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Year I Semester I Introduction to Medical Laboratory Sciences  Course overview............................................................................................5 Course Schedule.........................................................................................

Year I Semester I Introduction to Medical Laboratory Sciences  Course overview............................................................................................5 Course Schedule.......................................................................................................8 Textbook for references.............................................................................................9 Chapter I: fundamentals of medical laboratory...........................................10 Introduction to medical laboratory...........................................................................10 Safety in laboratory................................................................................................11 Professional ethics...................................................................................................14 Quality assurance in laboratory...............................................................................17 CHaPater II: Microbiology............................................................................23 Microscopy.............................................................................................................23 Bright eld microscopy............................................................................................25 Microscopy terminology..........................................................................................28 Classi cation of micro-organism...............................................................................30 Bacterial anatomy...................................................................................................34 Gram stain............................................................................................................36 Chapter III: haematology.............................................................................37 Anticoagulant.........................................................................................................38 Processing of blood sample.....................................................................................41 The Collection of Blood (Phlebotomy).......................................................................42 Collection tubes for blood specimens........................................................................51 Blood and its components........................................................................................51 Blood cells morphology...........................................................................................54 Basic haematology tests...........................................................................................57 Haematological tests reference range.......................................................................58 Chapter IV: Clinical chemistry.......................................................................59 Laboratory glassware and pipette............................................................................59 Spectrophotometry.................................................................................................63 Chemistry pro le.....................................................................................................65 Blood glucose estimation.........................................................................................66 Blood glucose in Plasma..........................................................................................67 fi fi fi  Centrifuge..............................................................................................................68 Chapter V: immunology and transfusion sciences........................................72 Immune system.......................................................................................................72 The development of blood transfusion.......................................................................74 Abo and rh blood group.........................................................................................77 Rh Blood Group System..........................................................................................79 Review....................................................................................................................81 Chapter VI: Histology and Cytology.............................................................83 Histological Tissue Processing..................................................................................85 Basic Automation in Histology Laboratory................................................................88  COURSE OVERVIEW Course title: Introduction to Medical Laboratory sciences Theory 2 Credits allocated: Practical 1 Theory 2 hours per 15 weeks = 30 hours Contact hours/week: Practical 2 hours per 15 weeks = 30 hours Placement in curriculum: Professional year I semester I Pre-requisites: - Co-requisites: Practical Mrs. Nuha Al Jabri Name of tutors: & MLS clinical tutors Course description: IMLS course is an introductory course for first year MLS students. It provides basic knowledge on different laboratory sections and tests done in each laboratory. The course will equip the students with skills required in medical laboratory through the practical sessions. The students will learn about importance, principles and uses of lab equipment and instruments. As well as, students will learn about professional ethics and safety procedures and precautions required to work safely in the lab. Objectives: The course is divided into different sections depending on laboratory departments. At the end of each section, student will gain certain knowledge and skills. 1. General knowledge about lab sections, safety and health rules, ethical rules and quality control: · Adhere to safety procedures and regulation (OSHA, COSHH and MSDS). · Justify the need to practice professional ethics · Know division of laboratory department and organisation of lab staffing. · Describe the principles of QA. 2. Microbiology: at the end of this section student should be able to · List different investigations (routine and special) done in micro lab · Learn about common micro samples and how they are processed. · Classify microorganisms · List different microscopes used in the laboratory. · Describe compound microscope and demonstrate the ability to use it.  · Perform gram stain and identify bacteria under microscope. · Know how to deal with critical results in micro lab 3. Clinical chemistry: at the end of this section student should be able to · List different investigations (routine and special) of clinical chemistry lab. · Know test profiles, normal values, how chemistry tests are performed and how to deal with critical results in chemistry lab. · Describe different glassware and their uses. · Briefly describe the principle and uses of spectrophotometer and centrifuge. · Perform and report any chemistry test · Know different pipettes used in lab and perform the pipetting techniques. 4. Haematology: at the end of this section student should be able to · List different investigations (routine and special) of haematology lab. · Know about sample collection and processing in haematology lab. · Discuss different types and uses of anticoagulant. · Perform phlebotomy · List blood components and identify normal morphology of blood cells under microscope. · Understand normal values in the Haematology lab · Perform blood smear and staining of slides. 5. Blood transfusion and immunology: at the end of this section student should be able to · Define different immunological terms. · Know principle of basic serological tests · Discuss the importance of blood donation, transfusion of compatible to patients, screening of donor blood and blood products. · Perform blood grouping and basic serological tests. 6. Histology: at the end of this section student should be able to · Discuss how samples are processed in histology lab. · Name different histological techniques · Gain a general knowledge on histology lab Teaching Strategies: Interactive lectures augmented by use of Audio-Visual aid. Explanatory notes with additional specific handouts. Practical sessions for hands-on experiences. Evaluation Scheme: Theory Internal exam - I 40% Week 8 (Topics covered from week 1 to 7) Final 60% Week 16 & 17 (Topics covered from 9 to 15) Practical Internal exam - I 40% Week 10 (practical from week 1 to 9) Final 60% Week 16 & 17 (All practical covered from week 1 to 15) Special conditions: · Missed assessments will be conducted within 5 working days after submission of an approved Leave Certificate (which should be submitted within 3 working days after return from the leave). · Mobile phones: Please note that use of mobile phones during theory and practical class is strictly not permitted. · Attendance: Attendance will be marked for all classes. If you exceed 15% of absenteeism without valid sick leave or 25% with valid sick leave, you will not be allowed to attend final exam. · The tutor will not repeat the class (theory & Practical) for individual student in case of absence. COURSE SCHEDULE Week Tutor/Clinical Theory Tutor Practical No. Tutor/Lab Orientation day Orientation day 1 Mrs. Nuha Mrs. Nuha Fundamentals of MLS Safety precautions in 2 Fundamentals of MLS Mrs. Nuha Mrs. Nuha laboratory. Ms. Zeyana &Mrs. 3 Microbiology Mrs. Nuha Microbiology Ilham. Ms. Zeyana &Mrs. 4 Microbiology Mrs. Nuha Microbiology Ilham Ms. Zeyana &Mrs. 5 Haematology Mr. Ajo Microbiology Ilham 6 Haematology Mr. Ajo Internal Exam I (Practical) – 40% 7 Haematology Mr. Ajo Haematology Mr. Ajo Internal exam I (Theory) 40% Mrs Hiba and 9 Clinical Chemistry Haematology Mr. Ajo Mrs Hiba Mrs Hiba and 10 Clinical Chemistry Haematology Mr. Ajo Mrs Hiba Mrs Hiba and 11 Clinical Chemistry Haematology Mr. Ajo Mrs Hiba Immunology and blood 12 Dr Mujtaba Clinical Chemistry Hiba & Hiba transfusion Immunology and blood 13 Dr Mujtaba Clinical Chemistry Hiba & Hiba transfusion Mr Amour & Dr 14 Histology (in Histology Lab) Mrs. Nuha Blood grouping Mujtaba 15 Revision TEXTBOOK FOR REFERENCES 1. RIDLEY, J. 2010. Essentials of Clinical Laboratory Science, Cengage Learning. 2. Victor Ho brand and Paul Moss, 2011, Essential Haematology, Wiley library, Oxford, UK. 3. Warren Levnsom, Review of Medical Microbiology and Immunology, Mc Graw Hill education, San Francisco, US. ff  C HAPTER I: FUNDAMENTALS OF MEDIC AL L ABORATORY INTRODUCTION TO MEDIC AL L ABORATORY Organisation of a medical lab: Department of medical laboratory: 1. Clinical chemistry: mainly the tests are performed on serum samples and some other samples such as plasma, urine and CSF. The department is subdivided into routine and special chemistry. The routine tests include; glucose, cholesterol, enzymes and electrolytes (such as sodium, potassium and chloride). Special chemistry tests include; hormones, vitamins, drugs and ions. 2. Haematology: whole blood is used for the majority of tests. Haematological procedures can be qualitative or quantitative. The quantitative tests include counting blood cells, estimating haemoglobin concentration and RBC indices. The qualitative tests include studying the shape of the blood cells and sickling test. Coagulation test is also done in haematology lab. Coagulation tests are performed to diagnose and monitor patients with blood clotting disorders.  3. Microbiology: is responsible for growing and identifying microorganisms such as bacteria obtained from patients’ blood, urine, sputum or wound. Susceptibility testing is performed to determine which antibiotics would be most effective in treating patient infection. Virology (study of viruses), mycology (study of fungus) and parasitology (study of parasites such intestinal parasites by examine stool samples) are part of microbiology lab. Urine analysis test is done in microbiology lab at which the physical, chemical and microscopic examinations of urine samples are performed. 4. Blood bank (transfusion services) or immune-haematology: several procedures are done in this department depending on the needs of the patient. The procedures include; ABO and Rh grouping, cross-matching, antibody screening test, blood donation and blood components separation. 5. Immunology or serology: might be subdivision form blood bank or microbiology departments, or it could be a separate department. In this department, specimens are tested using antigen-antibody methods. Serum is mainly used in serology and sometimes urine is used. Tests performed in serology include HIV, Hepatitis, mono-spot test, pregnancy test and other infectious diseases. 6. Histology and Cytology: is the study of body tissues and cells using cells, part of organ or a whole organ called biopsy. This two labs are mainly used for examination and diagnosis of the changes that occur to the morphology of the organs to diagnosis certain diseases like cancer. There are other specialised lab in big hospitals for advance testing like molecular, genetics, flow- cytometry, virology and other labs. Also, there is a section in the lab where samples are received and registered; this section is known as lab reception. SAFET Y IN L ABORATORY In medical lab, safety in the responsibility of all workers in the lab including the sharing area with patient and other health care workers where laboratory sta may collect specimen (in phlebotomy area) or receiving a sample collected by other health care workers like physicians or nurses. Safety is including handling the sample, processing the sample as well as disposal of the samples after process. Each lab has their own records for the right process of disposal of the samples and the right manner for each even may occur within the lab. Moreover any accidents occurred in the laboratory must be documented with the action taken. Lab hazards not only focus on samples and bacterial growth but also and others chemicals or physical hazards may face the laboratory workers like res. There are serval categories of possible hazards in the laboratory including; biological, physical and chemical hazards in order to ensure the complete safety of laboratory workers from any poetical threat within the lab. Biological hazards All organisms like bacteria, viruses, fungus and Protozoa which might invade the human body and lead to infectious process is considered as biological hazards. ff fi  Physical hazards Any man-made equipments and its surrounding considered as physical hazards including Bunsen burner, electricity, lab glassware, laboratory instruments and gas cylinders. Chemical hazards Any toxic, caustic and carcinogenic chemical including any chemicals that its fume could a ect the human respiratory system considered as chemical hazard. There are an international hazard symbols of a laboratory hazards used for all chemical in the lab. ff Exposure and prevention of infection 1. Pre-exposure: A list of vaccination is mandatory for all health care workers in all hospitals including hepatitis B and other vaccination. As well as, there is a records of storage and maintenance to avoid any exposure from the di erent categories. 2. Immediate Action: if any exposure occurs within a lab e.g: blood spill, according to the amount of sample and how it occurs the situation will be evaluated and action must be taken. 4. Post-exposure: in every lab there is a documentation process (incident report) after each exposure to avoid the occurrence of the same exposure in future. General safety precaution 1. Protective clothing/Personal protective equipment’s (PPE): All sta in the laboratory must wear protective clothing (coat) and gloves, which must be taken o before living the laboratory. 2. Washing facilities: Hand basins with wrist lever taps or foot pedals must be tted in each laboratory and also paper towels must be provided. 3. Fire equipment: All equipment for dealing with all minor res and controlling larger ones should be available in all the laboratories. Examples of re equipment are hoses, water and sand buckets, re blankets, and re extinguishers. In addition, all the sta should be familiar with the location of re apparatus in their own laboratory in cases of emergency. 4. Emergency treatment: First aid boxes should be kept in readily accessible positions. Emergency eye wash stations and showers should be available. Medical assistance should be sought from the medically quali ed sta or the injured person should be taken to the casualty department of the hospital. fi fi fi fi ff ff fi fi ff fi ff ff PROFESSIONAL ETHICS Ethics is a set of generally accepted practices or types of behaviour associated with a speci c group of people. This meaning has eventually evolved to that of a meaning more closely associated with “doing the correct or proper thing.” Ethics and medical law are inseparable. Medical law and ethics are inseparable in their approach to the rights of patients. Patients have an inherent entitlement to certain expectations in their medical care. Their rights as citizens do not end at the doors of medical facilities. The medical worker or student should know, through appropriate education and training, how to treat patients humanely, legally, and ethically and how to avoid any legal entanglements, before being assigned to a clinical facility. Moral refers to a system of conduct that is right and wrong, or what is virtuous. The science of morality relates to the principles of proper professional conduct concerning the rights and duties of health care professionals, their patients, and their colleagues. Code of ethics is the expectations of your stakeholder “e.g. Ministry of Health” from the health care worker. The Code of Ethics, along with the Standards of Practice, de nes professionalism in the practice of medical laboratory technology and an ethical member shall adhere strictly not only to the guidelines, but also to the underlying spirit and precepts. Every medical laboratory scientist owes a responsibility to the following: Patient, Public, College, and Profession. Code of professional Conduct for Medical Laboratory Personnel A code of professional conduct for medical laboratory personnel should include those practices and attitudes, which characterise professional and responsible laboratory personnel and are necessary to ensure a person works to recognised standards that patients and those requesting laboratory investigations can expect to receive. It also emphasises the professional status of medical practice. Adopting a code of professional conduct helps to remind district laboratory personnel of their responsibilities to patients, duty to uphold professional standards, and need to work with complete integrity. Code of conduct is Medical laboratory professionals are dedicated to serving the healthcare needs of the public. The welfare of the patient and respect for the dignity of the individual shall be paramount at all times. The oath of Hippocrates fi fi  All health care workers regardless their communication with patients have to oath before they starting the practice. The oath of Hippocrates is a formal statement of standard values and beliefs. Code of Professional Conduct for Medical Laboratory Personnel 1. Medical laboratory professionals are dedicated to serving the healthcare needs of the public. The welfare of the patient and respect for the dignity of the individual shall be paramount at all times. 2. Medical laboratory professionals work with other health care professionals, to provide e ective patient care. 3. Be dedicated to the use of clinical laboratory science to bene t humanity. 4. Be all times courteous, patient, and considerate to patients and their relatives. Safeguard the dignity and privacy of patients. 5. Place the well-being and service of patients above your own interests. 6. Respect and work in harmony with other members of your hospital sta or health centre team. 7. Be accountable for the quality and integrity of clinical services. 8. Exercise professional judgement, skill, and care while meeting established standards. 9. Do not misuse your professional skills or knowledge for personal gain, and never take anything your place of work that does not belong to you. 10. Promote health care and the prevention and control of disease. 11. Medical laboratory professionals shall practise safe work procedures at all times to ensure the safety of patients and co-workers and the protection of the environment. 12. Follow safe working practices and ensure patients and others are not put at risk. Know what to do should an accident or re occur and how to apply emergency rst aid. 13. Do not consume alcohol or take unprescribed drugs that could interfere with your work performance during laboratory working hours or when on emergency stand-by. 14. Use equipment and laboratory-ware correctly and do not waste reagents or other laboratory supplies. 15. Strive to improve professional skills and knowledge and adopt scienti c advances that bene t the patient and improve the delivery of the test results. 16. Ful l reliably and completely the terms and conditions of your employment. 17. Medical laboratory professionals shall promote the image and status of their profession by maintaining high standards in their professional practice and through active support of their professional bodies. ff fi fi fi fi fi fi ff 18. Do not disclose to a patient or any unauthorised person the results of your investigations and treat with strict con dentiality any personal information that you may learn about patient. 19. Medical laboratory professionals shall protect the con dentiality of all patient information. 20. Medical laboratory professionals shall take responsibility for their professional acts. 21. Medical laboratory professionals shall practise within the scope of their professional competence. 22. Medical laboratory professionals shall endeavour to maintain and improve their skills and knowledge and keep current with scienti c advances. They will uphold academic integrity in all matters of professional certi cation and continuing education. 23. Medical laboratory professionals shall share their knowledge with colleagues and promote learning. 24. Medical laboratory professionals shall be aware of the laws and regulations governing medical laboratory technology and shall apply them in the practise of their profession. fi fi fi fi  QUALIT Y ASSURANCE IN L ABORATORY All departments in any health care provider institute have programs for continuous monitoring of the facilities and system. Speci cally, in the medical laboratory there are documentation which stated the accuracy and validity of any tests provided within the lab. Quality control (QC) is an essential part of every laboratory's daily operations. It is often thought to be applicable only to testing procedures. However, a program of Quality Assurance (QA) should be in place to ensure the quality throughout the total testing process, from ordering the test to entering the result on the patient's record. This is called the turnaround time (TAT). Processing the samples in the laboratory, the test procedures, and procedural controls are parts of QC. Total Quality Management (TQM) is one name usually given to a program combining QC with QA. Quality assurance in clinical laboratories is such an important issue because laboratory results are used to aid- in diagnosing, prescribing treatment, and monitoring QC the progress of patients. Laboratory results must be reliable and the laboratory's QC procedures and QA results must be documented. Laboratory workers have the ethical and legal responsibility to ensure the TQM work performed in the laboratory is of the highest quality. This can be guaranteed by adherence to a comprehensive quality assurance program. Quality Control in The Clinical Laboratory Quality control is a system set up to ensure that certain limits for a test result or product are maintained. Assays of control materials and calibrating instruments are mandatory. In addition, if the laboratory performs non-waived procedures, it must subscribe to an external pro ciency testing (PT) program at regular intervals during the year, the pro ciency testing fi fi fi agency sends the laboratory control samples for testing. The laboratory then sends its results to the agency, the values are compared to those the agency obtained in multiple analyses, and a quality assessment report is sent to the laboratory. Instrument maintenance and repair records, comparison of test methods, and reagent preparation are also part of a QC program. The laboratory director must be certain that QC guidelines are followed for compliance with authenticated bodies such as CLIA ’88. Most hospital and larger laboratories have been largely una ected by CLIA '88 because they were already following strict QC procedures. The goal of CLIA '88 is to ensure that results from all laboratories are as reliable as those from a large reference or hospital lab. Standards and Control To validate results generated in a laboratory, all analytical instruments must be calibrated, and controls must be run with each set of patient samples. Calibration can be accomplished by analysing solutions of known composition called standards provided by the manufacturer. Standards A standard is a substance that has an exact known value and that, when accurately weighed or measured, can produce a solution of an exact concentration. Standards are also called reference materials. Standards are usually quite expensive and are not used on a daily basis; they are used to calibrate newly purchased instruments, to recalibrate instruments after repair, at manufacturer's recommended intervals, or if a method is out of control Controls A control serum is a solution that contains the same constituents as those being analysed in the patient sample. Most laboratories use commercially produced controls made from pooled sera. The manufacturer has already analysed each lot of serum for a variety of components; the expected range of assay values for each component is included when the controls are shipped to the laboratory. A single control serum might contain all the constituents on a certain chemistry pro le and therefore be suitable to include in all the chemistry assays the laboratory performs. If it does not, the laboratory will have to purchase a combination of controls. The control sera must be analysed along with patient samples, using the same methods, test conditions, and reagents. At least two levels of controls must be analysed, a normal value and an abnormal value. These must be, run with each set of patient samples, at least once per shift, and any time patients' results seem questionable. Each day's results are used to construct a QC record called a Levey Jennings chart. After an instrument has been repaired, the instrument must be recalibrated, and controls run. fi ff Safety The laboratory technician can be exposed to blood-borne pathogens while using instrument calibrators and running control sera samples. Many of these samples are of human origin and, although they have been screened for certain pathogens, may be infectious. Standard Precautions must be followed while performing all control procedures, just as when working with patient samples. Accuracy and Precision In any QC discussion, the terms accuracy and precision must be considered. Although accuracy and precision are sometimes used interchangeably, they do not have the same meaning. Accuracy refers to the closeness of an analytical result to the actual value. Results nearer to the real value are more accurate than ones further away from the real value. For example, if the real value for an individual's glucose is 90 mg/dL, analyses that yielded results of 88, 90, or 92 mg/dL would be more accurate than analyses that yielded values of 80, 75, or 82 rng/dL. The term Precision refers to reproducibility of results or the closeness of obtained values to each other. An example of precision would be results of 88, 87, 92, 91, 88, and 90 on the above glucose sample; these results vary little from each other. Another way of thinking about precision is to mentally picture a target with all the arrows or bullet holes closely grouped in one area of the target.If the values 78, 90, and 100 mg/mL are placed on the imagined target, the "arrows" will be scattered around the target, not grouped close together, indicating lack of precision. One can have precision without accuracy; in other words, one could produce results near in value to each other, but, because of an error, the values may be inaccurate, not near the true value. The worker's goal should be to achieve both precision and accuracy in the laboratory analyses performed. Precision Accuracy Quality Assurance in The Clinical Laboratory Errors can occur at several points during the specimen collection, test performance, and results reporting. Pre-analytical errors occur prior the testing procedures include misidentifying the patient, collecting a sample at the wrong time, and mishandling the specimen. Analytical errors occur during the test procedure and may be random or systematic. Systematic error is de ned as a variation that may make results consistently higher or lower than the actual value. Factors that may cause this type of error are deteriorated reagents, mechanical trouble in the instrument, or a peculiarity in worker methodology, such as manner of pipetting. The source of random error cannot be absolutely identi ed. This type of error causes variations on either side of the mean. Factors that may contribute to random error include an air bubble in a reagent line, di erences in technique among workers, and certain specimen characteristics. Post-analytical errors also can occur. A result may be accurate but may be transferred to another patient's records by entering the wrong patient identi cation number in the computer. When results are transcribed by hand, the wrong value may be copied from laboratory reports to a patient's chart. All these issues are addressed in a comprehensive QA program. The increased use of scan barcodes on request forms, patient identi cation bands, specimen containers, and patient charts, and interfacing laboratory computers with patient records, has eliminated many pre- and post- analytical errors. fi fi ff fi fi  Summary Term De nition Quality Control (QC) A system set up to ensure that certain limits for a test result or product are maintained. Quality Assurance (QA) A system set up to ensure the quality throughout the total testing process. Turn Around Time (TAT) The total timing of testing procedure from sample collection to releasing the report Pro ciency Testing (PT) An external program at regular intervals during the year, the pro ciency testing agency sends the laboratory control samples for testing. Accuracy The closeness of an analytical result to the actual value Precision The closeness of an analytical result from each other Pre Analytical error Any errors might occurs prior the testing procedure Analytical error Errors that occurs during testing procedure Post Analytical error Errors that occurs after testing procedure Systemic error A variation that may make results consistently higher or lower than the actual value Random error Error that occur for unknown reason; usually environmental or instrument generator. Levey Jenning chart A chart that used in laboratory to follow up the control variable. fi fi fi  C H A PAT E R I I : M I C RO B I O LO GY MICROSCOPY Microscopy is the technical eld of using microscopes to view samples and objects that cannot be seen with the unaided eye. Micro-organisms and cells of animals are so small that we cannot see them with the un-aided eyes; we need to magnify them so we can see them. We do not only need to see them, sometimes we want to observe any minute details in them and in the case of cells, any changes. Microscopy is the use of lenses of di erent magni cations to see objects which ordinarily we cannot see with our eyes. It was invented by a Dutchman called Anton van Leeuwenhoek. He was born in 1632 and started his studies of microbes at the age of twenty-one. He is the father of modern microscopy because he invented it in the 1670s. Microscopy today has many applications such as electron microscopy, X-ray microscopy, Phase contrast microscopy, and light microscopy. The light microscope, so called because it employs visible light to detect small objects, is probably the most well-known and well-used research tool in biology and medicine. For any medical laboratory scientist to be successful, he or she must adopt the microscope as his or her baby, or at least as one’s best friend! This is because you must use a microscope in haematology, histology, cytology, microbiology and to a lesser extent, in immunology. Types of the light microscopy 1. Bright eld microscopy 2. Dark ground (also called Dark eld) microscopy. 3. Phase contrast microscopy Apart from the above, we also have electron microscopy, Fluorescence microscopy. Bright eld microscopy. The bright eld microscope is best known to students and is most likely to be found in a classroom. Visible light is focused through a specimen by a condenser lens, and then is passed through two more lenses placed at both ends of a light-tight tube. The latter two lenses each magnify the image. It is good to note that the e cient working of a microscope depends on or is related to the magni cation, resolution, illumination, and contrast. Other than the compound microscope, you may see other variations like the dissecting microscope and the inverted microscope. fi fi fi fi fi fi fi ffi ff Part Function Eyepieces (Also called The eyepieces are made up of a series of lenses with a magnification of Oculars) usually 10x. Thumb wheel This is used for interpupillary adjustment, i.e. to adjust your eye span. This can rotate to change from one objective to another. In most binocular Revolving nosepiece. microscopes, it is fixed while in most monocular it is raised up and down when focussing. These are units of lenses with different magnification. As the name implies, they are focussed on the object. The magnifications used are: 10x for the low power objective; Objectives 40x for the high-power objective; and 100x for the oil immersion objective. Most microscopes have these three, but some have a fourth objective of 4x magnification called a scanning objective. This always allows the movement of the slide during focussing. It has a Mechanical stage. round opening, called the aperture that allows light to reach the slide. It carries also the vernier scales and the slide holders. The vernier scales are on the stage. They allow us to determine the position The vernier scale of an object on the slide. These help to hold the slide firm on the stage. One part of the slide holder is Slide holders spring loaded to give it the firm grip on the slide. This structure is located under the stage and its main function is to condense the light from the light source into a small cone of light. It ensures that most of the light waves reach the object and are not scattered. It also controls the Substage condenser intensity of the light with the help of the diaphragm which can be opened or closed with the help of a diaphragm lever. The condenser is held in place and centred for optimum working by two centring screws. The condenser may house a filter which absorbs some of the light. This is used to raise or lever the condenser depending on the amount of the Condenser adjustment knob. intensity of light required. This moves the stage in two horizontal planes; that is, back and forth and side Mechanical stage control. to side. The light source. The light source may be housed in the base or is a separate unit. The intensity of the light can be controlled by opening or closing the amount Light intensity control. of light coming from the light source. The condenser diaphragm is also used for the same purpose. This is used to move up the stage (or the body tube) in order to bring the The coarse adjustment. object under an approximate focus. This moves the stage up and down very slowly in order to bring the object The fine adjustment. under a sharp focus.  This is the tube that houses the eye pieces and the revolving nose piece. It is The body tubes. not very obvious in most microscopes, but it’s is importance is in the working out the magnification of a microscope. The arm. This supports the upper part of the microscope. The base supports the entire microscope. In most modern microscopes, the The base light source is fixed on the base. More sophisticated microscopes have a separate light source. BRIGHT FIELD MICROSCOPY How Microscope works? With a conventional bright field microscope, light from an incandescent source is aimed toward a lens beneath the stage called the condenser, through the specimen, through an objective lens, and to the eye through a second magnifying lens, the ocular or eyepiece. Some microscopes have a built-in illuminator, while others use a mirror to reflect light from an external source. The condenser is used to focus light on the specimen through an opening in the stage. After passing through the specimen, the light is displayed to the eye with an apparent field that is much larger than the area illuminated. The magnification of the image is simply the objective lens magnification (usually stamped on the lens body) times the ocular magnification. The coarse and fine focus knobs are used to sharpen the image of the specimen. Adjustments to the condenser affect the resolution and contrast. Some condensers are fixed in position, others are focusable, so that the quality of light can be adjusted. Usually the best position for a focusable condenser is as close to the stage as possible. The bright field condenser usually contains an aperture diaphragm, a device that controls the diameter of the light beam coming up through the condenser, so that when the diaphragm is stopped down (nearly closed) the light comes straight up through the centre of the condenser lens and contrast is high. When the diaphragm is wide open the image is brighter, and contrast is low.  Using bright eld microscopy First, what do I want to observe? Is it a stained specimen? Or is it a wet preparation? What magni cation do I need and therefore which of the objectives should I use? How much contrast/resolution do I require and therefore what should be the position of my condenser and how much light should I use? Am I comfortable with the eye span? Mount the specimen on the stage High magni cation objective lenses can't focus through a thick glass slide; they must be brought close to the specimen, which is why coverslips are so thin. The stage may be equipped with simple clips (less expensive microscopes), or with some type of slide holder. A mechanical stage allows precise positioning without touching the slide. Optimise the lighting A light source should have a wide dynamic range, to provide high intensity illumination at high magni cations, and lower intensities so that the user can view comfortably at low magni cations. Better microscopes have a built-in illuminator, and the best microscopes have controls over light intensity and shape of the light beam. If your microscope requires an external light source, make sure that the light is aimed toward the middle of the condenser. Adjust illumination so that the eld is bright without hurting the eyes. Adjust the condenser If the condenser is focusable, position it with the lens as close to the opening in the stage as you can get it. Start with the aperture diaphragm stopped down (high contrast). You can adjust this as you go on. Focus, locate, and centre the specimen. Start with the lowest magni cation objective lens to scan and locate the area you want to examine. It is rather easy to nd and focus on sections of tissues, especially if they are xed and stained. However, it can be very di cult to locate living, minute specimens such as bacteria. fi fi fi fi fi fi fi ffi fi fi  *Start with the specimen out of focus so that the stage and objective must be brought closer together. The rst surface to come into focus as you bring stage and objective together is the top of the cover slip. With smears, a cover slip is frequently not used, so the rst thing you see is the smear itself. If you are having trouble, focus on the edge of the cover slip or an air bubble, or something that you can readily recognise. The top edge of the cover slip comes into focus rst, then the bottom, which should be in the same plane as your specimen. Once you have found the specimen, adjust contrast and intensity of illumination, and move the slide around until you have a good area for viewing. Adjust eyepiece separation, focus With a single ocular, there is nothing to do with the eyepiece except to keep it clean. With a binocular microscope (preferred) you need to adjust the eyepiece separation just like you do a pair of binoculars. Binocular vision is much more sensitive to light and detail than monocular vision, so if you have a binocular microscope, take advantage of it. One or both of the eyepieces may be a telescoping eyepiece, that is, you can focus it. Select an objective lens for viewing The lowest power lens in some microscopes is usually 3.5 or 4x and is used primarily for initially nding specimens. We sometimes call it the scanning lens for that reason. The most frequently used objective lens is the 10x lens, which gives a nal magni cation of 100x with a 10x ocular lens. Typical high magni cation lenses are 40x and 100x. The latter magni cation is used exclusively with oil in order to improve resolution. Move up in magni cation by steps. Higher magni cation lenses must be physically closer to the specimen itself, which poses the risk of jamming the objective into the specimen. Be very cautious when focusing. But good quality sets of lenses are par-focal, that is, when you switch magni cations the specimen remains in focus or close to focused (granted your preparation is not too thick). All specimens have three dimensions, and unless a specimen is extremely thin you will be unable to focus with a high magni cation objective. The higher the magni cation, the harder it is to "chase" a moving specimen. Adjust illumination for the selected objective lens. fi fi fi fi fi fi fi fi fi fi fi fi fi  The apparent eld of an eyepiece is constant regardless of magni cation used. So, it follows that when you raise magni cation, the area of illuminated specimen you see is smaller. Since you are looking at a smaller area, less light reaches the eye, and the image darkens. With a low power objective, you may have to cut down on illumination intensity. With a high power you need all the light you can get, especially with less expensive microscopes. M I C RO S C O P Y T E R M I N O LO GY Magni cation Magni cation is the product of the separate magni cations of the objective and the eyepiece. If the eyepiece is 10x and the objective is 40x, the magni cation is 400. Factors a ecting magni cation are: I. The optical tube length (which is approximately the same as the mechanical tube length. II. The focal length of the objective III. The magnifying power of the eyepiece. Resolution The limit of useful magni cation is set by the resolving power of the microscope i.e. its ability to reveal closely adjacent structural details as separate and distinct. The resolution of a microscope makes it possible to distinguish between two neighbouring points as separate entities. The resolution of a microscope is a function of the wavelength of light used and the quality of the optics (the lenses) used. The gure shows examples of di erent levels of resolution from low to high. Working distance This is the clearance between the lowest point of the objective and the object under focus. It varies with di erent objectives. With x10 it is about 5 mm, for x40 it is about 1/3 mm, and for oil immersion objective, it is about 1/8 mm. Note that it is so low with the oil immersion objective that it is possible to break your slide while focussing if you are not careful. Note also that the working distance is less than the focal length of the objective. Adjustments of microscope: Low power: fi fi fi ff ff fi fi fi fi ff fi fi fi 10X objective is brought in line with the eyepiece lens. Condenser is fully lowered, iris diaphragm partially closed. Body tube is lowered till objective is little above the slide. Then looking through the eyepiece, raise the objective till clear view of the specimen is obtained. High power: 40X objective is used and brought in position, condenser is kept midway and the diaphragm ¾ opened. Oil immersion: Condenser raised, iris diaphragm fully opened, after bringing oil immersion objective in position Focus the required area under low power rst. Put a drop of cedar wood oil/ immersion oil over the slide, rotate the nose piece to make the oil immersion objective touch the drop of oil. Now, looking through the eyepiece, use the ne adjustment screw to focus correctly. How does oil immersion works Light rays after passing through glass will be di racted away from normal on passing into air. This means that some light rays that passed through an object will not reach the ff fi fi  objective lens and therefore the eye. By putting oil which has the same refractive index with glass, these rays will not diverge but will continue in a straight line into the objective. So by using immersion oil on top of stained preparations, light rays will not be scattered. Around 1676, when Anton von Leeuwenhoek discover the magni cation he saw unknown teeming colonies of creatures. He described them as vast populations of minute creatures, as he even saw some of them moving and assumed they were alive. Von Leeuwenhoek did not understand the signi cance to this discovery and did not realise the signi cant impact these images would have on the lives of everyone living. As early as the 1870s, Joseph Lister and oth- ers discovered that Petri dishes grew a species of bread mold called Penicillium sp. But it was not until 1945 that Alexander Fleming was awarded the Nobel Prize in Medicine for the discovery of penicillin; his original endeavors began in 1928 when he learned that a species of bacteria from the genus Staphylococcus were inhibited or killed by this mold. CL ASSIFIC ATION OF MICRO-ORGANISM What is microbiology? Microbiology is the study of all microorganisms. Microorganisms are types of living organisms capable of independent existence but are often not seen by the unaided eye. They can be seen under a microscope. There are many microorganisms. Some of them are of medical importance. The study of microorganism of medical importance is called medical microbiology. In medical microbiology, we study pathogens and the diseases they cause, and how the body de ance mechanisms ght against the pathogens. We also study the spread, treatment and prevention of the diseases caused by pathogens. Microorganisms of medical importance are Bacteria, Viruses, Protozoa, Fungi etc. Classi cation of microorganisms: They were classi ed under a third kingdom, the Protista. This includes the following groups of organisms: a) Bacteria (Including Rickettsia, c) Protozoa Chlamydia, Mycoplasma) d) Fungi b) Viruses Based on di erences in cellular organisation and biochemistry, the kingdom Protista has been divided into two groups prokaryotes and eukaryotes. Bacteria and blue green algae are prokaryotes, while fungi, other algae, slime moulds and protozoa are eukaryotes. 1- Prokaryotes: Have very simple cell structure. They do not have mitochondria. They do not have chromosomes. The genetic material of prokaryotes is the DNA (deoxyribonucleic acid) and situated in the cytoplasm. So, no true nucleus. They divide by simple binary ssion, fi ff fi fi fi fi fi fi fi  2- Eukaryotes: Have complex cell structure similar to those of higher animals. The genetic material (DNA) of a eukaryotic cell is di erentiated into chromosomes, which are contained in a nuclear membrane to form a de nite nucleus. They possess mitochondria. The divide by a process called mitosis. Characteristic Prokaryote Eukaryote DNA within a nuclear membrane No Yes Mitotic Division No Yes DNA associated with histone No Yes Chromosome number One More than one Membrane-bound to organles such as No Yes mitochondria Size of ribosome 70S 80S Cell wall containing peptidoglycan Yes No - Bacteria: Bacteria are prokaryotic microorganisms that do not contain chlorophyll. They are unicellular and do not show true branching, except by Actinomycetes. They form a large group made up of Pathogens: Which are disease-causing bacteria. Saprophytes: Which are free-living organisms that cause no disease. Most of the bacteria can be easily grown on laboratory culture media. They divide by binary ssion and are sensitive to antibiotics. - Viruses: They are neither eukaryotes nor prokaryotes. They are biochemically inert. They cannot synthesise their own macromolecules such as proteins and enzymes. They are completely dependent on host cells on which they grow for these molecules. Their genetic material is either DNA or RNA but never both. They cannot live or grow on ordinary laboratory media; they must be grown on living cells. Viruses are so small that they cannot be seen with the ordinary light microscope, the electron microscope is used to study them. Virology is the study of viruses. - Parasites: Some organisms live inside other living organisms. If an organism lives in another and derives bene t from the relationship such that it cannot capable of existing on its own without depending on the other partner, that relationship is called parasitism. The bene ting organism in this relationship is called parasites. The study of host parasite relationship is called Parasitology. Protozoa and Helminths are the parasites studied in parasitology. They are eukaryotes. - Fungi (Fungus): They are the biggest group of organisms. Unlike other microorganism, we can see them with our naked eye but internal structures can fi fi fi ff fi  only be seen with the aid of a microscope. They are eukaryotes. Most fungi are saprophytes. The study of fungus is known as Mycology Protozoa and Helminths Characteristic Viruses Bacteria Fungi (Parasite) Cells No Yes Yes Yes Size diameter (um)1 0.02 - 0.2 1-5 3 - 10 yeasts 15 - 25 trophozoites Nucleic Acid Either DNA or RNA Both DNA and RNA Both DNA and RNA Both DNA and RNA Type of nucleus None Prokaryotic Eukaryotic Eukaryotic Ribosome Absent 70S 80S 80S Mitochondria Absent Absent Present Present Nature of outer Protein capsid and Rigid wall contain Rigid wall contain chitin Flexible membrane surface lipoprotein envlope peptidoglycan Motility None Some None Most Method of replication Not binary ssion Binary ssion Budding or Mitosis Mitosis Size of bacteria Bacteria of medical importance generally measure 0.2-1.5 µm in diameter and about 3-5 µm in length. Bacteria are divided into groups based on their shape as determined by their microscopic appearance, bacteria may be classi ed into the following: Cocci- Spherical or oval shaped Spirilla- Rigid spiral forms Bacilli- Rod shaped Spirochetes- Flexuous spiral forms Vibrio’s- Comma shaped Cocci: these are usually round or oval in shape. We can divide them into many groups depending on how they appear after multiplying. A. In pairs i.e. appearing in twos, they are called diplococci Ex: Neisseria gonorrhoea and Neisseria meningitides B. In chains. They are called Streptococci Ex: Streptococci pyogenes. C. In bunch of grapes after dividing. They are called Staphylococci. Ex: Staphylococcus aureus fi fi fi  Bacilli (rods): These are rod-like or stick-like in shape and the end may be round or square or swollen. Examples are Salmonella, shigella etc. Vibrio’s: these are curved and described as comma shaped. Most vibrios are motile. An example of vibrio’s is Vibrio cholera. Spirilla: there are the name implies are rigid spirals but they have agella at both ends e.g. Spirillum minus Spirochaetes: These are exible coiled and motile organisms. Treponema e.g. T. pallidum that causes syphilis, Borrelia e.g. Borrelia Vincentia found in Vincent’s angina, and Leptospira e.g. Leptospira intergerns causative agent of leptospirosis that is transmitted by rats. fl fl  BACTERIAL ANATOMY The outer layer or cell envelope consists of two components – a rigid cell wall and beneath it a cytoplasmic or plasma membrane. The cell envelope encloses the protoplasm, comprising the cytoplasm, cytoplasmic inclusions such as ribosomes and mesosomes, granules, vacuoles and the nuclear body. Besides these essential components, some bacteria may possess additional structures. The cell may be enclosed in a viscid layer, which may be a loose slime layer, or organized as a capsule. Some bacteria carry lamentous appendages protruding from the cell surface which are: The agella which are organs of locomotion and The mbriae, which are organs for adhesion. 1. Bacterial cell wall: The cell wall accounts for the shape of the bacterial cell and confers on it rigidity and ductility. The cell wall contains lipoproteins, lipopolysaccharide and peptidoglycan. The main functions of the cell wall are to give the bacterium its shape, protects the cell membrane and cell contents and dictate gram reaction. The composition of the cell wall dictates what the staining reaction will be. Gram-positive bacteria have a large amount of peptidoglycan and teichoic acid. Gram negatives have a smaller amount of peptidoglycan and contain lipopolysaccharide molecules. 2. Slime layer and capsule: Many bacteria secrete a viscid material around the cell surface. When this is organized into a sharply de ned structure, it is known as the capsule. fi fl fi fi Ex: In Pneumococcus. When it is a loose under-level secretion, it is called the slime layer. Ex: In Streptococcus salivarius. The main function of capsules is to protect the bacteria from phagocytosis and from deleterious agents such as lytic enzymes found in nature. Examples of capsulated bacteria include Strep. pneumoniae, Klebsiella pneumoniae, and Bacillus anthracis. 3. Flagella: These are the organs with which some bacteria move from place to place. Motile bacteria possess one or more unbranched, long, sinuous laments called agella, which are the organs of locomotion. Flagella are made up of a protein ( agellin) similar to keratin or myosin. 4. Pili or Fimbriae: Some Gram-negative bacilli carry very ne, hair-like surface appendages called mbriae or pili. They are shorter and thinner than agella and project from the cell surface as straight laments. Classi able as either common or sex pili on the basis of their function. 5. Common pili or mbriae function as organs of adhesion. The mbriae are used by the bacteria to get attached to the surface of the cell that they enter or infect. Therefore, organisms that have pili are more virulent than those that do not have it. 6. Sex pili: A special type of mbria is the sex pili. These are longer and fewer in number than other mbriae. They are the sex organs of the bacteria. Through them, genetic material (DNA) is transferred to the receiving bacteria. They help in the attachment of bacterial cells during conjugation by forming hollow conjugation tubes through which genetic material is transferred. 7. Cytoplasmic membrane: The cytoplasmic (plasma) membrane is a thin (5-10 nm) layer lining the inner surface of the cell wall and separating it from the cytoplasm. It acts as a semi permeable membrane controlling the in ow and out ow of metabolites to and from the protoplasm. 8. Cytoplasm: The bacterial cytoplasm is a colloidal system of a variety of organic and inorganic solutes in a viscous watery solution. It di ers from eukaryotic cytoplasm in not exhibiting internal mobility (protoplasmic streaming) and in the absence of endoplasmic reticulum or mitochondria. The cytoplasm contains ribosomes, mesosomes, inclusions, vacuoles, nucleus and plasmids. 9. Ribosomes are centres of protein synthesis. fi fi fi fi fi fl fi ff fl fi fi fi fl fl fl  10. Mesosomes are vesicular, convoluted or multilaminate structures formed as invaginations of the plasma membrane into the cytoplasm. They are the principal sites of respiratory enzymes in bacteria and are analogous to the mitochondria of eukaryotes. 11. Intracytoplasmic inclusions: May be of various types, the chief of which are volutin, polysaccharide, lipid and crystals. 12. Nucleus: They have no true nucleus. It has no nuclear membrane or nucleolus. The genome consists of a single molecule of double-stranded DNA arranged in the form of a circle. DNA is not associated with basic protein. The bacterial chromosome is replicates by simple ssion instead of by mitosis as in higher cells. 13. Plasmids or epistomes: Bacteria may possess extra nuclear genetic elements consisting of DNA. These cytoplasmic carriers of genetic information are termed plasmids or epistomes. They are not essential for the life of the cell they inhabit but may confer on its certain properties like toxigenicity and drug resistance, which may constitute a survival advantage. GRAM STAIN.. Gram stain is the most important procedure in microbiology which was developed in 1884 by a Danish Physician called Christian Gram and it is used widely to date. It is most popular procedure that separates bacteria into gram-positive (blue in colour) and gram-negative (pink in colour). Both cocci and bacilli are described by their grams stain reaction. The basis of dividing bacteria into gram positive and negative lies in their grams stain reaction. The initial rosaniline dyes (e.g. crystal violet, methyl violet, or gentian violet) is allowed to act on the bacteria being stained and then a mordant (Iodine) is added before it is decolourised with alcohol or acetone. Then counter stained by dilute carbol fuchsin or safranin. Those that resist decolourization are called Gram-positive. Those that are decolourised are called Gram-negative. Grams positive organisms stain violet since they retain the primary stain i.e. crystal violet, due to the thick layer of peptidoglycan in their cell wall. While, Gram negative stain red or pink since they decolourised and then take up counter stain dilute carbol fuchsin or safranin due to the thin layer of peptidoglycan. NB: Please refer Gram stain note on your IMLS Practical book for details of gram stain. fi  C H A P T E R I I I : H A E M ATO LO GY Haematology is the study of the structure and function of blood and blood cells. Haematopoiesis is the process of blood cell production or formation that includes cell renewal, proliferation, di erentiation, and maturation. (haem= blood, poesies= synthesis) Under normal conditions, in an adult the production of blood cells is carried out by the bone marrow. Blood is described as a specialized connective tissue, which circulates in a closed system of blood vessels. Blood is one of the largest organs of the body. It is a ‘non-solid’ organ. Blood circulates throughout the body, providing support to the functioning of other organ systems in the body. Approximately 6 – 8 % of the human body’s total weight is made up of blood and this makes up approximately 5 – 6 litres of blood in an adult. For infants and children, the blood volume is more while for obese adults, the blood volume is less. Functions of blood I. Transportation: Blood carries important substances like water, nutrients, vitamins, electrolytes, hormones, oxygen and immune substances to cells and tissues. Blood carries waste products like carbon dioxide and other metabolic materials away from cells and tissues. Waste products (Carbon dioxide and toxic metabolism) ff  Cells and tissue II. Regulation: 1. Helps to regulate body temperature. 2. Helps to regulate acid base balance through bu ers. 3. Helps to regulate water content of cells by dissolved ions and proteins. III. Protection from diseases. IV: Protection from loss of blood (coagulation of blood). ANTICOAGUL ANT Anticoagulants are chemical substances that prevent clotting of blood when mixed with blood in proper proportion. If blood is collected into a plane specimen container, it will clot as the brinogen is converted into brin. After the blood has rmly clotted, the clot retracts, and the specimen can then be centrifuged. The top layer –SERUM can be removed and used for several laboratory tests. However, for several tests, it is necessary to prevent the blood from clotting by collecting the blood into specimen tubes containing anticoagulants and can be used for the investigations. If this specimen is centrifuged the top layer is the –PLASMA, which can be used for lab tests.For several reasons, di erent anticoagulants are used. I. EDTA [ Ethylenediamine Tetra-acetic Acid]: This can be obtained either the disodium salt or dipotassium salt. They are powerful anticoagulants. They bind and remove free calcium ions from blood, chelation, thereby preventing coagulation. EDTA preserves the morphology of the blood cells even up to 3 hours and cell counts up to 12 hours after collection of the blood. Therefore, it is the fi fi ff ff fi anticoagulant of choice for all cell counts and peripheral smear study. The internationally recommended anticoagulant is the dipotassium salt. The concentration is 1.5+/- 0.25 mg/ ml of blood. EDTA maintains the structure of both red and white cells and stops the clumping of platelets. The blood sample can then be used to count red blood cells, white blood cells and platelets. II. TRI SODIUM CITRATE. Sodium citrate binds with calcium ions in the blood to form a soluble calcium citrate complex thus preventing coagulation of blood. 1. Coagulation studies: Either 3.8% trisodium citrate pentahydrate or 3.2% trisodium dehydrate solution is used for coagulation studies.One part of anticoagulant is mixed with nine parts of blood (1:9 i.e., ratio 0.2 mL: 1.8mL) 2. ESR: In the Westgren method of ESR estimation, one part of anticoagulant mixed with four parts of blood (1:4). 3. Blood Bank: ACD solution (acid citrate dextrose solution) was used as an anticoagulant in blood bank. This has been replaced by citrate phosphate dextrose adenine (CPDA) Disadvantages: Since it is used as a liquid which results in dilution of cellular elements, it is not suitable for cell counts, haemoglobin, etc. III. HEPARIN It is used when almost instantaneous anticoagulation is required and to prevent any chances of haemolysis after the blood is drawn. Heparin acts by preventing conversion of prothrombin to thrombin. Heparin is used at a concentration of 1-5 mg (10-15 IU)/ml of blood. Syringe is heparinised by taking 1 drop of heparin to wet the inner surface of the syringe and the blood is collected. Heparin is used for serevel purposes: 1. Osmotic fragility test done for diagnosis of spherocytosis. 2. Red cell enzyme studies like G6PD and PK de ciency. fi  3. Clinical chemistry, genetic studies. Disadvantages: 1. Expensive. 2. Causes clumping of platelets and leucocytes, so it is not used for automated cell counters. 3. Imparts bluish discoloration to the background of peripheral blood smears. 4. Prevents coagulation for only a limited period and its e ect slowly disappears as the heparin is metabolised or neutralized in the blood. IV. FLUORIDE OXALATE Plasma or serum samples may be used for glucose analysis ONLY if the plasma/serum is separated from the cells within 1 hour of sample collection. Without an antiglycolytic agent, the blood glucose concentration decreases by approximately 0.56 mmol/l per hour at 25°C. Acts as an anti-glycolytic agent which inhibits the use of glucose by blood cells. Sodium uoride functions by stabilising the blood cell membrane and inhibiting the enzyme systems involved in glycolysis, which prevents red blood cells metabolising any glucose present in the sample. For this reason, it is the only suitable sample for accurate glucose analysis. Fluoride is a potent inhibitor of many enzymes, and the inhibition of glycolysis tends to cause uid shifts. Fluoride is a weak anticoagulant on its own, so potassium oxalate (another powerful enzyme inhibitor) is usually added to supplement its action. Fluoride/ oxalate samples are used for blood glucose (and lactate) estimation only. V. DEFIBRINATION OF BLOOD. De bration is the removal of brin from blood. This technique makes it possible to obtain blood samples in the liquid form without the use of an anticoagulant. This may be necessary when both serum and cells are required or when the use of an anticoagulant is not advised. Blood is de brinated by placing the blood in a specimen bottle containing glass beads or glass rods. The sample is thoroughly mixed, and the brin and platelets collected around the above materials. This leaves the red cells and white blood cells suspended in serum. The de brinated blood should not undergo any visible lysis. De brinated blood may be required for the investigation of certain types of haemolytic anaemia and for the preparation of bu y- coat smears. fl fi fi fl fi fi fi ff fi ff  Vacutainer Antocoagulant Uses Colour code CBC, peripheral smear, Sickling, G6PD, HPLC, Purple (Lavender stopper tube) EDTA-K2 Retics, Ammonia, lead, HBA1C, DCT, genetics testing.. etc. Plasma determination in chemistry, somatic Green stopper tube Sodium Heparin fragility, chromosomal studies Coagulation determination of plasma Light blue stopper tube Sodium Citrate (3.2%, 0.109M) specimens Glucose, toxicology determination. Gray stopper tube Sodium uoride/ potassium oxalate Antiglycolytic additives stabilise glucose values for up to 24 hours at room temperature Red/ Yellow stopper tube (without gel Serum determinations in chemistry, serology Clot activator seprator) and blood banking testing. PROCESSING OF BLOOD SAMPLE Sample recording on computer and sample preparation for testing. When your sample arrives in the laboratory, it is logged into the laboratory’s computer system. The sample is given a unique barcode number that tracks your sample throughout the entire testing and reporting process. To ensure accuracy, we should double check every sample entry into the computer system. This electronic audit process checks patient, requestor and test details before the sample is allowed to progress into the laboratory for testing. All the blood samples should be processed immediately after collection. Some tests require preparation of sample before testing can started. Safety Considerations: All blood specimens should be considered potentially infectious, the major risks being exposure to hepatitis and HIV. Universal precautions practices, including prior hepatitis vaccination, should be followed during blood collection and handling. Pre-centrifugation Handling - The rst critical step in the lab testing process, after obtaining the sample, is the preparation of the blood samples. Specimen integrity can be maintained by following some basic handling processes: Fill tubes to the stated draw volume to ensure the proper blood-to-additive ratio. Allow the tubes to ll until the vacuum is exhausted and blood ow ceases. Vacationeer tubes should be stored at 4-25°C (39-77°F). Tubes should not be used beyond the designated expiration date. fl fi fi fl  Mix all gel barrier and additive tubes by gentle inversion 5 to10 times immediately after the draw. This assists in the clotting process. This also assures homogenous mixing of the additives with the blood in all types of additive tubes. Serum separator tubes should clot for a full 30 minutes in a vertical position prior to centrifugation. Short clotting times can result in brin formation, which may interfere with complete gel barrier formation. If the blood is allowed to stand for more than one hour before lms are made, changes may occur to the morphology of the blood cells. These become more striking the longer the blood sample is left. If there is to be a delay in testing, the blood lms should be made immediately and xed in methanol and the whole blood sample should be refrigerated at 4o C. The whole blood should not be freeze. 1. Red blood cells: Crenation of cells and sphering occurs after storing at room temperature for more than 24 hours 2. White blood cells: Some but not all neutrophils may be a ected. The nuclear lobes may become separated with increased vacuolation. 3. Platelets: They swell and may get fragmented and loose granules. Blood Sample Centrifugation – It is recommended that serum be physically separated from contact with cells as soon as possible, with a maximum time limit of 2 hours from the time of collection. THE COLLECTION OF BLOOD (PHLEBOTOMY) The blood, the uid connective tissue should be collected for its investigations in a laboratory. When it is done well, it may appear to be extremely easy. However, it is a skilful job. You may nd yourself in a situation where you are expected to work quickly under possibly di cult circumstances. People do not like having needles struck into them, but they put-up with it because they realise that it is necessary. Therefore, a good phlebotomist (a person who collects blood) should put the patient at easy while the blood sample is being collected. Method of blood collection: The blood sample may be collected from a veins, capillaries, and arteries. ffi fi fl fi fi ff fi fi  Venipuncture: This may be carried out using a needle and syringe or a commercial evacuated system such as vacationer system. Types of veins 1. Large prominent veins: A phlebotomist must be careful not to insert the needle too far. Once inserted the needle should run along the vein never through it. 2. Large deep veins: By experience in blood collection, you will be guided by what you can feel rather than what you can see. This skill is described as palpation of the veins. Veins may lie below the surface of the skin and can only be located by touch. Take your time locating the exact position of the vein until you are con dent of where to insert the needle. 3. Small thready veins: May be more di cult to deal with. You may have to use a ne bore needle. If the blood is drawn out too quickly, the veins atten and become useless to get blood. They also bleed for a longer time so care should be taken to stop the bleeding after the blood sample has been collected. 4. Super cial veins: Tiny veins on the surface of the arms. These are not suitable for blood collection. 5. Floating veins: These may look promising but may move when you try to insert the needle. You will recognise these as you gain experience. You will need to anchor the vein about 3-5 cm below the puncture site, with the thumb or fore nger. Once the needle is in the vein the sample is then taken in the usual way. 6- Thrombosed veins: These are veins that have been overused and are hard and lumpy to the touch. It needs a lot of skill to draw blood from these veins. A suitable alternative site should be chosen. 7- Very deep veins: Often found in obese patients. Use palpation to nd a vein. Ask for assistance if you are unable to nd a suitable vein. Sites for Venipuncture The usual sites of choice for venipuncture are the veins on the front of the elbow (the ante- cubital fossa). These can usually be seen easily or palpated. It is unlikely that the needles will hurt the brachial artery, but you should know its position. If necessary other sites can be selected: fi fi ffi fl fi fi fi fi 1. The vein on the back of the hand- wrist / dorsal surface of forearm, make sure that you are supervised. 2. The angle/ foot, this is not a regular site for the procedure unless all other sites are not suitable. Make sure that you are supervised]. Equipment: 1. Gloves 6. Spare needle of various sizes 2. Tourniquet 7. Specimen containers/ vacutainer tubes 3. Skin cleansers 8. Surgical adhesive tapes 4. Sterile gauze or cotton balls 9. Pen to label samples 5. Needle and syringe/ vacutainer system 10. Biohazard/ sharp disposal containers. Vacutainer system.. Along with the needle and syringe method the blood collection using evacuated system is also in practice. Here the blood is collected through a needle in the vein directly into a specimen tube under vacuum. The needle is a combined needle and valve. The end of the needle consists of a rubber sheathed valve. This is inserted into an open-ended plastic adapter and is screwed into place. The needle can then be unsheathed and inserted into the vein. Each specimen tube (vacutainer has a vacuum within it. The cap of the bottle has a rubber seal. The vacutainer is placed into the adapter and pushed on to the needle and valve. The correct amount of blood (up to the mark) is then drawn into the tube. The tube can then be removed and another tube on to the needle and valve without blood loss. This system is very popular because it is safer than using needle and syringe, it allows di erent sample types to be taken at one time. Ensure that you practice using a tourniquet, how to tighten and release it properly. Make sure that you familiarise yourself with handling the syringe. Procedure of blood collection.. 1. Ask the patient’s name, date of birth, and check it on the request form. This is for the correct patient identity. 2. Carefully check the request on computer, decide how much blood is to be collected and arrange the appropriate containers. Label the tubes with the patient details (number, name, date of birth, date, and time of sampling). 3. Check if there are any speci c preconditions before sampling. E.g.: fasting sample, high risk patients etc. 4. Wash your hands and make sure that your ngernails are clean. Wear gloves if you wish to do so (see note below concerning safety) 5. Prepare the needle and syringe or the evacuated system. Attach the needle to the barrel of the syringe or to the vacutainer but do not remove the guard. 6. Inform the patient about the procedure. 7. Ask the patient to sit or lie down comfortably. Make sure that legs or angles are not crossing. 8. Examine both arms and select the most suitable vein. 9. Apply the tourniquet 6 cm above the bend of the elbow. This has the e ect of partially occluding the venous return from the arm. Make sure that the tourniquet is tight enough fi fi ff ff  to distend the vein but does not interfere with arterial circulation. Once the blood is owing well you may wish to loosen the tourniquet to avoid haemo-concentration. 10. Encourage the patient to make a st, if possible. This will help you to identify the veins and aid in the rapid removal of blood. 11. Locate a suitable vein by palpation. Do not attempt to insert the needle until you are sure of the puncture site. 12. Clean the skin with an alcohol based cleaner. If you touch the area after cleaning the puncture site, the area will be contaminated and must be cleaned again. 13. Remove the needle guard and discard it. Check that the point of the needle is positioned with the bevelled edge uppermost. 14. Fix the vein in position by pulling on the tissues with the thumb, just below the intended puncture site. 15. Insert the needle at an angle of 30o into the selected vein using a sliding rather than stabbing motion. Take care not to pass the needle through the vein. 16. Withdraw the required amount of blood without moving the needle. 17. Ask the patient to unclench his/her st. 18. Release the tourniquet. NEVER REMOVE THE NEEDLE BEFORE RELEASING THE TOURNIQUET. This will lead to bleeding into the tissues and the development of a haematoma (a swelling that contain blood). 19. Place a gauze piece or cotton wool swab over the puncture site, but do not press down. Carefully withdraw the needle and press the swab on to the wound. Ask the patient to keep the arm extended and to take over applying pressure to the wound. 20. Make sure the pressure is kept on the puncture site for 3-5 minutes to prevent the development of a haematoma. Patients taking anticoagulant medication such as warfarin will bleed for longer. Therefore, the pressure must also be maintained for a long period of time. 21. Remove the needle from the syringe. Never dispense the blood through the needle. Discard the needle into a sharp disposal box. NEVER RESHEATH THE NEEDLE BEFORE DISPOSAL. fl fi fi 22. Dispense the blood into the correct containers. 23. Gently, but thoroughly mix the anti-coagulated tubes. 24. Check the tube label with the patient details (number, name, date of birth and date of sampling). 25. Check that the ow of blood has stopped. If not apply further rm pressure. 26. Ask the patient if he/ she is allergic to plasters. Dress the wound with either a plaster or clean dry swab held in position with surgical adhesive tape. Recommended order for blood collection.. 1. Blood culture bottles, (this will ensure sterility). 2. Tubes with clotted specimens (no additives). This makes sure that there is no chance of contamination by anticoagulants from another tubes. 3. Coagulation tubes. 4. Tubes with additives. Safety.. 1. Always wear a clean white coat buttoned-up to protect your own clothes. If you spill blood in your coat change it. 2. Work with clean hands and ngernails. 3. The wearing of gloves is optimal, but always be sure that any cuts or abrasions on your hands are covered with a clean waterproof dressing. However, if you are working with high-risk patients (Hepatitis B, C, HIV) or performing blood cultures, gloves must always be worn. 4. If any spill blood or liquid on your hands, wash them as soon as possible in soap and water or with disinfectant cleanser. 5. Any spill blood in a working surface, clean it up immediately. fl fi fi 6. Any blood spill on patients, apologise and clean them up immediately. 7. Make sure that all sharps, syringes, and swabs are disposed in the appropriate containers. 8. Never use any blood-letting device more than once, even on the same patient. 9. Consult a senior sta before dealing with a high-risk patient. 10. Do not remove needle guards until the last moment, do not put unguarded needle on to work surfaces and never presheath a needle. 11. If you prick yourself with a needle, wash the site immediately with soap and water. Encourage the area to bleed freely. Clean the wound with spirit or antiseptic and cover with a waterproof dressing. Note all the details of the patient and obtain a blood sample from the patient to test for a infectious diseases. Inform the Head of the department who should complete an accident record form or note it in an accident book. 12. Avoid aerosol production from blood samples and never mouth pipette blood. Capillary bl