Clinical labrotaries

1. Clinical labrotaries MALIK ALQUB MD. PHD.

2. Steps in the Investigation of a Patient • Patient History • Physical Examination • Laboratory Tests • Imaging Techniques • Diagnosis • Therapy • Evaluation

3. Medical testing Doctor requires information Patient sample collection Laboratory Testing Report generation Computer system maintenance Sample received & processed

4. Laboratory Medicine • A discipline of medicine that functions to provide diagnostic tests which are utilized by physicians to assess the health of an individual. • Must more than just a “service”. Dynamic interaction with all hospital departments (Emergency (ER), Intensive Care Unit (ICU), Cardiac Care Unit (CCU) as well as physicians outside of the hospital to maximize health care through: • Consultation regarding tests to be requested • Education • Medical students, residents • Medical Technologists • Medical Staff • Development, Evaluation and Implementation of New Diagnostic Assays • Supporting Clinical and Basic Research • Interaction with all departments to maintain and/or improve the flow and accuracy of information (i.e test results)

5. Laboratory Medicine, disciplines. • Clinical biochemistry • Histopathology • Microbiology • Cytology • Blood Transfusion • Immunology • Haematology • Virology

6. CLINICAL CHEMISTRY Measurement of amounts of specific elements transported in the biological samples • Proteins • Sugars • Cellular breakdown products • Hormones • Toxins • Etc...

7. Biochemical tests • Are divided in three main categories • Core biochemistry • Urgent tests • Specialized tests

8. Core Lab • Many tests where abnormal values are incompatible with life and therefore of critical value to the physician • Electrolytes: sodium (Na), potassium (K), Chloride (Cl) • Blood gases: pO2, pCO2, pH, HCO3, oxygen saturation • Endocrine: Thyroid hormones • Cardiac markers • Liver enzymes • Amylase • Glucose • Toxicology Ethanol, methanol Drugs of abuse generally conducted as a screen

9. Uregent tests • Done on emergency basis • Continous service 24/24hr • Electrolytes: sodium (Na), potassium (K), Chloride (Cl) • Blood gases: pO2, pCO2, pH, HCO3, oxygen saturation • Cardiac markers • Glucose

10. Specilized tests • Hormones • Specificproteins • Vitamins • Drugs • Lipids • DNA analysis • Rare tests • Only larger centres have Special Chemistry Lab because • Requires the volume of specimens to justify the test • High cost of equipment to relative few specific tests

11. Specilized test Instrumentation and Analytical Methods • Electrophoresis • Used to separate serum proteins into 5 distinct bands • Used to separate Lipoproteins into 4 distinct bands • Often used to separate isoforms of enzymes • HPLC • Used to measure vitamins and hemoglobin variants • Infrared Spectroscopy • Used to analyze components of Kidney stones • Radioimmunoassay (RIA) • Used less and less but still employed for those analytes present in minute amounts (pmol) in the blood (ie. testosterone) • GC-MS (Gas chromatography-mass spectroscopy) and/or LC-MS (liquid chromatography- mass spectroscopy. • Used for quantitative drug measurement

12. Analysis of biological samples • Pre analysis • Prescription • Biologicalsamples • Sampling/Conditions • Transport • Reception and identification • Conform to laboratory guidance • Pretreatment of biological samples • Analysis • Quantitative and qualitative measurements • Automation • Control of quality • Post analysis • Technical validation • Biological validation • Reporting • interpretation

13. Pre analysis

14. Prescription • Good communication between medical staff and lab • To avoid unnecessary tests • To precise the condition of sampling • To inform the medical staff of scheduled analysis • To Aid in interpretation of results • To prepare the lab in critical situation

15. Biological samples • Blood • Urine • Cerebrospinal Fluid • Amniotic Fluid • Duodenal Aspirate • Gastric Juice • Gall stone • Kidney Stone • Stools • Saliva • Synovial Fluid • milk • Tissue Specimen Comprise the majority of all specimens analyzed

16. Sampling/Conditions • Collection Tubes • Timing, 24hr urine collection, dynamic tests • Temperture. cryoglobulines

17. Collection Tubes(Vacutainers) Separator Gel Serum Separator Gel Serum Separator Tube (SST) Clot

18. Collection tubes • Gold (and “tiger”) top tubes contain a gel that forms a physical barrier between the serum and cells after centrifugation • No other additives are present • Gel barrier may affect some lab tests

19. Collection tubes • Used for Glucose measurement. • After blood collection, glucose concentration decreases significantly because of cellular metabolism • Gray-top tubes contain either: • Sodium fluoride and potassium oxalate, or • Sodium iodoacetate • Both preservatives stabilize glucose in plasma by inhibiting enzymes of the glycolytic pathway • NaF/oxalate inhibits enolase • Iodoacetate inhibits glucose-3-phosphate dehydrogenase

20. Collection tubes • Green-top tubes contain either the Na, K, or lithium (Li) salt of heparin. Most widely used anticoagulant for chemistry tests. • Should not be used for Na, K or Li measurement • Can effect the size and integrity of cellular blood components and not recommended for hematology studies • Heparin accelerates the action of antithrombin III, which inhibits thrombin, so blood does not clot (plasma) • The advantage of plasma is that no time is wasted waiting for the specimen to clot

21. Collection tubes • Lavender-top tubes contain the K salt of ethylenediaminetetraacetic acid (EDTA), which chelates calcium (essential for clot formation) and inhibits coagulation • Used for hematology, and some chemistries • Cannot be used for K or Ca tests

22. Collection tubes • Blue-top tubes contain sodium citrate, which chelates calcium and inhibits coagulation • Used for coagulation studies because it is easily reversible.

23. Collection tubes • Brown and Royal Blue top tubes are specially cleaned for trace metal studies • Brown-top tubes are used for lead (Pb) analysis • Royal blue-top tubes are used for other trace element studies (acid washed)

24. Transport • The delay between sample collection and analysis, the conditions of transport must be respected like • Time (blood gases) • Temperature (fasting glucose)

25. Reception and identification • Was the blood collected from the correct patient? • Was the blood correctly labeled? • Patient name, ID, date, time of collection, phlebotomist

26. Conform to laboratory guidance • Look if the condition of Pre analysis are respected • Patient name, ID, date, time of collection • Volume

27. Pretreatment of biological samples • Centrifugation • Dilution • Storage

28. Analysis • Quantitative and qualitative measurements • Quantitative, Na, K, total Protein, etc… • Qualitative, Protein electrophoresis of urine

29. Automation Why automation; • Increase the number of tests by one person in a given period of time • Minimize the variations in results from one person to another • Minimize errors found in manual analyses – equipment variations – pipettes • Use less sample and reagent for each test

30. Types Of Analyzers • Continuous Flow • Tubing flow of reagents and patients samples • Centrifugal Analyzers • Centrifuge force to mix sample and reagents • Discrete • Separate testing cuvets for each test and sample • Random and/or irregular access

31. Continuous Flow • This first “AutoAnalyzer” (AA) was a continuous-flow, single-channel, sequential batch analyzer capable of providing a single test result on approximately 40 samples per hour. • Analyzers with multiple channels (for different tests), working synchronously to produce 6 or 12 test results simultaneously at the rate of 360 or 720 tests per hour.

32. Continuous Flow • In continuous flow analyzers, • samples were aspirated into tubing to introduce samples into a sample holder, • bring in reagent, • create a chemical reaction, • and then pump the chromagen solution into a flow-through cuvette for spectrophotometric analysis.

33. Continuous Flow

34. Continuous Flow • Continuous flow is also used in some spectrophotometric instruments in which the chemical reaction occurs in one reaction channel and then is rinsed out and reused for the next sample, which may be an entirely different chemical reaction.

35. Centrifugal Analyzers • Discrete aliquots of specimens and reagents are piptted into discrete chambers in a rotor • The specimens are subsequently analyzed in parallel by spinning the rotor and using the resultant centrifugal force to simultaneously transfer and mix aliquots of specimens and reagents into radially located cuvets. • The rotary motion is then used to move the cuvets through the optical path of an optical system

36. Discrete analyzers • Discrete analysis is the separation of each sample and accompanying reagents in a separate container. • Discrete analyzers have the capability of running multiple tests on one sample at a time or multiple samples one test at a time. • They are the most popular and versatile analyzers and have almost completely replaced continuous-flow and centrifugal analyzers.

37. Discrete Analyzers • Sample reactions are kept discrete through the use of separate reaction cuvettes, cells, slides, or wells that are disposed of following chemical analysis. • This keeps sample and reaction carryover to a minimum but increases the cost per test due to disposable products.

38. What is Quality Control? • Quality Control in the clinical laboratory is a system designed to increase the probability that each result reported by the laboratory is valid and can be used with confidence by the physician making a diagnostic or therapeutic decision.

39. CONTROL OF QUALITY • What is “normal” or “OK” • What makes something “weird” , “abnormal” or “deviant” and something to worry about? • When does a laboratory test result become “weird” or “abnormal” ? • At some point we have to draw a “line in the sand” … on this side of the line you’re normal … on the other side of the line you’re abnormal. Where and how do we “draw the line” ? • In the laboratory, we have to be concerned with these issues because we have to give meaning to our “observations” or test results – Are they normal or abnormal ? • Statistics is used to draw “lines in the sand” for patient specimens, control specimens and calibrators • If the results are “normal” we ‘re comfortable about them and don’t worry • But if they’re abnormal, we’re uncomfortable and we fear that there is something wrong with the patient or the test procedure .

40. To answer these questions we need a little statistics • There are two main ideas we need to concern ourselves with • Central tendencyhow numerical values can be expressed as a central value ) • Dispersal about the central value ( how spread out are the numbers ? ) • Using these two main ideas we can begin to understand how basic statistics are used in clinical chemistry to define normal values and when our instruments are ( or are not ) generating expected numerical results

41. Common Descriptive techniques • Mean Average value (expression of central tendency ) • Median Middle observation (expression of central tendency ) • ModeMost frequent observation (expression of central tendency ) • Observations can be grouped together into smaller groups. The frequency of each smaller group can be expressed graphically as a bar-chart or histogram • Standard Deviation ( SD ) : mathematical expression of the dispersion of the observations … how spread out the observations are from each other • Coefficient of Variation ( CV ) : A way of expressing the Standard Deviation in terms of the average value of the observations that were used in its calculation

42. Accuracy versus Precision • Accuracy : Observations that are close to the “true” or “correct” value • Precision : Observations that are reproducible or repeatable • The laboratory must produce results that are both accurate and reproducible

43. target ! Right on target ! Close enough ? In the laboratory we need to report tests with accuracy and precision, but how accurate do we need to be? It’s not possible to hit the bulls-eye every time. So how close is “close enough?”

44. 3 possible testing outcomes - Hitting the target x x x Lacks precision and accuracy x x Has good precision but poor accuracy x x x x x x x Good precision and good accuracy A lab must report the correct results all the time !!! x x x

45. Formulas for Statistical Terms Mean = Median : List all the observations in order of magnitude and pick the observation that’s in the middle Odd # of observations = Middle observation Even # of observations = Average of the 2 middle values Mode : The observation that occurs most frequently … There may be more than one, or none at all All three of these are expressions of a “central” observation, but they don’t say anything about the observations as a whole … Are they close together? Although we can look at all the individual observations, the mean, median and modes by themselves do not give us any indication about the dispersion of the observations

46. Formulas for Statistical Terms Standard Deviation : n = the number of observations (how many numerical values ) Σ = the sum of … in this case, the sum of all the = the mean value X = the value of each individual observation … this means that the value of will have to be calculated for every value of x The Standard Deviation is an expression of dispersion … the greater the SD, the more spread out the observations are

47. Discussion of the Standard Deviation (SD) • As the name suggests, it is a measurement of “deviation” • More specifically, it can measure “deviation” in terms of an individual observation or a group of observations • In both cases, the “deviation” is measured in terms of how far away the observation(s) are from the mean value • The SD is a measurement of dispersion • We use the SD to draw our “lines in the sand” • For most considerations, laboratories will define “normal” or “acceptable” results as being within ± 2.0 SD from the mean value • If results are greater than 2.0 SD from the mean, then we say they are “abnormal” or “out of control” • This means that they are unlike the other observations and they may be the results of faulty laboratory testing

48. Example of the Standard Deviation Establishment of Normal Values: A minimum of 20 observations should be sampled in order to obtain valid results ( but I’ll use just 6 to save time ) Lets determine the normal range for fasting plasma glucose using 6 people: John’s glucose = 98 mg/dl Average = 109 mg/dl Paul’s glucose = 100 mg/dl SD = 20.0 mg/dl George’s glucose = 105 mg/dl 2 SD = 40.0 mg/dl Ringo’s glucose = 150 mg/dl Mick’s glucose = 102 mg/dl Eric’s glucose = 101 mg/dl That means that the normal range for this group is from 109 ± 40, or 69 - 149 which is ± 2.0 SD from the mean Ringo is considered abnormal if we use this commonly accepted criteria to define normal and abnormal By the way, the CV for this group of observations is about 18% - a fairly big dispersal about the mean

49. Example of Control Specimens Every test in the laboratory requires that “control specimens” be performed on a regular basis to ensure the testing process is producing accurate results. Running controls is a “check” on the lab tech’s technique, reagents and instrumentation. Suppose you are running the glucose normal control and you get the following results : Glucose = 100 mg/dl … Is this acceptable? To answer this question you need to known what the previously established acceptable range for this control specimen is ( done like the normal range ) Let’s say the acceptable range for this control specimen is : Mean = 104 mg/dl SD = 5 mg/dl … That means that 2.0 SD = 10 mg/gl The acceptable range for this control is 104 ± 10 = 94 - 104 mg/dl In other words, 94 – 104 mg/dl is considered to be acceptable dispersion. So, your control value of 100 mg/dl is well within the acceptable range. Everything seems to be working OK !!!

50. Example of a Levy – Jennings chart A Levy – Jennings chart is a graph that plots QC values in terms of how many Standard Deviations each value is from the mean