Automation in blood banking

Uses of automated methodology in blood banks

1. AUTOMATION IN BLOOD BANKING PRESENTER - Dr Shreya Prabhu MODERATOR - Dr V Mahanthachar 1

2. WHY FULLY AUTOMATION IN BLOOD BANK ? To achieve “safe & timely blood transfusion” Faster TAT =>Faster blood transfusion Reduce labour intensive manual test procedure Test results directly upload to LIS (reduce transcription error) Thus, better quality of blood transfusion 2

3. Personnel shortages, TAT requirements and greater regulatory demands have provided incentive for blood services to seek automation Automated equipments provide the level of quality assurance required by new regulatory standards Barcode reduces identification errors by providing accurate patient identification 3

4. Automated systems from different manufactures use advanced technologies like CAT (Column Agglutination Technology) and SPRCA (Solid Phase Red Cell Adherence) Popular names for CAT and SPRCA are Gel and Solid Phase technology respectively Within the last several years automated methods for typing blood, screening blood antibodies have been developed. 4

5. Blood Typing Auto Analyzer is a reliable instrument for routine ABO blood grouping, Rh and other antigen typing. U se of an Auto Analyzer for the routine screening of blood sera for irregular antibodies has been reported Several manufacturers are in various stages of perfecting centrifuges that will automatically wash erythrocytes for Coombs testing 5

6. The automated methods have led to a remarkable improvement in blood safety The application of automation is now growing in hospitals and transfusion services, because human errors can lead to life threatening consequences for the patients 6

7. 7 FULLY AUTOMATED ANALYZER

8. Two basic Automation methodologies in blood bank I)GEL TECHNOLOGY (column agglutination using a gel matrix) II)SOLID PHASE TECHNOLOGY (solid-phase red cell adherence) 8

9. The equipments that have been fully automated and currently available in market are Galileo ( Immucor , Inc )- uses SPRCA Techno ( Diamed )- uses CAT Auto Vue (Ortho Clinic Diagnostics Ltd)- uses CAT (beads) 9

10. In the Solid phase method, a solid coating of Protein A or RBC stroma is placed in a microplate well for the antigen-antibody reaction, and anti-sera is placed in a well for ABO grouping. Gel methodology consists of plastic cards with six to eight integrated microarray “columns”—basically, reaction chambers over a gel matrix—where agglutinates are trapped. 10  

11. GEL TECHNOLOGY Gel technology is currently approved for ABO forward and reverse grouping, Rh typing, DAT, antibody screen, antibody identification and compatibility testing. HISTORY: In 1985,the gel test was developed by Dr Yves Lappierre of France Media used- Gelatin , acrylamide gel, glass beads Gel appeared to be ideal material for trapping agglutinates Compared with traditional tube technology, the gel test provides a more stable endpoint. 11

12. PRINCIPLE: Performed in a specially designed microtube . Based on controlled centrifugation of RBC through a dextran-acrylamide gel that contains predisposed reagents 12

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14. MICROTUBE 14

15. GEL CARDS 15

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18. CENTRIFUGATION 18

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20. Measured volumes of serum or plasma or RBCs are dispensed into the reaction chamber of the microtube The card is incubated and then centrifuged . The reaction chamber is actually a miniature test tube, providing an area for the sensitization of RBCs during incubation. The shape and length of the column provides a large surface area for prolonged contact of the RBCs with the gel particles during centrifugation . 20

21. The gel particles are beads of dextran-acrylamide that make up 75% of the gel-liquid mixture that is preloaded into each microtube . The gel particles are porous, they serve as a reaction medium, filter , sieving the RBC agglutinates according to size during centrifugation. Large agglutinates are trapped at the top of the gel and are not allowed to travel through the gel during the centrifugation of the card . 21

22. Agglutinated RBCs remain fixed or suspended in the gel, while unagglutinated RBCs travel unimpeded through the length of the microtube , forming a pellet at the bottom following centrifugation . The gel test reactions are stable, allowing observation for up to 3 days. 22

23. 23 Photograph of agglutinated RBC’s trapped above the gel matrix

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27. MIXED-FIELD REACTION 27

28. Negative reactions may appear mixed field when incompletely clotted serum samples are used in the gel test Fibrin strands may trap unagglutinated RBCs Before interpreting mixed-field, clinical history should be considered Ex- recently transfused or bone marrow transplant recipients are expected to have mixed populations of RBCs and commonly produce this 28

29. TESTS APPROVED BY FDA Gel technology is currently approved for ABO forward and reverse grouping Rh typing DAT A ntibody screen A ntibody identification C ompatibility testing 29

30. The ABO blood grouping card contains gels that include anti-A, anti-B, and anti-A,B for forward grouping. Microtubes with buffered gel are used for ABO reverse grouping. The Rh typing card uses microtubes filled with gel containing anti-D . Microtubes filled with gel containing anti- IgG are used for compatibility testing, antibody detection, and identification . 30

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32. ADVANTAGES Standardization is one of the major advantages, (there is no tube shaking to resuspend the RBC button) It includes simple standardized procedures, no wash steps. Decreased sample volume needed for testing. Provides stable, well defined end points of the agglutination reaction More objective, consistent and reproducible interpretation of the test results 32

33. DISADVANTAGES Need to purchase special incubators and centrifuges to accommodate the microtube cards used for testing . A specific pipette must be used to dispense 25 mL of plasma or serum and 50 micro L of a 0.8 percent suspension of RBCs into the reaction chambers of the microtubes . Ideally suited to individuals who have been cross trained to work in the blood bank 33

34. SOLID-PHASE TECHNOLOGY TESTS APPROVED BY FDA: Antibody screening Antibody identification Compatibility testing 34

35. In 1978 Rosenfield and coworkers were the first to apply the principle of solid-phase immunoassay to RBC typing and antibody screening tests . SPRCA (solid phase red cell capture assay) was developed commercially for the detection of RBC and platelet related antibodies . In these test systems,one of the test reactants (either antigen or antibody) is bound to a solid support (usually a microtiter well) before the test is started. The ability of plastics, such as polystyrene to absorb proteins from solutions and bind them irreversibly made SPRCA possible. 35 HISTORY

36. SPRCA was developed by trade name Capture® by Immucor In the Capture ® immucor technology, tests were adapted to microplate wells , either as full 96-well U-bottomed plates or as 1x8 or 2x8 strips of U-bottomed wells Capture-R® for the detection of RBC antibodies and Capture-P® for the detection of platelet antibodies. To perform these tests, a laboratory centrifuge capable of holding 96-well microplates is required, along with a microplate incubator and an illuminated reading surface or microplate reader 36

37. PRINCIPLE Based on SPRCA The tests uses chemically modified microplate test wells in which intact reagent RBCs are bound to the microwells before starting the test. Patient serum or plasma and low ionic strength saline are added to the RBC-coated microwells and incubated at 37 c to allow time possible for antibodies to attach. After incubation, the wells are washed with pH-buffered isotonic saline to remove unbound proteins 37

38. Wells are added with an indicator of anti- IgG –coated RBCs, and the microwells are centrifuged. Indicator cells are AHG coated RBCs Centrifugation forces the indicator RBCs to contact the immobilized sensitized reagent RBCs . Positive test show adherence of indicator RBCs to part or the entire well bottom, depending on the strength of the reaction 38

39. In second generation tests, RBC membranes are bound to the microplate test wells and air dried Capture R® immucor is first generation solid phase test, Capture R® Ready-Screen/Capture-R® Ready-ID® are second generation tests Solidscreen ® is a solid phase test that detects antibody using microplate wells that are coated with polyspecific antihuman globulin 39

40. TEST REACTIONS If antibody has attached to antigen , the indicator cells will form a monolayer of RBCs No antibody, nothing attached to antigen and indicator cells will form a clearly delineated button at the center of microplate well Positive tests show adherence of indicator RBCs to part or all of the well bottom, depending on the strength of the reaction 40

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43. ADVANTAGES Standardization is the major advantage of solid-phase technology. Solid-phase technology provides stable, well-defined endpoints of the reaction . E ase of use No predilution of reagents is required. It is possible to test hemolyzed , lipemic , or icteric samples. The enhanced sensitivity makes the detection of weak alloantibodies easier. 43

44. DISADVANTAGES The major disadvantage is the need for a centrifuge that can spin microplates , a 37°C incubator for microplates a light source for reading the final results . Increased sensitivity may also be a disadvantage in as much as solid phase may detect weak autoantibodies that other systems miss 44

45. Solid phase technology for immunohematology Capture has proved to be most dependable technology as it has following features: ACCURACY: IgG specific technology- detects clinically significant antibodies Sensitivity and specificity are higher Automated washing of hemolyzed , icteric, lipemic samples assures validity Controls with each run assures validity 45

46. EASE OF USE: Antigen are pre coated on test wells No pre-dilution of samples or reagents Same method and procedure for all assays 46

47. COST EFFICIENCY: Higher lab productivity Longer shelf life of coated antigens Cost benefit of single antibody screen of patients vs. number of crossmatches Competitively priced Easily automated or upgraded 47

48. PARAMETER ‘CAPTURE’ SPRCA OTHER TECHNOLOGIES SENSITIVITY Highly for IgG antibodies Less SPECIFICITY More False positivity is higher SHELF LIFE 3-4 months 4-5 weeks STANDARDIZATION Standardized Not APPROVALS US FDA approved Non- US FDA approved PLATELET ANTIBODY SCREENING AND CROSSMATCH available Not available 48

49. CURRENT TESTS AVAILABLE Test Gel test Solid Phase ABO- Forward Yes No ABO- Reverse Yes No Rh typing Yes No Antibody screen Yes Yes Crossmatch Yes Yes Antibody identification Yes Yes 49

50. Traditional Gel Solid phase Reaction chamber Tube Agglutination Microtube card Microplate wells Reaction patterns Agglutination Agglutination Solid-phase immune adherence Reaction matrix None dextran-acrylamide gels polystyrene microplate wells Testing detection AHG AHG Anti- IgG -coated red cells Washing required Yes No Yes Centrifugation Yes Yes  Yes 50

51. Reaction readings Quantitative: 1 to 4 Quantitative: 1 to 4 Semiquantitativeg : strong positive, positive, Negative, no MF Stable reactions No yes (2–3 days) Yes (2 days) Quality control Positive and negative control Lot number of cards and diluent on day of use LISS color change, positive and negative control Special equipment No Yes Yes Automation No Yes Yes 51

52. SENSITIVITY Technologies has been shown to detect at least as many IgG Ab as conventional test tube methods Sensitivity can be modified by pretreating the test cells or monolayer with enzymes Increased sensitivity is advantage when low titered clinically significant antibody present 52

53. QUALITY CONTROL GEL TEST Uses special dispensers to prepare the RBC suspensions and special pipettes to add measured volume of plasma/serum Each lot number of cards and diluent should be tested on the day of use to confirm that the test cards and diluted reagent RBC’s are reacting as expected 53

54. SOLID-PHASE TECHNOLOGY Recommends including a positive and a negative control with each batch of tests LISS formulated to detect the addition of plasma by a color change from purple to blue when plasma is added 54

55. AUTOMATION Automated instruments use Gel and solid phase technologies to increase the efficiency and productivity of donor testing They include ABS2000, ROSYS Plato(SPRCA), Galileo,Diamed and ProVue by ortho clinical diagnostics These systems are capable of performing ABO/Rh and antibody screens in the processing of donor blood 55

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57. The ABS2000 is credited as being the first fully automated walk away system It performs ABO/Rh using hemagglutination and antibody screens/ crossmatches using solid phase technology The ABS2000 uses a bardode scanner to log reagents and samples, transfer specimens by automated pipetting, prepare RBC suspensions, incubate, wash, centrifuge, read and interpret results Performs low to medium volume workloads 57

58. ROSYS Plato, ABSHV perform medium to high volume testing Dynex dias Plus by Immucor performs medium to high volume testing and uses robotic system and closed washing system thus minimizing biohazard exposure New addition to immucor line of automated instruments is the Galileo 58

59. GALILEO (IMMUCOR INC.) 59

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61. Galileo is a fully automated walk away system which uses solid phase microplate technology to perform all tests. It uses SPARC technology for all coombs based testing like AHG cross matching antibody screening antibody identification DAT For routine blood grouping testing, it uses hemaglutination technology 61

62. Special and unique feature is availability and automation of Platelet Antibody screening and Platelet Cross matching Manufactured by Immucor Inc , USA and used in many countries 62

63. It has different components like Centrifuge Incubator Washer Camera Sample and reagent loading bay Microplate loading tower w hich all are coordinated with each other to process testing and no manual intervention is required It has a sample capacity of 224 samples and number of reagents can be loaded depending on nature of tests performed 63

64. All reagents are bar-coded which helps in easy identification of the same by instrument itself There are various incubators of different temperature available to carry different nature of testing like blood grouping and antibody screening simultaneously Thus useful in institutions where different parameters are performed simultaneously Test results with images of microplates and all other relevant details are continuously stored in memory of instrument which can be reviewed as and when required 64

65. Advantages High loadging capacity- efficient to carry out heavy workload The capture technology used in Galileo is IgG specific and hence provides high specificity The capture technology used in Galileo is highly sensitive It has option to perform extended phenotyping for all clinically significant antigens like Rh,Duffy,MNS etc . 65

66. Automation of Platelet Antibody screening and Platelet Cross matching makes it unique The high processing speed of Galileo helps in high through put of large batch testing within short time The cell panels used for antibody screening and identification in Galileo are in dried form coated on microplate which increases the shelf life of these panels for around 10-12 weeks The washing step involved in assay procedure enables the use of lipemic , icteric and hemolysed samples 66

67. The system liquid used is 0.9% normal saline and it can be replenished even during the test run It has an option of online remote access through internet for technical support and it can be used for diagnostic purpose Test results can be reviewed in four different patterns by plate images, by grading of reactions, by reaction strength only and by straight result as positive and negative The inbuilt quality control run as positive and negative controls ensures validity of test results 67

68. Disadvantages Closed system- no other reagents can be used other than those by manufactures If test run not managed properly, can lead to wastage of consumables Cost effectiveness must be considered as the consumables are usually priced more Internet connection is required for remote technical support 68

69. The indicator cells used in coombs testing has onboard shelf life of 24 hours Archiving process must be done after regular interval of 10-15 days to store all test results 69

70. TECHNO (DIAMED) 70

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72. ADVANTAGES Has two working stations which function independently and can be useful if one station is not working Gel technique is used is more sensitive Interpretation of test results are done automatically and image cards are available to review the results The STAT position is available for urgent sample processing 72

73. DISAVANTAGES Not true continuous access and no sample can be loaded during centrifugation of cards Sample lodging capacity is of 36 samples The blood grouping card used does not include Anti-Ds from two different sources as recommended by NACO, NBTC, BCSH and AABB neither it has O cells in reverse grouping The blood grouping card used also does not include Anti-A,B which is essential for weak subgroup of A group 73

74. Instrument lacks the criteria of automated validation of test results as proper negative and positive controls are not available during test run Cards have to be manually placed- ? Fully automated It is not possible to determine the presence of clinically significant IgG antibodies when they are present with cold antibodies as they mask the presence of IgG reactivity in Gel Cards The blood grouping profiles are fixed in gel format and cannot be modified or changed according to different needs of different blood banks 74

75. AUTOVUE 75

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77. Uses Bead cards ProVue is a walk-away instrument with capacity for 48 samples and 16 reagents Safety features includes bar-code tracking system, 3 camera that record sample, reagent and card identification A camera in the instrument performs image analysis and uses a mathematical algorithm to interpret the results 77

78. ADVANTAGES STAT Position is available for urgent testing of any sample Bead cards are sensitive Partially continuous random access makes it better in handling nature of testing simultaneously Few reagents used 78

79. DISADVANTAGES The Weak D testing not available Loading capacity- 36 All samples scanned manually by hand barcode scanner to feed samples in instrument All samples centrifuged manually prior to loading samples in instrument 79

80. The sample must be at least 1 ml for processing in machine which is practically not possible in cross matching for donor samples from tube segments If any error occurs like waste container full, the instrument stops working and waits for technician to take required action The machine produces many equivocal reactions which must be edited manually by operator by visual interpretation of cards seen on screen 80