Automation in the Clinical Lab

Introduction of Automation of the Analytical Process
Unit Operations
Specimen identification
Specimen preparation
Specimen delivery
Specimen loading and aspiration
Specimen processing
Sample induction and internal transport
Reagent handling and storage
Chemical reaction phase
Measurement approaches
Signal processing, data handling and process control
Applications of automation in clinical lab

1. Automation in the Clinical Laboratory Course: Clinical Laboratory Principle (SIMS-443) ZA School of Medical Technology 1 Dr. Ali Raza Senior Lecturer SIMS-SIUT

2. Automation in the clinical laboratory Introduction Automation of the A nalytical Process Unit Operations Specimen identification Specimen preparation Specimen delivery Specimen loading and aspiration Specimen processing Sample induction and internal transport Reagent handling and storage Chemical reaction phase Measurement approaches Signal processing, data handling and process control Applications 2

3. Automation in the clinical laboratory 3

4. Automation in the clinical laboratory Definition: “ The process whereby an analytical instrument performs many tests with only minimal involvement of an analyst” “ Controlled operation of an apparatus , process, or system by mechanical or electronic devices without human intervention” 4

5. Automation in the clinical laboratory Intelligent automation : Built in systems to self-monitor and respond appropriately to changing conditions . Instrument performs a repetitive task by itself , Instrument performs a variety of different tasks . 5

6. Advantages of automation Automated instruments enables laboratories to P rocess much larger workloads Reduce number of staff Reduction in the variability of results and errors of analysis Significant improvement in the quality of lab tests Cost reduction 6

7. Advantages of Automation Assist the laboratory technologist in test performance Processing and transport of specimens Loading of specimens into automated analyzers Assessment of the results of the performed tests . 7

8. Principle: Automation in the clinical laboratory Automated analyzers generally incorporates mechanized version of basic manual laboratory techniques and procedures M odern instrumentation is packaged in a wide variety of configurations. C ommon configuration is the Random-Access A nalyzer . 8

9. Random-Access Analysis A nalyses are performed on a collection of specimens sequentially , with each specimen analyzed for a different selection of tests. This approach permits measurement of a variable number and variety of analytes in each specimen 9

10. Random-access analysis Profiles or groups of tests are defined for a specimen at the time the tests to be performed are entered into the analyzer A keyboard by instruction from a laboratory information system C onjunction with bar coding on the specimen tube by operator selection of appropriate reagent packs 10

11. Unit Operations (11) Steps required to complete an analysis are referred to collectively as unit operations 11

12. 1-Specimen Identification The identifying link (identifier) between Patient and specimen is made at the patient's bedside Maintenance of identifier should remains throughout Transport of the specimen to the laboratory , Specimen A nalysis, 3) Preparation of a Report 12

13. 1- Specimen Identification Automatic Identification and Data Collection (AIDC ) E lectronically detect a unique characteristic or unique data string associated with a physical object . Example of identifiers : Serial number P art number Colour Manufacturer P atient number 13

14. Automatic Identification and Data Collection 14

15. Labeling 15

16. Bar Coding I ncorporation of bar coding technology into analytical systems . Initiating bar code identification at a patient's bedside ensures greater integrity of the specimen's identity in an analyzer. 16

17. Labeling Electronic entry of a test order for a uniquely identified patient generates a specimen label bearing a unique laboratory Accession Number . Laboratory station Nursing station The unique label is fixed to the specimen collection tube when the blood is drawn . 17

18. labeling A- Primary labeling B-Secondary labeling 18

19. A- Primary labeling Arrival of the S pecimen (log-in) Patient I dentification and Collection Information L aboratory Requisition F orm An A ccession N umber 19

20. A- Primary labeling Proper alignment of the label on the collection tube 20

21. B-Secondary labeling: B earing essential information from the original label must be affixed to any secondary tubes created . E .g : Serum r emoval from the original tube 21 A number may be handwritten on the specimen cup, or a coded label may be affixed to the original tube or to a specimen cup.

22. Bar Coding System Composition: B ar code P rinter B ar code Reader/ S canner . 22 Bar code Reader Bar code Printer

23. Bar Coding System Symbology : describe as the rules specifying the way the data are encoded into the bars and spaces . S pecification for that symbology.: The width of the bars and spaces, The number of each bar Different combinations of the bars and spaces represent different characters . 23

24. Bar Coding System 24

25. Bar Coding System When a bar code scanner is passed over the bar code, the light beam from the scanner is absorbed by the dark bars and not reflected ; But the beam is reflected by the light spaces. A photocell detector in the scanner receives the reflected light converts that light into an electrical signal that then is digitized. 25

26. Bar Coding System One-dimensional bar coding systems Two-dimensional bar coding systems 26

27. One-dimensional bar coding systems is an array of rectangular bars and spaces arranged in a predetermined pattern following unambiguous rules to represent elements of data referred to as characters . 27

28. Two-dimensional bar coding systems The data is encoded based on both the vertical and horizontal arrangement of the pattern, thus it is read in  two dimensions . doesn't just encode alphanumeric information . use patterns of squares, hexagons, dots, and other shapes to encode data. Example: Data Matrix QR   Code PDF417   or 28 PDF417

29. C hoice for automatic identification: Bar Code Technology “Decrease in identification Errors ” 29

30. Errors “The state or condition of being wrong in conduct or judgement .” “ A measure of the estimated difference between the observed or calculated value of a quantity and its true value .” 30

31. Errors Human misreading of either specimen label or loading list may cause misplacement of specimens, calibrators , or controls. 31

32. Identification Errors Error risks begin at the bedside Compounded with each specimen processing step between collection and analysis. High risks with hand transcription : Accessioning, labeling and relabeling , Creation of load lists. 32

33. 2- Specimen Preparation 33

34. 2- Specimen Preparation Manually Specimen Preparation process results in a delay Examples : Clotting of blood Centrifugation T ransfer of serum to secondary tubes 34

35. 2- Specimen Preparation W hole blood assay system: specimen preparation time essentially is eliminated . Automated or semi automated ion-selective electrodes : M easure ion activity in whole blood rather than Ion concentration 35

36. 3 - Specimen delivery 36

37. 3- Specimen Delivery Methods are used to deliver specimens to the lab Courier Service Pneumatic tube systems Electric track vehicles: 4) Mobile robots 37

38. Courier Service Collection site to the lab and between lab At a given pick up point and specified time. Arrangement of immediate pick up adds cost to the analytical process Specimen breakage 38

39. Pneumatic tube systems Propel cylindrical containers through networks By compressed air or by partial vacuum . used for transporting solid objects 39

40. Electric track vehicles   C onveyor system for light goods transport . utilizes independently driven vehicles traveling on a monorail track network 40

41. Mobile Robots 41 Successful to transport lab specimen both within lab and outside lab. Various sizes and shapes of specimen containers Programmable Cost effective

42. 4- Specimen loading and Aspiration 42

43. 4- Specimen Loading and Aspiration Automatic analyzer directly analyzed serum/plasma from primary collection tube Serum transferred from the specimen tubes to cups CUP features: P ermit required volume for testing M ade of inert material D isposable M inimize cost M inimize Evaporation 43

44. 4- Specimen Loading and Aspiration Specimens may undergo Evaporation D egradation Thermo-labile A nalytes: Temperatures Photo labile Analytes : Photo degradation. E.g.: Bilirubin Specimens and calibrators are held at refrigerated loading zone for Thermo-labile Analytes Reduced Photo-degradation by Semi-opaque cups smoke- or orange-colored plastic covers The loading zone: area in which specimens are held in the instrument before they are analyzed . 44

45. 4- Specimen Loading and Aspiration Contamination Splatter of serum Stoppers of primary containers are opened Decant serum into specimen cups 45

46. 4- Specimen Loading and Aspiration Contamination Closed container sampling systems Initially penetrates the primary container's rubber stopper, followed by the specimen probe passes through a hollow needle Prevents damage or plugging of the specimen probe After the specimen probe is withdrawn , the outer hollow needle also is withdrawn so that the stopper reseals and no specimen escapes . Examples : Automated hematology and chemistry analyzers. 46

47. 5 - Specimen Processing 47

48. 5 - Specimen Processing Automation of analytical procedures requires removal of Proteins Other interferants To automate this separation step , several automated immunoassay analyzers use bound antibodies or proteins in a solid phase format . 48

49. 5 - Specimen Processing Binding of antigens and antibodies occurs on a solid surface to which the antibodies or reactive proteins have been adsorbed or chemically bonded . S olid phases are ( 1) Beads ( 2) coated tubes ( 3) Microtiter plates ( 4) M agnetic & Nonmagnetic Microparticles (5 ) Fiber matrices 49

50. 6- Sample introduction and internal transport 50

51. 6- Sample Introduction and Internal Transport S ample introduction into the analyzer and its subsequent transport within the analyzer A) Continuous-flow Systems Discrete Processing Systems 51

52. Continuous-flow Systems A type of sample analysis in which each specimen in a batch passes through the same continuous stream at the same rate and is subjected to the same analytical reactions 52

53. Discrete Processing Systems A type of analysis in which each specimen in a batch has its own physical and chemical space separate from other specimen. 53

54. Continuous-flow Systems: Peristaltic Pump S ample is aspirated through the sample probe into a stream of flowing liquid, whereby transported to analytical stations in the instrument To ensure proportionality between calibrators, controls, and specimens, the pump and roller speed must remain constant . Peristaltic pump 54

55. Discrete Processing Systems Positive-liquid-displacement pipettes Specimens , calibrators, and controls are delivered by a single pipette to the next stage in the analytical process . A positive-displacement pipette designed for 1- to dispense only aspirated sample into the reaction receptacle 2- to flush out sample together with diluent. 55

56. 6- Sample Introduction and Internal Transport Carry-Over: Transport of a quantity of analytes or reagent from one specimen reaction into and contaminating a subsequent one . 56

57. Carry-Over M inimized by Adequate flush-to-specimen ratio and incorporating wash stations for the sample probe . Wiping t he outside of the sample probe to prevent transfer of a portion of the previous specimen into the next specimen cup . Using New pipette tip for each pipetting 57

58. 7 - Reagent handling and storage 58

59. 7 - Reagent Handling and Storage Reagent Handling: Labels on reagent containers include information such as ( 1) Reagent identification ( 2) Volume of the contents or number of tests (3) Expiration date ( 4) Lot number Storage Plastic or glass containers used for reagents storage 59

60. Automated Analyzers are classified as Open-system analyzer Closed-system analyzer 60

61. Open Versus Closed Systems Open system change the parameters related to an analysis prepare "in-house" reagents use reagents from a variety of suppliers . Less expansive Longer stability Closed system reagent to be in a unique container or format provided by the manufacturer . E xpansive Shorter stability 61

62. 8- Reagent delivery 62

63. 8- Reagent Delivery Liquid reagents are acquired and delivered to mixing and reaction chambers either by Pumps (through tubes) P ositive-displacement syringe devices 63

64. 9 - Chemical reaction phase 64

65. 9 - Chemical Reaction Phase Sample and reagents react in the chemical reaction phase . Factors are important in this phase V essel in which the reaction occurs C uvet in which the reaction is monitored Timing of the reaction(s) M ixing and transport of reactants T hermal conditioning of fluids. 65

66. 9 - Chemical Reaction Phase Reaction vessels: R eused in many instruments . Time before reusable must be replaced depends on their composition . E.g.: 1 month for plastic 2 years for standard glass vessels Not replaced unless physically damaged. Pyrex glass Cuvet : disposable cuvets simplified automation eliminated carryover and maintenance of flow cells . development of improved plastics ( acrylic and polyvinylchloride ) and manufacturing technology . 66

67. Timing of the reaction(s): The time allowed for a reaction to occur depends on a variety of factors . Reaction time depends on the rate of transport of reaction mixture through the system to the measurement station. Mixing and transport of Reactants 1 . Forceful dispensing 2. Magnetic stirring 3. Vigorous lateral displacement 4. A rotating paddle 5. U se of ultrasonic energy 67

68. Thermal Regulation: E stablishment of a controlled temperature environment in close contact with the reaction container E fficient heat transfer from the environment to the reaction mixture. 68

69. 10- Measurement Appoaches 69

70. 10- Measurement Approaches P hotometers Spectrophotometer Fluorometers Luminometers 70

71. 10- Measurement Approaches ( Photomety or Spectrophotomety ) The measurement of absorbance requires the following three basic components An optical source: R adiant energy sources used in automated systems E.g : T ungsten , quartz-halogen, deuterium, mercury, xenon lamps , and lasers . Spectrum wavelengths 300 to 700 nm. 71

72. 2 . Spectral Isolation S pectral isolation is achieved by Interference filters . F ilters have peak transmissions of 30 - 80% and bandwidths of 5 to 15 nm F ilters are mounted in a filter wheel, Appropriate filter is moved into place under command of the system's computer 72

73. Spectral Isolation Monochromators with gratings and slits provide a continuous choice of wavelengths. Coupled with a stationary photodiode array, to isolate the spectrum. These two elements also are coupled with fiber-optic light guides to transfer the passage of light energy through cuvets at locations convenient for mechanization . 73

74. 3. A detector Photometric Detectors: Photodiodes used as detectors in many automated systems P rovide a high signal to noise ratio and fast detector response times for fluorescent and chemiluminescent measurements . 74

75. 3. A detector Notes: Proper alignment of cuvets with the light path(s) is important in both automated and manual analyzers . S tray energy and internal reflections must be kept to acceptable levels . If the light path is not perpendicular to the cuvet , inaccuracy and imprecision may occur, particularly in kinetic analyses . 75

76. 76

77. Reflectance Photometry In reflectance photometry diffuse reflected light is measured . The reflected light results from illumination, with diffused light , of a reaction mixture in a carrier or from the diffusion of light by a reaction mixture in an illuminated carrier. The intensity of the reflected light from the reagent carrier is compared with that reflected from a reference surface . 77

78. Fluorometry emission of electromagnetic radiation by a species that has absorbed exciting radiation from an outside source . Intensity of emitted (fluorescent) light is directly proportional to concentration of the excited species used widely for automated immunoassay. It is approximately 1000 times more sensitive than comparable absorbance spectrophotometry, but background interference due to fluorescence of native serum is a major problem. 78

79. Turbidimetry and Nephelometry Turbidimetry and nephelometry are optical techniques Are applicable to methods measuring the precipitate formation in antigen-antibody reactions These techniques are used to measure plasma proteins and for therapeutic drug monitoring. 79

80. Chemiluminescence and Bioluminescence Chemiluminescence and bioluminescence differ from fluorometry in that the excitation event is caused by a chemical or electrochemical reaction and not by photo-luminescence The applications of chemiluminescence and bioluminescence have increased significantly with the development of automated instrumentation and several new reagent systems . Because of their attamole -to- zeptomole detection limits , chemiluminescence and bioluminescence reactions have been used widely as direct and indicator labels in the development of immunoassays. 80

81. Electrochemical The most widely used electrochemical approach involves ion-selective electrodes . These electrodes have replaced flame photometry in the determination of sodium and potassium. Electrochemical detectors also have been used for the measurement of other electrolytes and indirect application in the analysis of several other serum constituents The relationship between ion activity and the concentration of ions in the specimens must be established with calibrating solutions, and such electrodes need to be recalibrated frequently to compensate for alterations of electrode response . 81

82. 11- Signal Processing, Data handling and Process Control 82

83. 11- Signal Processing , Data handling and Process Control The interfacing and integration of computers into automated analyzers and analytical systems has had a major impact on the acquisition and processing of analytical data. Analogue signals are converted to digital forms by analog-to-digital converters . The computer and resident software then process the digital data into useful and meaningful output . Data processing has allowed automation of such procedures as nonisotopic immunoassays and reflectance spectrometry because computer algorithms readily transform complex, nonlinear standard responses into linear calibration curves . 83

84. 11- Signal Processing , Data handling and Process Control Several functions performed by integrated computers in automated analyzers C ommand and phase the electromechanical operation of the analyzer are performed U niformly R epeatable Correct Sequence Control of operational features of automated equipment , calculation of results, monitoring of operation contribute to the increased reproducibility of results. 84

85. 11- Signal Processing , Data handling and Process Control Computers acquire, assess, process, and store operational data from the analyzers . Monitor instrument functions for correct execution and react to improper function by recording the site and nature of the malfunction. Computers enable communication interactions between the analyzer and operator. Diagnostic computer messages to the user describing the site and type of problem enable quick identification of problems and prompt correction . Graphical displays provide detailed and interactive troubleshooting guidance to instrument operators and visual display of the status of each specimen and associated quality control data . 85

86. P ermit interactive communication between computer systems in the modem laboratory analyzer and the Laboratory Information System (LIS ). I nstrument manufacturers have been developing ethernet interfaces for networked connections with TCP/IP (Transmission Control Protocol/ lnternet Protocol ). 86

87. W orkstation S erves as the point of interaction with the instrument operator Accepts test orders Monitors the testing process ( 4) Assists with analysis of process quality ( 5) Provides facilities for review and verification of test results 87

88. W orkstation The workstation is usually directly interfaced with the LIS host, accepting downloaded test orders, and uploading test results. Most workstations have facilities to (1) display Levy-Jennings quality control charts, ( 2) monitor the progress of each test order, and ( 3) troubleshoot the analyze 88

89. Integrated automation for clinical laboratory Chemistry Hematology Immunoassay Coagulation Microbiology Nucleic acid testing Provide efficient and cost-effective operation with a minimum of operator input. 89

90. Instrument Cluster To reduce labor costs, instrument manufacturers are developing approaches that will allow a single technologist to simultaneously control and monitor the functions of several instruments . 90

91. Automation P rocesses have been automated and used in the clinical laboratory. Urine analyzers Cell counters Nucleic acid analyzers Microtiter plate systems Automated pipetting stations Point of- care testing analyzers. 91

92. Automation Urine Analyzers Many of the same analytical principles are used for the quantification of serum and urine constituents. It is more difficult to automate testing of urine than serum because of the broad range of concentrations of many urine constituents . This requires a low limit of detection to measure low concentrations, and expanded linearity to permit measurements of high concentrations without dilution. This requirement , together with the relatively low demand for urine tests compared with that for serum tests, has restricted the development of analyzers designed specifically for urine constituents. 92

93. Cell Counters: Analyzers that perform a complete blood count have been automated through the use of the "Coulter principle,'' which is based on (1) Cell conductivity (2) light scatter ( 3) Flow cytometry . 93

94. Cell C ounters The Coulter principle is based on changes in electrical impedance produced by nonconductive particles suspended in an electrolyte as they pass through a small aperture between electrodes . In the sensing zone of the aperture, the volume of electrolyte displaced by the particle (cell) is measured as a change in voltage that is proportional to the volume of the particle. By carefully controlling the quantity of electrolyte drawn through the aperture, several thousand particles per second are counted and sized individually . Red blood cells, white blood cells, and platelets are identified by their sizes. Alternating current in the radiofrequency range short-circuits the bipolar lipid layer of the cell membrane , allowing energy to penetrate the cell . Information about intracellular structure, including chemical composition and nuclear volume, is collected with this technique . 94

95. Flow Cytometry Cells stained with a fluorescent dye that travel in suspension one by one past a laser light source. (Unstained cells also are measured.) Scattered light and emitted light are collected in front of the light source and at right angles, respectively. Information derived through measurement of light scatter when a cell is struck by the laser beam is then used to estimate (1) Cell Shape (2) Size (3) Cellular granularity (4) Nuclear lobularity (5) Cell surface structure 95

96. Automation Nucleic Acid Analyzers: Automation of the analysis of nucleic acids developed rapidly as an outgrowth of the Human Genome Project .“ Several manufacturers have developed automation to assist with the isolation of nucleic acids and with analysis of nucleic acids using several amplification schemes and nucleic acid sequencing . Many of these techniques have been miniaturized using chip technology Microfluidic chip 96

97. Microtiter Plate S ystems Commonly used in immunoassays and nucleic acid analyses. As used for enzyme-linked immunosorbent assay (ELISA) assays, microtiter plates usually are made of polystyrene and have 48 or 96 wells coated with antibody specific for the antigen of interest . After incubation of serum in the microtiter plate well, the well is washed to remove unbound antigen, and a second antibody with conjugated indicator enzyme is added. 97

98. Microtiter plate systems After a second incubation period, the well is washed to remove the unbound conjugate. A color producing product is developed by the addition of enzyme substrate and the reaction is terminated at a specific time. With the development of automated pipetting stations, the liquid handling steps required for microtiter plate assays have been fully automated to make microtiter plate assays a viable technology for carrying out large numbers of immunoassays. Automated pipetting stations have a cartesian robot with a pipette fixed to the end of a probe that moves about a rectangular space . The probe is capable of moving in the X, Y, and Z axes . Liquids may be aspirated and dispensed in any location within the rectangular space . 98

99. Automatic Pipetting stations used to automate an analytical procedure for which an automated analyzer does not exist or cannot be justified . Pipetting robots are Easy to program Rarely malfunction delivering aliquots with precision and accuracy. Multiple-channel pipetting R obots: allow parallel processing of specimens with 8- or 12-channel probes to handle microtiter plates. 99

100. Point-of-Care Testing Analyzers (POCT Analyzers ) known by a variety of names "near-patient" "decentralized" " off-site" testing Rapidly growing component of laboratory testing 100

101. 101 Reference: Tietz Fundamentals of Clinical Chemistry, Sixth Edition. Automation in the clinical laboratory, Chapter 11, pg . 171-187

102. 102 Thank You