2. Infectious diseases  infectious diseases are distinctive enough to be identified clinically.  Most pathogens, however, can cause a wide spectrum of clinical syndromes in humans. Conversely, a single clinical syndrome may result from infection with any one of many pathogens. Influenza virus infection, for example, causes a wide variety of respiratory syndromes that cannot be distinguished clinically from those caused by streptococci, mycoplasmas, or more than 100 other viruses.

3. 1. It is necessary to use microbiological laboratory methods to identify a specific etiologic agent. 2. Diagnostic medical microbiology is the discipline that identifies etiologic agents of disease. The job of the clinical microbiology laboratory is to test specimens from patients for microorganisms that are, or may be, a cause of the illness and to provide information (when appropriate) about the in vitro activity of antimicrobial drugs against the microorganisms identified

5.  The staff of a clinical microbiology laboratory should be qualified to advise the physician as well as process specimens.  The physician should supply salient information about the patient, such as age and sex, tentative diagnosis or details of the clinical syndrome, date of onset, significant exposures, prior antibiotic therapy, immunologic status, and underlying conditions.  The clinical microbiologist participates in decisions regarding the microbiologic diagnostic studies to be performed, the type and timing of specimens to be collected, and the conditions for their transportation and storage. Above all, the clinical microbiology laboratory, whenever appropriate, should provide an interpretation of laboratory results.

6. Manifestation of Infection ( An indication of the existence, presence of something, especially an illness.)  The manifestations of an infection depend on many factors, including  the site of acquisition or entry of the microorganism;  organ or system tropisms of the microorganism;  microbial virulence;  the age, sex, and immunologic status of the patient; underlying diseases or conditions;  and the presence of implanted prosthetic devices or materials. The signs and symptoms of infection may be localized, or they may be systemic, with fever, chills, and hypotension. In some instances the manifestations of an infection are sufficiently characteristic to suggest the diagnosis; however, they are often nonspecific.

7. Microbial Causes of Infection  Infections may be caused by bacteria (including mycobacteria, chlamydiae, mycoplasmas, and rickettsiae), viruses, fungi, or parasites.  Infection may be endogenous or exogenous.  In endogenous infections, the microorganism (usually a bacterium) is a component of the patient;s indigenous flora. Endogenous infections can occur when the microorganism is aspirated from the upper to the lower respiratory tract or when it penetrates the skin or mucosal barrier as a result of trauma or surgery.  In contrast, in exogenous infections, the microorganism is acquired from the environment (e.g., from soil or water) or from another person or an animal. 1.Although it is important to establish the cause of an infection, the differential diagnosis is based on a careful history, physical examination, and appropriate radiographic and laboratory studies, including the selection of appropriate specimens for microbiologic examination. 2. Results of the history, physical examination, and radiographic and laboratory studies allow the physician to request tests for the microorganisms most likely to be the cause of the infection.

9. Specimen Selection, Collection and Processing  Specimens selected for microbiological examination should reflect the disease process and be collected in sufficient quantity to allow complete microbiologic examination.  The number of microorganisms per milliliter of a body fluid or per gram of tissue is highly variable,  Swabs, although popular for specimen collection, frequently yield too small a specimen for accurate microbiologic examination and should be used only to collect material from the skin and mucous membranes.

10. contamination  Because skin and mucous membranes have a large and diverse indigenous flora, every effort must be made to minimize specimen contamination during collection.  Contamination may be avoided by various means. The skin can be disinfected before aspirating or incising a lesion. Alternatively, the contaminated area may be bypassed altogether. Examples of such approaches are  transtracheal puncture with aspiration of lower respiratory secretions or suprapubic bladder puncture with aspiration of urine. It is often impossible to collect an uncontaminated specimen, and decontamination procedures, cultures on selective media, or quantitative cultures must be used. Specimen Selection, Collection and Processing cont……..

11.  If possible, specimens should be collected before the administration of antibiotics. Above all, close communication between the clinician and the microbiologist is essential to ensure that appropriate specimens are selected and collected and that they are appropriately examined. Specimen Selection, Collection and Processing cont……..

12.  Sample Collection, such as handling, labeling, processing, aliquoting, storage, and transportation, may affect the results of the study  If case sample are handled differently from controls samples, differential misclassification may occur Specimen Selection, Collection and Processing cont……..

13. Information linked to Sample  Time and date of collection  Recent diet and supplement use,  current medication use  History of any disease  Recent medical illness  Storage conditions

15. Quality Assurance  Adoption of standardized operation procedures ( SOP ) for each aspect of biospecimen handling  Stored specimens should be tested on a regular basis  Aliquoting material into multiple small vials

17. Quality Assurance  Storing  Storing each person’s specimen in at least two different physical locations to avoid the likelihood of loss of a large volume of specimen as a result of accidental thawing due to freezer failure or electronic blackout.

18. Storage It is critical to maintain careful records of the identity and location of all materials, with  particular attention to storage history,  occurrence of temperature fluctuation  monitoring of stored control specimen in order to check the effects of storage duration.

19. Storage Samples stored on the top of the freezer may be exposed to more extreme temperature fluctuation then those stored at the bottom.

20. Timing  For studies of hormones, which have  hourly,  daily and  monthly timing of sample collection is critical.  It is critical to obtain information at the time of specimen collection, e.g.,  time and date of draw,  volumes and type of specimen,  medical illness,  medication use etc…….

21. Processing  The sample processing depends on the marker needed. Investigator must design studies to fit the requirement of the critical biomarkers.

22. Types of Biospecimens individuals who already diagnosed  Collection of biospecimens from individuals who already diagnosed as having illness to characterize the history of the disease. Many collections of tissues including tumor and normal tissues. It will be much better if the related epidemiological data are also collected.

23. Types of Biospecimens: Blood skilled technicians  The use of skilled technicians and precise procedures when perform phlebotomy are important because painful, prolonged or repeated attempts at venepuncture can cause patient discomfort or injury and result in less than optimum quality or quantity of sample.

24. Types of Biospecimens: Blood  Plasma  Serum  Lymphocytes  Erythrocytes  Platelets

25. Blood Sample Collection  When a large amount of blood sample needed, an evacuated tube system with interchangeable glass tubes can be used to avoid multiple venepunctures.  Evacuated tubes are commercially prepared with or without additives and with sufficient vacuum to draw a predetermined blood volume per tube.

26. Sterile Blood needles; Sterile Syringes; Plain Vacutainer; Blood Tubes; Alcohol Prep Pads; Tourniquet

27. Blood Collection: Color-code Tubes  When you use the red-top tubes, the sample can be placed for 1-2 hours so that the serum and blood clots will be separated. Blood clots can be used for DNA analysis. Red-top tubes contain no additives. These tubes are used for tests performed on serum samples and DNA.

28. Blood Collection: Color-code Tubes  Lavender-top tubes contain EDTA, commonly used clinically for complete blood cell counts.  This is the way to obtain  for DNA extraction,  plasma Biochemistry  red blood cells for other assays.

29. EDTA EDTA is a anticoagulant. It works by calcium chelation and is used clinically in heamatology studies. It is well suited  to DNA-based assays,  but has problems for cytogenetic analysis.

30. Blood Collection: Color-code Tubes  Green-top tubes contain heparin  Heparin is an anticoagulant. There are some reports of problems with heparin in PCR assays, studies generally find that there are no major difference in the use of EDTA or heparin

31. Blood Collection: Color-code Tubes cont….  Blue-top tubes contain sodium citrate and citric acid  Citrate also works by calcium chelation and is used in  coagulation studies and  blood banking.

32.  Black-top tubes contain sodium oxalate Blood Collection: Color-code Tubes cont….

33.  Yellow-top tubes contain acid-citrate- dextrose (ACD) solution. Blood Collection: Color-code Tubes cont….

34.  Grey-top tubes contain a glycolytic inhibitor. Blood Collection: Color-code Tubes cont….

35. Dried Blood Spot Dried blood spot specimens:  Small quantities of blood adequate for the characterization of DNA.  Not require venepuncture or low temperature condition during collection, processing and storage  Can be from whole blood or antocoagulated with EDTA

36. Dried Blood Spot  Blood specimen is spotted onto clean slides or paper or cotton cloth.  Transported and stored at room temperature  Serves as a good source of high-molecular- weight DNA  A quantity of 50 ul of dried blood can provide 0.5 ug DNA, sufficient for multiple PCR-based assays

37. Processing Serum fatty acids should be measured  within 2 weeks at 4 degree C,  within a few months at –20 degree C, and  within a year at –80 degree C

38. Urine Collection Urine is an ultrafiltrate of the plasma. It can be used  to evaluate and monitor body metabolic disease process,  exposure to xenobiotic agents,

39. Urine Collection  Urine collection is not readily obtainable. However, it is more inconvenient than blood collection.  The type of urine selected and the collection procedure used to depend on the tests to be performed.

40. Urine Collection  First morning  Random  Fractional  timed

41. Urine Collection  Clean and dry plastic or glass containers (50- 3000 ml capacity)  A preservative may be needed depending on the proposed assay  Total volume must be recorded  The specimen well mixed to ensure homogeneity  Aliquots for specific assays

42. Urine Collection  Time frame for measurement of proposed assay.  Microbial contamination  Cost of storing large volumes of material  effect of long-term storage on quantitative or qualitative detection

43. Tissue Collections  Confirming clinical diagnosis by histological analysis  Examining tumor characteristics at chromosome and molecular level

44. Tissue Collections  It requires to collect more materials than it is necessary for pathological evaluation  When possible, the tissue sample should contain both tumor and normal tissues to permit to study different characteristics of the two tissues.

45. Tissue Collections  Formalin-fixed paraffin-embedded tissue specimens  The tissue is embedded in frozen section support media and stored at –70 degree C  Snap frozen tissues.

46. Hair  Easy available biological tissue whose typical morphology may reflect disease conditions within the body  Provides permanent record of trace elements associated with normal and abnormal metabolism  A source for occupational and environmental exposure to toxic metals

47. Hair Hair analysis provides long-term information from months to years, concerning both the severity and pattern of drug use.  Hair roots can be optimal source of DNA for PCR analysis and  permit easy collection,  transportation and  low overall costs.

48. Nail Clippings  Toenail or fingernail clippings are obtained in a very easy and comfortable way.  They do not require  processing,  storage and  shipping condition and thus suitable for large epidemiological studies

49. Nail Clippings  Trace elements  Selenium levels  Arsenic levels  Less likely to be contaminated by environmental factors

50. Saliva  It is an efficient, painless and relatively inexpensive source of biological materials for certain assays  It provides a useful tool for measuring endogenous and xenobiotic compounds

51. Saliva Measurements  Corticosteroids  Antibodies to HIV-1

57. Feaces  Certain cells of interest  Infectious markers  Oncogenes

58. Shipping  Sample shipping requirements depends on the  time,  distance,  climate,  season,  method of transport,  type of specimen and markers to be assayed.  Polyurethane boxes containing dye ice are used to ship and transport samples that require low temperature. For samples require very low temperature, liquid nitrogen container can be used. The quantity of dry ice should be carefully calculated, based on estimated time of trip.

59. Safety  Protect specimen from contamination  Workers safety, HIV, HBV

60. Procedures  Standardized approaches in order to ensure quality control  Biological specimen collection manual  Manuals for field trip preparation, packing and shipping samples  Protocols for lab assays