Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Medical Laboratory technology Lab Manual for MLT students

MLT II lab manual for MLT students

Demonstration of working of spectrophotometer
Demonstration of maintenance of equipments and reagents
Sample formats for reporting test result
Demonstration of policies and procedures for infection control
Demonstration of mock diagnostic lab for learning & understanding patients right
Demonstration of mock environment to learn and understand conducive patient environment
Collection and handling of specimen for histopathology/cytopathology examination
Demonstration of working of Microtome
Demonstration of sharpening methods of microtome knife
Demonstration of tissue processing
Demonstration of PAP staining
Demonstration of PAS staining
Collection and handling of specimen for cytopathology examination
Demonstration of Mounting technique Demonstration of Mounting technique
Demonstration of maintaining record of inventory, test results etc

  • Be the first to comment

Medical Laboratory technology Lab Manual for MLT students

  1. 1. LAB MANUAL Medical Laboratory Technician Lab-II BATCH - B SUB CODE: BMLT6052 DEPARTMENT OF MEDICAL LAB TECHNOLOGY SMAS FACULTY INCHARGE: Mr. A. VAMSI KUMR (Asst Prof)
  2. 2. SYLLABUS List of Experiments Specific Instructional Objectives This program is aimed at training candidates for the job of a “Medical Laboratory Technician”, in the “Healthcare” Sector/Industry and aims at building key competencies Catalog Description On completion of this course student will able to Demonstrate the ability to perform clinical skills essential in providing basic diagnostic services such as Correctly collect, transport, receive, accept or reject and store blood /urine/stool and tissue samples, etc.; Conduct analysis of body fluids/ samples; Maintain, operate and clean laboratory equipment; Provide technical information about test results; Prepare and document medical tests and clinical results; etc. COURSE OUTCOMES On completion of this course the students will able to: 1. Demonstrate working of various instruments used and laboratory process and formats in Medical Laboratory Technology Laboratory. 2. Demonstrate automation and quality control in Medical Laboratory Technology Lab. 3. Diagnose different diseases and report it. 4. Demonstrate and apply computers and its applications in laboratory field.
  3. 3. List of Experiments Experiment No Experiment Name 1. Demonstration of working of spectrophotometer 2. Demonstration of maintenance of equipment’s and reagents 3. Sample formats for reporting test result 4. Demonstration of policies and procedures for infection control 5. Demonstration of mock diagnostic lab for learning & understanding patients right 6. Demonstration of mock environment to learn and understand conducive patient environment 7. Collection and handling of specimen for histopathology/cytopathology examination 8. Demonstration of working of Microtome 9. Demonstration of sharpening methods of microtome knife 10. Demonstration of tissue processing 11. Demonstration of PAP staining 12. Demonstration of PAS staining 13. Collection and handling of specimen for cytopathology examination 14. Demonstration of Mounting technique Demonstration of Mounting technique 15. Demonstration of maintaining record of inventory, test results etc 16. Demonstration of automation in clinical biochemistry laboratory 17. Demonstration of automation in hematology laboratory 18. Demonstration of automation in pathology laboratory 19. Demonstration of automation in microbiology laboratory 20. Demonstration of FNAC 21. Demonstration of laboratory diagnosis of HIV 22. Demonstration of laboratory diagnosis of HCV 23. Demonstration of maintenance of IQA 24. Demonstration of maintenance of EQA 25. Demonstration of advanced techniques in the field of Molecular biology 26. Demonstration of Computer and its applications 27. Demonstration of operating systems 28. Demonstration of MS –Word 29. Demonstration of MS-Excel 30. Ethics of hospital setup
  4. 4. Course Name/ code Medical Laboratory Technician Lab-II / BMLT6052 Experiment No 1 Title Demonstration of working of spectrophotometer Faculty In charge Mr. A. Vamsi Kumar (Asst Prof) Department / School Medical lab technology, SMAS Semester VI 1. DEMONSTRATION OF WORKING OF SPECTROPHOTOMETER History: For millions of years, light has defined the life of Homo sapiens. Through photosynthesis, light has given us food, energy, and atmosphere. And using light we communicate information, see the big objects far from us through the telescope and small objects through the microscope. From where does light get this transcending power? It took nearly a millennium until James Clark Maxwell in 1864 told the world that light is made of waves of disturbances of electric and magnetic fields. Introduction: • A spectrophotometer is an instrument that measures the amount of light absorbed by a sample. • Spectrophotometer techniques are mostly used to measure the concentration of solutes in solution by measuring the amount of the light that is absorbed by the solution in a cuvette placed in the spectrophotometer. • Scientist Arnold J. Beckman and his colleagues at the National Technologies Laboratory (NTL) invented the Beckman DU spectrophotometer in 1940. Principle of Spectrophotometer When a light at a particular wavelength is passed through a solution (incident light), some amount of it is absorbed and, therefore, the light that comes out (transmitted light) is diminished. The nature of light absorption in a solution is governed by Beer-Lambert law. Beer’s law states that the amount of transmitted light decreases exponentially with an increase in the concentration of absorbing material (i.e. the amount of light absorbed depends on the concentration of the absorbing molecules). According to Lambert’s law, the transmitted light decreases exponentially with increase in the thickness of the absorbing molecules (i.e. the amount of light absorbed is dependent on the thickness of the medium). By combining the two laws (Beer-Lambert law), the following mathematical derivation can be obtained:
  5. 5. Diagrammatic representation of the components in a spectrophotometer. Working Principle of spectrophotometer: The spectrophotometer technique is to measure light intensity as a function of wavelength. It does this by diffracting the light beam into a spectrum of wavelengths, detecting the intensities with a charge-coupled device, and displaying the results as a graph on the detector and then on the display device. 1. In the spectrophotometer a prism (or) grating is used to split the incident beam into different wavelengths. 2. By suitable mechanisms, waves of specific wavelengths can be manipulated to fall on the test solution. The range of the wavelengths of the incident light can be as low as 1 to 2nm. 3. The spectrophotometer is useful for measuring the absorption spectrum of a compound, that is, the absorption of light by a solution at each wavelength. Difference b/w Colorimeter & Spectrophotometer: The spectrophotometer primarily differs from colorimeter by covering the ultraviolet region (200-400 nm) of the electromagnetic spectrum. Further, the spectrophotometer is more sophisticated with several additional devices that ultimately increase the sensitivity of its operation severalfold when compared to a colorimeter. A precisely selected wavelength (say 234 nm or 610 nm) in both ultraviolet and visible range can be used for measurements. In place of glass cuvettes (in colorimeter), quartz cells are used in a spectrophotometer. Instrumentation of Spectrophotometer The essential components of spectrophotometer instrumentation include:
  6. 6. 1. A table and cheap radiant energy source • Materials which can be excited to high energy states by a high voltage electric discharge (or) by electrical heating serve as excellent radiant energy sources. 2. A monochromator, to break the polychromatic radiation into component wavelength (or) bands of wavelengths. • A monochromator resolves polychromatic radiation into its individual wavelengths and isolates these wavelengths into very narrow bands. PRISMS: • A prism disperses polychromatic light from the source into its constituent wavelengths by virtue of its ability to reflect different wavelengths to a different extent • Two types of Prisms are usually employed in commercial instruments. Namely, 600 cornu quartz prism and 300 Littrow Prism. GRATINGS: • Gratings are often used in the monochromators of spectrophotometers operating ultraviolet, visible and infrared regions. TRANSPORT VESSELS (cuvettes), to hold the sample • Samples to be studied in the ultraviolet (or) visible region are usually glasses (or) solutions and are put in cells known as “CUVETTES”. • Cuvettes meant for the visible region are made up of either ordinary glass (or) sometimes Quartz. A PHOTOSENSITIVE DETECTOR and an associated readout system • Most detectors depend on the photoelectric effect. The current is then proportional to the light intensity and therefore a measure of it. • Radiation detectors generate electronic signals which are proportional to the transmitter light. • These signals need to be translated into a form that is easy to interpret. • This is accomplished by using amplifiers, Ammeters, Potentiometers and Potentiometric recorders. APPLICATIONS OF SPECTROPHOTOMETER Some of the major applications of spectrophotometers include the following: 1. Detection of concentration of substances 2. Detection of impurities 3. Structure elucidation of organic compounds 4. Monitoring dissolved oxygen content in freshwater and marine ecosystems 5. Characterization of proteins 6. Detection of functional groups 7. Respiratory gas analysis in hospitals 8. Molecular weight determination of compounds 9. The visible and UV spectrophotometer may be used to identify classes of compounds in both the pure state and in biological preparations. Reference: 1. https://www.biochemden.com/spectrophotometer-instrumentation-principle/ 2. https://microbenotes.com/spectrophotometer-principle-instrumentation-applications/ 3. http://simulab.ltt.com.au/5/Laboratory/StudyNotes/snTheSpectrophotoM.htm 4. U.Satyanarayan, Textbook of Medical Biochemistry
  7. 7. Course Name/ code Medical Laboratory Technician Lab-II / BMLT6052 Experiment No 2.0 Title Demonstration of maintenance of equipment’s and reagents Faculty In charge Mr. A. Vamsi Kumar (Asst Prof) Department / School Medical lab technology, SMAS Semester VI 2. DEMONSTRATION OF MAINTENANCE OF EQUIPMENT’S AND REAGENTS Introduction: The care and maintenance of laboratory equipment is an integral part of quality assurance in the lab. Well- maintained lab equipment ensures that data is consistent and reliable, which in turn impacts the productivity and integrity of the work produced. Furthermore, since laboratory equipment generally takes up a big cut of the budget, good maintenance contributes to cost-cutting measures, by lowering the chances of premature repurchases and replacement. In addition, routine maintenance ensures that lab equipment is safe for use through highlighting and repair of faulty equipment and equipment parts. Various procedures and routines will ensure that your laboratory equipment is well-maintained and cared for, this includes; 1. Developing standard operating procedures for all lab equipment. 2. Preparing documentation on each specific equipment, outlining the repairs and maintenance undertaken. 3. Outlining a preventive maintenance program for each equipment. 4. Training both technical and managerial staff on proper use and care of lab equipment 1. STANDARD OPERATING PROCEDURE FOR MAINTENANCE OF LAB EQUIPMENT Standard operating procedures (SOPs) are a must for all complex lab equipment. This ensures that the correct use and maintenance of the equipment is integrated within routine work. Detailed instructions of equipment use should be sourced from the manufacturer’s operator manual. The SOP can be written by the lab manager, an equipment officer, or staff that frequently works with the specific equipment. The SOP should also be easily accessible at the workbench. A proper SOP should contain the following; ➢ The title and description of the content/scope of the SOP. ➢ Definitions of all abbreviations used. ➢ An outline of the personnel responsible for the equipment or involved in its use, including their qualifications and training requirements. ➢ Detailed instructions for the use of equipment, containing the do’s and don’ts of operating them. ➢ A description of quality control and maintenance. ➢ Instructions on waste management, where applicable. ➢ Reference documents, such as manuals used to prepare SOP and manufacturer’s websites, should be outlined for use when further information is required.
  8. 8. 2. EQUIPMENT MAINTENANCE DOCUMENTATION This is a centralized collection of all the information regarding a particular equipment. It is a reference archive for equipment maintenance that can be used to understand the history of the equipment. It is usually organized by the lab manager or the lab’s equipment officer. The maintenance log outlines equipment identification and descriptions like equipment name, model number, manufacturer, purchase date, warranty, model, etc as shown in Table 1. It also contains description of repair work, parts replacements, tests, measurements, adjustments, or deep cleaning done on the equipment. Item identification Equipment: Brand: Purchase date: Model: Storage/position in lab: Serial No. Warranty expiration: Manufacturer: Tel. No. Address: Contact person: Tel. No. Table 1: Example of an identification and maintenance log. A regularly updated equipment maintenance log can help to; • Highlight trends like repair costs and equipment durability and efficiency. Therefore, helping lab managers to make decisions on equipment models and brands that are best suited for the lab. • Point out the equipment that undergoes wear and tear frequently. If the cause of malfunction is operation related, it can highlight the need for re-training of laboratory staff. 3. PREVENTIVE MAINTENANCE PROGRAM A preventive maintenance program ensures that the equipment is functioning with minimal interruptions and within the manufacturer’s specifications. It maximizes the equipment operational efficiency and reduces overall costs. It is mainly recommended for equipment with moving parts, gas or liquid flow, optical systems and filters. The maintenance and quality control is performed under an outlined schedule and results are documented. A preventive maintenance file should detail; • Error alerts on the equipment and subsequent action to be taken. • Basic troubleshooting when the equipment malfunctions. • Logs for error reports and failure events; see example in Table 2. • The servicing and calibration done on the equipment and the dates for subsequent calibrations. Stickers should be used for equipment labelling to summarize the preventive maintenance actions undertaken, the date, and the personnel involved. Failure Events Date Event Corrective action Operator Table 2: Example of a failure event log for a preventive maintenance program.
  9. 9. 4. TRAINING LABORATORY STAFF ON EQUIPMENT MAINTENANCE Training of both technical and managerial staff is not a onetime activity. It should be regular with additional courses given when new equipment or improved models are bought. The initial induction training should be elaborate with an expert-guided discussion and demonstration, while follow-up training can be done in-house to refresh the staff technique. The lab manager or lab quality control officer are responsible for ensuring all staff are well trained. For proper staff training on equipment care and maintenance; • Provide all necessary documentation including SOPs, maintenance logs, service manuals etc. • Ensure that the staff have, along with theoretical presentation, a practical on-the-job training on use and maintenance of lab equipment. • Train all staff on preventive maintenance, where they learn the general care of equipment like lubrication and checking for possible damage. • At the end of the training, a scoring system should be availed to evaluate the effectiveness of the training. 6. GENERAL CARE TIPS FOR LAB EQUIPMENT 1. Cleaning Regular cleaning of lab equipment ensures that it is ready for use when needed, that stubborn stains/substances do not get a firm hold, and that experiments are not contaminated by impurities carried over from previous experiments. Make certain that; • The equipment is always cleaned before and after each use. • Cleaning reagents and cleaning aids used are specific for laboratory equipment care. • In addition to cleaning lab equipment before and after each use, a schedule is required for more in-depth cleaning. This might involve dissembling certain machines to clean hard-to-reach parts. • Always follow instructions from the manufacturer on cleaning policy. Certain parts of the equipment might require very specific solvents, cleaning materials, or drying procedure. 2. Calibration Calibration involves comparing the measurements of an equipment against the standard unit of measure, for the purpose of verifying its accuracy and making necessary adjustments. Regular calibration of laboratory equipment should be done because over time, biases develop in relation to the standard unit of measure. This guards against invalid data and ensures safety during experimentation. An independent specialist, that can provide calibration certificates where necessary, should be engaged in the process. Calibration should be done when; • The recommended time by the manufacturer elapses. • The equipment is hit by a force, dropped on the ground, or involved in any accident or an event of safety concern. • There are unusual patterns or sounds while the equipment is in use. • Measurements obtained are questionable. • Highly critical measurements, where data accuracy is of utmost importance, are to be carried out. 3. Repairs and Refurbishments Lab equipment is generally costly and repairs and refurbishment prolong the lifespan of equipment, saving the lab the expense of new purchases. The following are points to consider; • Repair and/or refurbish faulty or worn out lab equipment without any delay. Faulty machines may stop working suddenly in the middle of an experiment leading to loses and they can also be a source of safety concerns. • Minor repairs can be done by a dedicated staff, while major repairs should be directed to specialist with knowledge on the specific machine or equipment. • Refurbish old equipment to give them a new lease of life by cleaning thoroughly, polishing where necessary, lubricating movable parts, and replacing small worn out bits.
  10. 10. 4. Quality Replacement Equipment that cannot be repaired or refurbished should be replaced. It is advisable to buy equipment from well known sources that can guarantee quality and offer technical support. High-quality lab equipment is easier to maintain and its durability translates to reduced costs in the long term. Non-faulty equipment that is too old should also be replaced, while some wear and tear might not be noticeable during its operation, outdated machines are not reliable and technical support in terms of servicing and acquisition of spare parts may be limited. The care and maintenance of laboratory equipment should be a routine and embedded within the standard operating procedure of the lab. This will ensure that the life span of the equipment is prolonged and data collected within the laboratory is reliable. Reference: 1. https://www.labce.com/spg572652_reagent_maintenance_and_temperature_checks.aspx 2. https://conductscience.com/laboratory-equipment-care-and-maintenance/ 3. https://www.mynewlab.com/blog/laboratory-equipment-maintenance-101/ 4. https://www.slideshare.net/drgomibasar/quality-control-in-clinical-laboratory
  11. 11. Course Name/ code Medical Laboratory Technician Lab-II / BMLT6052 Experiment No 2.1 Title Quality Control Faculty In charge Mr. A. Vamsi Kumar (Asst Prof) Department / School Medical lab technology, SMAS Semester VI 2.0 QUALITY CONTROL Introduction: Many tools are used for quality control like: 1. Procedure manuals. 2. Maintenance schedules. 3. Calibrations. 4. Quality assurance program. 5. Training. Quality control will be different for the different disciplines of pathology: 1. Quality assurance for the blood transfusion. 2. Quality assurance for the microbiology. 3. Quality assurance for the biochemistry. 4. Quality assurance for the surgical pathology. Quality control depends up on: 1. The time between the collection and the performance of the test e.g. 1. Leukocytes and RBCs utilize glucose and cause a steady decrease in the concentration of glucose. 2. Specimen storage also causes an error in the result. 3. Evaporation of the sample may cause the wrong result like electrolytes. 4. Exposure to light effect Bilirubin level. 5. Refrigeration will affect lactate dehydrogenase (LDH). 6. The clerical mistake may occur at any stage.
  12. 12. Analytic factors can be minimized which depends upon instrumentation and reagents. 1. A schedule of daily and monthly preventive maintenance is needed for each instrument. 2. Keep a check on water quality, power supply, calibration of electrical balance, and calibration of glassware and pipettes. 3. Reagents and kits should be dated when received and when opened. 4. Run new lots of the reagents with the old lot in parallel before being used for analysis. 5. The primary standard is the most highly purified substance. 6. The secondary standard is one whose concentration is determined by analysis and compared with the primary standard. 7. Post-analytic errors are due to the recording and reporting of the results. Quality control purpose is: 1. Monitor analytic process. 2. Determine analytic errors during analysis. 3. Prevent incorrect patient values. 1. Monitoring of the analytic values is compared with known standards and compared with their expected values. Analytic errors are separated into: 1. Random errors affect precision and are the basis for varying differences between repeated measurements. 2. Systematic error component indicates a constant difference either increased or decreased. This may be caused by: 1. Poorly made reagents or standards. 2. Instrumentations defects 3. Poorly written procedures. Quality control material, it should be: 1. Available in sufficient quantity. 2. It should be stable during a period of a minimum of one year. 3. Keep in small volumes. 4. Its concentration should vary minimally. 5. Their composition should not vary from the vial to vial. 6. Control material should be tested like the test sample. 7. External quality assessment: 1. This is the programme in which the specimens are subjected to other laboratories for the analysis and the results of the individual laboratory are compared. 8. Total quality management focuses on: 1. Customer. The users are doctors and nurses while customers are patients. 2. Management commitment. 3. Training of the workers. 4. Measurement through quality improved tools. • Quality improvement occurs when the problems are permanently eliminated. Problems arise from imperfect procedures which are 85 %. Remaining 15% problems needs action and performance improvements of individual employees. So the main problems are the management problems and management has the power to change the work process. Control of the preanalytical mistakes 1. Patients identification and labelling are very important. Barcode technology has reduced these mistakes which are common in the handwritten labels. 2. Keep the record of the sample received and then when the report is ready. 3. Check the request form and the test tube name and the tests requested. 4. Check the adequate amount of the sample. 5. Observe if there is haemolysis or lipemic serum.
  13. 13. 6. take the history food intake, alcohol, drugs, smoking, stress, sleep, and the posture because these factors may influence the result. 1. Explain all instruction to the patient for the collection of the sample. 7. Incorrect containers and incorrect preservatives will affect the result. 8. Transport of the sample is very important and may be critical to some of the tests. 9. Processing of the sample as separation of the serum, where centrifuge speed, temperature, and the person are important. Reference: • http://www.labtestsblog.com/quality-control-in-biochemistry-laboratory/ • https://www.labce.com/spg572652_reagent_maintenance_and_temperature_checks.aspx • https://conductscience.com/laboratory-equipment-care-and-maintenance/ • https://www.mynewlab.com/blog/laboratory-equipment-maintenance-101/ • https://www.slideshare.net/drgomibasar/quality-control-in-clinical-laboratory
  14. 14. Course Name/ code Medical Laboratory Technician Lab-II / BMLT6052 Experiment No 2.2 Title Quality Assurance Faculty In charge Mr. A. Vamsi Kumar (Asst Prof) Department / School Medical lab technology, SMAS Semester VI 2.2 QUALITY ASSURANCE Introduction: Quality Assurance in a biochemistry laboratory is intended to ensure the reliability of laboratory tests. The objective of quality assurance is to achieve reliable test results by: • Accuracy • Precision Accuracy This refers to the closeness of the estimated value to that considered to be true. Accuracy can, as a rule, be checked only by the use of reference materials which have been assayed by reference methods. Precision This refers to the responsibility of the result, but a test can be precise without being accurate. Precision can be controlled by replicate tests and by repeated tests on previously measured specimens. The test result or value which we get should be closer to the previous one. Inaccuracy and/or imprecision occur as a result of using unreliable standards or reagents, incorrect instrument calibration, or poor technique, e.g consistently faulty dilution or the use of a method that gives a reaction that is incomplete or not specific for the test. According to Edward Demming: Improved quality = increased productivity at a lower cost. This can be done by: • Eliminating re-work • Saving time • Saving labour • Saving material e.g. reagents, specimens etc. • Patient care. Quality Assurance Programme includes: • Internal quality control (IQC) • External quality assessment (EQC) • Proficiency surveillance • Standardization Internal quality control This is based on monitoring the biochemistry test procedure that is performed in the laboratory. It includes measurement on specially prepared materials and repeated measurements on routine specimens, as well as statistical analysis day-by-day of date obtained from the test which has been routinely carried out. There is thus continuous evaluation of the reliability of the work of the laboratory. Hence IQC primarily checks the precision of lab work. External quality assessment This is the evaluation by an outside agency of the performance of a number of laboratories on specially supplied samples. Analysis of performance is retrospective. The objective is to achieve lab and method compatibility, but this doesn’t guarantee accuracy unless the specimens have been assayed by a reference lab alongside a reference preparation of known value. Schemes are usually organized on a national or regional basis. Hence, EQA is mainly concerned with the analytical part of the test.
  15. 15. Proficiency surveillance This is concerned with various aspects of the laboratory, apart from analysis- this ensures adequate control of the pre- and post- analytical stages of test. It implies critical supervision of all the aspects of laboratory tests, such as: 1. Sample collection 2. Labelling 3. Delivering 4. Storage 5. Reading 6. Reporting 7. Establishment of normal reference values. 8. Maintenance and control of apparatus and instruments, etc. Standardization This refers to both materials and reference methods. A material standard or reference preparation is used to calibrate analytic instruments and to assign a quantitative value to calibrators. A reference method is an exactly defined technique which provides sufficiently accurate and precise data for it to be used to assess the validity of other methods. There are certain agencies/organization who look after these things. The material prepared by these authorities are international standards (international reference preparation) and are of primary standard. These international standards are not freely available and are not intended for routine use, but serve as standards for assigning values to commercial standard which is of secondary standard. Things that can go wrong: 1. Writing the order 2. Transcribing the order to lab 3. The requisition from floor to the lab 4. Collecting the sample (IQC, PS, and Std.) 5. Handling after collection (PS) 6. Running the test (predominantly to IQC) 7. Sending the data back to the floor 8. Putting the data on the chart Awareness in quality control can be divided into 2 groups: Primary Goals • To report out all correct data. • Not to report the incorrect patient values. Error Detection • To detect error before it leaves the lab. Errors in a quantitative system There are two types of errors in a quantitative system: • Random error • Systematic error Random Error (Inherit Error, Noise Error, Background Error) This is the error in which there will be variations in the test result /data on either side of the mean- in other words, the values obtained will be low as well as high to the mean value or true value. This may be due to: • Slight variations in line voltage, lamp output or temperature • Slight variations in pipettors and dispensers This error is measured by standard deviation (SD) and coefficient variation (CV). Systemic Error This is the error in which variation occurs in one direction away from the true value i.e. either value goes up or down. The difference between the measured value and true value is called Bias. Systemic errors are errors within the test system or methodology.
  16. 16. • Assigned value to calibrators • Reagent composition • Antibody specificity Six components of an error detection system 1. Patient identification, sample collection and handling 2. Analytical method 3. Instrument maintenance 4. Control material: In build error detecting test, it is run as per the test and its value is known so helps to detect whether our test is correct or not 5. Quality control monitoring 6. Clerical Quality control material It is a known sample whose range of values has been established prior to the test either by international authorities or by commercial firms. This control sample is inserted into the testing process, being exposed to the same condition as the patient sample and the value is measured. If the values of control material are within the range then it is said that the test procedure of the error detectors. Standard deviation If values are assigned to a same specimen a number of times by repeating the test, the dispersion of results around the mean will indicate the error of reproducibility. Deviation of values from the mean is called standard deviation. This gives an idea about precision (random error). 95% of results should be within ±2 SD. 99.7% of results should be within ±3 SD. To determine SD, 10- 20 identical tests are carried out on the same sample. Gaussian distribution It is a bell-shaped curve resulting from events or data which occur symmetrically about the mean when frequency and data are plotted. Extent of spread of measurement about mean is SD. Note: Mean gives an idea about the accuracy of result i.e. systematic error. Stewart Levey Jennings chart • Runs or days are plotted on x axis. • Values obtained from analytic run are plotted on Y axis. • Samples of control specimens are included in every batch of patient’s specimen. • Mean and standard deviation are then established. Satisfactory results are obtained only when: • Sequential results oscillate about the mean • Less than 5% of results will fall outside of ±2 SD Error If 2 or more results are outside ±2 SD: • Consecutive values may get increased or decreased • Consecutive values shouldn’t on one side of mean • Cumulative summethod (CUSUM) • For this 20 identical tests are run • Any slight error can be detected with CUSUM chart • Any change in the calibration will be detected as an alteration in the slope of this line Reference: http://www.labtestsblog.com/quality-control-in-biochemistry-laboratory/
  17. 17. Course Name/ code Medical Laboratory Technician Lab-II / BMLT6052 Experiment No 3 Title Sample formats for reporting test result Faculty In charge Mr. A. Vamsi Kumar (Asst Prof) Department / School Medical lab technology, SMAS Semester VI 3. SAMPLE FORMATS FOR REPORTING TEST RESULT Introduction: In order for medical professionals to know a patient’s progress or medical status, creating medical reports are what they need. A medical report is an updated detail of a medical examination of a certain patient. It is a vital written document that describes the findings of an individual or group of people. A medical report template should contain nothing but accurate and credible data. Elements of a typical laboratory report Despite the differences in format and presentation, all laboratory reports must contain certain elements as mandated by federal legislation known as the Clinical Laboratory Improvement Amendments (CLIA). (CLIA '88 REGULATIONS, Section 493.1291; for more on regulation of laboratories. Some items included on lab reports deal with administrative or clerical information: 1. Patient name and identification number or a unique patient identifier and identification number. These are required for proper patient identification and to ensure that the test results included in the report are correctly linked to the patient on whom the tests were run. 2. Name and address of the laboratory location where the test was performed. Tests may be run in a physician office laboratory, a laboratory located in a clinic or hospital, and/or samples may be sent to a reference laboratory for analysis. 3. Date report printed. This is the date this copy of the report was printed. Often, the time that the report was printed will also be included. The date of printing may be different than the date the results were generated (see below), especially on cumulative reports. This report is an example of a cumulative report which is a report that includes results of several different tests run on different days. 4. Test report date. This is the day the results were generated and reported to the ordering physician or to the responsible person. Tests may be run on a particular patient’s samples on different dates. Since a patient may have multiple results of the same test from different days, it is important that the report includes this information for correct interpretation of results. 5. Name of doctor or legally authorized person ordering the test(s). This information enables the lab to forward your results to the person who requested the test(s). Sometimes a report will also include the name of other health practitioners requesting a copy of your report. For example, a specialist may order tests and request that a copy of the results be sent to your primary healthcare provider. 6. Other elements found on reports deal with the specimen that was collected and with the test itself:
  18. 18. 7. Specimen source, when appropriate. Some tests can be performed on more than one type of sample. For example, protein can be measured in blood, urine or cerebrospinal fluid, and the results from these different types of specimens can indicate very different things. 8. Date and time of specimen collection. Some test results may be affected by the day and time of sample collection. This information may help your health practitioner interpret the results. For example, blood levels of drugs are affected by the time a dose of the drug was last taken, so results of the test and its interpretation can be affected by when the sample was collected. 9. Laboratory accession number. Number(s) assigned to the sample(s) when it arrives at the laboratory. Some labs will have a single accession number for all your tests and other labs may have multiple accession numbers that help the lab identify the samples. 10. Name of the test performed. Test names are often abbreviated on lab reports. You may want to look for abbreviated test names in the pull down menu on the home page of this site or type the acronym into the search box to find information on specific tests. 11. Test result. Some results are written as numbers when a substance is measured in a sample as with a cholesterol level (quantitative). Other reports may simply give a positive or negative result as in pregnancy tests (qualitative). Still others may include text, such as the name of bacteria for the result of a sample taken from an infected site. 12. Abnormal test results. Lab reports will often draw attention to results that are abnormal or outside the reference range (see "Reference intervals" below) by setting them apart or highlighting them in some way. For example, "H" next to a result may mean that it is higher than the reference range. "L" may mean "low" and "WNL" usually means "within normal limits." 13. Critical results. Those results that are dangerously abnormal must be reported immediately to the responsible person, such as the ordering physician. The laboratory will often draw attention to such results with an asterisk (*) or something similar and will usually note on the report the date and time the responsible person was notified. 14. Units of measurement (for quantitative results). The units of measurement that labs use to report your results can vary from lab to lab. It is similar to the way, for example, your health practitioner chooses to record your weight during an examination. He or she may decide to note your weight in pounds or in kilograms. In this same way, labs may choose to use different units of measurement for your test results. Regardless of the units that the lab uses, your results will be interpreted in relation to the reference ranges supplied by the laboratory. 15. Reference intervals (or reference ranges). These are the ranges in which “normal” values are expected to fall. The ranges that appear on your report are established and supplied by the laboratory that performed your test. They are made available to the health practitioner who requested the test(s) and to other health care providers to aid in the interpretation of the results. For more on this, see the article on Reference Ranges and What They Mean. 16. Interpretation of results. In certain circumstances, the lab may note on the report what certain test results may indicate. 17. Condition of specimen. Any pertinent information regarding the condition of specimens that do not meet the laboratory's criteria for acceptability will be noted. This type of information may include a variety of situations in which the specimen was not the best possible sample needed for testing. For example, if the specimen was not collected or stored in optimal conditions or if it was visually apparent that a blood sample was hemolyzed or lipemic, it will be noted on the report. In some cases, the condition of the specimen may preclude analysis (the test is not run and results are not generated) or may generate additional comments directing the use of caution in interpreting results.
  19. 19. 18. Deviations from test preparation procedures. Some tests have specific procedures to follow before a sample is collected or a test is performed. If such procedures are not followed for some reason, it may be noted on the report. For example, if a patient forgets to fast before having a glucose test performed, the report may reflect this fact. 19. Medications, health supplements, etc. taken by the patient. Some tests results are affected by medications, vitamins and other health supplements, so laboratories may obtain this information from the test request form and transcribe it onto the lab report. Reference: https://labtestsonline.org/articles/how-to-read-your-laboratory-report
  20. 20. Course Name/ code Medical Laboratory Technician Lab-II / BMLT6052 Experiment No 4 Title Demonstration of policies and procedures for infection control Faculty In charge Mr. A. Vamsi Kumar (Asst Prof) Department / School Medical lab technology, SMAS Semester VI 4. DEMONSTRATION OF POLICIES AND PROCEDURES FOR INFECTION CONTROL INTRODUCTION According to the Centers for Disease Control (CDC), on any given day in the U.S., one in 25 hospital patients has at least one hospital-acquired infection (HAI). All healthcare facilities take this issue seriously and, as a result, infections have decreased markedly since 2006. With the implementation of effective infection control policies, and the advancement of disease prevention solutions, there has been a 25% to 50% decrease in some of the most common types of HAIs: • Central line-associated bloodstream infections • Catheter-associated urinary tract infections • Surgical site infections (CDC). While it’s important that the right policies and procedures be implemented, it is equally important that policies are regularly promoted to maintain momentum. What’s more, having an automated process that enables staff to access policies at the point of care quickly and easily can empower staff to take charge of adhering to hospital policies. Often times, teaming up with your Quality and Risk Department(s) to promote a culture of safety is a key step that shouldn’t be overlooked, as creating a safe hospital is equally important to them as it is to you. 1. Hand Hygiene This one seems like a no-brainer, we all know that good hand hygiene can reduce the risk of flu, food poisoning, and other HAIs. But it’s worth mentioning again as it can’t be stressed enough that this is perhaps one of the most important policies to have in place for your healthcare organization. • If using soap: wash for 40-60 seconds, rub all areas with soap and use single use towel to rub dry. Don’t forget to use a towel to turn off the faucet and if you have to, open the door with it. • If using alcohol rub: use enough product to cover hands, rub until dry. To be more thorough, it may be helpful to include notes as to specify the instances and how often hands should be washed. 2. Wear Gloves Similar to hand hygiene, wearing gloves and properly disposing of them plays an important part in reducing the spread of HAIs. Be sure to mention the following in your policy: • Wear gloves when handling any body related materials, including blood, secretions, excretions, membranes, body fluids, etc. • Change gloves between tasks • Properly discard gloves after completion of task and perform proper hand hygiene protocol 3. Wear a Gown
  21. 21. It’s important to protect your skin and avoid soiling your clothing, as it is likely that clothing cannot be discarded if it should happen, and that’s where gowns come in. Wearing a gown is an easy way to not only ensure that your clothing lives to see another day away from the cleaners but as they can easily be removed and will help avoid the spread of infectious diseases. • Wear gowns in any instances where splashes or spraying of secretions, excretions, blood or bodily fluids might be present. • Remove gowns as soon as possible and perform hand hygiene. 4. Protect your Face Our faces are portals for infectious diseases to make their way into our bodies. This is why it is important to protect our eyes, nose, and mouth against splashes or sprays of blood, fluids, secretions, etc. 5. Prevention of needlestick injuries Just like hand hygiene policies are a no-brainer, this one may seem like one too. However, sometimes it’s good to just be reminded. Being around surgical instruments and needles can be pretty dangerous as well. Having a set of policies in place outlining how to handle instruments or dispose of needles may be the refresher some people need to stay safe. 6. Respiratory hygiene and cough etiquette Flu season isn’t the only time we should be reminding staff and patients about this policy. Having a set of instructions in place may be the gentle nudge in the right direction people need to keep others healthy. Beyond covering up your nose and mouth with tissues or a mask when coughing and sneezing and performing proper hand hygiene frequently, hospitals should: • Place patients with respiratory ailments away from common areas • Post signs alerting people entering these areas to take extra care and remind them to perform hand hygiene when leaving • Post signs instructing those with ailments of proper hygiene etiquette • Make preventative tools available for staff and patients to take advantage of 7. Regular cleaning Ensure policies are in a place where common areas and areas with infectious patients are regularly cleaned and frequently touched surfaces are disinfected once it is recognized that they have been infected. 8. Linens They may seem harmless, but the truth is they could be carrying the pathogens that we have been trying so hard to avoid. Creating a policy where staff must wear gloves, gowns, and facial protection when handling linens, as well as disposing of the protective clothing immediately after use can go a long way in protecting staff from contracting an HAI. 9. Waste Disposal Having a waste management policy in place is also a crucial procedure to ensure staff are aware of. Most importantly, your policy should include how to properly dispose of waste that has been contaminated with blood, human tissues, and bodily fluids safely. 10. Patient Care Equipment Often times the equipment used to care for patients can become soiled. Having a policy in place where staff must clean and disinfect this equipment regularly and upon recognizing that the equipment has been soiled is another small step in the right direction of infection control and prevention. Reference: 1. https://www.hepacart.com/infection-control 2. https://www.policymedical.com/10-must-infection-control-policies/
  22. 22. Course Name/ code Medical Laboratory Technician Lab-II / BMLT6052 Experiment No 5 Title Demonstration of mock diagnostic lab for learning & understanding patients right Faculty In charge Mr. A. Vamsi Kumar (Asst Prof) Department / School Medical lab technology, SMAS Semester VI 5. DEMONSTRATION OF MOCK DIAGNOSTIC LAB FOR LEARNING & UNDERSTANDING PATIENTS RIGHT Introduction: A medical laboratory or clinical laboratory is a laboratory where clinical pathology tests are carried out on clinical specimens to obtain information about the health of a patient to aid in diagnosis, treatment, and prevention of disease.[1] Clinical Medical laboratories are an example of applied science, as opposed to research laboratories that focus on basic science, such as found in some academic institutions. Medical laboratories vary in size and complexity and so offer a variety of testing services. More comprehensive services can be found in acute-care hospitals and medical centers, where 70% of clinical decisions are based on laboratory testing.[2] Doctors offices and clinics, as well as skilled nursing and long-term care facilities, may have laboratories that provide more basic testing services. Commercial medical laboratories operate as independent businesses and provide testing that is otherwise not provided in other settings due to low test volume or complexity. Departments In hospitals and other patient-care settings, laboratory medicine is provided by the Department of Pathology, and generally divided into two sections, each of which will be subdivided into multiple specialty areas. The two sections are: • Anatomic pathology: areas included here are histopathology, cytopathology, and electron microscopy. • Clinical pathology, which typically includes the following areas: o Clinical Microbiology: This encompasses several different sciences, including bacteriology, virology, parasitology, immunology, and mycology. o Clinical Chemistry: This area typically includes automated analysis of blood specimens, including tests related to enzymology, toxicology and endocrinology. o Hematology: This area includes automated and manual analysis of blood cells. It also often includes coagulation. o Blood Bank involves the testing of blood specimens in order to provide blood transfusion and related services. o Molecular diagnostics DNA testing may be done here, along with a subspecialty known as cytogenetics. o Reproductive biology testing is available in some laboratories, including Semen analysis, Sperm bank and assisted reproductive technology. The following is an example of a typical breakdown of the responsibilities of each area: 1. Microbiology includes culturing of clinical specimens, including feces, urine, blood, sputum, cerebrospinal fluid, and synovial fluid, as well as possible infected tissue. The work here is mainly concerned with cultures, to look for suspected pathogens which, if found, are further identified based on biochemical tests. Also,
  23. 23. sensitivity testing is carried out to determine whether the pathogen is sensitive or resistant to a suggested medicine. Results are reported with the identified organism(s) and the type and amount of drug(s) that should be prescribed for the patient. 2. Parasitology is where specimens are examined for parasites. For example, fecal samples may be examined for evidence of intestinal parasites such as tapeworms or hookworms. 3. Virology is concerned with identification of viruses in specimens such as blood, urine, and cerebrospinal fluid. 4. Hematology analyzes whole blood specimens to perform full blood counts, and includes the examination of Blood films. Other specialized tests include cell counts on various bodily fluids. 5. Coagulation testing determines various blood clotting times, coagulation factors, and platelet function. 6. Clinical Biochemistry commonly performs dozens of different tests on serum or plasma. These tests, mostly automated, includes quantitative testing for a wide array of substances, such as lipids, blood sugar, enzymes, and hormones. 7. Toxicology is mainly focused on testing for pharmaceutical and recreational drugs. Urine and blood samples are the common specimens. 8. Immunology/Serology uses the process of antigen-antibody interaction as a diagnostic tool. Compatibility of transplanted organs may also be determined with these methods. 9. Immunohaematology, or Blood bank determines blood groups, and performs compatibility testing on donor blood and recipients. It also prepares blood components, derivatives, and products for transfusion. This area determines a patient's blood type and Rh status, checks for antibodies to common antigens found on red blood cells, and cross matches units that are negative for the antigen. 10. Urinalysis tests urine for many analytes, including microscopically. If more precise quantification of urine chemicals is required, the specimen is processed in the clinical biochemistry lab. 11. Histopathology processes solid tissue removed from the body (biopsies) for evaluation at the microscopic level. 12. Cytopathology examines smears of cells from all over the body (such as from the cervix) for evidence of inflammation, cancer, and other conditions. 13. Molecular diagnostics includes specialized tests involving DNA analysis. 14. Cytogenetics involves using blood and other cells to produce a DNA karyotype. This can be helpful in cases of prenatal diagnosis (e.g. Down's syndrome) as well as in some cancers which can be identified by the presence of abnormal chromosomes. 15. Surgical pathology examines organs, limbs, tumors, fetuses, and other tissues biopsied in surgery such as breast mastectomies. Specimen processing and work flow. In a hospital setting, sample processing will usually start with a set of samples arriving with a test request, either on a form or electronically via the laboratory information system (LIS). Inpatient specimens will already be labelled with patient and testing information provided by the LIS. Entry of test requests onto the LIS system involves typing (or scanning where barcodes are used) in the laboratory number, and entering the patient identification, as well as any tests requested. This allows laboratory analysers, computers and staff to recognize what tests are pending, and also gives a location (such as a hospital department, doctor or other customer) for results reporting. Once the specimens are assigned a laboratory number by the LIS, a sticker is typically printed that can be placed on the tubes or specimen containers. This label has a barcode that can be scanned by automated analyzers and test requests uploaded to the analyzer from the LIS. Specimens are prepared for analysis in various ways. For example, chemistry samples are usually centrifuged and the serum or plasma is separated and tested. If the specimen needs to go on more than one analyzer, it can be divided into separate tubes. Many specimens end up in one or more sophisticated automated analysers, that process a fraction of the sample to return one or more test results. Some laboratories use robotic sample handlers (Laboratory automation) to optimize the workflow and reduce the risk of contamination from sample handling by the staff.
  24. 24. The work flow in a hospital laboratory is usually heaviest from 2:00 am to 10:00 am. Nurses and doctors generally have their patients tested at least once a day with common tests such as complete blood counts and chemistry profiles. These orders are typically drawn during a morning run by phlebotomists for results to be available in the patient's charts for the attending physicians to consult during their morning rounds. Another busy time for the lab is after 3:00 pm when private practice physician offices are closing. Couriers will pick up specimens that have been drawn throughout the day and deliver them to the lab. Also, couriers will stop at outpatient drawing centers and pick up specimens. These specimens will be processed in the evening and overnight to ensure results will be available the following day. Reference: 1. https://en.wikipedia.org/wiki/Medical_laboratory 2. https://en.wikipedia.org/wiki/Laboratory_information_management_system 3. https://explorehealthcareers.org/career/allied-health-professions/clinical-laboratory-scientist-technician/ 4. https://academic.oup.com/labmed/article/40/2/105/2504825 5. https://wiki.ihe.net/index.php/Laboratory_Testing_Workflow 6. http://www.jssbilaspur.org/diagnostic-laboratory/
  25. 25. Course Name/ code Medical Laboratory Technician Lab-II / BMLT6052 Experiment No 6 Title Demonstration of mock environment to learn and understand conducive patient environment Faculty In charge Mr. A. Vamsi Kumar (Asst Prof) Department / School Medical lab technology, SMAS Semester VI 6. DEMONSTRATION OF MOCK ENVIRONMENT TO LEARN AND UNDERSTAND CONDUCIVE PATIENT ENVIRONMENT Introduction: Each year, nearly 444,000 individuals die due to avoidable hospital errors. Fortunately, care providers, support staff, and consumers acting in unison can improve patient safety outcomes. Through safety focused team initiatives, organizations can improve team performance. Patient safety involves avoiding errors, limiting harm, and reducing the likeliness of mistakes through planning that fosters communication, lowers infection rates, and reduces errors. Care providers, patients, and support staff share the same goal; the best possible treatment outcome. The following seven principles outline tips that some health organizations implement to achieve this goal. 1: Establish a Safety and Health Management System The Assessment Tool for Hospitals, published by the Occupational Safety and Health Administration (OSHA), suggests that care providers should formulate guidelines that determine enterprise safety and health management system performance. [1] To encourage compliance with safety protocols, it is important that administrators include all managers and employees in appropriate decision-making processes and perform regular organizational performance reviews. Regular reviews provide a dynamic indicator of whether an organization has achieved intended outcomes. Furthermore, administrators can use this information to adjust organizational policies as needed. 2: Build a Rapid Response System To aid organizations in planning rapid response systems (RRSs), the Agency for Healthcare Research and Quality (AHRQ) has developed TeamSTEPPS™ ((Team Strategies and Tools to Enhance Performance and Patient Safety)), Rapid response teams (RRTs) comprise one vital part of an RRS. The AHRQ suggests that health organizations determine the overall RRS framework using STEP Assessment: Status of the patient Team members Environment Progress toward goal TeamSTEPPS™ also outlines appropriate decision-making models for varying scenarios, such as Failure Modes and Effect Analysis (FMEA), Probabilistic Risk Assessment (PRA), and Root Cause Analysis (RCA). 3: Make Sure That Employees Know and Understand Safety Policies Employees and employers must understand their roles in organizational safety. [1] In addition to training each new employee about hospital safety, administrators should update staff members regularly about related policy changes.
  26. 26. Additionally, employees must understand the duties involved with upholding patient safety. Furthermore, every medical organization should clearly outline safety policies and procedures. Employees must feel safe to voice concerns. Therefore, along with a clearly outlined procedure for managing and reporting issues, effective safety training includes reassurance that administrators will receive information with impartiality. 4: Develop a Safety Compliance Plan Hospital administrators continually monitor and evaluate how employees follow established policies. Institutional governing boards and boards of directors use this information to adjust organizational policies as needed. [3] Compliance programs benefit health organizations in many ways, including but not limited to: ● Building community trust as a responsible organization ● Developing compliance standards suitable for the community and organization ● Establishing a framework to evaluate employee and vendor compliance ● Maintaining insurance claim integrity ● Mitigating or eliminating illegal activity ● Promoting positive treatment outcomes ● Providing a centralized compliance outlet By developing and maintaining a safety compliance plan, organizations—small and large—promote safe treatment environments. 5: Practice Patient-Centered Care Patient-centered care is a hot topic among debates about service quality. [4] Health administrators, hospital media communication, and legislators use the catch phrase often. In fact, insurers linked payouts, in part, to the degree that care facilities adopted patient-centered care well before the implementation of the Affordable Care Act. In the past, health advocates worried that the philosophy might undermine efforts to provide evidence-based treatments. Today, however, evidence-based treatment supporters view patient-centered care as a critical framework for establishing and promoting desired wellness outcomes. 6: Communicate Safety Information to Patients Historically, consumers played a passive role in their recoveries and, with vague comprehension, followed treatment plans unquestioningly. [5] In this environment, patients placed absolute trust in care providers. Today, however, practitioners understand that educated patients can assist in reducing medical errors. Additionally, with the wealth of information available online, it is important that patients understand what health-related facts apply to their unique circumstances. Contemporary patients increasingly participate in their own recovery planning. As educated consumers, they receive safer treatment, because care providers and health advocates have empowered them with the ability to ask the right questions and notice potential problems. 7: Incorporate Safe Hospital Design Traditional hospital design focused on operational efficiency rather than patient safety, designating interconnected work areas in close proximity. [6] However, patient-centered building design includes structural characteristics such as air quality, critical information proximity, noise dampening, and standardized feature locations, as well as fixtures that reduce contagion spread, such as employee hand sinks, in all treatment areas. Additionally, engineers design modern hospitals with wiring that supports advanced technology that reduces errors, with extra emphasis placed on areas designated as drug dispensaries. Most importantly, safe building designs incorporate planning to measure and benchmark facility conditions and characteristics, such as ease of information access, noise levels, scalability, and other factors.
  27. 27. Patients, employees, and administrators can eliminate most hospital errors by working as a team. However, it takes planning, commitment and work to maintain a safe hospital environment. Reference: 1. https://online.regiscollege.edu/blog/7-tips-ensuring-patient-safety-healthcare-settings/ 2. http://www.healinghealth.com/wp/wp-content/uploads/2013/07/susan-mazer-patient-safety.pdf
  28. 28. Course Name/ code Medical Laboratory Technician Lab-II / BMLT6052 Experiment No 7 Title Collection and handling of specimen for histopathology/cytopathology examination Faculty In charge Mr. A. Vamsi Kumar (Asst Prof) Department / School Medical lab technology, SMAS Semester VI 7. COLLECTION AND HANDLING OF SPECIMEN FOR HISTOPATHOLOGY/CYTOPATHOLOGY EXAMINATION Introduction: (Histopathology) Definition: Histopathology is a branch of pathology which deals with the study of disease in a tissue section. The tissue undergoes a series of steps before it reaches the examiners desk to be thoroughly examined microscopically to arrive at a particular diagnosis. To achieve this, it is important that the tissue must be prepared in such a manner that it is sufficiently thick or thin to be examined microscopically and all the structures in a tissue may be differentiated. The objective of the subsequent discussions will be to acquaint the staff with their responsibility; the basic details of tissue handling, processing and staining. Responsibility of a technician The technician is responsible for 1. Specimen preservation. 2. Specimen labeling, logging and identification. 3. Preparation of the specimen to facilitate their gross and microscopy. 4. Record keeping. To obtain these aims the following points need consideration. 1. As soon as the specimen is received in the laboratory, check if the specimen is properly labeled with the name, age, Hospital Registration No. and the nature of tissue to be examined and the requisition form is also duly filled. 2. Also check if the specimen is in proper fixative. Fixative should be fifteen to twenty times the volume of the specimen add fixative if not present in sufficient amount. 3. Check if the financial matters have been taken care off. 4. Make the entries in biopsy register and give the specimen a pathology number called the accession number. Note this number carefully on the requisition form as well as the container. This number will accompany the specimen every where. 5. If the specimen is large inform the pathologist who will make cut in the specimen so that proper fixation is done. Container should be appropriate to hold the specimen without distorting it.
  29. 29. 6. Blocks of tissues taken for processing should be left in 10% formalin at 60°C till processing. These would be fixed in 2 hours. 7. Slides should be released for recording after consultation with the pathologist. 8. Specimens should be kept in their marked container and discarded after checking with pathologist. 9. Block must be stored at their proper number the same day. Note the blocks have to be kept preserved for life long. Slides should be stored in their proper number after 3 days. It gives time for the slides to be properly dried
  30. 30. Handling & Processing of Cytopathology specimens: Cytology is the study of body cells that are either exfoliated spontaneously from epithelial surfaces or are obtained from various body tissues and organs by different techniques. Accordingly, cytology has following branches: A. Exfoliative cytology B. Aspiration cytology C. Imprint cytology I. EXFOLIATIVE CYTOLOGY This is the study of cells which are spontaneously shed off from epithelial surfaces into body cavities or into fluid. The cells can also be obtained by scraping, brushing or wash of body surfaces. The principle of this technique is that there is increased rate of exfoliation of cells in diseased states. APPLICATIONS OF EXFOLIATIVE CYTOLOGY Exfoliative cytology is applied in diagnosing diseases of the following: 1. Female genital tract 2. Respiratory tract 3. Gastrointestinal tract 4. Urinary tract 5. Body fluids (pleural, peritoneal, pericardial, CSF and semen) 6. Buccal smears for sex chromatin 1. Female Genital Tract Smears from female genital tract are known as ‘Pap smears’. These smears are prepared by different methods depending upon the purpose for which they are intended: i. Cervical smear is obtained by Ayre’s spatula from portio of the cervix by rotating the spatula through 360° to sample the entire cervix. The scraped material is placed on a clean glass slide and smear prepared. It is ideal for detection of cervical carcinoma. ii. Lateral vaginal smear (LVS) is obtained by scraping upper third of lateral walls of the vagina and is ideal for cytohormonal assessment. iii. Vaginal pool smear is obtained by aspirating material from posterior fornix of vagina and is done for detecting endometrial and ovarian carcinoma. Combined (fast) smears are a combination of vaginal pool and cervical scrapings and are used for routine population screening. 2. Respiratory Tract Material from respiratory tract may be obtained during bronchoscopic procedures as expectorant (sputum), or by brushing (BB), by washing (BW) and bronchioalveolar lavage (BAL). Sputum examination is advantageous as samples are easily obtained and cellular content is representative of entire respiratory tract. At least three samples of sputum, preferably early morning samples, should be examined. 3. Gastrointestinal Tract Lesions in the oral cavity can be sampled by scraping the surface with a metallic or wooden spatula. Samples can be obtained from the oesophagus, stomach, small and large intestine either by brushing or lavage during fibreoptic endoscopy. 4. Urinary Tract Samples from lesions in the urinary tract are either urinary sediment examined from voided urine/catheterised urine or washings of the urinary bladder obtained at cystoscopy.
  31. 31. 5. Body Fluids Fluid from pleural, peritoneal or pericardial cavity is obtained by paracentesis. At least 50-100 ml of fluid is aspirated. The sample is examined fresh but if delay is anticipated then fluid should be anticoagulated either in EDTA 1 mg/ml or 3.8% sodium citrate 1 ml/10 ml. Fluid should be centrifuged and smears are prepared from the sediment. If amount of fluid is less (less than 1 ml), then it can be subjected to cytospin centrifuged smear preparation. FIXATION OF SMEARS IN EXFOLIATIVE CYTOLOGY Methods of fixation depend upon type of staining employed. Pap smears are wet-fixed (i.e. smears are immersed in fixative without allowing them to dry). Smears to be stained by Romanowsky stains are air-dried as fixation is affected during the staining procedure. Fixative used is either equal parts of ether and 95% ethanol, or 95% ethanol alone, 100% methanol, or 85% isopropyl alcohol. Fixation time of 10-15 minutes at room temperature is adequate. Smears may be left in fixative for 24 hours or more. Smears should be transported to the laboratory in fixative solution in coplin jars. II. ASPIRATION CYTOLOGY In this study, samples are obtained from diseased tissue by fine needle aspiration (FNA). APPLICATIONS OF FNA FNA is applied for diagnosis of palpable as well as nonpalpable lesions. I. Palpable Mass Lesions in 1. Lymph nodes 2. Breast 3. Thyroid 4. Salivary glands 5. Soft tissue masses 6. Bones II. Non-Palpable Mass Lesions in 1. Abdominal cavity 2. Thoracic cavity 3. Retroperitoneum
  32. 32. Procedure for FNA Materials. For performing FNA, a Franzen’s handle, syringe with needles, clean glass slides and suitable fixative are required Method No anaesthesia is required. Ask the patient to lie down in comfortable position exposing the target area. Palpate the target area. Clean the overlying skin with spirit. Fix 10/20 ml disposable syringe in Franzen’s handle. Insert 20-25 gauge disposable needle into syringe. Fix the mass by palpating hand and insert needle into target area(A). Apply suction while moving needle back and forth within the lesion and change the direction of the needle. (B) Terminate the aspiration when aspirated material or blood is visible at the base/hub of the needle. Release the suction before withdrawing the needle to equalise pressure within the syringe (D) After withdrawal of needle, apply pressure for 2-3 minutes at the site of puncture to arrest bleeding and prevent haematoma formation. Aspirated material from the needle is expressed on to clean glass slides by first detaching the needle and filling the syringe with air and expressing it with pressure. Smears are prepared as for blood smears. If the material is semi-solid, it is first crushed by gentle pressure with a glass slide and smears prepared.
  33. 33. III. IMPRINT CYTOLOGY In imprint cytology touch preparations from cut surfaces of fresh unfixed surgically excised tissue are prepared on clean glass slides. These are fixed, stained and examined immediately. It is considered complementary to frozen section. APPLICATIONS OF IMPRINT CYTOLOGY Imprint cytology is useful in following situations: i. Lymph node biopsy ii. Surgically resected tumours ADVANTAGES OF IMPRINT CYTOLOGY i. Tissue and cell architecture is retained ii. Useful as an intraoperative pathologic consult Reference: 1. Text book of Medical Laboratory Technology by Godkar 2. Introduction to Medical laboratory technology by J. Baker, R.E. Silverstone 3. Anatomy for Nurses By Asha Latha 4. Hand book of Health care quality & patient safety 5. Damodaran K, Practical Biochemistry 6. Textbook of Parasitology by CP. Baveja 7. Textbook of Microbiology By Presscott 8. Textbook of Pathology by Harshamohan 9. Textbook of Immunology by S.K gupta 10. Textbook of Haematology & Clinical pathology by Ram das naik
  34. 34. Course Name/ code Medical Laboratory Technician Lab-II / BMLT6052 Experiment No 8 Title Demonstration of working of Microtome Faculty In charge Mr. A. Vamsi Kumar (Asst Prof) Department / School Medical lab technology, SMAS Semester VI 8. DEMONSTRATION OF WORKING OF MICROTOME Introduction: Microtomes These are mechanical devices for cutting uniform sections of tissue of appropriate thickness. All microtomes other than those used for producing ultra thin sections for election microscopy depend upon the motion of a screw thread in order to advance the tissue block on knife at a regulated number of microns. Motion of screws can be direct or through system of gears or levers to magnify the movement. Types of microtome 1. Hand microtomes – limited for use in botanical sections 2. Rocking microtome 3. Rotary microtome 4. Freezing microtome 5. Base sledge microtome 6. Vibrating knife microtome Procedure of Section cutting of paraffin embedded tissue Fixing of block 1. Fix the block in the block holder on the microtome knife in such as position that it will be clear of the knife when it is in position, block may be fixed directly or it may be fixed to a metal carrier which in turn is fixed to the microtome. 2. Insert the appropriate knife in the knife holder and screw it tightly in position. Adjust if required. The clearance angle should be set at 3-4 degree and angle of slope should be set permanently at 90 degree. It is important to tighten the knife clamp screw securely and block clamp screws most also be firm. ( The exposed ends of the knife must all the times be protected by magnetic or clip on knife guards to avoid any accidents.) 3. Trimming of tissue block : Move the block forward so that he wax block is almost touching the knife. To trim away any surplus wax and to expose a suitable area of tissue for sectioning, the section thickness adjusters are set at 15 microns. 4. On exposing a suitable area of tissue the section thickness is set to the appropriate level for routine purposes to 4-6 microns.
  35. 35. 5. Apply ice to the surface of the block for a few seconds and wipe the surface of block free of water. This step is optional but makes sections cut easily. 6. Note that the whole surface of the block will move parallel to the edge of the knife in order to ensure a straight ribbon of sections. 7. The microtome is now moved in an easy rhythm with right hand operating the microtome and left hand holding the sections away from the knife. The ribbon is formed due to the slight heat generated during cutting, which causes the edges of the sections to adhere. If difficulty is experienced in forming the ribbon it is sometimes overcome by rubbing one of the edges of the block with finger. 8. During cutting the paraffin wax embedded sections become slightly compressed and creased. Before being attached to slides the creases must be removed and the section flattened. This is achieved by floating them on warm water. Thermostatically controlled water baths are now available with the inside coated black. These baths are controlled at a temperature 4-6ºC below the melting point of paraffin wax. It is easy to see creases if the inside of water bath is black. 9. The action in floating out must be smooth with the trailing end of ribbon making contact with water first to obtain flat sections with correct orientation, floating out with the shiny surface towards the water is essential. When the ribbon has come to rest on water the remaining wrinkles and folds are removed by teasing apart by using forceps or seeker. 10. Picking up sections – The ribbon of sections floating on water is split into individual or groups of sections by use of forceps or seekers. Picking up a section on slide is achieved by immersing the slide lightly smeared with adhesive vertically to three fourths of its length bringing the section in contact with the slide. On lifting the slide vertically from the water, the section will flatten on to the slide. The sections are then blotted lightly with moistened blotting paper to remove excess water and to increase contact between section and slide. For delicate tissues or when several ribbons of sections are placed on the slide, omit the blotting instead keep the slide in upright position for several minutes to drain. 11. Drying of section : Sections are then kept in incubator with a temperature 5-6ºC above the melting point of wax i.e. at 60ºC for 20- 60 minutes. It is better to overheat than underheat. If the sections are not well dried they may come off during staining. The sections should not be allowed to dry without a good contact with the slide ,such sections will come off during staining.
  36. 36. TROUBLE SHOOTING FOR POOR SECTIONS There are times when proper section cannot be cut. Main reasons are either: 1. Faults occurring during section cutting or 2. Faults due to poor processing. Below are given the various defects, reasons for the defect and the remedy for the same. 1. Faults occurring during section cutting S.No Fault Cause Remedy 1. Tear or scratch across the section or splitting of ribbon. ➢ Jagged knife edge ➢ Dirt or hair on knife edge. ➢ Sharpen the knife ➢ Clean the knife 2. Tear or scratch across part of section ➢ Calcium, Carbon, or Suture etc., in the tissue or wax ➢ Examine block under magnifying glass. If calcium is present, decalcify block. Remove suture from the tissue with scalpel point. If dust is in wax - Re-embed 3. Holes in the section. ➢ Air bubbles in the tissue or wax ➢ A piece of hard material in tissue ➢ A soft piece of tissue in block ➢ Re-embed ➢ Remove hard material if possible ➢ Reprocess specimen 4. Cracks across the section parallel to knife ➢ A blunt knife ➢ Knife tilt too small. ➢ Block too hard for thickness of specimen ➢ Sharpen knife ➢ Adjust tilt ➢ Warm block slightly or re-embed in soft wax. 5. Section shows thin and thick horizontal lines (chatters) ➢ A loose knife ➢ A loose block ➢ A blunt knife ➢ Extremely hard tissue ➢ Tighten knife and/or block ➢ Sharpen the knife ➢ Soften the tissue if possible or embed in harden wax. 6. section cut thick and thin alternative ➢ Knife tilt is too great and is compressing the block ➢ Adjust tilt. 7. Section compress at one end. ➢ Blunt spot on the knife ➢ A soft spot in the wax, due to presence of clearing agent ➢ Move block along the knife or sharp knife. ➢ Re infiltrate tissue and re-embed 8. Section curves to one end. ➢ Edge of block is not parallel to knife. ➢ A dull spot on knife. ➢ Trim edges ➢ Move block along knife or sharpen knife. 9. Section curl as the they are cut ➢ Blunt knife ➢ Sections too thick ➢ Too much tilt to knife ➢ Sharpen knife ➢ Adjust microtome ➢ Correct the tilt 10. Sections lift from knife on upward travel of block ➢ Blunt knife ➢ Too much tilt to knife ➢ A build up of wax debris behind knife ➢ A greasy knife. ➢ Sharpen knife ➢ Correct the tilt ➢ Clean the knife
  37. 37. 11. Knife bites deeply into block ➢ A loose knife ➢ A loose block ➢ Tighten the knife and block 12. The block no longer feeds towards knife ➢ Forward feed mechanism had expired ➢ Release the safety locking catch, man back off feed ➢ mechanism and readjust knife holder 13. Sections crumble on cutting ➢ Knife is blunt ➢ Wax is too soft; has crystallized due to slow cooling or ➢ contamination with water or clearing agent. ➢ Defective processing e.g. incomplete fixation, dehydration, clearing or embedding. ➢ Sharpen knife. ➢ Re-embed and block with fresh wax ➢ Reprocess 14. Failure of block to ribbon ➢ Block not parallel to ribbon ➢ Paraffin too hard. ➢ Knife tilted too much ➢ Sections too thick ➢ Correct the alignment ➢ Re-embed ➢ Correct the tilt ➢ Adjust the section thickness 2. Faults due to poor processing. S.No Fault Cause Remedy 1. The tissue is shrunken away from wax ➢ Insufficient dehydration ➢ Reprocess 2. The tissue is too soft when block is trimmed ➢ Insufficient fixation ➢ Reprocess 3. Specimen crumbles and drops out of the wax leaving a rim of wax as a section ➢ Insufficient infiltration ➢ Overheated paraffin bath causing tissue to become hard ➢ and brittle ➢ Re infiltrate and re-embed ➢ Service the paraffin bath 4. Tissue is dried out or mummified ➢ Mechanical failure of tissue processing machine or a ➢ basket was out of balance and hung up. ➢ Place the specimen in the following rehydration solution ➢ for 18-24 hrs. ➢ Sodium Carbonate - 1.0 gm ➢ Dist. Water - 70.0 ml ➢ Absolute ethyl alcohol - 30.0 ml ➢ Re hydrate the reprocess Reference: 1. http://www.hh.um.es/pdf/Supplements/Suppl%201,%202011.pdf 2. Textbook of Pathology by Harshamohan
  38. 38. Course Name/ code Medical Laboratory Technician Lab-II / BMLT6052 Experiment No 9 Title Demonstration of sharpening methods of microtome knife Faculty In charge Mr. A. Vamsi Kumar (Asst Prof) Department / School Medical lab technology, SMAS Semester VI 9. DEMONSTRATION OF SHARPENING METHODS OF MICROTOME KNIFE Introduction: Microtome Knives : The knife is probably the greatest single factor in producing good sections. Types of microtome knives : Microtome knives are classified by the manner in which they are ground and seen in their cross section. 1. Plane wedge 2. Plano concave 3. Biconcave 4. Tool edge 1. Plane wedge : It is used for paraffin and frozen sections. 2. Plano concave: used for celloidin section since the blade is thin it will vibrate when for used for other harder materials. 3. Biconcave : It is recommended for paraffin section cutting on rocking and sledge type of microtome. 4. Tool edge : This is used with a heavy microtome for cutting very hard tissues like undecalcified bone. General description In the description of knives the expressions “Heel” and “Toe” are used to indicate to indicate which end of the cutting edge is referred to. The heel of the knife is the angle formed by the cutting edge and the end of the knife nearest to handle. The “toe” of knife is the angle formed by the cutting edge and the end of the knife farthest from the handle. Sharpening of microtome knives The cutting edge of an ideal microtome knife is a straight line formed by intersection of 2 planes, the cutting facets. The angle between the planes is called the bevel angle and is greater than the wedge angle between the sides of knife. The standard microtome knife has a wedge angle of approximately 15º and bevel angel varying between 27 and 32º. I. HONING Definition - Grinding of knife on a hone to restore straight cutting edge and correct bevel. There are various types of hones 1. Belgian black vein or Belgian yellow It is a yellow stone ½ inch thick and is backed with a black stone of
  39. 39. same thickness. Only yellow side should be used for honing. It is the best hone. It is quite a fast hone and may be used for coarse grinding and finishing. 2. Arkansas – Not very fast. 3. Aloxide – Fairly fast but coarse and not good for finishing a knife. 4. Carborundum – These hones can be obtained in a variety of grades only the finest of which should be used that too for coarse work. 5. Plate glass – May be used as a hone by applying an abrasive such as aluminium oxide to the surface and then using in the same way as ordinary hone. The advantage of such a hone is that it can be used for all types of honing by changing the abrasive powder. Lubricants for hone 1. Soap water 2. Liquid paraffin 3. Castor oil 4. Clove oil Method of honing 1. The hone is placed on a bench on a non skid surface. 2. A small quantity of light lubricant oil is poured on the centre of the hone and lightly smeared over the surface. 3. The knife complete with handle and backing sheath is laid on the hone with the cutting edge facing away from the operator, and the heel in the centre of the nearest end of hone. Correct positioning of the fingers is achieved by holding the handle of the knife between the thumb and forefinger with the cutting edge facing away from the operator (so that the thumb in on the back). When the knife is on the hone the tips of finger and thumb of other hand rest on the other end of knife ensuring even pressure along the whole edge of knife during honing. 4. The knife is pushed forward diagonally from heel to toe, turned over on its back and moved across the hone until the heel is in the centre with the cutting edge leading, and then brought back diagonally. It is turned to its original position, thus completing figure of 8 movement. 5. The process is continued until all jagged edges have been removed. The knife is ready for stropping. II. STROPPING Definition : It is the process of polishing an already fairly sharp edge. It removes burrs formed during honing.
  40. 40. Fine quality leather is used leather strops may be either flexible / hanging or rigid. In stropping usually firm surface is preferred. Action is reverse of honing toe to heel direction of stropping is also opposite. Assessment of the sharpened knife edge. Examine the edge the knife by reflected light and under microscope to assess the honing and stropping. Automatic microtome knife sharpeners There are many automatic knife sharpeners available Shandon type is most commonly used which consists of a glass plate on which fairly coarse abrasive powder like alumina powder is applied. First matting is done followed by lapping to remove all finer scratches. In all stages of use of abrasive powder care must be taken to remove by thorough washing any traces of abrasive powder from both knife and plate. Reference: 1. Text book of Medical Laboratory Technology by Godkar 2. Introduction to Medical laboratory technology by J. Baker, R.E. Silverstone 3. Anatomy for Nurses By Asha Latha 4. Hand book of Health care quality & patient safety 5. Damodaran K, Practical Biochemistry 6. Textbook of Parasitology by CP. Baveja 7. Textbook of Microbiology By Presscott 8. Textbook of Pathology by Harshamohan 9. Textbook of Immunology by S.K gupta
  41. 41. Course Name/ code Medical Laboratory Technician Lab-II / BMLT6052 Experiment No 10 Title Demonstration of tissue processing Faculty In charge Mr. A. Vamsi Kumar (Asst Prof) Department / School Medical lab technology, SMAS Semester VI 10. DEMONSTRATION OF TISSUE PROCESSING
  42. 42. Reference: Textbook of Pathology by Harshamohan
  43. 43. Course Name/ code Medical Laboratory Technician Lab-II / BMLT6052 Experiment No 11 Title Demonstration of PAP staining Faculty In charge Mr. A. Vamsi Kumar (Asst Prof) Department / School Medical lab technology, SMAS Semester VI 11. DEMONSTRATION OF PAP STAINING Introduction: Papanicolaou stain (also Papanicolaou’s stain or PAP stain) is the most important stain utilized in the practice of Cytopathology. It is a polychromatic stain containing multiple dyes to differentially stain various components of the cells. This technique was developed by George Papanicolaou, the father of Cytopathology. This method is used to differentiate cells in the smear preparation of various gynecological specimens (pap smears), materials containing exfoliative cells and material from fine needle aspiration. OBJECTIVES OF PAPANICOLAOU STAIN Papanicolaou described three chief objectives for staining of cytological smears: • Definition of nuclear details : Because of the widespread nuclear abnormalities of cancer cells and their diagnostic significance, good staining of the nucleus is of primary importance. • Transparency of cytoplasm: This is of particular importance because of the varying thickness and the frequent overlapping of cells. • Differentiation of cells: Differences in the staining reaction such as that between acidophilic and basophilic cells help greatly in the identification of certain cell types found in smears. PRINCIPLE OF PAPANICOLAOU STAIN Papanicolaou stain includes both acidic and basic dyes. Acidic dye stains the basic components of the cell and basic dye stain the acidic components of the cell. The polychromatic PAP stain involves five dyes in three solutions. 1. Hematoxylin : Natural dye hematoxylin is the nuclear stain which stains cell nuclei blue. It has affinity for chromatin, attaching to sulphate groups on the D.N.A. molecule. Harris’ hematoxylin is the commonest cytologically although Gills’ hematoxylin and Hematoxylin S can be used. 2. Orange Green 6 : This is the first acidic counterstain (cytoplasmic stain) which stains matured and keratinized cells. The target structures are staine d orange in different intensities. 3. Eosin Azure : This is the second counterstain which is a polychrome mixture of eosin Y, light green SF and Bismarck brown. Eosin Y gives a pink colour to cytoplasm of mature squamous cells, nucleoli, cilia and red blood cells. Staining solutions commonly used in cytology are EA 31 and EA 50, while EA 65 Light green SF stains blue to cytoplasm of metabolically active cells like parabasal squamous cells, intermediate squamous cells and columnar cells. Bismarck brown Y stains nothing and sometimes it is often omitted.
  44. 44. PROCEDURE OF PAPANICOLAOU STAINING Both progressive and regressive nuclear staining techniques can be used in Papanicolaou stain. Before staining, Wet fixation immediately with Cytology spray fixative 96% ethanol for minimum 30 min is required. I. Procedure of Progressive Papanicolaou Staining Method In the progressive method, the nucleus is stained with hematoxylin to a intensity desired. The intensity of the nuclear staining is controlled by the immersion of the slide into a blueing agent. Most commonly used blueing agent is Sott’s tap water (pH 8.02). Step Reagent Time 1. 95% Alcohol (Fixation) 15-30 minutes 2. 80% Alcohol 2 minutes 3. 60% Alcohol 2 minutes 4. Distilled Water 5 dips 5. Distilled Water 5 dips 6. Hematoxylin stain 3 minutes 7. Distilled Water 3 minutes 8. 60% Alcohol 2 minutes 9. 80% Alcohol 2 minutes 10. 95% Alcohol 2 minutes 11. Orange G Stain 3 minutes 12. 95% ALcohol 2 minutes 13. 95% Alcohol 2 minutes 14. Eosin Azure Stain 3 minutes 15. 95% Alcohol 2 minutes
  45. 45. 16. 95% Alcohol 2 minutes 17. 95% Alcohol 2 minutes 18. 95% Alcohol 2 minutes 19. Absolute Alcohol 2 minutes 20. Absolute Alcohol 2 minutes 21. Absolute Alcohol 2 minutes 22. Absolute Alcohol+Xylene (1:1) 2 minutes 23. Xylene 2 minutes 24. Xylene 2 minutes 25. Xylene Till clear 26. Mount in D.P.X II. Procedure of Regressive Papanicolaou Staining Method When using the regressive staining method, the nucleus is deliberately over-stained with a non-acidified haematoxylin. The excess stain is removed with dilute hydrochloric acid solution (acid water). The decolourising process is then stopped by immersing the slide in running tap water. Timing is crucial in the regressive method as de- staining may lead to a hyperchromatic nucleus becoming hypochromatic. Step Reagent Time 1. 90% Alcohol (Fixation) 15-30 minutes 2. 80% Alcohol 2 minutes 3. 60% Alcohol 2 minutes 4. Distilled Water 5 dips 5. Distilled Water 5 dips 6. Hematoxylin stain 3 minutes 7. Distilled Water 10 seconds 8. 1% Acid Alcohol 10 seconds (1 dip) 9. Distilled Water 10 seconds 10. Scott’s Tap Water 2-3 minutes 11. Running Tap Water 2 minutes 12. 60% Alcohol 2 minutes 13. 80% Alcohol 2 minutes 14. 95% Alcohol 2 minutes 15. Orange G Stain 3 minutes 16. 95% ALcohol 2 minutes 17. 95% Alcohol 2 minutes 18. Eosin Azure Stain 3 minutes 19. 95% Alcohol 2 minutes 20. 95% Alcohol 2 minutes 21. 95% Alcohol 2 minutes 22. 95% Alcohol 2 minutes 23. Absolute Alcohol 2 minutes 24. Absolute Alcohol 2 minutes 25. Absolute Alcohol 2 minutes 26. Absolute Alcohol+Xylene (1:1) 2 minutes 27. Xylene 2 minutes 28. Xylene 2 minutes 29. Xylene Till clear 30. Mount in D.P.X
  46. 46. 60 RESULTS AND INTERPRETATION OF PAPANICOLAOU STAINING 1. Nuclei : Blue 2. Acidophilic cells : Red 3. Basophilic cells : Blue Green 4. Erythrocytes : Orange-red 5. Keratin : Orange-red 6. Superficial cells : Pink 7. Intermediate and Parabasal Cells : Blue Green 8. Eosinophil : Orange Red 9. Candida : Red 10. Trichomonas : Grey green
  47. 47. 61
  48. 48. 62 Reference: 1. Text book of Medical Laboratory Technology by Godkar 2. Introduction to Medical laboratory technology by J. Baker, R.E. Silverstone 3. Anatomy for Nurses By Asha Latha 4. Hand book of Health care quality & patient safety 5. Damodaran K, Practical Biochemistry 6. Textbook of Parasitology by CP. Baveja
  49. 49. 63 Course Name/ code Medical Laboratory Technician Lab-II / BMLT6052 Experiment No 12 Title Demonstration of PAS staining Faculty In charge Mr. A. Vamsi Kumar (Asst Prof) Department / School Medical lab technology, SMAS Semester VI 12. DEMONSTRATION OF PAS STAINING Introduction: Periodic Acid Schiff (PAS) staining is one of the most commonly performed special staining technique in histopathology laboratory which is used to highlight molecules with high percentage of carbohydrate content such as mucin, glycogen, fungi and basement membrane in skin. Principle of PAS Staining PAS method works by exposing the tissue to periodic acid. Periodic acid acts as oxidizing agent which oxidizes compounds having free hydroxyl group (-OH group) or amino/alkylamine group resulting in dialdehydes. These dialdehydes when exposed to Schiff’s reagent, an insoluble magenta colored complex is formed. A suitable basic stain is used as counter stain. Preparation of staining solutions 1) Periodic Acid Solution : • Periodic Acid : 1 gram • Distilled water : 100 ml 2) Schiff’s Reagent : • Fuchsin Basic : 1 gm • Distilled water : 100 ml • Sodium metabisulphite : 2 gm • Conc. HCl : 2 ml • Charcoal activated : 0.3 gm [Note: Dissolve basic fuchsin in boiling water, cool at 50°C and filter. Add sodium metabisulphite and HCl. Store at dark room at room temperature overnight. Add charcoal, shake for one minute and filter] Procedure of PAS Staining 1. Bring sections to distilled water. 2. Treat with periodic acid for 5 minutes. 3. Rinse well in distilled water. 4. Cover with Schiff’s reagent for 5-15 minutes. 5. Wash in running tap water for 5-10 minutes 6. Counter stain with Herri’s hematoxylin for approximately 15 seconds. 7. Differentiate (if necessary) with acid alcohol and bluing as usual. 8. Wash in tap water.
  50. 50. 64 9. Rinse in increasing concentration of alcohol (70, 80, 95 and 100%) 10. Clear in xylene and mount as usual. Result Formation of insoluble magenta colored complex denotes positive result. Uses of PAS Staining Additional Notes Differentiation is the process of removing excess dyes from tissues. It is similar to decolorizing, but infers with high degree of selectivity. Differentiation can be accomplished by: Solvents (e.g. tap water in H&E staining), pH control, Mordants, Oxidizers, Other Dyes Bluing step converts the initial soluble red color (of hematoxylin) within the nucleus to an insoluble blue color. Some examples of bluing solution (alkaline pH) are: ammonia water, dilute lithium carbonate, Scott’s tap water (potassium carbonate, magnesium sulphate and water PAS stain is mainly used to highlight the molecules (structures) with high percentage of carbohydrate content such as glycogen, glycoproteins, and proteoglycans typically found in connective tissue, glycocalyx and basal laminae. PAS staining can be used to assist in the diagnosis of several medical conditions such as: • Glycogen storage disease (vs. other storage disease) • Adenocarcinoma which often secretes mucin • Paget’s disease of breast • Alveolar soft part sarcoma • Staining macrophages in Whipple’s disease • Erythroleukemia, Leukemia of immature RBCs • Fungal infection (cell wall stain magenta) Reference: https://laboratoryinfo.com/periodic-acid-schiff-pas-staining-technique-for-carbohydrates/
  51. 51. 65 Course Name/ code Medical Laboratory Technician Lab-II / BMLT6052 Experiment No 13 Title Collection and handling of specimen for cytopathology examination Faculty In charge Mr. A. Vamsi Kumar (Asst Prof) Department / School Medical lab technology, SMAS Semester VI 13. COLLECTION AND HANDLING OF SPECIMEN FOR CYTOPATHOLOGY EXAMINATION Handling & Processing of Cytopathology specimens: Cytology is the study of body cells that are either exfoliated spontaneously from epithelial surfaces or are obtained from various body tissues and organs by different techniques. Accordingly, cytology has following branches: A. Exfoliative cytology B. Aspiration cytology C. Imprint cytology I. EXFOLIATIVE CYTOLOGY This is the study of cells which are spontaneously shed off from epithelial surfaces into body cavities or into fluid. The cells can also be obtained by scraping, brushing or wash of body surfaces. The principle of this technique is that there is increased rate of exfoliation of cells in diseased states. APPLICATIONS OF EXFOLIATIVE CYTOLOGY Exfoliative cytology is applied in diagnosing diseases of the following: 1. Female genital tract 2. Respiratory tract 3. Gastrointestinal tract 4. Urinary tract 5. Body fluids (pleural, peritoneal, pericardial, CSF and semen) 6. Buccal smears for sex chromatin 1. Female Genital Tract Smears from female genital tract are known as ‘Pap smears’. These smears are prepared by different methods depending upon the purpose for which they are intended: i. Cervical smear is obtained by Ayre’s spatula from portio of the cervix by rotating the spatula through 360° to sample the entire cervix. The scraped material is placed on a clean glass slide and smear prepared. It is ideal for detection of cervical carcinoma. ii. Lateral vaginal smear (LVS) is obtained by scraping upper third of lateral walls of the vagina and is ideal for cytohormonal assessment. iii. Vaginal pool smear is obtained by aspirating material from posterior fornix of vagina and is done for detecting
  52. 52. 66 endometrial and ovarian carcinoma. Combined (fast) smears are a combination of vaginal pool and cervical scrapings and are used for routine population screening. 2. Respiratory Tract Material from respiratory tract may be obtained during bronchoscopic procedures as expectorant (sputum), or by brushing (BB), by washing (BW) and bronchioalveolar lavage (BAL). Sputum examination is advantageous as samples are easily obtained and cellular content is representative of entire respiratory tract. At least three samples of sputum, preferably early morning samples, should be examined. 3. Gastrointestinal Tract Lesions in the oral cavity can be sampled by scraping the surface with a metallic or wooden spatula. Samples can be obtained from the oesophagus, stomach, small and large intestine either by brushing or lavage during fibreoptic endoscopy. 4. Urinary Tract Samples from lesions in the urinary tract are either urinary sediment examined from voided urine/catheterised urine or washings of the urinary bladder obtained at cystoscopy. 5. Body Fluids Fluid from pleural, peritoneal or pericardial cavity is obtained by paracentesis. At least 50-100 ml of fluid is aspirated. The sample is examined fresh but if delay is anticipated then fluid should be anticoagulated either in EDTA 1 mg/ml or 3.8% sodium citrate 1 ml/10 ml. Fluid should be centrifuged and smears are prepared from the sediment. If amount of fluid is less (less than 1 ml), then it can be subjected to cytospin centrifuged smear preparation. FIXATION OF SMEARS IN EXFOLIATIVE CYTOLOGY Methods of fixation depend upon type of staining employed. Pap smears are wet-fixed (i.e. smears are immersed in fixative without allowing them to dry). Smears to be stained by Romanowsky stains are air-dried as fixation is affected during the staining procedure. Fixative used is either equal parts of ether and 95% ethanol, or 95% ethanol alone, 100% methanol, or 85% isopropyl alcohol. Fixation time of 10-15 minutes at room temperature is adequate. Smears may be left in fixative for 24 hours or more. Smears should be transported to the laboratory in fixative solution in coplin jars.
  53. 53. 67 II. ASPIRATION CYTOLOGY In this study, samples are obtained from diseased tissue by fine needle aspiration (FNA). APPLICATIONS OF FNA FNA is applied for diagnosis of palpable as well as nonpalpable lesions. I. Palpable Mass Lesions in 1. Lymph nodes 2. Breast 3. Thyroid 4. Salivary glands 5. Soft tissue masses 6. Bones II. Non-Palpable Mass Lesions in 1. Abdominal cavity 2. Thoracic cavity 3. Retroperitoneum Procedure for FNA Materials. For performing FNA, a Franzen’s handle, syringe with needles, clean glass slides and suitable fixative are required Method No anaesthesia is required. Ask the patient to lie down in comfortable position exposing the target area. Palpate the target area. Clean the overlying skin with spirit. Fix 10/20 ml disposable syringe in Franzen’s handle. Insert 20-25 gauge disposable needle into syringe. Fix the mass by palpating hand and insert needle into target area(A). Apply suction while moving needle back and forth within the lesion and change the direction of the needle. (B) Terminate the aspiration when aspirated material or blood is visible at the base/hub of the needle. Release the suction before withdrawing the needle to equalise pressure within the syringe (D) After withdrawal of needle, apply pressure for 2-3 minutes at the site of puncture to arrest bleeding and prevent haematoma formation. Aspirated material from the needle is expressed on to clean glass slides by first detaching the needle and filling the syringe with air and expressing it with pressure. Smears are prepared as for blood smears. If the material is semi-solid, it is first crushed by gentle pressure with a glass slide and smears prepared.
  54. 54. 68 III. IMPRINT CYTOLOGY In imprint cytology touch preparations from cut surfaces of fresh unfixed surgically excised tissue are prepared on clean glass slides. These are fixed, stained and examined immediately. It is considered complementary to frozen section. APPLICATIONS OF IMPRINT CYTOLOGY Imprint cytology is useful in following situations: i. Lymph node biopsy ii. Surgically resected tumours ADVANTAGES OF IMPRINT CYTOLOGY i. Tissue and cell architecture is retained ii. Useful as an intraoperative pathologic consult Reference: 1. Text book of Medical Laboratory Technology by Godkar 2. Introduction to Medical laboratory technology by J. Baker, R.E. Silverstone 3. Anatomy for Nurses By Asha Latha

×