear).

• Advantages: – – – – –

Provides a quick diagnosis; It is cheap; A little traumatizant; Easy of repeated; Useful in screening methods.

CYTODIAGNOSIS Techniques of collection of pathological samples 3. 4. 5. 6. 7. 8.

Exfoliating (sputum, prostate, breast, vaginal and cervical secretion); Aspiration puncture of serosal fluids (pleural, peritoneal, articulation fluid),cyst content; Scratching / brushing (uterine cervix, respiratory tract); Cavitary organ lavage (respiratory tract, gall bladder); Printing (skin lesions, lymph nodes); Fine needle aspiration puncture (thyroid, prostate, mammary glands, pancreas), deep solid tumors, bone marrow.

Ticket send to Anatomo Pathological Laboratory Medical service that serve for collecting the sample ............ Nr. Registration / Date ........... To Anatomo Pathological Laboratory, Name and surname of the patient…. Age…….Profession ...... Residence Clinical diagnosis Physiological and pathological history....... Type of samples used for smear preparation ……….. Smear number ................. The physician/ doctor signature and seal who performed the sample collection

CYTODIAGNOSIS Time of execution of the smears 1. Displaying • Directly: natural secretions, pus, samples obtained by aspiration puncture from solid organs, printing; • After Centrifuge 5-10 minutes, then displaying of the sediment: Liquids from serosal fluids, cystic fluids, cysts, urine and lavage. 2.Fixation • Agitation in the air a few seconds and drying (for staining with MayGrunwald-Giemsa); • Alcohol 96% (for staining of Papanicolau coloration-Pap); 3.Staining

• • • •

MGG-very simple Pap for cervicovaginal and bronchial cytology, Special for recording of lipids, glycogen, mucine Imunocitochimy

CYTODIAGNOSIS Medical service / Anatomo Pathological Lab …… Registration Nr …………… Date of report......... To unit who requested citodiagnosis Name and surname of the patient …. Age……Profession………..Residence… Description of the changes found at microscopic examination Conclusion (synthetic diagnosis) Recommendations Clinical diagnosis ........ The cythologist signature and seal

Special techniques for diagnosis

1. Immunohistochemistry -

Immunofluorescence for the detecting (by marking with of the fluorescent staining/dye) of autoantibodies, antigens, antigen-antibody complexes or complement fractions. Imunoperoxidasis (by peroxidase-antiperoxidase method and avidin-biotin complex) using monoclonal or polyclonal antibodies for detecting: • The origin of neoplastic cells (differential diagnosis between carcinoma-sarcomalymphoma): – Citokeratine - carcinoma; – LCA / leucocitar common antigen-lymphoma; – myoglobin-rabdomyosarcoma; – vimentin-connective tumors; – Factor VIII antigen-vascular tumors. • The tumoral oncofetal antigens (α-feto-protein, carcinoembrionar antigen); • The hormonal receptors; • Oncogens, tumor suppression gene (p53); • Establishing of immunophenotype pattern of malignant lymphomas

Special techniques for diagnosis 2. Histoenzimology - detecting enzymes; 3. Electron microscopy for differenciating between: - Carcinoma-melanoma-sarcoma; - Adenocarcinoma-mesothelioma; - Thymoma-lymphoma-seminoma; - Small cell tumors and fusiform cell tumors; - Endocrine and non-endocrine tumors; - Glomerular Nephropaties with immune mechanism; 4. Techniques of molecular biology: - PCR (polymerase chain reaction); - Cytometry in flux for DNA analysis (ploidy); - In situ hybridization to identify DNA fragments; - Analysis of gene rearrangement.