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					Standard Operating Procedure for Histopathology Technique
1. Scope
The present SOP manual on histopathology technique outlines the various methods used for the histological study of diseases/conditions. It recommends routine staining procedure as well as the special staining procedure for the study of various types of lesions or other anomalies. This document would work as national reference method for a veterinary diagnostic laboratory using histopathology procedure. The various methods and the specific procedures for a method outlined herein this manual shall be applied to the histopathology laboratory unit of central veterinary laboratory.

2. Introduction
Disease process starts at sub-cellular level in a living body. After that, deterioration is precipitated in the cell. The study of cellular level lesion is a matter of electron microscopy. If the causative agent is not eliminated and the process goes uninterrupted, the collective outcome would appear as a lesion at tissue level. We can study the tissue lesions under light microscope. If the etiological agent is not removed and the situation proceeds, the lesion would appear at organ level and subsequently a particular organ system would start functioning in an unnatural way. The histopathological study under the scope of compound microscope helps explore the lesions precipitated at tissue level.

Various types of pathological changes intended for the micro-anatomical study require various methods of tissue processing and staining techniques; collectively known as special processing and staining technique. However, there is a routine processing and


staining procedure which all the diagnostic laboratory involved in histological examination follows.

3. Code of Conduct
3.1. Personal Hygiene
3.1.1. Purpose
  To maintain a hygienic working environment in the laboratory room; and to secure general health status of the personnel working in with the laboratory.

3.1.2. Requirements
Personal protective equipments. Please refer to the annex for a detailed listing.

3.1.3. Procedure  Protective clothing should be worn all times while working in the laboratory. Carelessness would be considered not excusable.  The personal protective equipments are not allowed to take away to home. These things would be kept in the laboratory after finishing the work.  Eating, drinking, smoking, applying cosmetics will be strictly prohibited in the laboratory working area.  Personal vices like handling contact lens/spectacles, poking nose/ear and finger trimming with mouth should be improved.  No visitors would be allowed to meet the technician at the place working in the laboratory.

3.2. Occupational Health and Safety
3.2.1. Purpose To ensure occupational health by carefully exploring and adopting personal safety measures at various steps of procedures involved in the histopathology laboratory. 3.2.2. Requirements No specific equipment is required rather a laboratory staff requires to be well aware of possible injuries, accidents and other unsafe situation identified across the working place and procedure.


3.2.3. Procedure  Procedures will be stated in the guidelines of central veterinary laboratory for the technicians working in various laboratory units.  A technician (preferably veterinarians) would be specified for the purpose. He/she would be responsible to formulate procedures involving identification, reduction, elimination and control of risk factors.  If necessary technicians would be provided with necessary training package. The topics to be accommodated in the periodic training package are listed in the annex.  Any sort of accident would be promptly learned by the supervisor and he/she would immediately inform chief of CVL to manage the injured person for immediate health care.

3.3. Materials/equipment safety data-sheet
3.3.1. Purpose To provide safety notes regarding various chemicals/solutions/reagents and various equipments and electrical appliances. 3.3.2. Requirements Equipment catalogue, Manufacturers’ instructions etc. These data-sheets would be made available on the site of laboratory works. 3.3.3. Procedure  Read the equipment catalogue or manufacturers’ instruction sheet carefully to understand the operation of particular equipment.  Work with the equipment or chemicals as per the test protocol.  Replace the containers or bottles of the chemicals in its original place or stop running the equipment when the work is finished.  Note any deviation or abnormal operation of the equipment to the laboratory authority to ensure immediate repair. 4. Maintenance and Calibration 4.1. Purpose To ensure nonconforming situations of the testings as well as test results and to avoid accidents met with the equipments’ operation.


4.2. Requirements Services of the equipment maintenance unit and calibration service providing laboratory. 4.3. Procedure  Enlist the equipment available in the histopathology laboratory unit.  Identify the equipment that needs maintenance and calibration services.  Specify the time interval when the equipment needs maintenance and calibration.  Maintain the record of maintenance and calibration, the report of which is duly signed by the in-charge of the histopathology laboratory unit and the technician responsible for maintenance and calibration.


Histopathology laboratory Technique
5. Receiving the specimen The sources of specimen to CVL are private practitioners and government veterinarians, hatcheries, farms, farmers, government as well as non-government agencies and the other stakeholders, and PMLU (Post mortem laboratory unit) of its own. The histological specimen collected by any party must meet the requirements stated in the Histological Specimen Collection Protocol (Histo/doc 1) developed for the provision of this reference. 5.1. Recording the specimen  Any kind of specimen brought to or received by CVL shall be initially registered in the main entry of the laboratory.  The main entry register shall code the specimen.  The specimens shall be distributed to the relevant laboratory unit of CVL and the histopathology laboratory unit shall reregister the specimen in its specimen register record maintaining its privacy. 5.2. Physical Examination of the Specimen A specimen shall be examined against the following criteria:  Condition of the specimen; any departure of the specimen from the normal condition shall be conveyed to the test requesting party.  Accompanying documents  Information of the documents in line with the specimen.  Preservation technique employed  Sufficiency of the specimen 5.3. Trimming and Fixation of the Specimen


If the specimen is collected at PMLU of CVL, the tissues or organs collected shall be preserved in 10% neutral buffered formalin in general. A separate document for the purpose of Trimming and Fixation of the Specimen (Histo/doc 2) shall be developed with reference to this requirement. 6. Processing of the Tissues

The various fixed tissue samples shall be set for processing technique. It is also known as the process of dehydrating the tissues. The laboratory shall use series of alcohols of various concentrations for the purpose of dehydration. A separate tissue processing protocol shall be developed (Histo/doc 3).

7. Clearing of the tissues

The dehydrated samples shall be transferred to the clearing agent. A separate procedure manual for the clearing of tissues (Histo/doc 4) shall be developed for the purpose of this reference. 8. Impregnation of the tissues

The tissues after clearing shall be set into paraffin wax baths of melting temperatures as required. A procedure manual for impregnation of the tissues (Histo/doc 5) shall be developed with reference to this requirement. 9. Block Making


This step is necessary to shape the impregnated tissues in the form of blocks. The frames required to make the tissue blocks are provided in shapes or managed their shapes locally per the arrangement of the chuck part of the microtome. The method of block making shall be documented in the form of a procedure manual (Histo/doc 6). 10. Section cutting and obtaining the sections over glass slides

10.1. Setting the alignment

It becomes necessary to align the tissue block in a manner that it is parallel to the microtome blade/knife. The chuck is secured in position in the microtome assembly by means of knobs. The loosening of one knob and simultaneous tightening of opposite knob would ensure the parallel positioning of the blocks and the knife or blade. 10.2. Cutting serial sections

 Set the alignment of the microtome.  Secure the tissue block in the chuck.  Adjust the thickness of the tissue sections at 5 mµ..  Expose the tissue of the block by carefully and successively trimming the wax layer of the tissue block.  Cut the exposed tissues in serial sections with uniform motion of the microtome.


10.3. Obtaining sections over the glass slides The procedure of collection of thin sections over the glass slides shall be prepared with reference to this requirement (Histo/doc 7). 11. Staining Technique  In general the various sections shall be stained per routine staining method. A separate staining manual shall be prepared with reference to this requirement.  Similarly, tissue sections shall be stained employing special staining technique to explore the specific micro-pathological lesions. A series of procedure documents shall be prepared for various special staining techniques for the purpose this requirement. 12. Study of slides and Interpretation of test results  Various permanent slides shall be studied first under low magnification of the microscope and gradually under medium and high magnifications.  The lesions translated after the study of slides shall be prepared into a test report. A test report format has been provided in the annex of this manual.  The test format shall mention the histological lesions explored under the microscope. It shall specify name of any disease/condition rather an opinion shall be suggested for a particular disease or condition.  The test report shall be duly signed by technician and in-charge pathologist interpreted the results and the technical manager of the laboratory. 13. Referring the test reports to report delivering desk of the laboratory 14. Record keeping A back up (official copy) of the test reports prepared and handed over to the report delivery desk shall be maintained in the form of record allotting a dispatch number. 15. Ensuring the receipt of test results by the clients  It would be rather a duty of HPLU to ensure the fact that the client has received the test report from the report delivery desk of the laboratory.


 The HPLU shall maintain the dispatch number and date of delivery of the test report to the clients from the laboratory. 16. Feedback  HPLU shall collect, study, process and make corresponding improvement in its routine work to address the feedback and critical comments of the clients. It would be considered as an endeavour in the way of quality improvement of the HPLU.  The HPLU shall let it know to its clients the necessary corrective actions taken to improvement in its works.


Shared By:
banshi sharma banshi sharma Chief Molecular biologist