Thanks for your download. If you have completed your report, kindly put as reference Jimoh, A.A. 2010. A Technical Report on Student Industrial Work Experience Scheme in a Medical Diagnostic Laboratory. www.academia.edu/3651724/Industrial_Training_Report Correponding Author JIMOH Abdullahi Adekilekun (B.Sc. Ife, M.Sc. Ibadan) jimohabdullahi1@gmail.com 1 A TECHNICAL REPORT ON STUDENT INDUSTRIAL WORK EXPERIENCE SCHEME (SIWES) UNDERTAKEN AT STANDARD MEDICAL DIAGNOSTIC LABORATORY, EDE, OSUN STATE. SUBMITTED TO THE SIWES COORDINATOR DEPARTMENT OF MICROBIOLOGY FACULTY OF SCIENCE OBAFEMI AWOLOWO UNIVERSITY ILE-IFE BY JIMOH ABDULLAHI ADEKILEKUN MCB/2006/119 COURSE CODE: MCB 399 IN PARTIAL FULFILLMENT OF THE AWARD OF A BACHELOR OF SCIENCE DEGREE (B.SC) IN MICROBIOLOGY OBAFEMI AWOLOWO UNIVERSITY ILE-IFE. MAY, 2010 2 DEPARTMENT OF MICROBIOLOGY, OBAFEMI AWOLOWO UNIVERSITY, ILE-IFE,OSUN-STATE. 16TH MAY, 2010. THE COORDINATOR, STUDENT INDUSTRIAL WORK EXPERIENCE SCHEME, DEPARTMENT OF MICROBIOLOGY, OBAFEMI AWOLOWO UNIVERSITY, ILE-IFE, OSUN- STATE. DEAR SIR, LETTER OF TRANSMITTAL In partial fulfillment of the requirement for the award of a Bachelor of Science degree {B.SC} in microbiology. I, Jimoh Abdullahi Adekilekun hereby submit a copy of the report of the industrial training undergone at STANDARD MEDICAL DIAGONOSTIC LABORATORY,EDE ,OSUN STATE. Yours Faithfully, Jimoh Abdullahi Adekilekun +2348065381185 jaoladasdblue@yahoo.com, jimohabdullahi1@gmail.com DEDICATED TO… 3 The ALMIGHTY ALLAH for his grace upon my life and also my mom, brother, sister, my friends and big daddy for being mine. ACKNOWLEDGEMENT 4 I would want to genuinely appreciate my mom for her persistence on my behalf, patience, love and financial support. I also would want appreciate my brother, sister and my friends for being just the best. Sincere thanks to big daddy for his love and support. A big thank you to the organizers of this SIWES program, it was indeed an educating program. I would also like to thank the scientists at Standard Medical Diagnostics Laboratory for their patience in answering our questions and also for giving necessary explanations when due and my classmates and friends with whom I underwent this SIWES program. Thanks to Almighty Allah for making all this possible, I am very grateful TABLE OF CONTENTS 5 Title Page Letter of Transmittal………………………………………………………...i Dedication…………………………………………………………………..ii Acknowledgement………………………………………………………….iii Table of Content…………………………………………………………….iv Chapter 1 1.1 Brief history of SIWES and Objectives of SIWES……………………1-2 1.2 Structural organization of STANDARD MEDICAL DIAGONOSTIC LABORATORY, EDE, OSUN STATE……………………………………………………….3 Chapter 2 2.1 General Laboratory Equipments………………………………………3-4 2.2 Care and Safety in the Laboratory…………………………………….5-6 Chapter 3 3.1 Microbiology laboratory…………………………………………….7-21 Chapter 4 4.1 Hematology…………………………………………………………...21Chapter 5 5.1 Chemical Pathology…………………………………………………. Chapter 6 6.1 Experience gained and problems encountered during the period of the SIWES program……………………………………………………………………37 6.2 Recommendation……………………………………………………... 6.3 Conclusion……………………………………………………………. 6.4 Appendix…………………………………………………………….. 6.5 References…………………………………………………………… 6 CHAPTER ONE 1.1 STRUCTURAL ORGANISATION OF STANDARD MEDICAL DIAGNOSTIC LABORATORY, EDE, OSUN STATE. Chief Medical Director Administrative Services Reception Chemical pathology Biomedical Services Laboratories Microbiology 7 Health Records Hematology 1.2 BRIEF HISTORY OF S.I.W.E.S. SIWES was established in 1973 by the Industrial Training Fund (ITF) as one of her programmes. It was designed to give Nigerian students studying occupationallyrelated courses in higher institutions the experience that would supplement their theoretical learning in order to solve the problem of lack of adequate practical skills preparatory for employment in industries by Nigerian graduates of tertiary institutions. The Scheme exposes students to industry based skills necessary for a smooth transition from the classroom to the world of work. It affords students of tertiary institutions the opportunity of being familiarized and exposed to the needed experience in handling machinery and equipment which are usually not available in the educational institutions. Participation in SIWES has become a necessary pre-condition for the award of Diploma and Degree certificates in specific disciplines in most institutions of higher learning in the country, in accordance with the education policy of government. Usually there are three modules: The first module is for two months and this is taken by all 200- level Engineering and Food Technology students in University. This module of industrial Training is designed to expose the students to engineering and technology operations at the shop floor level. The second module is for three months. This is for the 300-level students of Engineering, Food Technology, Geography, Biochemistry, Nursing, Pharmacy, Geology, Physics, and Library Science. The third module is however for six months and it is taken by 400-level students of Engineering, Food Technology, Botany, Microbiology, Industrial Chemistry, Computer Science, Zoology, Agriculture and Physiotherapy. SIWES is operated by the ITF, the coordinating agencies (NUC, NCCE, NBTE), employers of labor and the institutions concerned (universities and polytechnics).Funded by the Federal Government of Nigeria. Beneficiaries-Undergraduates students of the following: Agriculture, Engineering, Technology, Environmental, Science, Education, Medical Science and Pure and Applied Sciences. 8 Duration - Four months for polytechnics and Colleges of Education , and six months for the Universities. A SURVEY OF THE INSTITUTIONS PARTICIPATING IN SIWES Universities 59 Polytechnics 85 Colleges of Education 62 Total 206 This survey was carried out year 2008. 1.3 OBJECTIVES OF SIWES SIWES is a program organized for students of higher institutions to acquire practical knowledge of their various discipline in a real standard establishment different from the kind of experience or knowledge gained within the four walls of the classroom or school laboratory. 9 CHAPTER TWO 2.1 GENERAL LABORATORY EQUIPMENTS THE LIGHT MICROSCOPE The microscope employs a hollow, extremely intense cone of light concentrated on the specimen. The field of view of the objective lens lies in the hollow, dark portion of the cone and picks up only scattered light from the object. The clear portions of the specimen appear as a dark background, and the minute objects under study glow brightly against the dark field. This form of illumination is useful for transparent, unstained biological material and for minute objects that cannot be seen in normal illumination under the microscope. AUTOCLAVE The autoclave is effective equipment used for steam sterilization at pressures above the atmospheric pressure. Thus, it is possible to steam at higher temperature then the boiling point, which a lot of microorganisms cannot withstand. Autoclaving is the most effective method for sterilizing culture media. When sterilizing culture media with autoclave, we do so at 1.05Kg per square centimeter for 15 minutes to eliminate contaminations. REFRIGERATOR This is used to preserve samples, reagents etc, which are used for daily analysis and cannot be exhausted at once. The refrigerator helps provide optimum environment for materials to be preserved. INCUBATOR The incubator is mainly used to incubate culture media as microbes have different optimum temperatures for growth and reproduction. The temperature of an incubator can be set to the preferred temperatures. WATER BATH This is required to incubate bottle of culture media, liquids in flasks or other large Containers, and when incubating samples in the test tube racks. WEIGHING BALANCE This is a delicate instrument used for weighing essential, reagent, stains and culture 10 media that requires adequate weighing. STRAIGHT WIRE It is made up of a thick metallic lower part and a straight thin upper metallic part usually made up of platinum. This straight wire is used for stab culture and for picking discrete colonies. Usually sterilized before, during and after usage. This is achieved by flaming on Bunsen burner red hot and allowed to cool a bit before use. WIRE LOOP Made up of a thick metallic lower part and a straight thin upper metallic part curved into a small circle usually made up of platinum. Wire loop is used generally for inoculating samples and picking colonies sterilized by flaming red hot before, during and after use. It is always better to use the sides of the loop rather than the apex during inoculation. MYCOLOGY NEEDLE It is made up of a thick metallic lower part and a short straight thin upper metallic part usually made up of platinum. Used for needle mount preparations of fungi and fungi inoculation. It is usually sterilized by flaming. GLASS SLIDES Used for preparation of slides for microscopy. Sterilization is by flooding with alcohol and flaming off excess alcohol. COVER SLIPS This is use for covering wet smears of preparations. It is sterilized by flooding with alcohol and flaming off excess alcohol. PETRI DISH Used for the preparation of culture media. It is usually bought sterilized. The disposable type cannot used a second time while the glass ware type can be reused be usually sterilized by autoclaving. FORCEPS A pair of forceps is a metallic object used for handing hot object or contaminated materials. It is sterilized by flaming red hot. Others include: 11 Other Laboratory equipments include includes sterilized slide, Giemsa Stain, needle, syringe, ethanol, sterilized bottle, agar (MacConkey or Chocolate), Gram positive, Gram negative sensitivity kit , cotton wool, EDTA, microscope, oil immersion, ethanol, sterilized slides, swab sticks, cotton wool, spirit, giemsa stain, lancets, surgical blades, oil immersion, pipette, light microscope, hot plate (dryer), centrifuge, hand gloves, microhaematocrit centrifuge, capillary tubes for measuring PCV, sealant, microhaematocrit reader, anaerobic jar, test tubes, bottles, water bath, weighing balance, microscope, pipette, beakers, bio safety cabinet, cotton 12 2.2 SAFE WORKING PRACTICES IN A MEDICAL LABORATORY The following are some of the important points which apply when working with infectious materials: 1. Never mouth-pipette. Use safe measuring and dispensing devices. 2. Do not eat, drink, smoke, store food, or apply cosmetics in the working area of the laboratory. 3. Use an aseptic technique when handling specimens and cultures. 4. Always wash your hands after handling an infectious material in the laboratory, when leaving the laboratory and before attending to patients. Cover any open wound with a water proof dressing. 5. Wear appropriate protective clothing when working in the laboratory. Ensure it is decontaminated and laundered correctly. 6. Wear protective gloves and when indicated a face mask, for all procedures involving direct contact with infectious materials. When wearing gloves, the hands should be washed with the gloves on, particularly before doing ant clerical work. 7. Centrifuge safely to avoid creating aerosols. Know what to do should a breakage occur when centrifuging. 8. Avoid practices which could result in needle stick injury. 9. Do not use chipped or cracked glassware and always deal with a breakage immediately and safely. 10. Avoid spillages by using racks to hold containers, work neatly and keep the bench surface free of any unnecessary materials. 11. Decontaminate working surfaces at the end of each day’s work and following any spillage of any infectious fluid. 12. Report to the laboratory officer in charge, any spillage or other accident involving exposure to infectious material. 13. Know how to decontaminate specimens and other infectious materials. 14. Use and control an autoclave correctly. 13 15. Dispose laboratory waste safely. CHAPTER THREE ACTUAL ACTIVITIES CARRIED OUT IN THE UNITS In Standard Medical Diagnostics Laboratory, where I underwent my SIWES program, we have the following sections: Reception, Chemical Pathology, Hematology, Microbiology laboratory. 3.1 AT THE RECEPTION The receptionist on seat, collects samples from patients waiting to be transferred to the laboratory, put bills on the patients cards depending on the kind of tests to be done, register the patients cards and then also register results before they are given out to patient, they also give out universal, anticoagulant bottles to patient and give them necessary instructions on how to collect into the bottles that is being given to them. Some of the laboratory materials are stored in the reception. Listed below are a few steps to follow when dispatching microbiological specimens: 1. Keep a register of all specimens dispatched. Record the name, number, and ward or health centre of the patient, type of specimen, investigation required, date of dispatch, and the method of sending the specimen. When the report is received back from the microbiology laboratory, record the date of the receipt in the register. 2. Check the specimen container is free from cracks, and the cap is leak-proof. 3. Use sufficient packaging material to protect a specimen especially when the container is a glass tube. When the specimen is fluid use sufficient absorbent material to absorb it should a leakage or breakage occur. 4. Mark all specimens that may contain highly infectious organisms. 3.2 MICROBIOLOGY LABORATORY In this laboratory the following tests are carried out Malaria parasite test 14 3.21 • Urine Analysis, MCS • Malaria Parasite Test • Stool microscopy • Semen Analysis • Blood Microfilaria URINE ANALYSIS, MCS URINE BENCH Pathogens that could be found Bacteria • Gram positive: Staphylococcus, Haemolytic Streptococci • Gram negative: Escherichia coli, Proteus species, Pseudomonas, Aeruginosa, Klebsiella strains, Salmonella typhi, Neisseria gonorrheae The following activities are carried out on the urine bench: Urine macroscopy i.e. Appearance which includes the color, turbidity etc. The microscopy to check out for possible parasite. Then culture of urine samples. URINE MACROSCOPY AND MICROSCOPY Some other urine parasites include Wucheraria brancoftii, Onchocerca etc. Collection of urine Urine is collected in clean universal bottles. The mid part of the first early morning sample is preferred. MACROSCOPIC EXAMINATION Appearance: the normal urine color should be either amber or yellow. Other colors could be red brown black or white. Turbidity: it could be slightly turbid, turbid or clear. MICROSCOPIC EXAMINATION Note: You culture before you spin the urine samples in the centrifuge to avoid contamination the samples. The urine samples are poured inside test tubes and labeled with the laboratory number of the patient. It is then arranged inside the centrifuge and allowed to spin for 10minutes, so as to separate the urine into layers. 15 The supernatant part of the spinned urine is then disposed off into a container containing a disinfectant and then the sediment is placed on the glass slide. The sediment of urine sample on the slide is covered with a cover slip and then examined under the microscope. The following could be seen under the microscope: bacteria cells, epithelial cells, cellular casts, red blood cells and white blood cells. HOW TO CULTURE URINE Culture on Cysteine Lactose Electrolyte Deficiency Agar (C.L.E.D) and MacConkey agar (both are differential agar) which differentiate between lactose and non-lactose fermenting organisms. C.L.E.D does not allow swarming of Proteus. A sterile wire loop is used to culture urine. PROCEDUREDip a wire loop inside the universal tube containing the urine (open the cork of the tube with the side of your palm and keep holding the cover while you dip the wire loop into the urine) and then inoculate your plate. The inoculation is done by introducing urine into the plate and making a smear, from the inoculums a primary and secondary streaking is made. To make the primary streaking, spread from the inoculums at angle 90 and the secondary streaking is done by spreading from the primary streaking. Then incubate overnight -that is putting inoculated plate in the incubator at 350C for 18-24 hours. The plate can then be read the next day. On CLED, the lactose and non-lactose fermenting organisms are checked and then confirmed on MacConkey agar. For lactose fermenting organisms, the colonial appearance is recorded and then the gram staining is done. If it is gram negative, the organism present could either be Klebsiella or Escherichia coli. Biochemical test can then be done by inoculating citrate, urea and peptone water. The peptone water is used for sensitivity test on nutrient agar (DST); the plate is then incubated at 370C and also the urea and citrate for 12-18 hours (overnight). Klebsiella is evident if citrate is positive or urea is positive. Citrate is positive when it is blue in color whereas urea is negative when it is yellow and positive when it is red. Citrate is negative and urea is negative when Escherichia coli is evident. 16 FOR NON-LACTOSE FERMENTERS, oxidase test is done. Positive oxidase test shows evidence of Pseudomonas species in urine while negative oxidase test shows evidence of Salmonella, Shigella, Proteus, Vibrio cholerae. Biochemical test is then done for these organisms. 3.22 MALARIA PARASITE TEST Some parasites that could be detected in the blood are: Plasmodia, Trypanosomes, Leishmania, filarial worms. SPECIMEN- A one meter in blood diameter of the blood film of the patient. Specific identification of parasites requires a permanent stain. For permanent staining, two types of blood films can be prepared. Thick films allow a larger volume of blood to be examined, thus making it easier to detect light infections with fewer parasites, while species identification is difficult. Thin films are necessary to see the morphological characteristics of the parasites and to identify them. PROCEDURES PRECAUTION: It is necessary for one to be very careful while collecting and preparing blood samples. A number of parasitological, bacterial and viral diseases can be transmitted through blood. Blood film should be prepared preferably within one hour of collection. The time of collection should be mentioned on the specimen as well as on the result sheet and also the laboratory number for correlation. It is preferable to prepare blood films with fresh blood without anticoagulant. If it is not possible, blood pant coagulated with EDTA (10mg/5ml) should be used. Step1 An absolutely clean, grease-free slide, well-washed slide cleaned with 70% ethanol is recommended (at Standard Medical Diagnostics Laboratory new slides are used). The slide is labeled with the patient’s laboratory number. Step 2 Swab the top of the patient’s third finger or thumb with cotton wool soaked with ethylated spirit to disinfect and clean the possible micro organisms present on the surface of the skin. Step 3 17 Prick the point cleaned with a sterilized lancet and discard immediately. Step 4 Apply pressure on the lower side of the top with your own hand so the blood would be able to come out in few trickles as a drop or two will be placed on either sides of the slide since the laboratory number labeled on the slide will be in the middle. Step 5 You prepare a thick blood film for the malaria parasite test. To make a thick blood film, place two or three small drops of fresh blood without anticoagulant on a clean slide with a sterilized end of another slide. Mix the drops in a circular motion over an area about two centimeters in diameter, (continue mixing for about thirty seconds to prevent formation of fibrin strands that may obscure the parasites after staining, if anticoagulated blood is used, it is not necessary to continue mixing for thirty seconds). Step 6 Allow the film to dry in air at room temperature on a dryer (hotplate) to fix the film. Step 7 After drying, the slide is placed directly into an aqueous stain called Giemsa stain to make the thick blood film to lyses the red blood cells and to remove hemoglobin so that the parasites can be easily detected GIEMSA STAINING TECHNIQUE Giemsa stain is a Romanaosky that requires dilution in buffered water or buffered saline before use. Giemsa stain (stock solution) Giemsa stain powder 0.6g Methanol, absolute (acetone-free) 50ml Glycerol 50ml Dissolve the Giemsa stain in methanol in a brown bottle containing a few glass beads. Add glycerol, mix and place the bottle in a water bath at 50-60 degree centigrade for two hours to dissolve the stain. Shake gently at half-hour intervals. The stain should stand at room temperature for three weeks and should be filtered before use. If kept air-tight, the stain is stable for several months. Preparing a working solution of Giemsa stain 18 For thick films the commercial stock solution is diluted with the ratio 1:50 with a neutral or slightly alkaline buffer (7.0 to 7.2) e .g phosphate or tap water if the pH is satisfactory. TECHNIQUE USED The labeled slide with the blood film on either end of it is directly stained in diluted Giemsa stain for like 15 minutes. It is then brought out and rinsed properly with tap water, gently flushing the stain off the slide with water. Dip the slide briefly in the buffer or rinse under gently running tap water. • Wipe the under surface of the slide to remove excess stain. • Allow it to air-dry in a vertical position. • View under the light microscope with the oil immersion lens. • A drop of oil is placed on each dried, stained and fixed blood film and then viewed for malarial parasites. TROPHOZOITE OF MALARIAL PARASITE AS VIEWED UNDER THE LIGHT MICROSCOPE. Trophozoite is the growing form of the parasite in the peripheral blood of man after the invasion of the red blood cells by merozites. When the mature schizonts rupture the merozites penetrate the red blood cells and develop into trophozoites. Immature trophozoites are concave disc appearing as ring forms in stained preparation. It consists of; 1. A rod-shaped nucleus (chromatin dot) stained red. 2. A peripheral rim of cytoplasm that stains blue and 3. An unstained clear area or vacuole in the centre that pushes the chromatin dot to the periphery of the cytoplasm. 4. Three stages of the asexual life cycle occur in man, namely the trophozoites, schizont and the gametocytes. The parasites reside in the peripheral red blood cells. Each species is identified on two basic parameters. 1. Appearance of the infected red blood cells. 2. Appearance of the parasite. 19 Results: Malaria Parasite Chromatin of parasite: Dark red Cytoplasm of parasite: Blue Schuffner’s clots: Red Maurer’s dots: Red-mauve 3.23 BLOOD MICROFILARIA TEST • In this test, blood is gotten from the patient’s vein. • A rubber tube rope is tied on the upper part of the patient’s arm. • A vein is located between the middle fold of the arm and the upper section of the arm. When the vein is located, the spot where it is found is swabbed with a cotton wool soaked in spirit. • A sterilized needle is used to prick the vein and blood is drawn and immediately transferred into the small anticoagulant bottle gotten from the reception, it is corked tightly. • Immediately you are through drawing the blood you loosen the rubber rope on the arm to reduce pressure and the blood stops coming out. • You clean the spot on the person’s arm after all these with a clean sterilized cotton wool * Note the blood gotten can be used for either microfilaria test or malaria parasite test. TEST Blood Microfilaria can be detected in the direct wet mount of fresh blood by their characteristics, shape and motility. For identification of microfilaria worm in stained blood films, the following characteristics are looked for: (i) Presence or absence of a sheath. (ii) Presence or absence of nuclei at the tip of the tail (iii) Size of microfilaria worm (iv) Size of cephalic space in sheathed microfilaria worm. 20 3.24 STOOL MICROSCOPY Three protozoan parasites which may be found in human stool are: - Rhizopodea (amoebae) e.g. Entamoeba histolytica - Zoomastigophora (flagellates) e.g. - Ciliatea (ciliates) e.g. Balantidium coli Giarelia Intesinalis EXAMININATION OF FAECES It is viewed under light microscope at x10 and x40 First you view macroscopically for the following: Form, color, Smell, consistency, presence of blood, and mucus, nematode, tapeworm, and segments. When viewed under the microscope in normal saline • You place a drop of normal saline on a thin slide with the pipette in the normal saline bottle. • Pick a tiny bit of the stool sample and make a smear in the normal saline with it. • View under the light microscope for cellular exudates such as helminthes egg, protozoa cyst, and actual larva of nematode worms. • When viewed under the microscope in iodine it is the same process as listed in the first two steps above, just use iodine in this case and not normal saline. When viewed under the light microscope, stained protozoa cysts are more visible. Other things that could be seen under the microscope are: fat globules, undigested starch, vegetable cells, and air bubbles. Cysts can be concentrated by the formal ether technique or by a simple floatation in concentrated zinc sulphate 3.25 SEMEN ANALYSIS Semen Analysis with Microscopy This involves the analysis of semen by culturing and performing sensitivity test. Part A (i) Physical examination • Volume: 1ml, 2ml and above • Viscosity: Watery or Normal 21 • (ii) Appearance: creamy, whitish or Creamy – whitish Microscopy • Motility • High power • Normal • Abnormal N.B. The best sperm count is about 90x106 total counts but normal count is 45 x 106, but when the total count is 25 x 106 the diagnosis could be infertility. (i) After the examination in part A, sterilize inoculating loop by flaming, culture the semen sample on MacConkey and Chocolate agar. NOTE* to always culture on chocolate agar you cut the agar in the middle and throwing of this cut part into the waste to prevent organisms from swarming in the agar. (ii) Incubate for 24 hours (iii) Examine the colony if there is growth, gram stain, set up biochemical tests (iv) Inoculate peptone water for flooding of DST (sensitivity test using the right Antibiotic disc). HOW TO CARRY OUT SENSITIVITY TEST: Flood the nutrient agar with inoculated peptone water, place the antibiotic disc on the flooded plate and incubate overnight for 12-18 hours. At the end of the stipulated time any antibiotic surrounded by a region where no microorganism grew can proof useful against the microorganism discovered present. Procedures for gram stain 4.7 (i) Crystal violet solution (ii) Iodine solution (functions as a mordant) (iii) Acetone (decolorizes) (iv) Safranin (counter stain) Procedure 1. Prepare a heat fixed smear from a 18-24 hour old culture 2. Stain with crystal violet solution for 1 – 2 minutes and rinse off the solution. 3. Rinse off with iodine solution for 1 minute 22 4. Rinse off the iodine solution and wash the slide with acetone until the crystal violet dye no longer runs from the slide and this will last only 5-15 seconds. 5. Rinse under gentle – running tap, and counter stain with safranin for 30 seconds. 6. Wash with water, blot dry and examine under microscope. Observation 1 Gram-positive cell appear purple, or crystal violet iodine complex 2 Gram negative cells are red or pink Note* Cells could be either bacilli or cocci. 23 CHAPTER FOUR 4.1 HAEMATOLOGY LABORATORY In this laboratory, the following tests are carried out • Blood Group • Pregnancy Test • Widal Agglutination Reaction • Packed Cell Volume • Human Immune Deficiency Virus (HIV) Screening 4.11 BLOOD GROUP TEST Analysis A needle is inserted into the vein and blood into a tube. During the procedure, the elastic band used is reserved to restore circulation. Once the blood has been collected, the needle is removed and a band aid or gauze is applied. Procedures • Venous blood is collected into EDTA sample bottle • Antiserum A, B, and D were placed on the white tile separately in three spots • Three separate drops of blood were dropped unto each of the spots • Each spot was then mixed together with the tip of a clean glass slide or an inverted rubber pipette • The tile was rocked for three minutes to view agglutination 24 Results Blood type (or blood group) is determined, in part, by the ABO blood group antigens present on red blood cells. A blood type (also called a blood group) is a classification of blood based on the presence or absence of inherited antigenic substances on the surface of red blood cells (RBCs). These antigens may be proteins, carbohydrates, glycoproteins, or glycolipids, depending on the blood group system. Some of these antigens are also present on the surface of Red blood cell compatibility • Blood group AB individuals have both A and B antigens on the surface of their RBCs, and their blood serum does not contain any antibodies against either A or B antigen. Therefore, an individual with type AB blood can receive blood from any group (with AB being preferable), but can donate blood only to another type AB individual. • Blood group A individuals have the A antigen on the surface of their RBCs, and blood serum containing IgM antibodies against the B antigen. Therefore, a group A individual can receive blood only from individuals of groups A or O (with A being preferable), and can donate blood to individuals with type A or AB. 25 • Blood group B individuals have the B antigen on the surface of their RBCs, and blood serum containing IgM antibodies against the A antigen. Therefore, a group B individual can receive blood only from individuals of groups B or O (with B being preferable), and can donate blood to individuals with type B or AB. •Blood group O (or blood group zero in some countries) individuals do not have either A or B antigens on the surface of their RBCs, but their blood serum contains IgM anti-A antibodies and anti-B antibodies against the A and B blood group antigens. Therefore, a group O individual can receive blood only from a group O individual, but can donate blood to individuals of any ABO blood group (i.e. A, B, O or AB). If anyone needs a blood transfusion in a dire emergency, and if the time taken to process the recipient's blood would cause a detrimental delay, O Negative blood can be issued. RBC Compatibility chart In addition to donating to the same blood group; type O blood donors can give to A, B and AB; blood donors of types A and B can give to AB. Red blood cell compatibility table Recipient[1] Donor[1] O− O+ A− A+ B− O− O+ A− A+ B− B+ AB− AB+ 26 B+ AB− AB+ Table note 1. Assumes absence of atypical antibodies that would cause an incompatibility between donor and recipient blood, as is usual for blood selected by cross matching. Recipients can receive plasma of the same blood group, but otherwise the donor-recipient compatibility for blood plasma is the converse of that of RBCs: plasma extracted from type AB blood can be transfused to individuals of any blood group; individuals of blood group O can receive plasma from any blood group; and type O plasma can be used only by type O recipients. Plasma compatibility table Recipient Donor[1] O A B AB O A B AB Table note 1. Assumes absence of strong atypical antibodies in donor plasma Rh D antibodies are uncommon, so generally neither D negative nor D positive blood contain anti-D antibodies. If a potential donor is found to have anti-D antibodies or any strong atypical blood group antibody by antibody screening in the blood bank, they would not be accepted as a donor (or in some blood banks the blood would be drawn but the product would need to be appropriately labeled); therefore, donor blood plasma issued by a blood bank can be selected to be free of D antibodies and free of other atypical antibodies, and such donor plasma issued from a blood bank would be suitable for a recipient who may be D positive or D negative, as long as blood plasma and the recipient are ABO compatible. 27 4.12 PREGNANCY TEST PRECAUTION It is necessary for one to be very careful while collecting and preparing blood samples. A number of parasitological, bacterial and viral diseases can be transmitted through blood.. The time of collection should be mentioned on the specimen as well as on the result sheet and also the laboratory number for correlation. STEPS INVOLVED 1. Select a sterile, dry plastic syringe of the capacity required, e.g. 2.5ml,5ml or 10ml 2. Apply a soft tubing tourniquet or Velcro arm bound to the upper arm of the patient 3. Using the index finger feel for a suitable vein, selecting a sufficiently large straight vein that does not roll and with a direction that can be felt. 5. Cleanse the puncture site with 70% ethanol and allow drying. 6. When sufficient blood has been collected, release the tourniquet and instruct the patient to open his or her fist. 7. Centrifuge for 3-5 minutes (RCF 12000-15000xg), using the shorter time when the RCF is 15,000xg 8. Immediately after centrifuging, first check that there has been no leakage of blood from the bottle or breakage. 9. Pregnancy Test is therefore carried out by inserting a pregnancy strip in the bottle containing blood. Most chemical tests for pregnancy look for the presence of the beta subunit of hCG or human chorionic gonadotropin in the blood or urine . hCG can be detected in urine or blood after implantation, which occurs six to twelve days after fertilization.[1] Quantitative blood (serum beta) tests can detect hCG levels as low as 1 mIU/mL, while urine tests have published detection thresholds of 20 mIU/mL to 100 mIU/mL, depending on the brand.[7] Qualitative blood tests generally have a threshold of 25 mIU/mL, and so are less sensitive than some available home pregnancy tests. Most home pregnancy tests are based on lateral-flow technology. RESULTS The strip shows whether the patient is pregnant or not if 28 • Positive (double line): the patient is pregnant • Negative (single line): the patient is not pregnant • Invalid: No visible band at all. The test is repeated NOTE: For detection of hCG in urine, same procedure is followed Precautions • Test kit must not be beyond expiry date • The test device must not be reused • The test kit is for in vitro diagonostic use only 4.13 WIDAL AGGLUTINATION REACTION Materials Widal kit, white tile, pipette, test tube, centrifuge, stop watch, blood sample Procedure • Venous blood is collected into sample bottle and spun at 3000rpm for 5 minutes • The serum is taken with the aid of a pipette and put on white tile in different spots of 4 per row making two rows • First row is labeled O, OA, OB, OC and the second row H, HA, HB, HC respectively. • Antiserum from the widal kit for each spot are released on top of the pipette blood. • The tile is then rocked for 3 minutes Results Expected results ratio: • Highly reactive……….1:320(positive) • Very reactive…………1:160(positive) • Weak reaction………..1:80(positive) 29 • Non Significant………1:40(negative) • Non Significant………1:20(negative) 4.14 PACKED CELL VOLUME Value of Test: The packed cell volume also called haematocrit, is used to calculate the mean cell haemaglobin concentration (MCHC) and mean cell volume (MCV). These red cell indices are used in the investigation of anaemia. The PCV is also used to screen for anaemia when it is not possible to measure haemaglobin, and to diagnose polychaemia vera and to monitor its treatment. It is suitable for screening large clinic populations’ e.g. antenatal clinics. Specimen: To measure the PCV, either well mixed well oxygenated EDTA anti coagulated blood can be used or capillary blood collected into a heparinzed capillary. Equipment: Microhaematocrit reader, centrifuge, needle, syringe, capillary tube. Test Method 1 .About three quarters fills either • a plain capillary with well mixed EDTA anticoagulated blood (tested within 4 hours of collection), or • a heparinised capillary with capillary tube 2. Seal the unfilled end, preferably using a sealant material. If unavailable, heats seal the capillary using a small flame from a sprint or a pilot flame of a bursen burner, rotating the end of a capillary in the flame. 3. Carefully locate the capillary in one of the numbered slots of the microhaematocrit rotor with the sealed end against the rim gasket (to prevent breakage). Write the number on the patient form. 4. Centrifuge for 3-5 minutes (RCF 12000-15000xg), using the shorter time when the RCF is 15,000xg 30 5. Immediately after centrifuging, read the PCV. First check that there has been no leakage of blood from the capillary or breakage. To read the PCV in a hand held, align the base of the red cell column on the 0 line and the top of the plasma column on the 100line.Read off the PCV from scale. The reading point is the top of the red cell column, just below the buffy coat layer (consisting of WBCs and platelets). Results Above the packed red cells is a white layer of platelets. Plasma is usually straw colored, but if bright yellow; it is jaundiced, when colorless; it is iron deficient, when red; haemolysis has occurred The normal PCV range for male is 39% - 53%. The normal PCV range for female is 35% -49%. Factors that affect PCV result • Quality of capillary tube • Time and speed of centrifugation • Spectrum collection: quantity of anticoagualant 4.15 HUMAN IMMUNE DEFICIENCY SCREENING HIV tests are used to detect the presence of the human immunodeficiency virus in serum, saliva, or urine. Such tests may detect HIV antibodies, antigens, or RNA. Terminology The window period is the time from infection until a test can detect any change. The average window period with HIV-1 antibody tests is 22 days for subtype B. Antigen testing cuts the window period to approximately 16 days and NAT (Nucleic Acid Testing) further reduces this period to 12 days. Performance of medical tests is often described in terms of: • Sensitivity: The percentage of the results that will be positive when HIV is present • Specificity: The percentage of the results that will be negative when HIV is not present. 31 All diagnostic tests have limitations, and sometimes their use may produce erroneous or questionable results. • False positive: The test incorrectly indicates that HIV is present in a non-infected person. • False negative: The test incorrectly indicates that HIV is absent in an infected person. Nonspecific reactions, hypergammaglobulinemia, or the presence of antibodies directed to other infectious agents that may be antigenically similar to HIV can produce false positive results. Autoimmune diseases, such as systemic lupus erythematosus, have also rarely caused false positive results. Most false negative results are due to the window period; other factors, such as post-exposure prophylaxis, can rarely produce false negatives. Rapid Antibody Tests are qualitative immunoassays intended for use as a point-of-care test to aid in the diagnosis of HIV infection. These tests should be used in conjunction with the clinical status, history, and risk factors of the person being tested. The specificity of Rapid Antibody Tests in low-risk populations has not been evaluated. These tests should be used in appropriate multi-test algorithms designed for statistical validation of rapid HIV test results. If no antibodies to HIV are detected, this does not mean the person has not been infected with HIV. It may take several months after HIV infection for the antibody response to reach detectable levels, during which time rapid testing for antibodies to HIV will not be indicative of true infection status. For most people, HIV antibodies reach a detectable level after two to six weeks. Materials Blood serum, Abort determine HIV-1 and HIV-2 test kit, and centrifuge Procedure • Venous blood is collected into EDTA sample bottle • The blood is spun at 3000rpm for 10 minutes • The strip is then immersed into blood serum with the narrow end pointing towards the blood 32 • It must be immersed past the mark line. The strip is taken out after 3 seconds and laid on a flat clean dry non absorbent surface. • Water for colored band to appear Results Readings should be taken within 10 minutes • Positive: Distinct color band appear on the control and test regions. This indicates the presence of HIV-1 and HIV-2 • Negative: Only one color band appears on the control region. No apparent band on the test region. This indicates that the patient is HIV negative • Invalid: No visible band at all. The test is repeated 33 CHAPTER FIVE 5.1 CHEMICAL PATHOLOGY The following tests are carried out in Chemical pathology laboratory • Fasting Blood Sugar • Random Blood Sugar 5.11 FASTING BLOOD SUGAR The fluctuation of blood sugar (red) and the sugar-lowering hormone insulin (blue) in humans during the course of a day with three meals. One of the effects of a sugar-rich vs a starch-rich meal is highlighted. The blood sugar concentration or blood glucose level is the amount of glucose (sugar) present in the blood of a human or animal. Normally, in mammals the body maintains the blood glucose level at a reference range between about 3.6 and 5.8 mM (mmol/L). It is tightly regulated as a part of metabolic homeostasis. Materials Blood sample, glucomter NOTE: Glucometer is an instrument used to measure the glucose (sugar) level of a patient. Procedure • Blood is collected from the thumb of the patient • The blood is made to drop at the tip end of the glucometer and then left for few minutes(about 3-5minutes) • The reading is then taken and written down Results The normal range is 70-100mg/dL. If the result from the reading is very much less than 70mg/dL, the patient is said to be hypoglycemic and needs sugar transmission, if the result is far higher than 100mg/dL the patient is said to be hyperglycemic and needs insulin transfusion. 34 5.12 RANDOM BLOOD SUGAR This test is similar to fasting blood sugar, the difference being that the test can be carried out anytime on a patient (that is, whether the patient has or has not eaten is irrelevant) and it is useful in the case of emergency. Materials Blood sample, glucometer Procedure • Blood is collected from the thumb of the patient • The blood is made to drop at the tip end of the glucometer and then left for few minutes (about 3-5minutes) • The reading is then taken and written down. Results The normal range is 100-180mg/dL. If the result from the reading is very much less than 70mg/dL, the patient is said to be hypoglycemic and needs sugar transmission, if the result is far higher than 100mg/dL the patient is said to be hyperglycemic and needs insulin transfusion 35 CHAPTER SIX 6.1 EXPERIENCE GAINED ➢ I learnt almost all the practical aspects involved in medical microbiology and also microbiology in general. ➢ I got to know about and learnt the use of the laboratory equipment. ➢ I learnt to obey all laboratory rules for my safety and that of the patients. ➢ I learnt to relate properly with other co-workers. 6.2 ▪ PROBLEMS ENCOUNTERED In most cases, safety rules are not taken into consideration and the necessary safety gadgets and equipment are not usually in place. ▪ It is suggested that some form of allowance should be given to the students by the employers as a form of encouragement and to assist in their cost of living, basically feeding, transportation and accommodation especially in areas far from the students’ neighborhood. ▪ I also would want to say that more time should be given to students for their SIWES program. 6.3 • RECOMMENDATIONS I propose that more time should be given to the students of microbiology for SIWES activities • I recommend that government should provide placements for students undergoing SIWES in the several fields of Nigerian Economy. • I recommend that more preference should be given to the power sector so as to provide adequate light to various Medical laboratories in the country. 36 6.4 CONCLUSION In conclusion this program has enabled students to gain a lot and many can now practice the applied aspects of their various disciplines and other related areas on their own. The program has really being. 6.5 APPENDIX CLED- Cysteine Lactose Electrolyte Deficiency DCA- Deoxycholate citrate agar SIWES- Student industrial work experience scheme. 6.6 • REFERENCES My industrial attachment experience at Standard Medical Diagnostic Laboratory Textbooks • District laboratory practice in tropical countries (part 2) by Monica Cheesbrough o www.google.com 37