Medical Laboratory Technology Reference Book PDF Vocational Higher Secondary Education (VHSE) Second Year 2016

Vocational Higher Secondary Education (VHSE) Second Year MEDICAL LABORATORY TECHNOLOGY Reference Book Government of Kerala Department of Education State Council of Educational Research...

Vocational Higher Secondary Education (VHSE) Second Year MEDICAL LABORATORY TECHNOLOGY Reference Book Government of Kerala Department of Education State Council of Educational Research and Training (SCERT), KERALA 2016 Reference Book List of Contributors Participants 1. Dileep R.K Vocational Teacher in MLT, Victory VHSS Olathanni, Neyyattinkara,Thriuvananthapuram 2. Sanjeev Kumar P Vocational Teacher in MLT, GVHSS Arimbra, Malappuram 3. Sabeerali kundukavil Vocational Teacher in MLT, GVHSS Omanoor, Malappuram 4. Shyju V S Vocational Teacher in MLT, GVHSS Kondotty, Malappuram 5. Abdullakuty K Vocational Teacher in MLT, BYK VHSS, Valavannur, Malappuram 6. Jahfar P Vocational Instructor in MLT, Calicut Girls VHSS, Kozhikkode Experts 1. Dr. Pradeepkumar M Asst. Prof. in Biochemistry, Medical College, Thiruvananthapuram 2. Gopakumar T Asst. Prof. in Microbiology, Medical College, Thiruvananthapuram Academic Co-ordinator Smt. Bindu C Research Officer, SCERT Prepared by : State Council of Educational Research and Training (SCERT) Poojappura, Thiruvananthapuram 695012, Kerala Website : www.scertkerala.gov.in e-mail : [email protected] Phone : 0471 - 2341883, Fax : 0471 - 2341869 Typesetting and Layout : SCERT © Department of Education, Government of Kerala 2  Medical Laboratory Technology FOREWORD Dear Learners, This book is intended to serve as a ready reference for learners of vocational higher secondary schools. It offers suggested guidelines for the transaction of the concepts highlighted in the course content. It is expected that the learners achieve significant learning outcomes at the end of the course as envisaged in the curriculum if it is followed properly. In the context of the Right- based approach, quality education has to be ensured for all learners. The learner community of Vocational Higher Secondary Education in Kerala should be empowered by providing them with the best education that strengthens their competences to become innovative entrepreneurs who contribute to the knowledge society. The change of course names, modular approach adopted for the organisation of course content, work-based pedagogy and the outcome focused assessment approach paved the way for achieving the vision of Vocational Higher Secondary Education in Kerala. The revised curriculum helps to equip the learners with multiple skills matching technological advancements and to produce skilled workforce for meeting the demands of the emerging industries and service sectors with national and global orientation. The revised curriculum attempts to enhance knowledge, skills and attitudes by giving higher priority and space for the learners to make discussions in small groups, and activities requiring hands-on experience. The SCERT appreciates the hard work and sincere co-operation of the contributors of this book that includes subject experts, industrialists and the teachers of Vocational Higher Secondary Schools. The development of this reference book has been a joint venture of the State Council of Educational Research and Training (SCERT) and the Directorate of Vocational Higher Secondary Education. The SCERT welcomes constructive criticism and creative suggestions for the improvement of the book. With regards, Dr P.A. Fathima Director SCERT, Kerala 3 Reference Book CONTENTS PART - A About the Course................................................. 05 Major skills (with sub skills)................................. 06 Syllabus of Module 3 & 4..................................... 07 PART - B Over view-- Module 3........................................... 15 Unit 3.1. Laboratory Management......................... 15 Unit 3.2. Clinical pathology.................................. 29 Unit 3.3 Clinical Biochemistry.............................. 49 Extended Activities - Module 3............................. 81 List of Practical - Module 4.................................... 81 Over view-- Module 4........................................... 83 Unit 4.1. Diagnostic Microbiology.................... 83 Unit 4.2. Histo technology and Cytology....... 135 Extended Activities - Module 4........................... 145 List of Practical - Module 4.................................. 145 References........................................................ 148 4  Medical Laboratory Technology ABOUT THE COURSE Medical Laboratory Technology is fast developing along with growing population and technological advancement. It is the most sought job titles in the global Health Care System. Medical Laboratory Technology is a broad area comprising of different disciplines like Clinical Pathology, Hematology, Biochemistry, Bacteriology, Immunology, Virology, Mycology, Parasitology, Histopathology, Cytology, and Cytogenetics & Molecular biology. In a country like ours, where fast and tremendous technological advancement and population growth happens, the demand and supply of trained man power is not on par. Introduction of a certificate course in Medical Laboratory Technology at higher secondary level is the remedy to this major skill gap in the country. Medical Laboratory Technology plays a crucial role in the diagnosis of diseases, prognosis and treatment. Apart from the laboratory diagnosis, application of Medical laboratory technology extends to detection of genetic disorders, epidemiology of infection diseases, detection of metabolic disorders and even to answer unraveled questions in forensic medicine. The course is designed to provide multi skilled competent personnel in the field of medical laboratory technology to meet the increasing demand. On completion of the course students acquire basic skills in the branches of medical laboratory technology which cater to entry level jobs. The course also provides inroads for students to undergo higher education including research in disciplines of laboratory medicine. The structure of the course is designed in such a way that the first module of First Year Curriculum familiarizes the learners to the basics of Human Anatomy & Physiology and gives an idea about the important units and features of a Diagnostic Laboratory. The topic also envisages the understanding of proper use and handling of common Laboratory Equipment and Glassware. A proper know - how about Blood, the commonest sample of any laboratory is given as part of the First module so that the learner will have a clear idea about the components, composition and collection of blood. The second module deals with the common Hematological investigations done in a laboratory. The practical and theoretical exposure will make the learners competent in the field. The second module also covers the topic Blood Banking which has attained much relevance nowadays due to the regular need for blood transfusions. The third module of the curriculum focuses on the effective management of a Laboratory, various analytical methods and recent advances in clinical biochemistry 5 Reference Book and in clinical pathology. The unit familiarises students with different instrument used in Clinical biochemistry from the simplest, micropipette to the most advanced fully automatic STAT Analyser. Fourth module of the course introduces the learners to the fascinating world of microorganisms and familiarises both traditional and recent trends in microbiology to provide a basic knowledge and imparts skill in diagnostic microbiology. It also covers Histo techniques and cytological techniques, so the learner gets a basic idea about the various steps involved in the preparation of tissue for microscopy. This will help in their future studies or career. The Curriculum also provides introduction to the automated machineries and techniques which can be experienced during the field visits or as part of OJT (On the Job Training). The laboratories as well as PTCs attached to schools provide ambient atmosphere for attaining perfection in performance for the students. The curriculum of VHSE which gives prime importance to practical is further skill enhanced with the scheduled 'On the Job Training Programs conducted in laboratories both on the government as well as private sector. The school curriculum is further enriched with introduction of ICT enabled teaching-learning methodologies as well as learning activities like survey, camps, expo etc. Major Skills Phlebotomy skill Skill in Haematological techniques Blood Banking Skill Laboratory Management Skill Skill in Biochemical techniques Skill in Clinical Pathological techniques. Skill in Microbiological techniques Sub Skills Measurement of BP and Pulse Skill in Handling and operation of common laboratory equipments Skill in safe handling of various chemicals Skill in First aid practice Skill in pipetting Skill in reagent preparation Tissue processing skill in histopathology 6  Medical Laboratory Technology SYLLABUS LABORATORY MANAGEMENT, CLINICAL PATHOLOGY & CLINICAL BIOCHEMISTRY Module 3 Unit 1 Laboratory Management 40 periods Unit No. Unit Period 3.1.1 Lab safety 20 Introduction Signs and symbols used in a laboratory Handling and storage of chemicals in a laboratory. Laboratory Hazards-Physical, Chemical, Biological, Electrical, Fire, Radiation Laboratory Safety Precautions-Personal Hygiene Fire Extinguishers Biomedical Waste Management First Aid Practice in Laboratory 3.1.2 Laboratory Management 20 Introduction Code of Ethics of a laboratory Professional Role of communication in laboratory Organization of a Laboratory Components of a Laboratory Lay out plan of a multi-room laboratory Organizational pattern of a Laboratory Familiarization of Request forms and report forms. Ordering and Utilization of supplies Maintenance of Stock Registers- Consumables, Non-consumables Accreditation and Certification of Laboratories. Accrediting Agencies- NABL, ISO, CAP, CRISIL - Bar coding and Total Laboratory Automation (TLA) Familiarization of Common Laboratory Software 7 Reference Book Module 3 Unit 2 Clinical Pathology 100 periods Unit No. Unit Period 3.2.1 Introduction 10 Importance, Common specimens, General guidelines for sample collection 3.2.2 Urine Analysis 44 - Importance, Types of urine samples Methods of collection, preservatives - Physical Examination - Chemical Examination-Sugar, Protein, Blood, Ketone bodies, Bile pigments, Bile salts, Urobilinogen - Microscopic Examination - hCG test in Urine 3.2.3 Sputum Examination 5 - Importance, Specimen collection - Physical examination - Microscopic examination 3.2.4 Stool Analysis 15 - Importance, Specimen collection - Physical examination - Chemical examination- Occult blood, Reducing substances - Microscopic examination- Saline & Iodine mount 3.2.5 Semen Analysis 16 - Importance, Specimen Collection - Physical Examination, Liquefaction Time, - Microscopy- Total Sperm Count, Motility, Morphology - Chemical Examination-Fructose, Acid phosphatase CSF and other body fluids 10 3.2.5 - CSF- Introduction - Specimen collection - Physical & Microscopic Examination - Chemical Examination- protein, glucose ,chloride (Name of method of estimation & clinical significance only) - Other body fluids - Recent advances in Clinical pathology 8  Medical Laboratory Technology Module 3 Unit 3 Clinical Biochemistry 200 periods Unit No. Unit Period 3.3.1 Introduction to Biochemistry 12 - Types of chemicals and preparation of solutions. - Types of specimens in clinical Biochemistry - Collection and processing of specimens for biochemical analysis - Types of assays- Endpoint and Kinetic (definition and example only) - Cleaning of glass wares for biochemical analysis 3.3.2 Instruments used in Biochemistry 8 - Familiarise with Colorimeter, Spectrophotometer, Flame photometer, Centrifuge, Electronic balance, Distillation apparatus, Deionizer 3.3.3 Blood Glucose Estimation 28 - Introduction to Diabetes - features, types, complications, - Types of samples- FBS, PPBS,RBS, Anticoagulant used - Methods of estimation- GOD-POD in detail - Normal value and Clinical Significance - Hyper and hypoglycaemia - Mention Glucometer Technique - GTT and GCT procedures, - Mention relevance HbA1C 3.3.4 Renal Function Tests 40 - Introduction, Common tests included Estimation of Blood Urea Mention common methods Urea-Berthelot method in detail, Normal value and Clinical significance Renal, Pre-renal, Post renal conditions of Uraemia Estimation of S. Creatinine. Mention common methods. Jaffe's method in details, Normal value and Clinical significance Estimation of Uric Acid. Mention common methods. Uricase method in detail. Normal value and Clinical Significance. - Mention Clearance tests- Urea and Creatinine - Mention Importance of Micro-albumin and Cystatin-C 3.3.5 Liver Function Tests 40 Introduction, Common tests included Bilirubin-Formation of Bilirubin 9 Reference Book Unit No. Unit Period Types of Bilirubin- conjugated and unconjugated Estimation of Bilirubin. Malloy- Evelyn method in detail. Normal value and Clinical Significance Estimation of Total protein- Biuret method in details Estimation of Albumin- BCG method in details Normal value and clinical significance of total protein and Albumin, A-G Ratio. Other LFT Parameters- ALP, ALT, AST in brief. 3.3.6 Lipid Profile 25 Introduction - Relevance, tests included in the Profile Estimation of S.Cholesterol. Mention common methods, CHOD-PAP method in detail, Normal value and Clinical Significance Mention Triglycerides, HDL, LDL 3.3.7 Other parameters of Diagnostic importance 21 Serum Electrolytes- Serum Sodium and Potassium Normal value and Clinical significance Clinically important Minerals- Calcium and Phosphorus (normal value and significance only) Name Diagnostically important Hormones T3, T4, TSH, FSH, LH, Prolactin, progesterone Name Clinically important enzymes- Acid Phosphatase, S. Amylase, GGT, Name Cardiac markers- Troponin-I, Troponin-T CPK, CK-MB, LDH, SGOT Name Tumour Markers- CA-125, CEA, AFP,CA-19.9, PSA, Beta hCG 3.3.8 Quality control in Biochemistry 10 - Introduction, Common terms used in Quality control, Errors - random and systemic , L.J. Chart, External QC and Internal QC 3.3.9 Automation and Recent advances 16 Need for Automation, Advantages of Automation Types of Auto Analysers-Semi and Fully automated Electrolyte Analyser (ISE) in brief Advanced Diagnostic Methods inbrief - C.L.I.A.,C.L.F.A, Turbidometry, Nephalometry, HPLC, Mention Point of care testing (POCT) 10  Medical Laboratory Technology Module 4 Unit 1 Diagnostic Microbiology 290 periods Unit No. Unit Period 4.1.1 Introduction to Microbiology 15 Classification of Microbes, pathogen, commensals, type of Infections, communicable diseases, Carriers Historical aspects in Microbiology 4.1.2 Structure and classification of bacteria 15 Structure- Cell wall, flagella, fimbriae, capsule, spore, plasmid Classification of bacteria based on morphology- Arrangement, Motility and oxygen requirement 4.1.3 Sterilization and disinfection 45 Importance of sterilization and Disinfection Methods of sterilization Physical methods- Dry heat, Moist Heat Chemical methods- alcohols, aldehydes, gases Mechanical methods- Filtration, Radiation Describe principle, parts, and use of - Hot air Oven, Autoclave Disinfectants and Antiseptics and their application 4.1.4 Growth &Cultivation of Bacteria 40 Bacterial growth and replication - Mention essential growth requirements- Temperature, PH, Gaseous requirements Culture media Classification of culture media with examples Preparation and use of common media Peptone water, Nutrient Agar, Blood Agar, Chocolate agar, Mac Conkey Agar Bacteriological wire loop, Straight wire - Inoculation of Culture media- Liquid and Solid Mention Streak, Stroke, Stab, Lawn culture - Mention Anaerobic techniques- Gaspak 4.1.5 Basic identification Techniques 50 Introduction Identification of bacteria Different methods Detection of motility - Name different methods - Hanging drop method in detail Staining 11 Reference Book Unit No. Unit Period - Principle, requirement, procedure and interpretation of Simple stain, Grams stain, AFB stain-Diagnostic significance Biochemical tests- Coagulase,Catalase, IMViC 4.1.6 Immunology and its diagnostic applications 35 Introduction - Types of Immunity, Antigen ,Antibody - Structure of antibody Types of antibody- Ig G, IgM, IgA, IgD, Ig E Antigen Antibody reactions- Specificity, Sensitivity, Avidity, Pro-zone ,post-zone, Titre Clinical applications of Agglutination, precipitation, flocculation, ELISA, Immuno-Fluorescence. 4.1.7 Laboratory Diagnosis of Common Bacterial diseases 30 Collection, Processing and transportation of common specimens-Urine, Blood, Sputum, CSF, Stool, Pus, body fluids, swabs General considerations- Macroscopy , Microscopy, Culture Mention common culture media and identification methods used. Antibiotic Sensitivity Testing (ABST)- Kirby Bauer Method Common Disease and pathogens encountered -Typhoid, Tuberculosis, Cholera, Dysentery, Syphilis, Leptospirosis, Tetanus, Meningitis& UTI Common Serological Techniques for diagnosis of Bacterial diseases- ELISA & its commercial preparations - Immunochromatographic technique WIDAL,RPR,-Procedure and interpretation 4.1.8 Laboratory Diagnosis of Common Viral diseases 20 Introduction to viruses Common viral diseases and pathogens encountered - AIDS, Hepatitis, Dengue, Chickun Guinia, Rabies, Infuenza, Mumps and Measles. Diagnostic techniques for viral infections - Mention common Serological tests used,Latex agglutination, Card tests, ELISA, Tissue culture, PCR Technique 4.1.9 Laboratory Diagnosis of Common Parasitic diseases 40 Introduction to parasites - Parasite, Commensal, Symbiosis, Host (Intermediate & 12  Medical Laboratory Technology Unit No. Unit Period Definitive host), Vector, Zoonosis Classification-Intestinal & Blood Parasites Common blood parasites and their lab diagnosis - Blood collection- - Time of collection - Preparation of smear-Thick and thin - Dehaemoglobinisation of thick smear Lab Diagnosis of Malaria - Disease,mode of transmission, hosts causative agent, types of malaria. - Examination of thick and thin smear-Morphological identification of different stages of parasite - Other stains used- JSB - Other methods- Card method , QBC Lab Diagnosis of Filariasis- - Disease, mode of transmission, host, and nocturnal habit - Lab diagnosis- wet smear examination, thick smear examination, Concentration technique. Lab Diagnosis of Intestinal parasites - Introduction -Helminthic infections and parasites - Amoebiasis -Entomoeba histolytica- Disease, Mode of Transmission, Trophozoite & Cyst Lab diagnosis -Macroscopic examination Microscopic examination -Stained & Unstained preparation Common Helminths- Tape worm, Round worm, Hook worm, Whip worm, Pin worm, - Lab diagnosis-Macroscopic & Microscopic examination - Concentration Techniques of Stool sample- Mention Floatation & Sedimentation methods 13 Reference Book Module 4 Unit 2 Histotechnology & Cytology 50 periods Unit No. Unit Period 4.2.1 Histotechnology 20 Introduction - Methods of examination of Tissues and cells - Gross examination - Microscopic examination Examination of Unfixed Tissue Examination of Fixed Tissue - Collection of specimens - Biopsy - Autopsy - Fixation - 10% Formalin - Decalcification 4.2.2 Tissue Processing 20 Steps in tissue processing - Dehydration - Clearing - Impregnation - Embedding Microtomes-Rotary Microtome,-Cryostat - Section cutting - Mention role of adhesives - Staining -H&E Staining - Mounting of Tissue sections - Filing and storage of tissue sections 4.2.3 Diagnostic cytology 10 Introduction Types of specimens Processing Fixation Staining Advantages and applications in diagnostic cytology 14  Medical Laboratory Technology PART B Laboratory Management, Clinical Pathology & Clinical Biochemistry Module 3 Over view The third module comprises of three vital areas of laboratory medicine, "Laboratory management, Clinical pathology and Clinical Biochemistry". Basic knowledge on laboratory management concepts helps a technician to uphold his professional quality and skill. Clinical Pathology laboratory pertains to analysis of body fluids which enables to reveal pathophysiological maladies and it receives more than 60% of specimens sent for investigations to a diagnostic laboratory. Clinical pathology Techniques are of historical importance in the evolution of medicine and are the cheapest methods with little discomfort and stress for specimen collection providing information of immense value. With the supporting and supplementing information and investigations, the clinicians still largely depend on clinical pathology to resolve diagnostic dilemma. Clinical biochemistry is the significant part of laboratory diagnosis as it helps to understand biochemical mechanism of the body in relation to diseases. Manual methods and principles of clinical chemistry have been replaced by automated techniques and recent methodologies. The scope of investigations has almost changed the diagnostic scenario from a level of general health assessment to organ function tests. 3.1 Laboratory Management The effective operation of a medical laboratory and proper delivery of laboratory results to clinician and their patients are integral part of a well defined health care system. An effective laboratory management is essential for providing an accurate, reliable and timely laboratory results which forms the basis of almost all of the medical decisions and diagnosis made in the modern era. The task of laboratory management involves integration and co ordination of organizational resources like personal, equipment, money, time and space so that standardized planning organization and operation of a laboratory happens. It essentiates management skills in ensuring laboratory safety, handling of laboratory wastes and observing laboratory ethics, 15 Reference Book protocols, accreditation and certification criteria. The unit familiarizes the learner with the Code of Ethics of Laboratory professional, safety measures to be taken in a laboratory, tips for personal hygiene and about the care and handling of chemicals in a laboratory. It also creates awareness about the different signs and symbols used in a laboratory, different types of hazards and about the segregation and management of laboratory waste. Software based laboratory management systems have been evolved over years that support laboratory operations. Most of them utilize web enabled sample management, data analyzing and reporting facilities.Introduction of Bar coding and Total Laboratory Automation has caused tremendous improvement in the patient identification and time management mechanism. Learning outcomes The learner: explains different laboratory safety precautions and first aid classifies different methods of biomedical waste management identifies Code of laboratory ethics and safe laboratory practice explains organization of a laboratory, its organizational pattern and the role of Communication in a laboratory identifies and prepare lay out of a Medical Laboratory formats various request forms, stock registers and order form identifies the importance of accreditation, certification of laboratories and identify different accrediting agencies identifies the importance of barcoding and Total laboratory Automation and use of Common Laboratory Software 3.1.1 Lab safety Introduction Laboratory safety measures are designed to encourage and promote safe and efficient working practices in a lab. It protects all laboratory personnel and other people with right of entry from illness or injury. All laboratory personnel have responsibility to adhere to and observe safety programme all the time. Occupational injury and illness are caused mostly by bad practices, inexperience, ignorance and failure to follow standard practices. Safe laboratory Practice mainly depends on, 16  Medical Laboratory Technology 1. Awareness on Signs & Symbols used in a laboratory 2. Identification of the possibilities for common laboratory hazards 3. Proper management of Bio medical wastes 4. First Aid practices Signs and symbols used in a laboratory To minimize accidents in the laboratory it has been made mandatory to use signs and symbols in the laboratory. These are used to indicate possible hazard during a procedure. A thorough knowledge on signs and symbols helps the laboratory personnel to deliver his duty properly. The symbols include hazard warning symbols, safety (mandatory) symbols and prohibitory symbols. Hazard warning symbols are black pictures in yellow or orange background. Safety symbols are round white pictures with blue background, prohibitory symbols are round black pictures in white background with a red edge and diagonal line. Handling and storage of chemicals in a laboratory Proper handling and storage of chemicals and reagents is necessary to prevent hazards. Chemicals are stored according to their physical and chemical properties Types of chemicals 1. Flammable Chemicals - eg :- Acetone, Ether, Xylene, Alcohol 2. Corrosive chemicals - eg :- Concentrated Acids, Alkalies,phenol etc 3. Oxidising Chemicals - eg :- Potassium dichromate, Chromic acid, Chlorites etc. 4. Explosive Chemicals - eg :- Picric Acid. 17 Reference Book 5. Radioactive chemicals- eg :- I 125 , I 131 , H3 6. Carcinogenic chemicals-eg :- Benzidine, O- toluidine, Selenite, etc 7. Toxic Chemicals -eg :- Potassium Cyanide Handling and Storage of chemicals 1. Label the chemicals with hazard symbol, simple instruction for use, strength and Expiry date (it is mandatory for manufacturers). 2. Flammable chemicals are stored in fire proof metal containers at ground level in a cool dry place and always handle them away from direct flame. 3. Corrosive chemicals are stored in amber coloured bottles at ground level. 4. Explosive chemicals are never left in dry state. 5. Radioactive chemicals are handled by properly trained persons only 6. Carcinogenic chemicals are stored in closed container. Exposure to such chemicals should be kept minimum. 7. Highly toxic chemicals like Potassium cyanide are kept locked in cupboards and proper documentation is made in stock register. 8. Do not open flammable chemicals near flame 9. Do not add water to acid 10. Avoid mouth pipetting. Laboratory Hazards Laboratory hazards are mainly Physical, Chemical, Biological, Electrical, Fire, Radiation hazards. Physical hazards include Cutting with broken glassware, sharps, Electric shocks,Falling on wet floor, Burns and scald. Burns usually caused by flammable chemicals, faulty electric equipment, Burners, corrosive chemicals, and hot dry objects. Scalds are caused by hot liquids. General precautions to prevent physical hazard in a laboratory are, 1. Proper storage and handling of glassware 2. Adopt Standard electrification methods 3. Proper storage and handling of chemicals 4. Proper handling of lab equipment, 5. Prevent spillage of hot fluids 6. Use quartz chips to prevent violent boiling of liquids 18  Medical Laboratory Technology 7. Use thick walled glass containers and round bottomed flask for heating purpose 8. Use hazard symbols Chemical hazards are caused by physical contact, ingestion and inhalation of chemicals, leads to from minor burns to even life threatening. General precautions to prevent chemical hazard in a laboratory are 1. Awareness on the physical and chemical properties of laboratory chemicals 2. Proper storage of chemicals 3. Mouth pipetting should be avoided 4. Dangerous chemicals (toxic chemicals) are kept in small amount for routine use 5. Use hazard symbols Biological hazards can be caused by infectious agents like Human immunodeficiency virus (HIV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Mycobacterium tuberculosis, Herpes simplex virus etc. Contaminated food and water, Needle pricks and cuts, Laboratory animal bite can also cause bio hazard. General precautions to prevent Biological hazard in a laboratory are 1. Consider all specimens in the laboratory as potentially dangerous 2. Use gloves, masks, apron and eye protectors in the laboratory 3. Proper waste disposal or management 4. Vaccinate laboratory staff against infectious diseases like HBV 5. Mouth pipetting should be avoided 6. Do not eat , drink, smoke or apply cosmetics in the laboratory 7. Proper washing of hands after work, Cleaning and disinfection of work bench on completion of work Electrical hazards are mainly caused by faulty operation and improper maintenance of electrical equipment. It may result in minor burns to severe injuries that may lead to death. General precautions to prevent electrical hazard in a laboratory are, 1. All Electrical equipment should be properly grounded 2. Overload circuits should be avoided, do not use extension cords 3. Electrical equipment should not be handled with wet hands 4. Do not disable any electrical safety features. 19 Reference Book 5. Repair or services should be done by authorized persons only 6. Do not use Equipment for a task not designed for it 7. Do not leave equipment switched on when not in use Fire in the laboratory may occur due to naked flames, electrical overloading, flammable reagents and smoking in the laboratory. General precautions to prevent Electrical hazard in a laboratory are, 1. Instead of open flames use hot plates 2. Store flammable and explosive chemicals properly 3. Install fire extinguisher in the laboratory. All lab personnel should know the location of fire extinguisher and how to use it 4. In case of fire, escape through fire exit route Fire Extinguishers A fire extinguisher is a fire protection device used to control small fires in emergency situations. Typically, a fire extinguisher consists of a hand-held cylindrical pressure vessel containing agents which can be discharged to extinguish a fire. Different type of fire extinguishers are now available for managing different classes of fires. Fire extinguishers for ABC type fires are commonly used in the laboratories. Radiation Hazards are caused by improper handling of radioactive chemicals. Various analytical procedures like RIA (Radio immunoassay) demand use of radioactive substances in a laboratory. X ray units and other nuclear medicine procedures are also cause radiation hazard. General precautions to prevent radiation hazard in a laboratory are, 1. Radioactive material should be stored in lead shielded container properly labeled with identity. 2. Only trained staff is permitted to handle radioactive chemicals 3. Use hazard symbols 4. The staff should wear safety spectacles, disposable gloves and personal dosimeters. 5. Accurate records are maintained for use and disposal of radio active chemicals. 20  Medical Laboratory Technology Laboratory Safety Precautions-Personal Hygiene 1. Wear gloves and apron 2. Use eye protector if necessary 3. Do not Eat, drink, apply cosmetics inside laboratory 4. Wear shoe/ chappal inside the laboratory 5. Avoid Mouth pipetting. 6. Wash hands properly before and after performing laboratory experiment 7. Disinfect the work bench before leaving the laboratory. 8. Girls must put-up hair inside the laboratory 9. Carry minimum personal articles inside laboratory. First Aid Practice in Laboratory. First aid is the help given immediately to an injured person. For example First aid helps an injured to avoid excessive bleeding from an injury until medical attention has been obtained. A quick and confident approach of first aider can save life from many emergency conditions. Common first aid procedures are given below Injuries caused by broken glass: Wash the wound immediately to remove any glass pieces. Apply mercurochrome or acriflavine ointment to the wound. Cover with gauze and adhesive tape Acid/Alkali splashes on the skin: Wash thoroughly; bath the affected skin with cotton wool soaked in 5% aqueous sodium carbonate if acid and 5% acetic acid or undiluted vinegar, if alkali. Acid/Alkali splashes in the eye: Water spray from a wash bottle or rubber bulb into the medial corner of the eye. Put 4 drops of 2% Aqueous Sodium bicarbonate into the eye, if acid, and saturated solution of boric acid, if alkali. Swallowing acid: Make the patient drink some 5% soap solution immediately. Make him gargle with the soap solution. Give him 3 or 4 glasses of ordinary water. If the lips and tongue are burned by the acid, rinse thoroughly with water. Bathe with 2% aqueous sodium bicarbonate. Swallowing alkalies: Make the patient drink 5% solution of acetic acid or lemon juice or dilute vinegar. Make him gargle with the same acid solution. Give him 3 or 4 glasses of ordinary water. If the lips and tongue are burned by the alkali, rinse thoroughly with water; bathe with 5% acetic acid. 21 Reference Book Poisoning :-Send for a physician or qualified nurse, specifying the toxic substance involved. Place the victim in open air while waiting for the physician. Minor burns: Plunge the affected part in cold water or ice-water to soothe the pain. Apply Mercurochrome or Acriflavine ointment to the burn. Apply dry gauze dressing loosely. If the burn becomes infected or does not heal, refer the patient to a physician. Never tear off the blisters that form over the burns. Severe burns: If the victim is on fire, roll him in a blanket or overall to smoothen the flames. Inform the physician. Lay the victim on the ground. Do not remove his clothing. Cover him if he is cold. Do not apply any treatment to the burns. This must be left to the physician. Unconsciousnes: If the victim is breathing then lay him face down with his head on one side and arm and the leg of that side in the bent position. This posture makes breathing easier and provides better blood circulation to all body parts. If the victim is not breathing start artificial respiration immediately. Artificial respiration: The procedure should be started quickly as brain infarction occurs within few minutes of oxygen deficiency. Steps of artificial respiration is 1. Lay victim on back 2. Clear any obstruction of the mouth 3. Place an object under the shoulder so the head is tilted back 4. Pinch the nostrils and apply mouth to mouth resuscitation 10 times a minute until breathing starts. Biomedical Waste Management in Laboratory Biomedical waste is generated during the diagnosis, testing, treatment, research or production of bio materials. Management of biological wastes consists of the collection, segregation and proper treatment of individual type of waste. The wastes are colour coded according to the nature of waste and their method of disposal and 22  Medical Laboratory Technology segregated at source itself. It is mandatory for each laboratory to adopt proper waste management systems of their own or to be associated with some agencies which do the work in a corporate manner. 3.1.2 Laboratory Management Code of Ethics of a laboratory Professional The laboratory personnel should be aware of the code of ethics which is the minimum standard about his professional skill. It includes the do's and dont's in the laboratory. It is a set of principles of right conduct. 1. Treat patients and colleagues with respect, care and thoughtfulness 2. Perform duties in an accurate, precise, timely and responsible manner 3. Safeguard patient information as confidential, within the limits of the law 4. Prudent use of laboratory resources 5. Advocate the delivery of quality laboratory services in a cost effective manner 6. Work within the boundaries of laws and regulations 7. Strive to improve professional skills and knowledge Role of communication in laboratory A laboratory must maintain a healthy relationship between patients, physicians, nursing staff, sales representatives, administrative staff etc. A trained laboratory technologist must know the following aspects of communication 1. Speak to patient clearly with pleasing manners so that his confidence towards the technician increases 2. Communicate correct knowledge to hospital staff on laboratory tests, results and significance of tests. 3. Communication with physicians on interpretation of lab requests and labora- tory results 4. Good interpersonal relationship to be observed with co workers. Organization of a Laboratory Only a well-designed laboratory can provide reliable and timely results for the diagnosis of diseases. The laboratory should be properly planned, so that it will be functional and convenient. The laboratory should have adequate space, ventilation, light, power supply, water supply, work benches, Reagents, Equipment and Laboratory personnel. 23 Reference Book An accessible space adequate for setting a desired laboratory is identified. Good ventilated laboratory has reduced risk of infection. Laboratory should have uninterrupted power supply, good lighting system. Good supply of running water and water drainage facility is essential in a laboratory. Work benches should be made at suitable heights with enough leg space. Surface polished Cement benches or wooden benches can be used. Wooden benches are acid proofed. Basic laboratory equipment, glassware and reagents should be made purchased from reputed suppliers. Well trained qualified staff is the back bone of laboratory and the quality of the result depends on the quality of workmanship. A laboratory should have staff pattern including administrator, laboratory technologists, technicians and laboratory assistants for its proper functioning. Components of a Laboratory: The components of a clinical laboratory include various departments like hematology, blood bank, microbiology, biochemistry, serology and clinical pathology. It also includes reception area, room for specimen collection, refreshment room and toilets Lay out plan of a multi- room laboratory: The working area for different departments as well as sample reception should be conveniently arranged making the necessary reagents and apparatus easily accessible. Request forms and report forms. A request form is a form for lab test signed by the doctor.The request form may carry the patients name, age, IP/OP no., provisional diagnosis, nature of specimen, and name of test required on that specimen. A report form is the form in which the laboratory issues the results of the tests asked for. Various types of report forms for various departments are used routinely. Preprinted and computerised report forms are currently used. Maintenance of Stock Registers- Consumables, Non-consumables.Every laboratory should maintain stock register and each purchase of commodities to the laboratory should be included in it. It is mandatory to maintain separate stock registers for consumables and non-consumables. Reagents chemicals and glass wares are included in consumables. Instruments and machineries used in laboratory and items which will not be exhausted on usage are included in non-consumables. 24  Medical Laboratory Technology Ordering and Utilization of supplies. A medical laboratory needs an uninterrupted supply of chemicals and reagents for the smooth functioning. Hence prompt ordering and purchasing is important. For this purpose every laboratory personnel should know the details of the item to be purchased and details of various suppliers in that area and merits and demerits of each brand, shelf life period etc. Accreditation and Certification of Laboratories. Accreditation is used to grade the laboratories having an appropriate quality management system and can properly perform certain test methods and calibration parameters according to their scopes of accreditation. Example for accreditation agencies are NABL, ISO, CAP, CRISIL etc. Bar coding A major advance in the automation of specimen identification in the clinical laboratory is the incorporation of bar coding technology into analytical systems. A bar coded label (often generated by the laboratory information system and bearing the sample accession number) is placed onto the specimen container and is subsequently "read" by one or more bar code readers placed at key positions in the analytical sequence. The identified information is then transferred to and processed by the system software. Total Laboratory automation involves the integration and automation of the individual steps of the analytical process. Some manufacturers have developed stand-alone automation system which sort, centrifuge, decap, aliquot, and label tubes. More advanced automation systems provide options such as conveyors to transport specimens, direct sampling interfaces to higher volume analyzers, refrigerated storage and retrieval systems. Now total lab automated system utilizes advanced technologies such as robotics too. Laboratory information management system (LIMS), often referred to as a laboratory information system (LIS) or laboratory management system (LMS), is a software based laboratory and information management system with features that support whole process of a modern laboratory including operation management, data/records management, quality control, and information sharing among stakeholders including technicians, Physicians & patients etc. 25 Reference Book DETAILING OF PRACTICALS Demonstration of signs and symbols used in laboratory Common laboratory signs and symbols collected are distributed for identification and is recorded in the practical log Preparation of different types of laboratory request forms Sample request form collected are distributed, prepare a model of it and record in practical log Preparation of different types of laboratory Report forms- Haematology, Biochemistry, Clinincal pathology, Serology, Mixed forms Different models of report forms collected are distributed, prepare a model of each and record in practical log Prepare a lay out plan of a multi room laboratory A lay out plan is prepared (Chart /model) and is kept as portfolio Preparation of models of stock registers- consumables, Non-consumable A model stock register is prepared. Purchase entry and issue of any two items are made and is recorded in the practical log Stock Register- Non consumable Name of item : Page No: Bill Date of Present Initial of Lab in Sl.no. no.& Rate Amount stock Balance Remarks purchase Quantity charge Firm 1 2 3 Stock Register- consumable Name of item : Page No: Bill Date of Present Initial of Lab in Sl.no. no.& Quantity Rate Amount stock Issued Balance Remarks charge purchase Firm 1 2 3 Preparation of model of Order form Prepare a model format of order form. It should include name of product , name of manufacturer/ supplier, volume/ pack size, quantity required, specification if any 26  Medical Laboratory Technology Demonstration of colour coding for biomedical waste segregation Chart showing colour coding for biomedical waste segregation are distributed for identification and is recorded in the practical log ASSESSMENT ACTIVITIES Assignment on Safe Laboratory Practice in Medical laboratory Seminar on Common Hazards in a Laboratory Chart Preparation on First Aid Practice Chart Preparation on different color labels for segregation of biomedical waste Model Preparation of Lay out of an ideal laboratory Collection of different types of report forms Preparation different report forms,& Stock registers Preparation of model of a Stock register Theory Evaluation Questions 1 Diseases like AIDS, Hepatitis B etc can be transmitted through 3 laboratory specimens if they are not properly handled. Enumerate any three steps adopted to prevent transmission of such diseases to laboratory personnel 2 Prepare a model Haematology report form for your PTC 4 Laboratory (Hints: Lab ID, Patient ID, Test Details) 3 Given are two important symbols used in a laboratory 4 Warning Symbol Prohibitory Symbol 27 Reference Book a) Differentiate between warning and prohibitory symbols 2 b) Explain the meaning of the two given symbols 2 4 I am a chemical who is out from the labs now a days. Technician 1 says it due to the fact that I am a carcinogen. Who am I 5 Match the following NABL Burning of Lab waste HBV infection Artificial respiration 4 CPR Lab Accreditation Incineration Biological Hazard 6 One of your friend hesitate to wear laboratory apron.Advice the 2 friend showing 2 important reasons to use lab apron 7 Your lab is having waste disposing bins of different colours. In which 1 bin you will put a used syringe and needle. (Red, Green, Black Blue) 8 Prepare and label the lay out for a model laboratory having 5 minimum five sections 9 Various personal safety precautions are taken by laboratory 4 personnel during work. Substantiate the statement with examples. 10 The different chemicals used in laboratories are hazardous if they 4 are not properly stored or handled with care. Substantiate the statement by giving some proper storing and handling instructions. 28  Medical Laboratory Technology Unit 3.2 CLINICAL PATHOLOGY A change that takes place in the human body during the process of disease is always reflected in the chemical composition of body fluids. Clinical examination of these fluids reveals the presence of abnormal constituents, altered cellularity, microorganisms and other physical evidences. These evidences from a clinical pathology lab provide endless support to a physician in reaching an early and accurate diagnosis. Apart from the common importance like that of any other laboratory investigation, its importance is paramount in the sense that it includes most of common clinical investigations that are routinely done in a clinical laboratory. Hence an adequate and appropriate understanding of the accurate procedure of these investigations is very essential for a technician. Lack of sufficient automation, decreased sensitivity in microscopy and less specific chemical reactions in the absence of enzyme chemistry are some of the inherent limitations of clinical pathology analysis. Even though the advances in fibro-optic technique enables a pinpoint observation of lower respiratory tract and gastro intestinal tract, the basic analysis of sputum and stool samples still remains in mainstay and so the case of other samples. Easy availability of samples, rapid results, and reasonable precision justifies the need of a clinical pathology lab in a hospital and in the curriculum too. Reporting of positive abnormal finding of the clinical pathology results are important equally to knowledge about the absence of abnormalities for correct diagnosis of a disease. The analytical tests discussed here are mostly manual types which utilize principles of basic chemical reaction and primarily focuses on physical examination of fluids, microscopy, and simple chemical screening. Learning outcomes The learner: identifies different specimens and describes the general guidelines for sample collection in clinical pathology discusses the importance of urine analysis identifies different type of urine samples and method of collection performs physical, chemical and microscopical examination of urine 29 Reference Book performs urine pregnancy test identifies the importance sputum analysis identifies the importance of stool examination discusses the importance of semen analysis and describes the method of semen analysis identifies the importance and describes the analysis of C.S.F and other body fluids. mentions the recent advances in clinical pathology Introduction Clinical pathology is the diagnosis of disease based on the laboratory analysis of body fluids. Blood, urine, stool, sputum, cerebrospinal fluid, semen, etc are specimens commonly submitted for analysis in a clinical pathology laboratory. Physical, Chemical, Serological and Microscopical examination of specimens are carried out in the laboratory General guidelines for sample collection Collect specimens in proper container ie wide mouthed, spill free, disposable containers Label the specimen such as name of patient, type of specimen, ID no., Date and Time of collection etc. Follow proper protocol for transportation of specimens Keep the samples ready for performing analysis. 3.2.2 Urine Analysis Urine is the most common specimen examined in clinical pathology laboratory. Urine is formed by the kidneys. The body removes water and many harmful waste substances from the body through urine. This is a normal physiological process of the body. Urine analysis provides mainly diagnostic and prognostic information on conditions like kidney diseases, liver diseases, metabolic disorders, urinary tract infections and parasitic infestations. In addition pregnancy, endocrine disorders and drug overdose are also investigated with a specimen of urine. Simplicity in obtaining the specimen and lesser cost of test makes urine analysis an popular tool in laboratory investigations. 30  Medical Laboratory Technology Types of urine specimen Random specimen 24 hour specimen. Early morning specimen Methods of collection Random specimen is a sample collected without any priority to the time or diet and is collected by the patient in a wide mouth dry container, after cleaning the external genitalia with mild soap and water. Urine specimen from catheter can be obtained in necessary conditions. Urine collection bags are used in case of infants and in geriatrics. A clean, early morning, fasting specimen is generally the most concentrated specimen and is preferred for qualitative, quantitative and microscopic examination for the detection of abnormal constituents. 24 hour urine specimen collection is done by collecting urine in a special container, 2.5 to 5 litre capacity with added preservative. Preferably morning 8 am to next day 8 am over a period of 24 hr. It helps to diagnose kidney problems. It is often collected to do tests like clearance tests and to measure protein, hormones, minerals, and other chemical compounds. The preservatives are added to prevent changes in the urine (pH, odour, bacterial growth etc) that may happen on standing longer periods. Preservatives commonly used are toluene, formaldehyde, chloroform , boric acid, hydrochloric acid etc. Physical examination of urine All routine urine analysis should begin with a physical examination of the urine sample. This examination includes assessment of appearance (colour and transparency), specific gravity, volume, reaction and odour. For best results of the physical examination of urine the specimen should be evaluated immediately after collection. Colour Normally the urine is straw coloured (due to urochrome). Colour depends on the concentration of urine and varies with the presence of constituents. Normal colour of urine changes in different disease conditions or in persons taking medications. Deep Yellow : Mild to severe dehydration, Jaundice, B complex therapy Red to brown : Haematuria, haemoglobinuria, myoglobinuria, porphyria Brown to black : Alkaptonuria, methaemoglobinuria. Milky : due to the presence of lymph (Chyluria) 31 Reference Book The colour of urine is usually described after visual inspection with common colour terms. Transparency Freshly voided urine is clear and transparent. It may become turbid if exposed for long time due to the urea being acted upon by bacteria or the presence of proteins, pus cells, blood, bacteria, urates, phosphates. Specific gravity It implies the capacity of kidney to concentrate urine. Normal specific gravity of urine is 1.010-1.025 Values more than 1.025 indicate severe dehydration, DM (diabetes mellitus), Adrenal insufficiency. Values less than 1.010 indicates increased water output as in Diabetes insipidus. A low fixed specific gravity usually found is chronic renal failure. It is measured by urinometer or refractometer. Volume Normal urinary output ranges between 1000 - 1500 ml / day. It depends upon fluid intake, solute load, loss of fluid by skin or otherwise, climatic condition etc A urine out put more than 3 litre/day is called Polyuria and is seen in- Diabetes Mellitus, Diabetes Insipidus, Recovery from acute renal failure (ARF), Diuretic therapy etc. A urine output Less than 500 ml/day is Oliguria and is seen in Acute renal failure, Vomiting, Fever, Burns. Anuria is the suppression of urine output (generally less than 50 ml per 12 hours is considered as anuria) Urine volumes can be measured usually with a volumetric cylinder Reaction (pH) Normal fresh urine is acidic approximately 6. Depending on the person's acid-base status, the pH of urine may range from 4.5 to 8. A Urine pH 8.5 or more may be found after heavy meals, Urinary tract obstruction,Chronic renal failure A pH 4.5 or less may be seen after heavy exercise, Metabolic Acidosis, Chronic Respiratory, Acidosis, Uncontrolled diabetes Extremely acidic or alkaline urine usually indicates a poorly collected specimen. 32  Medical Laboratory Technology Litmus Paper, Reagent strip testing(pH paper), are commonly employed for measurement of pH, Odour Freshly voided urine is slightly aromatic. Urine becomes more ammoniacal due to bacterial activity. A fruity odour is felt in case of severe diabetes due to presence of ketone bodies. A putrid or foul smell is felt in Urinary tract infections. Chemical Examination of Urine Chemical examination of urine is done to detect presence or absence of certain constituents which reflects disease state of body. The parameters include Protein, Sugar, Ketone bodies, Blood, Bile pigments, Bile salts and Urobilinogen. Conventional test methods are commonly employed.Reagent test strips (dip sticks) are commercially available and recently used. 1. Proteins Normally very small amount of protein (100 mg/day) is excreted and is not detectable by routine qualitative test methods. Proteinuria is the term used for increased protein excretion in urine. Commonly found protein in urine is albumin so it is often termed as albuminuria. Proteinuria is seen in kidney diseases such as Nephrotic Syndrome, Multiple myeloma, chemical poisons etc. Methods of detection The methods are based on the principle of precipitation of protein by chemical agents or coagulation by heat Heat and Acetic acid method Sulphosalicylic acid method Reagent test strips (dip sticks) like albustix 2. Sugar Glucose is the predominant sugar and is normally absent in urine. The renal threshold for glucose is 180 mg%. If the blood sugar rises above renal threshold level, the glucose will appear in the urine and is called glycosuria. Major cause of Glycosuria isDiabetes mellitus. Transient glycosuria is seen in pregnancy, stress etc. Methods of detection Benedicts test--When Benedicts qualitative reagent is heated with urine, glucose present in urine reduces cupric ions present in the reagent to cuprous ions. Alkaline medium is provided to the reaction by sodium 33 Reference Book carbonate present in reagent.The color changes to green, yellow, orange or red according to the concentration of glucose in urine. Reagent test strips (dip sticks) like glucostix. 3. Ketone Bodies Ketone bodies is a term used to describe three discrete but metabolically related chemicals-Acetone, Acetoacetic acid and  - hydroxy butyric acid.Ketone bodies are spilled into urine and the presence of Ketone body in urine is called ketonuria. Ketone bodies are detected in conditions such as diabetic ketoacidosis and starvation. Methods of detection Rothera's test ( Ketone bodies form purple coloured complexes with so- dium nitroprusside in alkaline medium) Reagent test strips (dip sticks) like ketostix A fresh urine specimen is always prefered for ketone body estimation. 4. Blood Presence of blood in urine is called Haematuria, seen in cases of Nephrolithiasis (urinary calculi) and in malignancy. A microscopic exmination of urine helps to confirm the presence of intact erythrocytes. Healthy individuals normally will have no detectable blood in their urine. Methods of detection Benzidine test - It is the test for occult blood. It is highly sensitive but as benzidine is considered as a potent carcinogen, this test is no longer used now. Reagent test strips ( Haem occult test) 6. Bile Pigments These are breakdown products of haemoglobin excreted in bile. The two most important bile pigments are bilirubin, which is orange or yellow, and its oxidized form biliverdin, which is green. Normally bile pigments are absent in urine. Presence of bile pigments in the urine indicates liver dysfunction such as obstructive jaundice. Fresh urine sample should be used for bilirubin determination because exposure of urine to light may cause loss of bile pigment by oxidation and leads to negative result. 34  Medical Laboratory Technology Methods of detection Fouchet's test.( Bilirubin is treated with ferric chloride in Tri chloro acetic acid to give greenish blue colour) Reagent test strips (dip sticks) 7. Bile Salts Bile salts are made in the liver from cholesterol and these help in the fats absorption. Sodium taurocholate and sodium glycocholate are the major bile salts. Normally bile salts are negative in the urine. The presence of bile salt in the urine indicates disease like Obstructive Jaundice. Methods of detection Hay's test. (Bile salts when present, lower the surface tension of urine. When sulphur powder is added to the urine, sulphur particles sink to the bottom of the tube indicates postive result.) 8. Urobilinogen Majority of Bile pigment derived from breakdown of hemoglobin is excreted in the stool, but small amounts are reabsorbed into the blood from the intestines and then excreted into the urine.Urobilinogen is formed in the intestines by bacterial action on bilirubin.Urobilinogen is normally present in urine in low concentrations. A fresh specimen is essential for the detection of urobilinogen, as it is a light-sensitive compound. Positive test results help to detect liver diseases such as hepatitis, cirrhosis and haemolytic anaemia Methods of detection Ehrlich's Test (Urobilinogen forms a cherry red complex with para dim- ethyl amino benzaldehyde in normal urine. On further dilution of sample (>1:20) a positive result is significant.) Microscopic examination of Urine Microscopic Examination of urine sediments provides a direct sampling of urinary tract morphology, which helps the diagnosis very much. The specimen used for microscopic examination should be as fresh as possible. Red cells and many formed solids tend to disintegrate upon standing, particularly if the specimen is warm or alkaline.Concentrated first morning, mid-stream, clean catch urine specimen, is preferred for microscopy. Urine is collected without faecal & vaginal contamination. 35 Reference Book The urine sediments assessed under microscope can be of two types Organized - Leukocyte (Pus cells), Erythrocytes, Casts, Epithelial cells, Bacteria, Parasites and fungi. Unorganized - Crystals and amorphous sediments. Pus cells - They are round or oval in shape and present normally, 0 to 3 leukocytes per high-power field will be seen on microscopic examination. The pus cells are leucocytes. More than 5 cells per high-power field probably indicate urinary tract infection. Erythrocytes - Red cells are not usually present in normal urine and appear as refractile bodies, seen in acute glomerulo nephritis, stones in urinary tract and malignancy etc. Estimate their number per high-power field and report it. Epithelial cells - Normal urine shows a few epithelial cells. Marked increase in the number of epithelial cells signifies some pathological conditions of the site of their origin. Bacteria - Presence of numerous bacteria in the centrifuged deposit of freshly voided urine signifies infection in the urinary tract. Casts - These are formed by coagulation of albuminous material in the kidney tubules. Casts are cylindrical and vary in diameter. The sides are parallel, and the ends are usually rounded. Casts in the urine always indicate some form of kidney disorder and should always be reported There are different types of casts. They are Hyaline casts, Red cell casts, Granular casts, Epithelial casts, Waxy casts and Fatty casts Crystals Crystals of various substances can be seen according to the reactions ( pH) of urine. Crystals found in normal acid urine- Amorphous urates- yellow and granular precipitate Uric Acid Crystals appear in several forms, Multi colored when polarized most commonly Diamond shaped Calcium Oxalate Crystals is most frequently observed in urine and octahedryl in shapes, often referred to as an 'envelope' shape Crystals found in Alkaline urine Amorphous phosphates-fine granular precipitates Calcium phosphate- stillete prism 36  Medical Laboratory Technology Calcium carbonates- colourless spheres or dumbbell shaped Triple Phosphate Crystals are Colorless, 4-6 sided prisms Referred to as 'coffin lid crystals Crystal found in abnormal urine Cystine - colourless, retractile, hexagonal Tyrosine - Fine needle in clumps, yellow and silky Leucine - yellow oily spheres Sulphonamide - yellow brown striated sheaves or round with radial striations Urine Pregnancy Test Most chemical tests for pregnancy look for the presence of the beta subunit of hCG, or human chorionic gonadotropin, in the blood or urine. hCG can be detected in urine or blood after implantation, which occur six to twelve days after fertilization. 37 Reference Book The common test done for detection of hCG in Urine is Card Test. It qualitatively detects the presence of hCG. It is a single step immunoassay and has high sensitivity. Paper strips coated with monoclonal anti-hCG antibody incorporated in a disposable card is used for the test. The urine is introduced in the sample window. Appearance of coloured bands in the control and test areas denote a positive test. Urine Analyser Automated machines are now available in the clinical laboratory to perform urine analysis called 'Urine Analyzer'. Using urine strip readers, the unit can detect and quantify a number of analytes including bilirubin, protien, glucose, red blood cells etc. The instrument works on the principle of Reflectance photometry and can process several hundred strips per hour. Different types of urine analysers include Cobas 6500, UF-1000-SIEMENS CLINITEK ANALYSER etc. DETAILING OF PRACTICALS Detection of Protein in Urine - Heat and Acetic Acid Method 1. Cloudy urine specimens are filtered or centrifuged 2. Fill clear urine 2/3 of a small test tube 3. Boil the upper portion of urine over a flame by keeping the tube in a slanting possition. (lower half serves as control) 4. If turbidity develops, add 1-2 drops of 3-5% acetic acid solution. Sometimes turbidity may be due to phosphate or carbonate precipitation. If it is so then glacial acetic acid clear up the turbidity. If it is due to protein then precipitation will be there after the addition of acetic acid. 5. Reboil the specimen 6. If turbidity is present protein is present.if there is no turbidity in upper portion then protein is absent. 7. Grade the turbidity as follows: 38  Medical Laboratory Technology Negative : No cloudiness Trace: Barely visible/ slight cloudiness. 1+ : definite cloud without granular flocculation 2+ : heavy and granular cloud without granular flocculation 3+ : dense cloud with marked flocculation. 4+ : thick curdy precipitation and coagulation Detection of sugar in Urine - Benedicts Test Procedure 1. Pipette 5ml of benedicts reagent in test tube 2. Add 8 drops of urine 3. Heat carefully over a flame for 1-2 minutes or place in boiling water bath for 5 minutes. 4. Cool under tap water. 5. Note the colour of solution and precipitate. Interpret as follows No change in color i.e. blue : Absence of sugar. Green : Trace green with yellow precipitate : 0.5% Yellow : 1% Orange : 1.5% Brick red :  2% Detection of Ketone bodies in Urine - Rothera's Test Procedure 1. Transfer about 5 ml of urine to a test tube 2. Add solid ammonium sulphate a little at a time with mixing to saturate the urine. 3. Add a pinch of sodium nitroprusside or 2-3 ml of solution 4. Mix well and add liquor Ammonia drop wise along the sides of the tube 5. Observe purple ring at the junction of two layers Appearance of purple ring indicates the presence of ketone bodies 39 Reference Book Detection of Bile pigment in Urine - Fouchet's Test Procedure 1. Take 5 ml of urine in test tube 2. Add 2 ml of barium chloride and mix well 3. Filter through filter paper 4. Unfold the filter paper and spread it on the dry filter paper. 5. Add 1- 2 drops of Fouchet's reagent on the precipitate 6. A green or blue color indicates presence of bilirubin. Detection of Bile salt in Urine - Hays test Procedure 1. Take 2ml of urine in a test tube or small beaker 2. Sprinkle little amount of fine sulphur powder 3. Observe the movement of sulphur powder without shaking the beaker 4. Sinking of sulphur powder indicates presence of Bile salts Microscopic examination of urine 1. Transfer urine sample to a conical centrifuge tube. 2. Centrifuge your sample at a moderate speed 1500 - 2500 rpm for 5 minutes. 3. Discard the supernatant by quickly pouring off fluid. 4. Tap tube with index finger to mix sediment with remaining fluid. 5. Make a wet mount of sample by transferring 1 drop of material to a slide and covering with a coverslip. 6. Examine the sample under the microscope under low and high power. 7. Identify the sediments observed and report their approximate number in Lpf/Hpf. hCG detection in urine- Urine Pregnancy card test 1. Open the sealed pouch of the test card and bring the card to room tem- perature 2. Add three drops of urine through sample window using dropper provided. Wait for coloured bands to appear. 3. Appearance of coloured bands in the control and test areas denote a positive test 40  Medical Laboratory Technology 3.2.3 Sputum examination Sputum is mucus that is coughed up from the lower airways of the respiratory tract i.e lungs, trachea and bronchi. Saliva and nasopharyngeal secretions are not part of the sputum. Sputum samples are used for microbiological investigations of respiratory tract infections and cytological investigations of cancer. Purulent sputum contains pus, composed of white blood cells, cellular debris, dead tissue, serous fluid, and viscous liquid (mucus). Collection: An early morning sample obtained by deep cough is a preferred sample for analysis, For the diagnosis of Tuberculosis, sample may be collected on three consecutive days. Wide mouthed disposable containers are preferred for the collection of sputum sample. The patient should take a deep breath, and empty his lungs in one breath and at the same time cough as hard and deeply as he can. Whatever he brings up by coughing, should be spit into the container and collected. Physical Examination It involves measurement of colour and consistency or appearance. Normal sputum is pale yellow in colour and contains mucoid materials. It may be red in Pulmonary Tuberculsis due to the presence of blood (Haemoptysis), dark yellow in bronchitis and brown in pneumonia. Normal sputum is viscous due to the presence of mucus, purulent in cases of bronchitis or acute upper respiratory tract infection, frothy in pulmonary oedema. Microscopic examination It involves mainly the examination of unstained and stained smears. For preparing sputum smears for microscopic examination, the purulent portion of the sample must be included. While handling utmost care should be taken in suspected cases of TB. Examination of Ziehl Neelson stained smears are most routinely used method in the diagnosis of Pulmonary Tuberculosis. Grams stained smears are commonly used for the detection of bacteria causing respiratory infections such as Pneumococci, Haemophilus and staphylococcus. Stained and unstained smears are used to detect the presence of puscells, epithelial cells, RBCs, malignant cells, Curshmann spirals, Charcot leydon crystals etc. 41 Reference Book DETAILING OF PRACTICALS Demonstration of Ziehl Neelsen Staining Technique Prepare a smear from the purulent portion part of the sputum using an applicator stick, heat fix and do ziehl nelson staining, air dry and look under oil immersion field of microscope for the presence of red coloured rod shaped organism in a blue back ground, number of organisms noted and graded as 1+to 4+ in the report. Presence of tubercle bacilli indicates tuberculosis infection. 3.2.4 Stool Analysis Stool is an important specimen for the diagnosis of diseases of gastrointestinal tract such as diarrhoea, dysentry, parasitic infection, gastrointestinal bleeding, peptic ulcer, carcinoma and malabsoption syndromes. Tests which reveal the presence of blood denotes an ulcerative lesion somewhere in the gastro intestinal tract, either inflammatory or as a result of cancer. Examination of stool shows the presence of parasites such as Entamoeba histolytica, various types of tape worms, pin worms, round worm and its egg. Collection of Specimen: A portion of fresh specimen is collected in a clean wide mouthed container with a spatula. Areas containing mucus, blood and pus should be incorporated in the specimen. Analysis includes Physical, Chemical and microscopic examinations Physical examination- Macroscopic examination of the stool involves consistency, colour, blood ,odour,pH etc. The presence of adult worms in a freshly passed stool (adult stages of Ascaris lumbricoides and Enterobius vermicularis, Proglottids of Taenia species) is also reported. Chemical examination It includes the tests for occult blood and reducing substances Blood may be present hidden in the stool called occult blood and is seen in malignancies, ulcers, haemorrhoids etc. Occult blood in stool were detected by Benzidine based tests, and is now not used due to the carcinogenicity of benzidine. It has been replaced by strip tests. The presence of reducing substances is usually done in children suspecting lactose intolerance. The unabsorbed sugars in stool are measured as reducing substances. Benedict's test is commonly employed for detecting reducing substances. 42  Medical Laboratory Technology Microscopic examination Microscopic examination of faeces can be done by saline preparation and iodine preparation. It helps detection of identification of cells, crystals, protozoa, ova of intestinal parasites Direct saline wet mount a. Place a drop of saline on the slide. b. Pick up a small amount of faecal material on the end of an applicator stick c. Emulsify in the saline and cover with a cover slip. Examine on low and high power. The smear should be thin enough to read a printed page through it. d The entire preparation must be examined for the presence of eggs, larvae and protozoa. Iodine wet mount Iodine preparation is useful for the identification and differentiation of protozoal eggs and cysts a. Place a drop of Lugol's iodine solution on a slide. b. Pick up a small amount of fecal material on an applicator stick c. Emulsify in the iodine solution and cover with a coverslip. d. Examine on low and high power as described in the previous procedure. 3.2.5 Semen Analysis Semen is a composite secretion formed by testes (male reproductive organ) as well as the accessory glands. Semen analysis is also called 'Seminogram' evaluates certain characteristics of semen and the sperm contained therein. It is done to evaluate male fertility or verifying the success of vasectomy or suspected cases of paternity in medico legal disputes. As a result of its relative simplicity, semen analysis is first requested before more complicated and expensive examination of the female in the infertility treatment. Specimen collection The sample should be collected after a minimum of 3-5 days of sexual abstinence. The sample should be obtained by masturbation and ejaculated into a clean, wide- mouthed container made of glass or plastic. It should be emphasized that the semen 43 Reference Book sample needs to be completely collected and reached in the laboratory within 30 minutes of collection, in order to limit the exposure of the semen to fluctuations in temperature. Semen analysis should begin with a physical (gross) examination soon after liquefaction,preferably at 30 minutes, but no longer than 1 hour after ejaculation. Physical examination includes volume, viscosity, colour, reaction and liquefaction time Liquefaction time: Semen is typically a semisolid coagulated mass. Within a few minutes at room temperature, the semen usually begins to liquefy and complete within 30 minutes. The time taken to liquefy the sample is noted. Macroscopic examination of semen Semen viscosity: The viscosity of the sample can be estimated by gently aspirating it into a plastic disposable pipette,allowing the semen to drop by gravity and observing the length of any thread. A normal sample leaves the pipette in small discrete drops. If viscosity is abnormal, the drop will form a thread more than 2 cm long. Colour: A normal liquefied semen sample has a grey-opalescent appearance. It may appear less opaque if the sperm concentration is very low. The colour may be red-brown when red blood cells are present (haemospermia), yellow in jaundice or in taking certain vitamins or drugs. Volume: Normal volume of semen is between 1.5-5 ml. Volume of sample is usually measured by collecting the sample directly into a modified graduated glass measuring cylinder. Reaction (pH):Reaction of semen is normally alkaline (pH 7.2-8.9), decrease in pH interfere with semen motility. Microscopic examination: During the microscopic examination of the sample, estimation of motility, sperm count and evaluation of sperm morphology is performed. The presence of cells other than spermatozoa and clumping of spermatozoa is also to be observed. 44  Medical Laboratory Technology Detection of Motility: A drop of well mixed liquefied semen is placed on a clean glass slide and put a cover glass. Then examine under high power objective for the motility of spermatozoa in at least 10 fields report in percentage and graded as Rapid progressive motile (Active). Slow progressive motile (sluggish) Immotile Sperm Count The number of sperms in sample can be estimated in a counting chamber after diluting the semen. After liquefaction, gently mix the specimen and draw it up to the 0.5 mark of a WBC pipette. Then draw the diluting fluid (Sodium bicarbonate- Formalin fluid) up to the 11 mark and mix well. Charge the counting chamber, keep for 5 minutes and then count the number of sperms in the four corner squares under low power objective. Count only those spermatozoa which are complete ie. with head body and tail. Calculate the number of spermotozoa/ml of the sample by multiplying the number of sperms counted by 50,000. The normal Sperm count is 80-160 millions /ml. Oligozoospermia refers to seminal fluid in which contain decreased number of spermatozoa. Azoospermia is a condition in which semen contains no sperm. Necrozoospermia refers to semen in which all sperm are non-viable or non-motile. Sperm morphology Sperm morphology is evaluated by examining the stained smear. After assessing approximately 200 spermatozoa, report the percentage of normal and abnormal forms. Normal semen contains less than 20% abnormal forms. Normal sperm morphology Spermatozoa consists of a head, neck, middle piece (midpiece), endpiece. For a spermatozoon to be considered normal, both its head and tail must be normal. All borderline forms should be considered abnormal. _ The head should be smooth and oval in shape _ The midpiece should be slender, regular and about the same length as the sperm head. 45 Reference Book _ The end piece should be thinner than the mid piece, and longer, about 10 times the head length. Chemical examination Chemical analysis of semen sample involves the detection of the following analytes: Acid Phosphatase - To evaluate the secretory function of the prostate and used in forensic analysis Fructose - since fructose is the source of energy to sperm, fructose con- centration indicates viability of sperms in sample. 3.2.6 CSF And Other Body Fluids CSF Examination CSF (cerebrospinal fluid) is a clear watery fluid circulating in the sub arachnoid space around the spinal cord and brain. It protects the brain and spinal cord from injuries, also acts as a transport medium for transport of substances between the tissues of brain spinal cord and blood. Examination of CSF is done for the diagnosis of various diseases like meningitis, tumours of central nervous system CSF is collected by lumbar puncture under aseptic precautions. A special type needle (Lumbar puncture needle) is used for specimen collection. Usually collected by an experienced clinician. The puncture site is cleaned with iodine and spirit. The needle is carefully introduced into the subarachnoid space. When the needle tip is reached the correct depth remove the stilette. CSF will flow through the needle. In adults the needle is usually administered between the spines of 3rd and 4th lumbar vertebra. In small children it is done between 4th and 5th lumbar vertibra.CSF pressure is also measured using a glass manometer attached to the needle. 6 to 8 ml CSF is usually collected for routine examination in three sterile containers. First few drops in the first container for bacteriological investigations, major portion in the second tube for bio chemical investigation and the rest for cytological investigation in the third tube. Examination of CSF should be done within 1 hour after collection. Physical Examination Normal CSF is clear having the specific gravity 1.003. Turbidity may be due to the presence of RBC leucocytes or bacteria, Yellow colour due to old haemorrhage, severe jaundice or spinal constriction. Bloody due to trauma during collection sub arachnoid haemorrhage. In tuberculosis meningitis the clot appears as a cob web. 46  Medical Laboratory Technology Microscopic Examination Done to evaluate the cellular constituents of CSF.Should be done soon after the collection. Normal CSF contains lymphocytes only. Polymorphs are present only in pathological conditions. normal cell count is 0-5 lymphocytes per cumm. Cell count is done by diluting 1in 10 with diluting fluid and counting is done using Improved Neubaur counting chamber or Fuchs Rosenthal counting chamber. The number of cells per ml of undiluted CSF is counted and calculated. Increased cell count is seen in meningitis, encephalitis, poliomyelitis, syphilis etc. Differential Cell Count Sample having an increased cell count is subjected to differential count. Prepare a smear using centrifuged sample dry and stain with Lieshman's stain, examine under microscope. Increased lymphocyte percentage indicates viral infection and increased neutophils indicates bacterial infection. Chemical Examination Chemical Examination includes glucose, protein and chloride. Normal protein content is 15-40mg%. Globulin is the major protein present. CSF protein is increased in acute meningitis, sub arachnoid haemorrhage etc. Sulphosalicylic acid test is usually used for CSF protein determination. Normal CSF glucose level is 40-70mg%, level is usually decreased in bacterial meningitis , small increase is noticed in poliomyelitis, encephalitis brain tumours etc. In diabetes the CSF glucose level is increased normally about 60-80% of the blood glucose level. Methods employed for blood glucose estimation can be used for CSF Glucose estimation also. Normal CSF Chloride level is 115-125 mEq/l and it is decreased in all types of meningitis, CSF Chloride is usually estimated by titrimetric method. Other body fluids include Pleural, pericardial, peritoneal fluid ,Ascitic fluid , synovial fluid, gastric juice etc. The general approach of laboratory study is the same as the CSF-physical examination, microscopic examination, bacterial culture and chemical examination.Synovial fluid is the fluid found around the joint examined in order to assist in the diagnosis of joint-arthritis, gout or infection of the joint (septic arthritis). 47 Reference Book ASSESSMENT ACTIVITIES Performance of urine analysis with the given sample and record the results Assignment given on importance of sputum examination in Tuberculosis Assignment given on how Microscopic examination of stool is performed Flow chart Preparation showing different procedures in semen analysis Chart Preparation showing precautions taken during collection of semen Theory Evaluation Questions 1 Your MLT Teacher asks your group to present a seminar on Semen Analysis a) Prepare sub topics for the members 3 b) Explain the procedure for determining the Total sperm count 5 c) With the help of a labeled diagram explain the structure of a 3 normal spermatozoa 2 CSF collection a complex process. Name the method used for this 1 3 The given diagram shows the result obtained while doing urine 1 pregnancy test for a patient T C 3 a) Interpret the result (1 score) b) Write the principle and procedure of the test( 3 score) 4 Give reasons for the following A. Just a positive test for urine urobilinogen doesn't have much clinical signific ance 1 B. Sulphur powder sinks to the bottom of urine sample positive for bile salts 1 5 Prepare an instruction notice to patients regarding sputum sample collection. 3 6 Find "me "using the clues i) I am an instrument used to check specific gravity. Temperature 2 correction is not needed ii) I am a hormone. I am normally found in the urine of pregnant woman 48  Medical Laboratory Technology Unit 3.3 CLINICAL BIOCHEMISTRY Clinical Biochemistry is one of the most rapidly advancing areas of a clinical laboratory which deals with various biochemical parameters of the body. The marked increase in the number and availability of biochemical determinations has evolved the advancement of laboratory medicine to a highly sophisticated molecular level. Advances in technique, practicing standards, and interpretation in this field have made the area, most multifaceted and complex.This unit of Clinical Biochemistry will make the learner familiar with the basic biochemical analytical procedures as well as to get aware of the recent trends in clinical biochemistry. This unit aims at the importance of emphasizing the application of clinical biochemistry to medicine. This unit gives the basic theoretical and practical information's in clinical biochemistry which are used for the diagnosis and treatment of diseases. In a clinical laboratory most of the investigations which the physicians rely on are from clinical biochemistry. Every disease has a biochemical origin, which may alter the biochemical parameters. These parameters are estimated from the body fluids by processing different specimens. Learners should know the basic requirements for the biochemical investigations including different biological specimens, their collection and processing of biochemical estimations and have brief knowledge of preparation of solutions and different types of assays. The learner should familiarize common instruments and the working procedures. The module suggests students activities a step by step guide to perform few of the biochemical estimation procedures to practice the procedures taught in the lessons. The module covers the routine biochemical investigations like blood sugar, renal function tests, Liver function tests, lipid profile and relevance of other clinical biochemistry estimations. Learning outcomes The learner: defines clinical biochemistry, grades of various chemicals and preparation of solutions differentiates different types of assays used in biochemistry. identifies different types of specimens, their collection and processing for biochemical analysis. 49 Reference Book performs cleaning of glass wares explains the parts, working, use and to operate common instruments used in biochemistry explains Diabetes and differentiates various blood samples used for blood sugar estimation identifies different blood sugar estimation methods and estimates blood glucose by GOD POD method explains GTT, GCT procedures, Glucometer technique and importance of HBA1c explains the relevance of renal function test and to identify various tests included in the RFT panel identifies common blood urea estimation methods and estimate blood Urea by Berthlot method identifies common creatinine estimation methods and to estimate S.Creatinine by Jaffes method discusses the importance of uric acid and estimation of uric acid by uricase method identifies the importance of microalbumin, Cystatin-C and clearance tests for the evaluation of renal function explains the relevance of Liver function test and to identify various tests included in the LFT panel explains jaundice