Gel tech

gel technology

1. GEL TECHNOLOGY GUIDE: DR. (MRS.) JADHAV M. V.

2. CONTENTS Historical Aspects Basics of immunohaematology Principle of Gel Technology Grading of reaction Applications / Uses Literature Advantages / Disadvantages

3. Historical Aspects

4. Historic aspects Ancient Egyptians bathed in blood and aristocrats consumed blood so as to cure disease. 1616 :Concept of circulation of blood Sir William Harvey 1665 : First canine transfusion Royal Society of London 1667 : Animal to human transfusion J Denis 1818 :Human to human transfusion James Blundell

5. 1869 :Use of nontoxic anticoagulant- sodium phosphate : Braxton Hicks 1901 :Discovery of ABO blood groups and transfusion reactions : Nobel prize awarded Karl Landsteiner 1916 :The first anticoagulent preservative (citrate-glucose) was used to preserve rabbits blood for 2 weeks. Rous-Turner’s solution was used for storage of blood during the First World War.

6. 1939 :Discovery of hemolytic disease of newborn: Levine and Stetson. 1940 : Discovery of Rh factor and anti Rh : Landsteiner and Weiner 1940 : discovery of human antibody similar to antiRhesus : Wiener and Psters World War II stimulated blood preservation research and developed techniques in blood transfusion. The pioneer work of Dr. Charles Drew during World War II led to the establishment of a world wide system of blood banks

7. 1950 : Freeze drying of plasma New concept of component therapy 1959 : First attempt of Bone Marrow Transplant in France 1978 : CPDA-1 was developed, ↑ shelf life of RBCs for 35 days 1988 : First umbilical cords blood transplant 1999 : NAT for HIV and HCV

8. Basic Concepts in Immunohematology

9. 14 nm 35 nm 24 nm IgG IgM Intracellular distance

10. IgM – Large pentameric structures can connect RBCs

11. 1 3 2 Agglutination

12. Effects of Normal Saline The RBCs are surrounded by an electron cloud Require longer incubation time for cells suspended in saline medium. Weak & incomplete antibodies get repelled and hence are unable to cause agglutination

13. Incubation time is 15 min Only. LISS Methods

14. GEL TECHNOLOGY

15. Gel Technology Innovative approach to red cell serology. Invented by Dr.Yves Lapierre of France in 1988. Developed to minimize problems associated with conventional techniques of blood grouping. Addresses the issues of standardization and documentation with unmatched sensitivity, specificity and efficiency. DiaMed -ID Microtyping system from DiaMed AG, Switzerland in assocition with Dr. Lapierre , is based on the Gel Tecnology .

16. Principle of Gel Technology Controlled centrifugation of RBCs through a gel column Serum and cell reaction takes place in a microtube . Six microtubes in a plastic card – easy handling. Microtube consists of a reaction chamber that narrows to become a column with a conical bottom. Reaction chamber is designed to allow prior incubation of test serum and RBCs. Each column contains Sephacryl gel suspended in a buffer solution. Depending on the configuration of card , the gel is premixed with antisera /AHG/other reagents.

17. Add Reactants Serum/plasma/ red cells Principle Reaction Chamber Gel and Reagent 6 Microtubes in Plastic Card

18. Principle of Gel Technology Sephadex gel matrix acts as a sieve. Large aggutinates remain on or near the top of gel interface. Smaller agglutinates pass partway through gel , depending on size. Unagglutinated cells pass to base of microtube to form a button. Cells are always added prior to serum so that serum does not come into contact with gel – this eliminates the ‘WASH PHASE’ as in conventional technique. Grading of reaction depending on the distribution of RBCs throughout the column.

19. Grading of Reaction

20. INTERPRETATION OF GEL TEST 4+ Solid band of red cells at top of gel 3+ Agglutinated red cells in upper half 2+ Red cell agglutinates through length 1+ Aggl. red cell in lower half of gel col. NEGATIVE

21. Incubator Centrifuge

22. Uses of Gel Technology Any immunohaematology test that has haemagglutination at its end point: ABO- Rh typing, typing for other blood group systems. Antibody screening and identification. Compatibility testing – crossmatching . DAT/IAT, other Coombs phase test. Antibody classification- IgG , IgM , IgA , complement… Specialised hematological tests: PNH, Sickle cell anaemia .

23. Forward & Reverse typing The process of identifying an individual’s bld. grp . Involves testing of red cells with known antisera (FORWARD TYPING) and plasma with known group red cells (BACK/REVERSE TYPING)

24. Forward & Reverse typing Reagents required i . ID DiaClon ABO/D + Reverse typing cards containing monoclonal anti- A, anti- B & anti- D suspended in the gel. The tube labeled “ Ctl ” is the negative control. Two tubes with “neutral” gel serve for reverse grouping with A1 and B cells. ii. ID NaCl /Enzyme card which contains six microtubes with neutral gel. iii. ID – Diluent 2: modified LISS for red cell suspension. iv. Test cell reagents: ID DiaCell A1, B, O (0.8± 0.1% suspension), ready to use.

25. Forward & Reverse typing For ABO/D determination (forward typing) Prepare a 5% red cell suspension in ID – Diluent 2 (LISS) as follows: Allow the Diluent to reach room temperature before use. 1. Dispense 500μl of ID – Diluent 2 (LISS) into a clean test tube. 2. Add 25μL packed cells & mix gently. For Reverse typing Use Plasma or serum

26. Forward & Reverse typing 50 µl Diacell A1 50 µl Diacell B 50 µl Patient Serum 50 µl Patient Serum 10 – 12.5µl Patients RBC suspension 5%

27. Forward and Reverse typing Incubate the card at room temp. for 10 minutes Centrifuge the card for 10 minutes.

28. D-HDN ( Rh HDN)

29. Determination of ABO/ Rh -antigen and DAT. DAT on newborn blood samples has become a standard procedure, since it is important to know if the newborn’s red cells have been sensitised with maternal antibodies in utero . Cord/ heel prick samples. ID-Card “ABO/ Rh for newborns”

30. Cross Matching Reagents required i . ID LISS/Coombs cards with six microtubes containing polyspecific AHG (Anti- IgG + C3d). ii. ID – Diluent 2: modified LISS for red cell suspension. Prepare a 0.8 -1.0% red cell suspension in ID– Diluent 2 ( LISS ) as follows: i . Dispense 1 mL of ID – Diluent 2 into a clean test tube. ii. Add 10μL of packed cells & mix gently.

31. Major Cross matching

32. Direct and Indirect Coombs Test Detection of Ig & complement bound to RBCs is critical in diagnosis of immune mediated hemolytic anaemia . DAT is used in inv. of Autoimmune &/ Alloimmune Hemolytic anaemia in which RBCs are coated with in vivo antibodies &/ complement. IAT detects alloantibodies in serum of blood donors, prospective transfusion recepients &prenatal patients.

33. Direct Coomb’s Test Patient’s RBC suspension Add 50 µl of above soln. Results

34. DAT +ve: Further options DC-Screening I DAT + with polyspecific AHG indicates RBCs coated with Ig /complement. To differentiate the reaction, monospecific AHG reagents-anti IgG,-IgA , - IgM , C3…. Results define a clinical condition : WAIHA- IgG ± C3 CAD- C3 ± IgM / IgA PCH- C3/ IgG Mixed AIHA- IgG + C3

35. DAT +ve: Further options….and more DAT IgG1/IgG3 Risk of hemolysis by AIHA /HDN depends on amount of IgG &/ complement coated on RBCs as well as on IgG subclasses involved. IgG1 & IgG3 activate complement. In DAT IgG1/IgG3 card 2 dilutions of both anti-IgG1 & anti-IgG3 are added to gel to differentiate betwn low &high risk for hemolysis .

36. DAT +ve: Further options….and more  DAT IgG -Dilution DAT IgG – Dilution card prvides an indication of clinical importance of DAT positive results. The number of IgG molecules per cell influence the red cell destruction seen in AIHA,HDN and Transfusion reactions.

37. Indirect Coomb’s Test O cell suspension Add 50 µl of above soln. Results Add 25µl of patient serum Centrifuge Incubate

38. Review of Literature Kaur et al at Christian Medical College and Hospital presented their experience with gel based DiaMed ID microtyping system for incompatibility testing over a year period and noticed a startling 65 fold rise in the reported no. of incompatibe units in 1 yr. which rose from 4 (0.02%) to 260 (1.6%) thus making gel technology more sensitive than CTT for crossmatching .

39. Review of Literature Role of Gel based technique for Coomb’s test M Jaiprakash , PK Gupta Harsh Kumar Dept. of Transfusion Medicine, AFMC, Pune AIM: Compare CTT & GT for Coomb’s test & to evaluate their sensitivity & specificity. Results: Conclusion: Gel Technology is a better alternative to CTT. POSITIVITY CTT GTT DAT 6.1% 8.6% IAT 5.4% 6.6% GT Sensitivity Specificity NPV DAT 100% 97.3% 100% IAT 100% 97.6% 100%

40. Review of Literature Gel card in diagnosis of autoimmune haemolytic anaemia . Renu Saxena et al at AIIMS, New Delhi. Aim: To compare the efficacy of Gel card Coomb’s test with conventional Coomb’s test in diagnosis of AIHA. Results: 13 out of 50 cases showed positive ICT & DCT by both CTT & GT. Out of 13 cases 3 showed (+)DCT & (-)ICT by CTT but GT showed (+)DCT & (+)ICT in all 3 cases. Conclusion: Simple, reliable and quick method for detection of autoantibodies , antibodies against RBCs.

41. Antibody Screening and Identification Performed by testing the unknown serum with panel(s) of phenotyped reagent red cells, at various phases, as may be required AHG, Enzyme, room temp., 4*C. Results obtained are plotted onto phenotype charts of the panel cells and antibody specificities deduced. A single crossmatch cannot detect all antibodies, for which it may be necessary to carry out further tests. Antibody screen & identification are procedures used for detection of unexpected antibodies to red cell antigens and determination of their clinical significance.

42. Various Gel Cards Rh -Subgroups Single Antigen Single Antigen Single Antigen

43. Particle Gel ImmunoAssay (PAGIA) Gel Technology now adopted with use of inert polymer particles for detection of: Syphilis Paroxysmal Nocturnal Haemoglobinuria Leishmaniasis Sickle Hb Screening

44. Paroxysmal Nocturnal Hemoglobinuria Patients with PNH have a defective gene called PIG-A, involved in the biosynthesis of glycosyl-phosphatidylinositol (GPI). Without GPI, important regulatory proteins (e.g. CD55 or “DAF”, and CD59 or “MIRL”) cannot bind to the cell surface and protect blood cells from attacks of complement. This may result in a break down of erythrocytes and release of hemoglobin which causes the urine to turn dark during an episode (or “paroxysm”) of hemolysis , though this is not found in all cases.

45. Paroxysmal Nocturnal Hemoglobinuria Reagent1 : ID-PNH-Gel card with rabbit antiserum against mouse immunoglobulines . Reagent 2 : Monoclonal antibodies directed against DAF and MIRL respectively, and negative control, ready-to-use, 1.4 mL. Additionally required: Diluent 2.

46. ID-PNH-Gel card with rabbit antiserum against mouse immunoglobulines . Monoclonal antibodies directed against DAF and MIRL respectively, and negative control, ready-to-use, 1.4 mL.

47. Principle of PNH Gel Technology Monoclonal anti-DAF and anti-MIRL are incubated with RBC suspension of suspected PNH cases and then centrifuged through a microtube containing rabbit antimouse antibody suspended in a gel column. After centrifugation through the gel, cells carrying antibodies, confirming the presence of MIRL or DAF, will show a positive reaction. This denotes that the patient does not have PNH. Negative results or double population confirm the presence of PNH.

48. ID-PAGIA Syphilis Antibody Test

49. ID-PAGIA Syphilis Antibody Test

50. ID-PAGIA Syphilis Antibody Test

51. ID-PAGIA Syphilis Antibody Test

52. Advantages of Gel Technology Improved sensitivity and specificity Easy to use, simple to read No wash phase in IAT Minimal training required Reliable, reproducible results Easy storage and long shelf life of reagents Easy disposal of biodegradable cards Widest range of reagents and instrumentation

53. Disadvantages of Gel Technology Special centrifuge to accommodate the microtubes cards. Special incubators to incubate the microtube cards. Pipette to dispose 25ul of serum. Expensive.

54. AUTOMATION IN BLOOD BANKING

55. INTRODUCTION Process whereby an analytical instrument performs many tests with only minimal involvement of analysts. - mechanization of steps in a procedure Started in 1940 ; to reduce manual errors due to fatigue/erroneous sample identification. Consolidate chemistry & immunoassay systems on one platform.

56. INTRODUCTION Factors necessitating Laboratory automation Turnaround times demands Staff shortages Less maintenance 24 /7 uptime Throughput of samples ↑ no. of analytes & methods on one system.

57. Automated identification methods Barcodes A barcode comprises a series of vertical bars and spaces arranged in various combinations to represent different characters. There are different barcode systems, each with different rules governing the representation of the characters. eg . CODABAR system with ABC symbols By combining the numbers, letters and other characters, a series of barcodes can be built up to represent donation numbers, blood groups and various blood products. In each instance an eye readable number or description is included with the machine- readable code. Device which will interpret barcodes pass a beam of light across the code making use of 2 levels of optical reflectance viz. The black bars and white spaces Optical character recognition – interpret the eye readable characters.

58. Blood bank information systems Blood bank information systems are computer systems that have been developed specifically to assist the blood bank professionals in management of the patients, donor and blood component information. Helps to assess trends and decide future policies by accessing the statistical information. Helps to correlate the laboratory data with donor records and help to trace the donor records following transfusion reactions.

59. BBIS consists of : Hardware Software: application software, operating software and interface software Users FDA, AABB and CPA have specific regulatory and accredation requirments for BBIS

60. Stages in Laboratory Investigations Pre – analytical stage Analytical stage Post – analytical stage

61. PRE – ANALYTICAL STAGES IN LAB. Sample Delivery - Blood drawers or runners / courier facility Pneumatic tube delivery system Conveyers or track system Mobile robots Sample processing – Three phases Pre-centrifugation :- all measurements in < 45 min. Centrifugation :- blood (Plasma/Serum), Urine & Other body fluids (conc. particulate matter) Post-centrifugation

62. PRE ANALYTICAL MODULES Labeling – Bar code label system. Sorting – stopper color, size, tests ordered, instrument design requirements, Decapping Aliquoting Recapping Storage / retrieval

63. SAMPLE IDENTIFICATION

64. CENTRIFUGATION & VOLUME DETERMINATION

65. DECAPING & ALIQUOTTING

66. SORTING

67. ANALYTICAL STAGE Tasks included – Sample introduction & transport to cuvet or dilution cap. Addition of reagent Mixing of sample & reagent Incubation Detection Calculation Read out & result reporting

68. POST – ANALYTICAL STAGE Electrical signal generated by detector Analyzers Microprocessor / computer Digital signal. Chemistry analyzer computers display graphical info. – calibration curves, flags.

69. POST – ANALYTICAL STAGE Data processing i.e. – data acquisition, calculation, Monitoring & displaying – charts & curves, Performing statistics on patient & control value – flags. Analyzer computers have capacity to link to – lab. Info. System internet – companies own manufacturing site – can see real time lab. data & can solve mechanical problem in short duration.

70. AUTOMATED SYSTEM DESIGNS Total Laboratory Automation - Japan,1980 Integration of several instruments = processing specimen management + transportation systems + analyzers + digital interpretation + dispatch of results. creates inclusive, continuous network making each step in testing automated.

71. AUTOMATED SYSTEM DESIGNS Chemical, hematological, coagulation & immuno - histochemical studies at the same time on a sample. Advantage – reduction in – labeling errors, turnaround times.

72. AUTOMATED SYSTEM DESIGNS Disadvantages – significant financial investment - Increased floor space. - highly technical personnel - Infrastructure remodeling, - personnel team building, - Software interfacing.

73. Automated blood grouping Groupometric : Matte in 1963 It is an automated blood grouping machine using multi-channel cuvettes for the reaction mixtures Electro-mechanical units: for agglutination reactions Electronic unit: for processing the results of agglutination tests and print out of the group. Autoanalyzer grouping machine with continuous flow system and laser scanner.

74. The ORTHO AUTOVUE INNOVA System is an automated imunohematology testing system used for blood typing, antibody screening and compatibility testing using ORTHOBIOVUE System cassettes. The ORTHO AUTOVUE INNOVA System is a computersoftware driven, fully automated system which provides automated liquid pipetting,cassette handling, incubation, centrifugation, reaction grading and interpretation. The software used with the AUTOVUE INNOVA can be interfaced with a laboratory information system for test data transfer.

75. APHRESIS Apheresis is collection of anti-coagulated whole blood from a donor, its separation into components, retention of desired component and return of remaining constituents back to the donor with the help of automated cell separator machines.

76. ADVANTAGES OF APHRESIS Reduced multiple donor exposure Reduced risk of alloimmunization Reduced incidence of transfusion transmitted diseases Full and effective transfusion dose Purer product: leucocyte reduced products High quality product Fewer donor reaction due to return of fluid

77. Types of cell separators Intermittent flow cell separator (closed system) Continuous flow cell separator Automated separation techniques by centrifugation Cell separation by membrane filtration Continuous magnetic cell separator ( immunomagnetic )

78. Automated separation technique by centrifugation: Centrifugal force separates blood into different component depending upon the specific gravity. Blood is drawn from an automatic pump Anticoagulant is added tube blood is pumped into roatating bowl,chamber in which layering of components occurs based on the density desired component retained and rest returned to donor either by continous flow or by intermittent flow.

79. Separation by Membrane Filtration: Filtration of plasma through membrane which allows collection of plasma from a healthy donor. Membranes are arranged as hollow fibres which expels the cellular elements in the flow of blood. Most commonly used apheresis devices are: Haemonetic corporation: Platelets, plasma, leucocytes. Baxter: Plasma, platelets, red cells, leucocyte Gambro: Plasma, platelets, leucocyte and peripheral blood stem cells.

80. TECHNOLOGY FUCTIONN HAEMONETIC Intermittent flow centrifuge separator. Anticoagulant blood is pumped into rotating bowl Incoming blood is separated , red cells move to the periphery and plasma to inside of rotating bowl and the white cells and plasma between red cells and plasma Using optical detectors and fluid surge elutriation process desired component retained. GAMBRO( Cobe ) Continuous flow centrifuge cell separator where two arm blood is drawn and returned. Here flat membrane is used to separate the cells of blood from plasma. Allows lower WBC and RBC contamination in platelets. BAXTER Continuous flow technology. CS 3000 has two separation containers firstly for collection of leucocyte reduced platelets and other for white cells (CS 3000 plus).

81. HAEMONETICS CENTRIFUGE

82. COBE Spectra – Automated Apheresis

83. Thank You

84. REFERENCES Transfusion Medicine-Technical Manual, 2 edition 2003,WHO www.DiaMed.com Indian J Pathol Microbiol 2003, Vol. 46, 48, 49