Concise Book of Medical Laboratory Technology: Methods & Interpretations Ramnik Sood
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LaboratoryCHAPTER 1

 
INTRODUCTION
The definition of health includes a state of complete and perfect physical, mental, social and spiritual well-being and not just the absence of disease or infirmity and good health is a fundamental right of every living human being on earth. However, modern world, though, has to an extent eliminated infectious diseases. But the focus has now shifted to lifestyle diseases. Pollution of every nature too has taken its toll. About half a century back, the predominant diseases used to be infective ones but now you may find individuals in mid-twenties waiting for their turn for open heart surgeries. Also, modern medicine has increased the longevity of life accompanied by attendant geriatric diseases like Alzheimer’s disease and malignancies. The polluted and toxic world has not spared the fetuses in utero and neonates. A new face of disease has emerged, diseases like HIV-AIDS and severe acute respiratory syndrome (SARS), are new entrants in the long list of infective diseases. We may have eradicated smallpox but tuberculosis and malaria have raised their heads with a vengeance. So, do what you might. Some forms of disease, mild or severe will strike every human being living. On getting sick, the patient first comes in contact with a clinician—medical or surgical. The clinician gives a patient hearing (if the patient is conscious) to his problems and symptoms and also takes note of various signs, which he sees or elicits. Sometimes, he may immediately arrive at a diagnosis and may under emergency circumstances institute treatment at first instances. In most cases, however, he will have a differential diagnosis in mind and to arrive at a specific diagnosis he usually orders for a battery of tests.
Various means of diagnosis are available.
  1. Most important: Clinical laboratory tests which include any tissue or fluid obtained from the body.
  2. Imaging sciences: X-rays, ultrasound, color Doppler, computerized axial tomography (CAT) scan, magnetic resonance imaging (MRI) scan and the latest positron emission tomography (PET) scan.
  3. Electrical signal processing techniques: ECG, EMG, EEG and nerve transmission techniques, etc.
  4. Direct visualization techniques: With the availability of fiberoptic-based technologies, the clinician is now capable of passing small tubes (called scopes) through natural passage ways of the human body (without actually surgically opening up the part), e.g. gastroscopy, cystoscopy, etc. These techniques, eventually culminate in taking small tissue samples (biopsies) which are sent to histopathology laboratories.
So, whenever, any sample from a human body is taken (either voided naturally or obtained by the clinician or the laboratorian), it is referred to the clinical laboratory for investigation. On receipt of a report from the laboratorian, the clinician, then, makes up his mind and starts a unidirectional or specific treatment against the disease thus diagnosed. It would not be wrong to designate medical laboratory personnel as the backbone of the clinicians. But, for these technologists, the clinicians would forever grope in the dark. Gone are the days when diabetes mellitus was presented with the classical triad of symptoms—increased thirst, hunger and urination; likewise, typhoid seldom presents with a step-ladder pattern fever. Blood testing is absolutely mandatory, to know that they exist, their severity and eventually, after treatment; to know that they are under control or cured. Investigations are diagnostic as well as prognostic tools.
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Clinical laboratory investigations nowadays are being utilized as future predictors. On getting warning signals, one can take necessary corrective measures (lifestyle and/or dietary) and can prevent diseases from striking or at least deferring or postponing their arrival.
HELP! AND YOU DID AND YOU ALWAYS WILL. When a clinician is lost, you shall show him the way in the best possible way, you lead him to a diagnosis and let him do his job thereafter. He may come back to you later to determine that his efforts have been fruitful.
The following pages within the covers of this book will show you the right path on how to be an excellent laboratorian. Do your best in serving mankind. As you yourself may be a patient tomorrow. This book shall also serve you well by providing interpretation of the results obtained by you. This book shall be true to its title “Concise Book of Medical Laboratory Technology: Methods and Interpretations”.
While physiology is the study of essentially normal structures and functions of a body, pathology deals with the study of a diseased organ or system of the body, its abnormal functions, their mode of origin, their progress to recovery or otherwise. All these studies come under the ambit of a clinical pathology laboratory. A clinical laboratory has further sub-branches such as: hematology, biochemistry, seroimmunology, microbiology, cytogenetics, histopathology, cytopathology, blood banking and last but not least—clinical microscopy.
A clinical laboratory can be manned by a qualified doctor specializing in clinical pathology, biochemistry, immunology, blood banking, histopathology, cytopathology, hematology, microbiology or cytogenetics. The pathologist is usually assisted by laboratory technicians or technologists (they are also qualified for the job) and lastly the cleaning and documentation staff. Only by collective efforts of the individuals mentioned above, a proper report can be generated. Be grateful to the clinician for having faith in you and give back nothing except an accurate and correct timely report. A delayed report may at times be too late. The patient may have lost his life by then. A timely correct report is the essence of running a good laboratory.
The cycle of health-disease with all intermediaries is given in Figure 1.1. Just as there are primary, secondary and tertiary health centers, there are also the primary, secondary and tertiary laboratories too. In India, there are no specific guidelines as to what or how much they can do and overlapping can occur. A superior laboratory may perform all functions of an inferior laboratory too.
 
Primary Laboratory
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FIG. 1.1: Health-disease-health cycle
In rural setups, for instance, a primary laboratory may provide only the basic investigations. These investigations are simple to perform and do not involve expensive 3machinery usage. Such laboratories are also attached to physician chambers nowadays, so that clinicians may obtain basic inputs right in their own premises. These primary laboratories may provide the following simple investigations:
  • Hemograms (hemoglobin estimation, total and differential counts, erythrocyte sedimentation rate and packed cell volume with basic peripheral smear study including the reporting of hemoparasites)
  • Routine and microscopic studies of urine and stool. Routine examination also entails chemical examination either by laborious and time-consuming old chemical methods or by new generation dipstick tests. These may include tests for glucose, bilirubin, ketones, hemoglobin, leukocytes, pH, nitrites, protein, urobilinogen and specific gravity in case of urine. For stool samples, reducing substances, pH and occult blood may be performed. Basic spot/latex/device tests (e.g. pregnancy test) may be conducted.
 
Secondary Laboratory
These are laboratories that assist a clinician to confirm a clinical suspicion or establish a diagnosis. Therapy and prognosis monitoring can also be provided from these laboratories. Such laboratories are staffed by qualified personnel who are trained and experienced to perform the tests. They also have a perfect knowledge of the equipment and machines they use. They should be aware of quality control essentials and be well versed with interpretational aspects of the reports generated by their laboratories. In addition to what has been mentioned under primary laboratories, secondary laboratories also perform:
  • Routine immunohematological tests.
  • Routine examination of all body fluids, e.g. semen, cerebrospinal fluid (CSF), sputum, etc.
  • Routine bacteriologic studies including stains, cultures and antibiograms. Routine mycological investigations would include—primary cultures, isolation and identification techniques along with microscopic evaluation.
  • Routine immunoserological tests. These can include tests like Widal, STS, ELISA or strip or device tests HIV I and II, hepatitis B and hepatitis C, etc.
  • Routine biochemistry investigation and organ profile tests, e.g. lipid, cardiac, liver and renal profiles.
  • Under hematology, these laboratories may also provide RBC indices, platelet, reticulocyte count and absolute eosinophil counts. They can also classify anemias and should be able to indicate hematologic malignancies. When headed by a pathologist, they should be in a position to report bone marrow smears/preparation too.
 
Tertiary Laboratory
These kinds of laboratories should be able to perform all kinds of sophisticated and delicate/precise investigations. The tertiary laboratories can branch out in very special fields and not cater to all aspects of specialized tests. Besides doing all investigations that are conducted in secondary laboratories, they also carry out the following:
  • Specialized hematological (e.g. leukemia type), coagulation profiles and immunohematological investigations. They are equipped with 18 parameter cell counters with differentials and flow cytometry
  • Complete biochemical assays, commonly referred to as SMA-12, SMA 27, etc. Also included are elemental assays, e.g. zinc, magnesium, iron, total iron binding capacity (TIBC), lithium, etc. special enzymes like HBDH, lipase and isoenzymes, etc.
  • Complete immunology based assays for hormones, cancer markers, hepatitis markers, rheumatic/autoimmunity etiology-based profiles, TORCH profiles, rare infectious diseases (e.g. brucellosis leptospirosis, cysticercosis, echinococcosis, etc.)
  • All microbiological processes, e.g. cultures—aerobic, anaerobic, fungal, tubercular, etc. with antibiograms.
The techniques for these investigations may vary. They may be ELISA, chemiluminescence, turbidimetry, PCR, etc. These laboratories are totally automated and have sizable workload. Furthermore, they also undertake all histopathology (simple H and E, special staining techniques, immunohistochemistry methods) and cytopathology processing and reportings. They may also undertake cytogenetic investigations, e.g. chromosomal analysis. The dissemination of reports from these laboratories is in keeping with recent trends in telecommunications, e.g. fax, e-mail, etc.
In the United States of America, these laboratories though classified differently (with a few differences) are covered under the Clinical Laboratory Improvement Act (CLIA) of 1988.
 
LABORATORY SET-UP
Unless the laboratory is hygienic and provides necessary physical and operative comfort, it would be wrong to expect perfect results. To get perfect results, one has to provide a perfect set-up for people to work in.
 
Laboratory Building and Space
Ample working space is absolutely essential. For smaller laboratories up to 25 square meters (Fig. 1.2), the working platforms can be arranged along the walls while the central area is kept free for movement.
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FIG. 1.2: A typical small laboratory
For larger areas, partitions can be made which would create separate spaces for different sections (Fig. 1.3). The chief pathologist must have casual access to all sub-units of the laboratory. If possible, he should be able to directly see into the cabins either through glass windows or through closed circuit cameras. In the cabins again, the central region should be kept free and benches be placed against the walls and away from the doors.
  • Hygiene is of utmost importance. The whole facility should be absolutely clean, uncrowded and devoid of any hindrances to movement of men and materials. Never, should a chance arise where two people would clash or contaminated material would be spilt all over
  • Scratch proof matt finish vitrified floor (slip resistant) should be provided. The walls should preferably have white ceramic tiles. Such provisions are resistant to chemicals and disinfectants
  • All benches should be preferably 2½ feet high and those to be used while standing should be at least 3 feet high. The bench surfaces should be solvent and acid proof.
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FIG. 1.3: A typical large/complete laboratory plan
Every laboratory and/or its section must have at least one sink and one hand wash basin. The hand wash basin should not be used for any other purpose, the sink can be utilized for laboratory purposes like washing off stains from slides or washing glassware or discharging non-contaminated laboratory refuse.
 
Physical Aspects of a Laboratory
  • The ambient temperature should be within the comfort zone of a human body. It should between 21 and 27°C. If the laboratory is in a cold zone, it must have heating provision, and conversely, if it is in a hot zone, it must have cooling or air conditioning. The environment control appliances like air conditioners or heaters must not directly discharge air at the working bench zone
  • A good exhaust system is a must for all laboratories. This removes dirty air (aerosols), which may at times be foul smelling. The sample collection zone too, must have excellent exhaust provision
  • Adequate ventilation is also essential but without strong currents of air
  • Lighting should be more than adequate and places where very delicate or fine processes are being conducted should have additional lighting provision. As far as possible, do not use excessive heat producing bulbs and lamps. The new CFLs are ideal
  • Windows that are exposed to bright sunlight can be internally fitted with reflective films or blinds
  • There should be sufficient running water for the laboratory and all must have sufficient number of sinks and hand wash basins
  • As most machines consume a lot of electricity, sufficient power load (a little in excess) must be available to the laboratory
 
Provisions and Precautions
Every working room or cabin should have adequately spaced provision of water, electricity, gas, sinks lighting and exhausts. All aspects, whether plumbing, electrical systems or gas connection must pass through regular inspections and a log book should be maintained of such preventive exercises. Preventive maintenance should be carried out by knowledgeable and qualified persons.
 
Fire Prevention
  • Install appropriate fire extinguishing system and timely testing of such a system be conducted at regular intervals
  • Color code and place firefighting equipment at an easily visible and reachable location. Check the working capability of all such systems at regular intervals
  • Provide adequate ventilation in zones where flammable chemicals are used. Before these substances reach combustible or explosive concentration, they should be removed by mechanical exhausts
  • 5Post “No-smoking” signs in zones where smoking can be hazardous
  • Lastly, mark clearly the emergency exit points. Keep the emergency exit route free from obstructions.
 
Electrical Installations
  • Hire a proper, qualified electrical engineer and explain to him the purpose of the premises being taken. As far as possible, all points where sparks can be generated should be kept out of room/cabins where explosive chemicals are likely to be used
  • Use earthing everywhere and install fire-resistant cables in the laboratory
  • Employ only certified products
  • Use one electrical socket for a single device or machine. Overloading is usually the cause of accidents.
 
Liquefied and Compressed Gases
  • Color code and identify each gas container. Check their valves regularly
  • Keep all such cylinders away from sources of heat and electrical sparks
  • When not in use, replace protection/safety caps back on the cylinder mouths.
 
Chemicals and Radioactive Substances
  • Label all bottles with proper names of contents and affix warning signs and symbols as applicable to them
  • Clearly display the warning charts (both chemical and radioactive) next to such containers. All staff members working in such areas should be well trained to handle accidents of any kind that can happen
  • A stringent record of stocks should be maintained of all persons and radioactive substances being used in the laboratory. A bottle lost or stolen is invitation to problem.
 
Stores
  • Every bottle/container should be labeled. Affix the hazard intensity on the bottle or the container
  • Ensure in every possible way that the containers cannot under any circumstances fall or spill. This can be done by placing the most dangerous chemical at the bottom or at the floor level
  • Proper ventilation should be ensured in storage zones that house flammable chemicals. Keep fire extinguishing equipment handy. Post “No smoking” signs that are clearly visible. Make sure that the place remains free from pests.
 
Staff Safety and Facilities
The most important asset of any institution is the manpower that works for it. It holds true for laboratories too. Absence of staff due to morbidity or mortality can stifle your working capacity, capability and reputation. Provide adequate facilities to your team. (Designate a room or space meant exclusively for retiring or resting and consuming foodstuffs).
  • Hot and cold running water with soap and disinfectants should always be provided. Clean hand towels should be replaced daily
  • A clean toilet for use by staff members is mandatory as are the changing rooms. If possible, separate units for male and female members should be provided
  • Biomedical wastes and non-biomedical wastes should be discarded properly and safely. Chemical treatment of liquid wastes and incineration of solid wastes should not be overlooked. Wastes handled properly ensures good health of your working team
  • Designate a room or space meant exclusively for retiring or resting and consuming foodstuffs. Under no circumstances, laboratorians should eat or drink on their workbenches. Provide safe drinking water to all
  • Each room/cabin must have a first-aid box kept at an identified place that is easily accessible. Every person working in the laboratory must be aware of all hazards that exist and must also know about the remedial measures that should be taken if something happens. What can be managed in house should be managed, when required, assistance of other specialists must be taken. Contact numbers of such institutions/specialists must be displayed prominently
  • All members of your team must be immunized as relevant to the laboratory work. Make sure no single person works alone in a room or cabin. Two compatible persons should work together always.
 
Basic Laboratory Safety
  • Use only certified safe equipment in the laboratory
  • Decontaminate all equipment regularly and before their servicing or maintenance, use appropriate disinfectants correctly
  • As far as possible, use disposable plasticware to avoid contamination (chemical, biological, etc.) and breakages with ensuing dangers
  • Regularly test and service biological safety cabinets and fume cupboards.
Appropriate safety measures taken by you will go a long way in enhancing productivity.
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As a rule, the place for receiving or withdrawing the specimens should be separate from the working compartment. To avoid specimen mixing (hazardous), each sample should be carefully labeled. The label should clearly mention the alloted specimen number, the date and time of receipt of specimen, the investigations to be done and most important the name of the patient.
Both, the clinical and the paraclinical workers are equally at risk of acquiring transmissible diseases through the patient or through the test samples. The risk of these can be lessened by taking appropriate vaccinations. In addition, one should attend to one’s general hygiene and prevent fomite transmission of any infectious disease. Disinfect the working benches and as far as possible autoclave (or chemically disinfect) various glassware used in the laboratory. Use a rubber teat for sucking/filling the pipettes. To avoid strain on the eyes, keep both eyes open while doing microscopic work. Before leaving the laboratory, one should thoroughly wash one’s hands with soap and water, and then rinse them well in a disinfectant lotion.
 
CODE OF CONDUCT FOR MEDICAL LABORATORY PERSONNEL
  1. Place the well-being and service of the sick above your own interests.
  2. Be loyal to your medical laboratory profession by maintaining high standards of work and strive to improve your professional knowledge.
  3. Work scientifically and with complete honesty.
  4. Do not misuse your professional skills or knowledge for personal gain.
  5. Never take anything from your place of work that does not belong to you.
  6. Do not disclose to a patient or any unauthorized person the result of your investigations.
  7. Treat with utmost confidentiality and personal information that you may learn about a patient.
  8. Respect and work in harmony with the other members of your hospital staff or health center team.
  9. Be at all times courteous, patient, and considerate to the sick and their relations.
  10. Promote health care and the prevention and control of disease.
  11. Follow safety procedures and know how to apply first aid.
  12. Do not drink alcohol during laboratory working hours or when on emergency stand-by.
  13. Use equipment and laboratory-ware correctly and with care.
  14. Do not waste reagents or other laboratory supplies.
  15. Fulfil reliably and completely the terms and conditions of your employment.
Always remember that you can be a patient tomorrow. Treat others as you would want them to treat you.
 
ACCIDENTS
 
Safety Measures in the Laboratory
You must remain alert and cautious while working in the laboratory. You must know that careless handling of reagents, glassware or specimens to be tested in the laboratory can cause serious injury and is dangerous to life.
 
Hazards in the Clinical Laboratory
Clinical laboratory workers may encounter three types of hazards:
  1. Physical,
  2. Chemical, and
  3. Biological hazards.
 
Physical Hazards
Physical hazards are present in ordinary equipment or surroundings. Electrical equipment, open flames, laboratory instruments and glassware can all be hazardous if improperly used.
 
Electricity
  • All electrical equipment must be properly grounded following the manufacturer’s instructions
  • Even minor repairs, such as replacement of the microscope bulbs, require that instrument be disconnected from the power supply before the work is begun
  • All electrical cords and plugs be kept in good shape and order with no frayed cords or exposed wires
  • Avoid overloaded circuits
  • Extension cords present several safety hazards and should not be used except in emergency.
 
Fire
Fire is a potential danger in the workplace:⊠
  • Though rare, they can occur when open flames are used in the vicinity of flammable liquids
  • Make sure that loose clothing and long hair do not catch fire
  • Instead of open flames, use hot plates, microwave ovens, electric incinerators and slide warmers
  • Store flammable chemicals in a flameproof cabinet, away from heat sources and well-ventilated area. A flameproof cabinet can protect flammable chemical from flames 7until firefighters arrive and also allow workers more time to escape
  • All laboratory workers must know about the escape route and procedure to follow if that exit is blocked
  • All workers must know the location of fire extinguishers and how to use them
  • Inspect all fire extinguishers periodically and log the date of inspection.
 
Usual Causes of Fire in the Laboratory
  • Naked flames (do not work with loose clothing and long hair near naked flames). Naked flames can also ignite flammable liquids and gases
  • Electrical overloading. Use one socket for one equipment only. Do not operate a 15 amp equipment from a 5 amp socket
  • Poor electrical maintenance. No frayed or open/exposed wires be ever used
  • Leaving equipment switched when not in use. Out of sight is out of mind
  • Deteriorated gas tubing. Leakage of gas is an open invitation to fire hazard. If you suspect gas leakage, do not operate any electrical equipment (do not ever switch on a light or a fan)
  • Smoking in the laboratory
  • Misusing matches. Use carbonized matches as far as possible
  • Storing flammable and explosive chemicals in an ordinary refrigerator.
 
When a Fire Occur
  • For tiny blazes; water, sand and a fire blanket can be employed to put out the fire. For larger blaze, a fire extinguisher can be used
  • Never use water on an electrical fire or one caused by organic solvents (ether, alcohol, petrol, etc.). For electrical fires, use carbon dioxide fire extinguisher. For organic solvents, use sand or halon
  • Escape via the fire exit route. Stay close to the floor, cover your mouth and nose with a damp cloth to filter out some of the harmful fumes
  • Inform firefighting department of your area if you feel the fire can go out of hand. Medium to large fires should be reported irrespective of your preparedness to handle them.
 
Laboratory Equipment (Table 1.1)
  • Use all laboratory equipment as per manufacturer’s recommendation
  • Any instrument with moving parts, such as a centrifuge, must be operated with a special regard for safety. Latch, the lid before turning it on. On turning it off, do not open the lid before it has come to a complete stop
  • Autoclaves present special hazards. Strictly adhere to manufacturer’s instruction to prevent explosions and burns. Use insulated gloves while removing hot items from the autoclave.
 
Glassware
  • Use glassware that is free of chips and cracks. Damaged glassware is weakened and may break, resulting in injury
  • Broken glass should be cleaned with a brush and dustpan and not with bare hands
  • Glass should not be discarded into regular trashcans, but into rigid cardboard or plastic containers
  • Wherever possible, replace glassware with plasticware.
 
Equipment Related Hazards
  • Hypodermic needles: Accidental inoculation, aerosol or spillage
  • Centrifuges: Aerosols, splashing and tube breakages
  • Culture stirrers, shakers, agitators: Aerosols, splashing and spillage
  • Refrigeration: If flammable chemicals are stored within them, the light switches, thermostats, etc. can provide sparks to ignite them
  • Water baths: Provide ground for microorganismal growth
(The risk of acquiring hepatitis B from a needle stick is 30%, hepatitis C is 2 to 10% and HIV is 0.3%).
 
Equipment/Materials Employed to Eliminate/Reduce Hazards
TABLE 1.1   Fire fighting equipment
Fire fighting material
Used for
Contraindicated for
Fire blanket
Clothing fire, G small blaze
Electrical fires, flammable liquids, a small blaze burning metals, alkali metal
Water
Paper, wood, fabric
Electrical fires, flammable liquids, burning metal, alkali metal
CO2 fire
extinguisher
Flammable liquids and gases, electrical fire
Dry powder
As above
Foam
Flammable liquids
Halon spray
All kinds of fires
  • Laboratory apron: Assists in diminishing skin contacts to a certain extent
  • 8Biological safety cabinets: Prevent dangers arising out of aerosols and splatters
  • Splatter shields: Provide protection from splatter of specimen and chemicals
  • Pipetting aids (teat or electromechanical devices). Prevent from hazards arising out of mouth pipetting
  • Goggles: Protect eyes from impacts and splashes
  • Face shields: Protect the face from impacts and splashes.
 
Safety with Chemicals/Reagents
Excepting just a couple of reagents, almost all chemicals/reagents used even in the most basic laboratory are lethal poisons if consumed by anyone. Even if they are splashed on the skin/eye, they can cause irreversible damage. There is an appropriate way of handling and storage of hazardous chemicals to avoid injury and damage to self and others. In our country (and other tropical nations), excessive heat can decompose many chemicals, cause explosions, or lead to the formation of toxic fumes.
 
Labeling of Hazardous Reagents/Chemicals
At appropriate places, display the prohibition signs; and on all dangerous reagents or chemicals, stick Hazard warning symbols. In the following pages, important signs and symbols as related to safety in the laboratory are given.
 
Incompatible Chemicals
Fair number of common laboratory chemicals react dangerously if they come in contact with specific chemicals. Ensure that you keep such chemicals away from each other. A few examples are listed below:
 
Acids
  • Acetic acid with chromic acid, nitric acid, hydroxyl compounds, ethylene glycol, peroxides and perman-ganates
  • Chromic acid—with acetic acid, alcohol, glycerol and other flammable liquids
  • Sulfuric acid—with chlorates, perchlorates, perman-ganates and water.
 
Vaporizing Substances
  • Acetone—with sulfuric acid and nitric acid
  • Flammable liquids—with chromic acid, hydrogen peroxide, nitric acid, ammonium nitrate and halogens.
 
Others
  • Alkali metals, e.g. calcium, potassium, sodium (these form hydroxides on coming in contact with water) and with other chlorinated hydrocarbons
  • Chlorine—with ammonia, hydrogen, benzene and other finely divided metals
  • Copper—with azides, hydrogen peroxide and acetylene
  • Cyanides—with all acids and alkalies
  • Hydrogen peroxide—with copper, iron, chromium and most other metals
  • Iodine—with acetylene and ammonia
  • Sodium azide—with lead, copper and other metals.
 
Flammable Chemicals
These include ether, xylene, toluene, methanol, ethanol, glacial acetic acid, acetic acid, acetone, acetic anhydride, alcoholic Romanowsky stains and acid alcohol, etc.
 
Storage
These should be stored in a fire-proof metal box at ground level, preferably in a cool store. A container well lined with tin foil can also be used. Store only small quantities of such solvents on the shelves.
 
Safe Use
Ensure that there is no open flame nearby while opening a bottle containing flammable solvent. Nearest flame should be at least 10 feet away. Never heat a flammable liquid over any flame. Use a water bath or electric hot plate.
 
Control of Fire Caused by Flammable Chemicals
Best controlled by smothering them. Use sand, thick blanket or the now available multipurpose fire extinguishers. Pouring water on such fires will spread them. Every laboratory should be equipped with the commercially available fire extinguishers. If these are not available, there should be sand buckets in accessible places.
 
Corrosive Chemicals
These include strong acids, e.g. concentrated sulfuric acid, hydrochloric acid, nitric acid, glacial acetic acid, trichloroacetic acid, orthophosphoric acid, and strong alkalies like sodium hydroxide and potassium hydroxide.
 
Storage
Store these at low levels.
 
Safe Use
Never attempt mouth pipetting. Accidental swallowing can be lethal as these chemicals cause destruction of living tissue. Always pour a corrosive chemical at below eye level, slowly, and with great care to avoid splashing. Wear protective eye glasses/eye shields while opening such containers. Always add the corrosive substance to water and that too slowly. The addition of small amount of water to sulfuric acid is enough to produce sufficient heat to break a glass container.
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Toxic, Harmful, and Irritating Chemicals
These are chemicals that can cause death or serious ill-health if swallowed or inhaled or if they come in contact with skin. Examples are potassium cyanide, mercuric nitrate, sodium azide, sodium nitroprusside, formaldehyde solution, chloroform, barium chloride and methanol. Iodine and sulfuric acid also fall in this category. Skin and mucous membrane irritants are xylene, formaldehyde and ammonia vapors.
 
Storage
Store highly toxic chemicals, e.g. potassium cyanide in a locked cupboard. Stock solutions should also be stored safely in a cupboard, not on an open shelf.
 
Safe Use
Always wear protective gloves and after working with them immediately lock them up. Always wash your hands after using a toxic or harmful chemical. Keep fume forming chemicals in a fume cupboard. Never mouth pipette them.
 
Oxidizing Chemicals
These include chlorates, perchlorates, strong peroxides, potassium dichromate, and chromic acid.
 
Storage
Keep these away from organic materials and reducing agents. They can produce much heat when in contact with other chemicals, especially flammable chemicals.
 
 
SIGNS FOR MEDICAL LABORATORIES
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FIG: General laboratory
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FIG: Laboratory cautionary
 
 
Safe Use
Handle them with utmost care. Most of them are dangerous to skin and eyes and when in contact with reducing agents.
 
Explosive Chemicals
These chemicals can explode on being heated or on getting exposed to flame or friction. A good example is picric acid, which must be stored under water. If picric acid is allowed to dry, it can explode.
 
Carcinogens
These chemicals can cause cancer by ingestion, inhalation, or by skin contact. Such chemicals include benzidine, O-toluidine, O-dianisidine, a and b naphthylamine, nitro-samines, nitrosophenols, nitronaphthalenes, and selenite. The carcinogenic risk is directly proportional to the length and frequency of exposure and the concentration of the chemical.
 
Storage
Label their containers “CARCINOGENIC” and handle with special precautions.
 
Safe Use
Must wear protective plastic or rubber gloves, a facemask and eyeshields when handling carcinogenic chemicals. Do not let them come in contact with skin. After handling a carcinogen, wash well in cold water all the apparatus, bench, bottles and protective gloves (before removing them) and change your overall. Rinse your hands in cold running water before using soap. Should a carcinogen come in contact with skin, wash the affected part in cold running water for 5 minutes.
 
ACCIDENTS IN THE LABORATORY
They may be caused by:
  1. Toxic substances
  2. Heat
  • Open flames
  • Hot liquids
  • Inflammable liquids
  • Explosions
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FIG: General prohibition
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FIG: General laboratory
  1. 12Broken glass
  2. Contamination by infected material
  3. Electric shock.
A suggested list of first aid equipment is given later in the chapter. The items should be readily available in the laboratory. They must not be kept in a locked cupboard.
 
First Aid in Laboratory Accidents
 
Acid Burns
Nitric, sulfuric, hydrochloric and trichloroacetic acids.
In all cases: Wash immediately with large quantities of water.
 
Acid Splashes on the Skin
  1. Wash thoroughly and repeatedly with water.
  2. Bathe the affected skin with cotton wool soaked in 5% aqueous sodium carbonate.
 
Acid Splashes in the Eye
  1. Wash the eye immediately with large quantities of water sprayed from a wash bottle or rubber bulb. Squirt the water into the corner of the eye near the nose (Figs 1.4 and 1.5).
  2. After washing, put 4 drops of 2% aqueous sodium bicarbonate into the eye.
  3. Refer the patient to a physician. Continue to apply bicarbonate solution to the eye while waiting for the doctor. Alternatively, hold the eye under the running tap.
 
Swallowing Acids
Accidental swallowing while using a pipette:
  1. Call a physician.
  2. Make the patient drink some 5% soap solution immediately. Alternatively, give him two whites of egg mixed with 500 mL of water or milk. If neither of these is available, he should drink ordinary water.
  3. Make him gargle with the soap solution.
  4. Give him 3 or 4 glasses of ordinary water.
  5. If the lips and tongue are burned by the acid:
    • Rinse thoroughly with water
    • Bathe with 2% aqueous sodium bicarbonate.
 
Alkali Burns
Sodium, potassium and ammonium hydroxide.
In all cases: Wash immediately with large quantities of water.
Important: Alkali burns are as serious as, and often more serious than, acid burns.
 
Alkali Splashes on the Skin
  1. Wash thoroughly and repeatedly with water.
  2. Bathe the affected skin with cotton soaked in 5% acetic acid (or undiluted vinegar).
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FIG. 1.4: Eye washing upright
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FIG. 1.5: Eye wash lying
 
Alkali Splashes in the Eye
  1. Wash immediately with large quantities of water sprayed from a wash bottle or rubber bulb. Squirt the water into the corner of the eye near the nose.
  2. After washing with water, wash the eye with a saturated solution of boric acid (apply drops repeatedly).
  3. Refer the patient to a physician at once.
 
Swallowing Alkalis
Accidental swallowing while using a pipette:
  1. Send for a physician.
  2. Make the patient drink at once:
    • 13A 5% solution of acetic acid or lemon juice or dilute vinegar (1 part vinegar to 3 parts water).
  3. Make him gargle with the same acid solution.
  4. Give him 3 or 4 glasses of ordinary water.
  5. If the lips and tongue are burned by the alkali:
    • Rinse thoroughly with water
    • Bathe with 5% acetic acid.
 
Poisoning
This can be caused by:
  • Inhaling toxic vapors or gases (e.g. chloroform)
  • Accidental swallowing while pipetting a poisonous solution.
 
In all cases
  1. Send for a physician or qualified nurse, specifying the toxic substance involved
  2. Place the victim in the open air while waiting for the physician.
 
Burns Caused by Heat
They fall into two categories:
  • Severe burns—affecting large areas of skin, e.g. burns caused when burning ether or boiling water is spilled over the victim
  • Minor burns—affecting a small area of skin, e.g. burns caused by hot glassware or a Bunsen flame.
 
Severe Burns
  1. If the victim is on fire, e.g. if splashed with burning ether or other inflammable solvent, roll him in a blanket or overall to smother the flames.
  2. Inform the physician on duty immediately.
  3. Lay the victim on the ground. Do not remove his clothing. Cover him if he is cold.
  4. Do not apply any treatment to the burns. This must be left to the physician.
 
Minor Burns
  1. Plunge the affected part into cold water or ice-water to soothe the pain.
  2. Apply mercurochrome or acriflavine ointment to the burn.
  3. Apply a dry gauze dressing loosely.
  4. If the burn becomes infected or does not heal, refer the patient to a physician.
Note: Never tear off the blisters that form over the burns.
 
Injuries Caused by Broken Glass
These are caused by broken test tubes, syringes or other glassware.
  1. Wash the wound immediately to remove any glass pieces.
  2. Apply mercurochrome or acriflavine ointment to the wound.
  3. Cover with gauze and adhesive tape.
  4. If the cut bleeds profusely, stop the bleeding by pressing down on it with a compress. Refer the patient to a physician.
  5. If the cut bleeds heavily with the blood spurting out at intervals, try to stop the bleeding with a compress and call a physician or qualified nurse.
  6. Continue to press on the wound while awaiting the physician’s or nurse’s arrival. He or she will decide whether a tourniquet should be applied.
 
Contamination by Infected Material
 
 
Wounds caused by broken glassware containing stools, pus, etc.
  1. Wash the wound immediately.
  2. Check whether the cut is bleeding. If not, squeeze hard to make it bleed for several minutes.
  3. Bathe the whole area, i.e. the edges of the cut and inside the cut, with antiseptic lotion.
  4. Wash thoroughly with soapy water.
  5. Bathe again with antiseptic lotion.
  6. Refer the patient to a physician, if the material involved is known to be very infective, e.g. pus.
 
If infected material is accidentally sucked into the mouth:
  1. Spit it out immediately.
  2. Wash out the mouth with diluted antiseptic lotion.
  3. Wash out the mouth thoroughly with large amounts of clean water.
 
Bodily Damage by Electric Shock
A low-voltage alternating electric current (220 V) is usually used in the laboratory and electric shocks are rare. They may occur when faulty equipment is being handled, particularly with wet hands. The symptoms are fainting and asphyxia.
  1. Before doing anything else, put off the main switch.
  2. Send for a physician.
  3. Begin giving mouth-to-mouth respiration immediately if required (Fig. 1.6).
 
Precautions for the Avoidance of Accidents
  1. Handling acids and alkalis
    1. Diluting sulfuric acid with water: Always add the sulfuric acid to the water drop by drop, stirring 14the mixture after each drop. Do this preferably in a sink. Never pour water into sulfuric acid (because of the danger of splashing).
    2. Bottles of acids and alkalis: Keep them on the lower shelves of the cupboards. When you take one out, hold it firmly upright with a dry hand. Do not keep acids and alkalis in bottles with ground glass stoppers as they may get stuck.
    3. Pipetting: Where possible, use small measuring cylinders for measuring acids and alkalis. If more accurate measurement is required, use a pipette plugged with non-absorbent cotton wool or with a rubber tube attached. Pipette slowly, watching the level of the liquid.
  2. Heating glassware and liquids
    1. Test tubes: Never heat the bottom of a test tube. The liquid inside might sputter. Heat the middle of the tube, shaking gently. The mouth of the tube should be facing away from the worker and any other person, towards an empty space or a sink.
    2. Ordinary glass and Pyrex: Only Pyrex glassware and porcelain receptacles can be heated over a Bunsen flame. Ordinary glass will break.
    3. Inflammable liquids: Only small quantities of inflammable liquids such as ether, ethanol, acetone, benzene, toluene and carbon disulfide should be kept in the laboratory.
      Warning: Ether will ignite at a distance of several meters from a flame. Never place a bottle of ether on a workbench where there is an open flame (Bunsen burner, spirit lamp, etc.). Carbon disulfide is even more dangerous.
      zoom view
      FIG. 1.6: Mouth-to-mouth respiration
    4. Butane gas: When lighting a gas burner, always light the match and hold it to the burner before turning on the gas tap. Turn off the main valves of all butane gas cylinders every evening. Replace the rubber connecting pipes once a year.
  3. Do not use broken, cracked or chipped laboratory glassware.
  4. Put clear labels on poisons. Keep them in a locked cupboard.
  5. Do not use nylon clothes while working as these are easily inflammable. Always use a laboratory apron.
  6. Always ensure that electrical wiring and electrical appliances are in good condition.
 
Suggested List of First Aid Equipment for Laboratory
  1. 5% aqueous sodium carbonate
  2. 2% aqueous sodium bicarbonate in an eye drop bottle
  3. 5% acetic acid
  4. Saturated solution of boric acid in an eye drop bottle
  5. Soap powder solution (5 g per liter of water)
  6. Acriflavine ointment
  7. Mercurochrome 2%
  8. Antiseptic lotion
  9. Cotton wool
  10. Gauze
  11. Roller bandage
  12. Adhesive tape
  13. Scissors.
 
Contamination from Infective Material
If contamination has occurred, then:
  1. Disinfect the part with the disinfectant available in the laboratory. Thoroughly clean the affected area with a stream of running water.
  2. Sucking the contaminated material: Spit out all that has been sucked. Use a disinfectant liquid (e.g. diluted dettol) for mouth washing. If the infected material has been swallowed accidentally, forced vomiting to be done, ascertain the kind of infection and take advise from a medical person.
  3. If skin is infected by highly virulent organisms, touch the involved part with pure carbolic acid.
 
Precautionary Measures
  1. A fire extinguisher should always be handy.
  2. Keep sand bucket in the laboratory.
  3. Take measures to prevent electrical short circuiting.
  4. No smoking in the working zone of the laboratory.
  5. 15Breakable items should be kept in proper racks and never at the edge of the working table.
  6. Do not suck anything with the mouth, use rubber teats and bulbs for sucking.
  7. Do not place eatables on the working bench.
  8. Keep fingernails short.
  9. At the end of the day, clean all working benches with a disinfectant. See that nothing except the required electrical appliance is on.
  10. Dispose all infected material properly. Can put such material in hypochlorite solution or in an acidic solution, e.g. diluted sulfuric acid (25%). Burn off all dried contaminated articles, e.g. filter papers.
  11. The glassware should be disinfected with a suitable disinfectant and be cleaned thoroughly with running water.
  12. Use rubber gloves and a nose mask while working with infective samples, e.g. serum of viral hepatitis patient.
 
UNIVERSAL WORK PRECAUTIONS (UWP) FOR LABORATORY PERSONNEL (ESPECIALLY IN RELATION TO HIV TRANSMISSION)
 
Introduction
Healthcare personnel (HCP) can acquire certain illnesses beyond those acquired by all others who live and work in our society, by virtue of their profession. HCPs are at risk of acquiring any of the whole gamut of infections from patients/specimens, which may be viral, bacterial, parasitic or fungal. However, this risk due to occupational exposure can be minimized if not obliterated altogether, if we follow universal work precautions (UWP).
Today, with the WHO estimates of above 5 million HIV positive persons in India, there is an urgent need to review UWP. Besides HIV, there is the very real danger of acquiring Hepatitis B and Hepatitis C in exactly the same way as HIV and could also be fatal. Hepatitis B is 100 times more infectious than HIV. Besides, Hepatitis B is also far more prevalent in India in comparison to HIV with estimated carriers being between 30 and 40 million, a considerable number being infectious. However, fortunately, effective vaccination is available for hepatitis B; therefore, it is strongly recommended for all levels of healthcare workers.
Much of the contamination in the laboratory occurs as a result of penetrating injuries caused by sharp objects and the spilling and splashing of specimen materials.
 
Components of UWP
  1. Handwashing.
  2. Barrier precautions (mask, cap, plastic apron and protection of feet).
  3. Careful handling of all kinds of sharps and needles.
  4. Effective infection.
  5. Sterilization.
  6. Correct disposal of different kinds of wastes generated in a health care facility.
 
Guidelines of Basic Practices and Procedures
  • Prevention of puncture wounds, cuts and abrasions and protection of existing wounds, skin lesions, conjunctiva and mucosal surfaces
  • Application of simple protective measures designed to prevent contamination of the person and his/her clothing
  • Good basic hygiene practices, including regular handwashing
  • Control of surface contamination by containment and disinfection procedures
  • Safe disposal of contaminated waste.
 
Biosafety Regulations for Laboratory Procedures
  • Wear gloves when handling infectious materials or where there is a possibility of exposure to blood and other body fluids. All laboratories that work with material that is potentially infected with HIV require a generous supply of good quality gloves.
  • Discard gloves whenever they are thought to have become contaminated or perforated, wash your hands and put on new gloves. Alternatively, where there are economic constraints, wash gloved hands whenever they get contaminated with blood/body fluids before collecting further samples
  • Do not touch your eye, nose, or other exposed membranes or skin with your gloved hands.
 
Sterilization (for Nondisposable Items)
  • For sharps, reusable blades, cystoscopy instruments, endoscopy instruments, use CIDEX (2% glutaraldehyde) or 5% Korsolex. Disinfection usually occurs in 30 minutes
  • Use autoclaving for other reusable items (e.g. needle holders, gowns, etc.)
  • Wherever, autoclaving is not possible, boiling must be for 30 minutes at the least.
 
Waste Disposal
Divide waste into three parts at source.
  1. Household type noninfectious waste:
    • Not to be decontaminated
    • To be disposed off as such.
  2. 16Infected sharp waste disposables (needles/surgical instruments):
    • Place in puncture-proof container containing disinfectant (1% bleach prepared every morning). Needles should ideally be burnt (machines are available that operate on electricity)
    • Final disposal.
  3. Infected nonsharp waste:
    • Is to be decontaminated
    • Placed in disinfectant 5 to 10% bleach as the case may be (left over blood, tissues, etc.).
 
Final Disposal
  • Purchase of needle destroyer if resources permit
  • Incineration of all infected waste
  • Deep burial in controlled land fill sites (protected from all sides)
  • Shredding of disposable plasticware waste.
 
Postexposure Care
  • Minor bleed with percutaneous inoculation, open skin wound, breached skin, exposed mucous membranes.
 
First Aid
  • Allow to bleed by squeezing
  • Wash with water
  • Antiseptic.
 
Report
  • Employee identification, date, time with place of accident
  • Circumstances around accident
  • Action taken.
 
Initial Consultation
  • Easy access to medical advice with counseling. Consult, physician for AZT prophylaxis regime if medication available.
 
Laboratory Testing
  • After consent with counseling within 2 weeks, 5 weeks, 12 weeks, or 24 weeks.
 
Clinical Follow-up
  • For fever, pharyngitis, rash, malaise, lymphadenopathy, myalgia and arthralgia within 6 months
  • Do not leave the workplace or walk around the laboratory while wearing gloves
  • Wash hands with soap and water immediately after any contamination and after work is finished. If gloves are worn, wash your hands with soap and water after removing the gloves. This is a vital and simple precaution that is often overlooked
  • Wear a laboratory gown or uniform when in the laboratory. Wrap-around gowns are preferable. Remove this protective clothing before leaving the laboratory
  • When work with material that is potentially infected with HIV is in progress, close the laboratory door and restrict access to the laboratory. The door should have a sign BIOHAZARD: NO ADMITTANCE
  • Keep the laboratory clean, neat and free from extraneous materials and equipment
  • Disinfect work surfaces when procedures are completed at the end of each working day. An effective all-purpose disinfectant is a hypochlorite solution with a concentration of at least 0.1% available chlorine (1 g/L, 1000 ppm)
  • Whenever possible, avoid using needles and other sharp instruments. Place used needles, syringes and other sharp instruments and objects in a puncture resistant container. Do not recap used needles and do not reuse needles from syringes for disposal
  • Never pipette by mouth
  • Perform all technical procedures in a way that minimizes the risk of creating aerosols, droplets, splashes or spills
  • Use a biosafety cabinet while working on aerosolizing specimen
  • Do not eat, drink, smoke, apply cosmetics or store food or personal items in the laboratory
  • Make sure that there is an effective insect and rodent control program
  • If a laboratory personnel has lesions on hand and feet, then:
    • If superficial, he or she should wear protective dressing and wear gloves over it
    • If wound is deep or raw then the concerned person should not handle samples till the wound heals.
  • If there is a pregnant healthcare worker then in view of the occupational risk to the woman and the developing fetus, on compassionate grounds, where possible she should be involved in clerical tasks or stay away from work for the duration of her pregnancy.
 
Containing Spills
  • Cover the spill immediately with absorbent material to avoid aerosolization
  • Soak the material by pouring disinfectant on it
  • Leave the area for 30 minutes
  • Mop with more adsorbent material after wearing gown, mask and gloves
  • 17Place material in appropriate bin for disposal (autoclaving or incineration).
 
Collection of Specimen
  • Always keep labeled bottle ready on the bedside
  • Wear disposable gloves
  • Keep adequate cotton with spirit at collection site
  • Keep a bucket full of disinfectant [CIDEX (glutaraldehyde)], one for at the most 5 beds.
 
Transport of Specimen
  • Specimens should be collected in plastic; screw-capped containers prelabeled with patient identification data, should be packaged and transported in puncture resistant containers in upright position with the sign of biohazard on the container.
 
MEDICOLEGAL ASPECTS OF CLINICAL PRACTICE
Under the Consumer Protection Act (CPA), India, 1986; any patient, registered consumer organization, state or central government or patient’s legal heirs can sue the undermentioned persons for shortcomings in “service” provided by them.
  • A technician, microbiologist, biochemist or pathologist running a laboratory
  • Any private polyclinic, nursing home or hospital, registered or otherwise
  • As government hospitals provide service without consideration (free of cost), they cannot be held responsible under CPA 1986
  • Doctors appointed by the government, however, can be held accountable under other civil and criminal laws for proven negligence
  • Medical practitioners delivering new service without any consideration in a charitable hospital or medical camps are exempted from the provisions under CPA
  • As per clause 2(d) (ii) of the CPA 1986, a consumer implies any person who hires or avails of any service for a consideration, which has been paid or promised, or partly paid and partly promised, or under any system of deferred payment, and includes any beneficiary of such services other than the person who hires or avails of the service for consideration paid or promised or partly paid and partly promised or under any system of deferred payment, when such services are availed of with the approval of the first mentioned person
  • The time limit stipulated for filing a complaint is 2 years from the date of alleged negligence
  • Patients can be dealt with severely if they file frivolous and false complaints just to harass the medical practitioner
  • Free services provided are exempted under CPA
  • A laboratorian is also a consumer as he buys various instruments, equipment, diagnostic kits/reagents/devices. He too can file a complaint under CPA for any defect or deficiency in service related to that purpose
  • Ignorance is not held as an excuse as an established legal principle. Concurrently, law does not expect a very high degree of knowledge but expects only average knowledge from a medical practitioner
  • Medical negligence is a civil wrongdoing classified as ‘tort’, where a medical practitioner fails to take proper care in respect of examination, diagnosis, investigation, treatment, etc. resulting in injury or mortality
  • Laboratorians are expected to keep all reports confidential (legally and ethically). The reports can be divulged to the referring clinician or to the patient or the relatives of the patient (with patient’s onsent). Reports pertaining to sexually transmitted diseases or HIV/AIDS should be handed over only to the patient
  • Legally, only authorized or registered blood banks can supply units of blood. All mandatory information must be clearly mentioned on the bottle label legibly
  • These days doctors have ‘Malpractice Insurance Covers’. In case a legal notice is received by such a doctor, he should immediately notify the insurance company. The insurance company must take all necessary actions in such a case. The company should appoint a lawyer to give reply or to take legal steps and inform the doctor about it. The doctor, by permission of the company, can appoint a lawyer of his choice
  • What constitutes a legal notice? Any letter received by a medical practitioner from a patient or a voluntary registered organization or an advocate, demanding explanation about treatment given or demanding some explanation about treatment for alleged injury or death constitutes a legal notice
  • Section 27 of the Civil Procedure Code provides that when a suit has been duly instituted, a summon may be issued to the defendant to answer the claim, and such summon is to be served in the prescribed manner. When a complaint is lodged before the commission or the forum, the defendant practitioner is informed by a registered letter by the office, which is called a summon in legal parlance. In this summon, time for the reply and date of hearing is mentioned. Usually, the time given for filing the reply is 30 days.
 
LABORATORY INSTRUMENTS
 
Microscope
Micro = Small, Scope = to view.
It magnifies the image of the object to be visualized through it. Normally, the laboratory microscopes provide a 18magnification of 40x (scanner), 100x (low power), 400x (high power) and 1000x (oil immersion). The total magnification is obtained by multiplying the magnification of the objective with that of the eyepiece.
 
Parts of the Microscope
It has three sets of parts. They are the:
  1. Stand,
  2. Mechanical adjustments, and
  3. Optics or the lenses.
 
Stand
It consists of:
  1. The tube—supports objectives and eyepiece.
  2. The body—gives support to the tube.
  3. The arm—gives correct height and angulation to the body and the tube.
  4. The stage with a pair of spring clips or a mechanical stage.
  5. The substage holds the condenser lens with its iris diaphragm and a holder for light filters and stops.
  6. The foot on which other parts rest, can be in tripod or horseshoe shape.
 
Mechanical Adjustments
 
Focusing Adjustments
These are coarse and fine adjustments.
 
Coarse Adjustment
Controlled by a pair of large knobs, one on each side of the body. On rotating this, the tube moves with its lenses. Some microscopes have this attached to the stage; so that instead of the tube, the stage moves up and down. Coarse adjustment is enough for low power lenses.
 
Fine Adjustment
Necessary for high power and oil immersion lenses. This is usually controlled by two smaller knobs on each side of the body. They may be graduated to indicate the movement in microns.
 
Draw Tube
It is used to adjust the distance between the objective lens and the eyepiece lens.
 
Inclination
The arm can be tilted upon the foot by a hinge.
 
Condenser Adjustments
Focusing of condenser is done by rotating a knob present on one side below the stage.
 
Aperture Adjustment
It is done by the iris diaphragm (made up of leaves).
 
Centering of Condenser
It is done to bring the light beam accurately through the instrument. In some microscopes, it is permanently fixed.
 
Mechanical Stage
It has knobs for moving the slide across or along the stage.
 
Monocular, Binocular and Digital Microscopes
Monocular—has only one eyepiece (Fig. 1.7).
 
Binocular—has 2 eyepieces, the only advantage it offers is that it causes less strain on the eyes (Fig. 1.8A). Nowadays digital microscopes are available, here digital image is projected onto a digital display device (Fig. 1.8B).
zoom view
FIG. 1.7: Monocular microscope with substage lamp
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FIG. 1.8A: Binocular microscope with substage lamp
19
zoom view
FIG. 1.8B: Digital microscope
 
Microscope Optics
 
Objective
On objective quality, depends, the quality of the image. These are usually made up of more than one lens. On each objective is engraved the magnification power.
 
Numerical Aperture
Numerical aperture (NA) of the objective is important, for on this, depends, among other things, the amount of light which the lens passes and the detail which it can make visible, on which it is said to resolve.
 
Oil Immersion Objectives
They are used to avoid bending of light beam (with higher magnification). The oil used should have the same optical properties as glass, e.g. cedar wood oil. Liquid paraffin can also be used.
 
Objective Aberrations
With increasing magnification certain optical aberrations creep in:
  1. Spherical aberration—edge of the lens gives slightly higher magnification than its center.
  2. Chromatic aberration—blue light is magnified slightly more than red.
These aberrations can be avoided by using a series of lenses made of special glass, carefully calculated and designed.
 
Objective Qualities
  1. Achromatic—are the usual average quality lenses and are good enough for routine laboratory work.
  2. Fluorite (Fi)—are highly corrected and expensive, have a wider field and are good for searching blood films.
  3. Apochromatic (Apo)—are very highly corrected and costly and are only of value in special work.
 
Spring-loaded Objectives
The high power objectives (40X and 100X) of most modern microscopes are spring loaded, i.e. the front mount of the objective will be pushed in rather than pushed through a specimen, if such an objective is accidentally pressed against a specimen when focusing (Fig. 1.9).
 
Working of Oil Immersion Objectives
A beam of light passing from air into glass is bent; and while passing from glass to air, it is bent back again. The bending effect and its limitations can be avoided by replacing the air between the specimen and lens with an oil which has optical properties similar to that of glass, i.e. immersion oil. When an appropriate oil is used, the light passes in a straight line from glass through the oil and back to glass as though it were passing through glass all the way. Whenever possible, the immersion oil recommended by the manufacturer of a microscope should be used (Fig. 1.10).
 
Eyepiece
The most commonly used eyepiece is known as Huygens eyepiece which has 2 lenses mounted at a correct distance apart, with a circular diaphragm between, which give a sharp edge to the image. These are available in different magnifications. Lesser the magnification, brighter and sharper is the image. For routine work, a 10X Huygens is good enough. The 15X eyepieces are also available, as are wide field ones.
 
Condenser and Iris
Condenser is a large lens mounted below the stage, with an iris and diaphragm. There may be 2 or more lenses. Its function is to deliver the light beam to the objective at a sufficiently wide angle.
zoom view
FIG. 1.9: Microscope objectives
20
zoom view
FIG. 1.10: Working of an oil immersion objective
 
The Mirror
It is placed below the condenser and iris, it can be turned in any direction. It reflects the light beam from the source to the iris and condenser. It usually has two mirrors mounted back to back, one flat and the other concave. Flat mirror is used in the presence of condenser and the concave without the condenser.
 
Light Source
 
Daylight
Use of direct sunlight is bad for the microscope and the eye. It is best to use reflected sunlight of a dull white background. It is not sufficient for oil immersion lens and it is not available during evening or night.
 
Electric Light
A 60 watt frosted electric lamp placed 18" away from the microscope is sufficient for most routine work. Many microscopes are now provided with built-in sources of illuminations. In the absence of electricity, a battery lamp or an oil lamp can be utilized. The light from these artificial sources is rather yellow but may be used. Best, however, are halogen lamps.
 
Special Applications of the Microscope
 
Phase Contrast Illumination
This is needed to visualize transparent microorganisms suspended in a fluid. Ray of light travels in a wave form in a straight line. Two such rays traveling together are said to be in phase, and they produce a brighter illumination. If, however, these rays are out of step with each other, they are said to be out of phase. They interfere and produce less bright illumination. Phase contrast microscopy makes use of this property of rays to help or hinder each other and thereby resulting increased contrast in the microscopic image.
The desired effect is brought about by placing an annulus in the condenser and a phase plate in the objective. A circle is engraved in the phase plate which matches the ring of beam coming through the condenser and annulus. This circle makes the wave take a longer or a shorter step, so becoming out of phase with those aves which pass through the rest of the plate.
Supposing that the specimen is suspension free fluid, the only light that reaches the eye is that which goes from the annulus through the phase plate. Whereas presence of organisms would diffract and scatter the light. The light passing through the fluid gets out of phase with the light that has the organisms stand out in contrast to their background.
 
Equipment Needed
An annulus, a phase plate and a telescope that is needed for adjusting the rings of both annulus and the phase plate.
 
Method
  1. Focus the specimen with the right objective after illuminating the microscope.
  2. Place the matching annulus at its position.
  3. Remove the eyepiece and put the telescope in its place, adjust it till the two rings, one bright and one dark are in focus.
  4. Adjust condenser screws till the bright annulus ring fits exactly into the darker ring of the phase plate.
  5. Remove the telescope, replace the eyepiece, focus and examine the specimen.
 
Importance
This method is made use of for examining live organisms, for examaple,
  1. Cholera vibrios
  2. Amebae
  3. Trypanosomes
  4. Trichomonas, and
  5. Other flagellates.
It can also be used for platelet counting and for examining routine urine specimens.
 
Demerits
  1. A halo is seen around each particle, it gives a false appearance of its structure.
  2. In addition, some resolution power is lost but this is more than compensated for by the increased contrast that is produced.
 
Dark Ground Illumination
This method too, is used for visualizing organisms suspended in fluid, both the structure and the motility of 21the organisms can be seen. In this method, the light enters the special condenser which has a central blacked-out area so that light cannot pass directly through it to enter the objective. Instead the light is reflected to pass through the outer rim of the condenser at a wide angle which illuminates the microorganisms by a ring of light surrounding them (Fig. 1.11).
In this method, the light that is seen comes only from the microorganisms themselves and not from the light source. Hence, the organisms are brightly illuminated against a dark background. Though useful, this method is rather cumbersome.
 
Equipment Needed
  1. An oil immersion dark ground condenser with the centering screws.
  2. A funnel stop for insertion in 100X objective to reduce its NA and exclude light coming directly from the source.
  3. A brightly illuminated microscope lamp.
  4. Scratchless slides not more than 1 mm thick.
 
Method
  1. Fit the dark ground condenser and raise it to stage level.
  2. Place the coverslipped specimen on the thin polished glass slide. Both, the coverslip and the slide should be absolutely clean.
  3. Place a drop of immersion oil between the condenser and the slide.
  4. Adjust light source and the mirror properly.
  5. Focus 10X objective and observe.
  6. Focus condenser up or low, so that the ring ultimately becomes just a spot of light. Focus this spot right in the center.
  7. Use 40X objective; if needed, use the 100X oil immersion by inserting the funnel stop into it.
zoom view
FIG. 1.11: The principle of dark ground illumination
 
Demerits
  1. Focusing and/or centering of condenser is difficult as is the alignment of the lamp.
  2. Difficulties may arise under the following circum-stances:
    • Smear traces on the slide or coverslip
    • If the specimen is dense
    • A bubble is present in the immersion oil
    • Insufficient oil contact below or above the slide.
 
Importance
  1. This method is of particular importance for the examination of Treponema group of organisms.
  2. It can also be of use for microfilariae, for the sheath of the pathogenic forms can be clearly seen which otherwise needs to be stained.
  3. For examining the rapid movement of Vibrio cholerae.
  4. In addition, this method can be used for:
    • Leptospira
    • Borrelia, and
    • Spirillum species.
    • The ideal objective for dark ground illumination is the 50X fluorite as this lens gives a clear, sharp and a well-illuminated image.
 
Fluorescence Microscopy
This method entails the illumination of particles/microorganisms (previously stained with a fluorescent dye) with ultraviolet (UV) light into visible light (yellow or orange), by lengthening their wavelength. This procedure is made use of for visualizing, besides other things, mycobacteria glowing against a black background.
All other wavelengths emitted by the lamp except the ultraviolet (UV) are to be filtered off (by using appropriate optical filters) and no harmful rays of UV light should reach the observer’s eye (by using an immersion dark ground condenser as described for previous method). Again, another filter is used to remove all unwanted fluorescent light by placing a secondary or a barrier filter above the eyepiece (Fig. 1.12).
 
Equipment Needed
  1. A fluorescent lamp (mercury vapor or quartz iodine, the latter is better, being cheaper, lighter and easier to use).
  2. A blue (primary or exciting) filter, generally a BG 12.
  3. A yellow (secondary or barrier) filter.
  4. An immersion dark ground condenser.
  5. A nonfluorescent immersion oil, e.g. liquid paraffin.
 
Importance
22
zoom view
FIG. 1.12: Components of fluorescence system
  1. For identifying mycobacteria.
  2. It is used extensively in fluorescent antibody techniques used in parasitology and bacteriology.
  3. It is also used widely in histopathology of kidney, skin, etc. where immune/autoimmune basis of disease is expected. In fact, anything can be confirmed with high degree of sensitivity and specificity, if antibodies against it (later tagged with a fluorescent dye) can be produced.
  4. Used widely in cytogenetics.
 
Electron Microscope
 
Basic Principle
The resolution of the light microscope has been shown to be limited by the NA and the wavelength of light employed. As the degree of correction in glass lenses is very high, the main limitation is imposed by the light (e.g. half wavelength of light), giving a normal resolution of approximately 250 nm; and when UV light is used, a resolution of about 100 nm. By the substitution of an electron beam for light rays, a much greater degree of resolution can be obtained; since at an acceleration of 50,000 volts, electrons have a wavelength of only 0.001 nm; therefore, a theoretical resolving power of 0.0005 nm could be attained, which would enable molecules to be seen. Unfortunately, the degree of correction that is currently feasible with transmission electron microscope (TEM) lenses will permit a resolution of only 0.25 nm, but this is still a thousand times greater than that possible with the light microscope. A further difficulty with the TEM is that, since electrons have poor penetrating power, the sections to be examined must be very thin, less than 50 nm thick. This necessitates the use of special hard embedding media plastics) and special ultra-microtomes to cut such thin sections. Steel knives cannot be used to cut these sections; either glass or diamond knives are used.
Weighing scales: For weighing large quantities. Analytical balance: For accurate weighing of smaller quantities.
 
Weighing Scales or Analytical Balance
 
Use and Care
  1. The weighing equipment must be placed on a firm bench, away from vibration, draughts, direct sunlight and dust.
  2. It should be kept perfectly horizontal by altering the screws on which the equipment stands.
  3. Chemicals, etc. should never be placed directly on the pans. Weigh them in a container.
  4. Never touch the weights with hands, handle them with forceps.
  5. The balance should be at rest before adding or removing the weights or chemicals.
  6. Before taking the reading, the glass window of the instrument should be closed.
Electronic analytical balances are also available. Made by various companies, these are very accurate.
 
Centrifuge
Centrifuge is used to sediment or deposit rapidly particles such as cells which may be suspended in a fluid. The speed is expressed as rpm, i.e. revolutions per minute.
 
Relative Centrifugal Force (RCF)
More important than rpm is relative centrifugal force (RCF). RCF is expressed as the acceleration due to gravity or G (dynes per cm). The formula is:
G = 0.00001118 × (r) × (n)2
where r = radius in centimeters
and n = revolutions per minute.
The time of centrifugation is equally important. The tubes should be spun for a definite period to obtain the desired effect.
 
Types of Centrifuge
 
Hand Centrifuge
Fixed to the bench, the handle is rotated manually. It gives low speeds only.
 
Motor-driven Centrifuge
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Operated through mains electricity supply. The tubes may be kept in a fixed angle head or in a swing out head (Figs 1.13 and 1.14).
 
Microhematocrit Centrifuge
Also motor driven for finding out packed cell volume (PCV) of red blood cells (RBCs). In this, blood-filled capillary tubes are spun and later the percentage of RBC-filled column is estimated (Figs 1.15 and 1.16).
 
Use and Care
  1. Use centrifuge tubes made of strong glass and they should not be too long.
  2. The opposite tubes should be balanced properly.
  3. The centrifuge speed should be increased gradually.
  4. The instrument should be kept clean. If something spills over inside, it should be cleaned and the instrument disinfected, if necessary.
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FIG. 1.13: Swing out head centrifuge(Courtesy: Yorco Sales Pvt. Ltd)
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FIG. 1.14: Motor driven centrifuge with rpm. indicator and auto (timed) shut off(Courtesy: Yorco Sales Pvt. Ltd)
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FIG. 1.15: Dual centrifuge routine centrifuge with microhematocrit attachment(Courtesy: Yorco Sales Pvt. Ltd)
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FIG. 1.16: Microhematocrit centrifuge and its parts(Courtesy: Yorco Sales Pvt. Ltd)
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Glassware (many items are now made of plastic)
  1. Flasks—are of different sizes and shapes.
    1. Erlenmeyer or conical flasks—for heating and boiling liquids (Fig. 1.17).
    2. Volumetric flasks—are graduated for getting exact volume of liquids (Fig. 1.18).
    3. Round and flat-bottomed flasks for preparing solutions (Figs 1.19A and B).
  2. Beakers—available in different sizes (Fig. 1.20).
  3. Bottles
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    FIG. 1.17: Conical flasks
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    FIG. 1.18: Volumetric flasks
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    FIGS 1.19A AND B: (A) Round bottomed flask and (B) Flat bottomed flask
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    FIG. 1.20: Beakers
    1. Specimen bottles—with top screws, e.g. the universal type containers.
    2. Reagent bottles—have ground glass or plastic stoppers, available in different sizes and may be made of amber colored glass (Figs 1.21A and B).
    3. Drop bottles—fitted with special tops through which drops can be delivered (Fig. 1.22).
  4. Funnels—used to hold filter papers when filtering fluids or for pouring liquids into narrow neck containers (Figs 1.23A and B).
  5. Cylinders—used for measuring liquids, they have a pouring spot (Fig. 1.24).
  6. Tubes—are of various sizes; of the test tube or centrifuge (conical) type, with or without a top rim (Figs 1.25 and 1.26).
  7. Pipettes—are used to measure and deliver a given volume of fluid.
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    FIG. 1.21A AND B: (A) Specimen bottles and (B) Reagent bottles
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    FIG. 1.22: Drop bottles
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    FIGS 1.23A AND B: (A) Separating funnel and (B) Funnel
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    FIG. 1.24: Measuring cylinder
    1. Volumetric pipettes—have a bulb shape in the stem. Each pipette is marked to show the given volume of fluid, it contains or delivers (Figs 1.27A and B).
    2. Graduated pipettes—are of various sizes. They may be of the non-blow out or the blow out type.
    3. Blood pipettes—have a white back and include the 0.02 mL pipette used for hemoglobin, red cell and platelet counts, and also the 0.05 mL pipette for white cell counts (Fig. 1.28A).
    4. Pasteur pipettes—have multiple uses. They are not graduated or marked. These can be bought or made in the laboratory (Fig. 1.28B).
 
Other Necessary Equipments
 
Serological Water Bath
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FIG. 1.25: Test tubes
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FIG. 1.26: Centrifuge tubes
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FIGS 1.27A AND B: (A) Volumetric pipette and (B) Measuring pipette
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FIGS 1.28 A AND B: (A) Blood pipettes and (B) Pasteur pipttes
It is electrically heated and has a thermostatic temperature regulator. It can provide temperature ranging from room temperature to 100°C. Various sizes to suit various workloads are available (Fig. 1.29).
 
Incubator
Works on electricity and regulates temperature thermostatically. Necessary for various investigations where body temperature 37°C (or otherwise) incubation is required (Fig. 1.30A).
 
Hot Air Oven
This is used for drying and sterilizing glassware. This too is thermostatically controlled and electrically heated. It looks like an incubator (Fig. 1.30B).
 
Reporting Laboratory Tests and Keeping Records
 
Standardization
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FIG. 1.29: Serological water bath(Courtesy: Yorco Sales Pvt. Ltd)
Standardization in the reporting of laboratory tests contributes to the efficiency of the laboratory service and is of great value when patients are referred from one place to another. Whenever possible, request forms and other laboratory printed stationery should be prepared and issued by a central stationery office.
 
Use of Rubber Stamps
When stationery is not supplied from a central source, standardization in presenting and reporting results can be achieved by the use of rubber stamps. Adequate ink must be used and the stamp must be positioned carefully.
 
Format
The top part of the report card must prominently give the name, address and telephone numbers of the laboratory. It should then have place for printing the patient’s name, age, sex, name of the referring doctor, the laboratory reference number and date. Next, the title of the report should be mentioned, e.g. urinalysis, stool examination, hematology, biochemistry, etc. After this, print the investigation name, leave space for patient’s values, print normal values followed by the units. The report must end with the signatures of the person in-charge of the laboratory.
 
Keeping Records in the Laboratory
A record of all test results must be kept by the laboratory as carbon copies, work sheets, or in simple exercise books. A day book is ideal as it has the necessary ruled lines. In your record put the date, reference number, patient’s name, name of the referring doctor, investigations asked for, reports given and payment status (if privately owned laboratory).
 
Laboratory Reporter
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FIG. 1.30A: Incubator(Courtesy: Yorco Sales Pvt. Ltd)
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FIG. 1.30B: Hot air oven(Courtesy: Yorco Sales Pvt. Ltd)
An ideal laboratory computer program helps in reporting and recording diagnostic center, pathology lab and other diagnostic imaging fields. The program should also keep history records of the patients. It must have facilities of making and reporting profiles, e.g. lipid, renal, cardiac, hepatic and diabetic profile. A program can be called ideal if:
 
It Reduces Overload
  • Avoids manual operations by printing booking slips, receipts, bills, envelopes, etc.
  • Prints daily register of patients
  • 28Prints rate lists
  • Reports only the tests required and not the whole group of tests
  • A comprehensive reporting option for day end operations including daily collection report and doctor wise daily collection.
  • It helps referencing doctor
  • Provides clear reports with normal values
  • Abnormal values are underlined or highlighted automatically
  • Prints history reports of the patients.
 
It Makes Working Easy
  • Reports as per your own method of grouping of tests, profiles, etc.
  • Automatic calculation of charges
  • Keeps list of referencing doctors
  • Maintains daily collection on referencing doctor/institution
  • Provides workload report.
 
The Computerized System is Easy to Operate
  • Simple menu-based operations and does not require any detailed knowledge of computers
  • Help facility at every stage of working for beginners
  • Can find a patient detail based on of reference number, name, date and referencing doctor.
 
Features and Provisions
  • Keeps the results for as long as you want
  • Keeps normal values for male/female and adult/child for all tests
  • Can change any normal value as per your equipment, techniques and methods
  • Provision for reporting by different doctors
  • Reports can be printed on simple paper or on preprinted letter heads (computer stationery).
 
Graphs
  • Prints graphs, e.g. GTT for to the point reporting
  • Can make/design your own graphs
  • Can see the graph on screen as well as print.
 
Accounts
  • Maintains your bank, cash accounts
  • Provides all ledgers
  • Makes trial balance for final accounts.
 
Address Manager
  • A mail list program, keeps address details.
  • Provides easy working on the basis of name
  • Keeps addresses for Labmate program of referencing doctors, patients and reporting doctors
  • Prints address directory with telephone numbers, etc.
  • Prints labels for sticking on your mail
  • Can group your addresses as per nature of address such as friend, relative, doctor, patient, etc.
The features given above are complete. All records can be retrieved date wise or name wise. Any program that provides the above-mentioned capabilities can be considered as an ideal laboratory reporter.
 
Caution:
All medical electronic diagnostic devices need a stable constant voltage, therefore, proper protective cover must be provided. CVT (constant voltage transformer), servo stabilizers, and UPS (uninterrupted power supply) should be installed in the mainline or with specific instruments.