Chapter 12 Other Technologies and Automation

• Gel tests and Solid-Phase assays emerged for accurate, reproducible blood bank testing
• These technologies increase safety, reduce bio-hazardous waste, are automatable, decrease human error, and free lab personnel

Automation

• Blood banks/transfusion services are the last areas of the clinical laboratory to move to automation
• Chemistry, hematology, and immunology have been using automation for many years
• Blood testing complexity and subjectivity hampered full automation
• Personnel shortages, turnaround time requirements, and cost containment provided automation incentives
• Automation equipment allows lab personnel to perform multiple tasks simultaneously
• Bar-coding reduces identification errors
• Standardized techniques minimize testing errors
• Automated infectious disease testing was first, then serologic testing
• The chapter is on equipment that automates serologic testing in blood centers and transfusion services, even though automated viral marker testing is performed in all blood centers

Gel Technology

• In 1985, Dr. Yves Lapierre of Lyon, France, developed the gel test using media like gelatin, acrylamide gel, and glass beads
• Gel particles trap RBC agglutinates during standardized sedimentation or centrifugation to separate agglutinated red blood cells
• Agglutinin testing doesn't require saline washes to remove unbound immunoglobulin
• No need for antiglobulin control cells to confirm the presence of antiglobulin reagent in negative tests
• Gel tests provide a more stable endpoint, more reproducible results, and reduces variability vs traditional tube technology
• In 1988, Dr. Lapierre worked with DiaMed A.G. to develop and produce the gel test in Europe
• In Sept 1994, the FDA granted Micro Typing Systems (MTS) a license to manufacture and distribute an antiglobulin anti-IgG gel card and a buffered gel card in the USA
• In Jan 1995, Ortho Diagnostic Systems Inc. (ODSI) and MTS signed an agreement giving ODSI exclusive rights to distribute the gel test in North America
• In March 2002, Ortho Clinical Diagnostics fully acquired MTS and became known as Micro Typing Systems
• Gel-based test is named the ID-Micro Typing System
• The FDA has approved gel technology for: ABO forward/reverse grouping, Rh typing, direct antiglobulin testing, antibody screening/identifying antibodies, and compatibility testing
• The ABO blood grouping card has gels that contain anti-A, anti-B, and anti-A,B for forward grouping
• Microtubes with buffered gel are used for ABO reverse grouping
• Rh typing card uses microtubes filled with gel containing anti-D
• The Rh phenotype card has gels that contain anti-D, anti-C, anti-E, anti-c, anti-e, and a control
• Microtubes filled with gel containing anti-IgG are used for compatibility testing, antibody detection, and identification
• The gel test is based on the controlled centrifugation of RBCs through a dextran-acrylamide gel containing predispensed reagents in a specially designed microtube
• Each microtube has an upper reaction chamber that is wider than the tube itself and a long, narrow column
• In the gel test, a plastic card (measures approximately 5 x 7 centimeters) with six microtubes is used instead of test tubes
• Each microtube contains predispensed gel, diluent, and reagents
• Measured volumes of serum/plasma or RBCs are dispensed into microtube's reaction chamber
• Incubate and centrifuge the card
• The Reaction chamber is where RBCs may be sensitized (antigen-antibody binding) during incubation
• Each microtube column has dextran-acrylamide gel particles suspended in a diluent with reagents
• The column shape/length provides a large surface area for prolonged contact of the RBCs with the gel particles during centrifugation
• Gel particles of beads of dextran-acrylamide make up 75% of the gel-liquid mixture preloaded into each microtube
• Gel particles are porous, serve as a reaction medium and filter, sieving the RBC agglutinates according to size during centrifugation
• Large agglutinates are trapped at the top of the gel and not allowed to travel through the gel during centrifugation
• Agglutinated RBCs remain trapped in the gel
• Unagglutinated RBCs travel unimpeded through microtube length, forming a pellet at the bottom after centrifugation
• Agglutination reactions in the gel test are graded from 1+ to 4+ (including mixed-field)
• A 4+ reaction is characterized by a solid band of agglutinated RBCs at the top of the gel column
• Usually, no RBCs are visible at the bottom of the microtube
• In a 3+ reaction, most of the agglutinated RBCs remain near the top of the gel column, with a few agglutinates staggered below the thicker band, and most of agglutinates are observed in the top half of the gel column
• In a 2+ reaction, RBC agglutinates are distributed throughout the upper/lower halves of the gel column, few agglutinates at the bottom of the microtube
• In a 1+ reaction, RBC agglutinates predominantly seen in lower half of gel column, some RBCs at the bottom of the microtube
• Weak reactions may only have a few agglutinates remaining in the gel area above the RBC pellet at the bottom of the microtube
• In a negative reaction, the RBCs form a well-delineated pellet at the bottom of the microtube, and the gel above the RBC pellet is clear and free of agglutinates
• In a mixed-field (mf) reaction, the layer of agglutinated RBCs is at the top of the gel and a pellet of unagglutinated cells is at the bottom of the microtube
• False-positive mixed-field reactions can occur when incompletely clotted serum is used
• Fibrin strands in such serum may trap unagglutinated RBCs, forming a thin line at the top of the gel
• Consideration of the patient's clinical history is relevant before interpreting if they are recently transfused or bone marrow transplant recipients expected to have mixed populations of RBCs that commonly produce mixed-field reactions
• Gel technology advantages: Standardization as there is no tube shaking to resuspend the RBC button
• Gel test has stable, well-defined endpoints of the agglutination reaction, observed/reviewed up to 3 days
• Includes simple standardized procedures, no wash step, no need for antiglobulin control cells
• Objective, consistent, reproducible interpretation of test results
• Is ideally suited to individuals who have been cross-trained to work in the blood bank
• Other advantages include decreased sample volume needed for testing and enhanced sensitivity/specificity of the results
• Improved productivity when compared with traditional tube testing
• Gel technology disadvantages: Sample restrictions and the need for special equipment
• Hemolyzed/grossly icteric blood samples should not used, because visual interpretation is difficult in the ID Micro Typing System.
• Grossly lipemic samples require clarification by centrifugation or filtration.
• Rouleaux may produce hazy reactions or false-positive results
• Special incubators/centrifuges are needed to accommodate the microtube cards
• Specific pipette must be used to measure & dispense the 25 µL of plasma/serum and the 50 µL of 0.8% RBCs into the reaction chambers of the microtubes, and pipette calibration must be regularly checked
• Automated equipment is successfully performing ABO and Rh testing, antibody screening, compatibility testing, and antibody identification.
• The ORTHO ProVue™ Analyzer is a walk-away instrument with a capacity for testing 48 samples & 16 reagents
• Instrument safety features: a bar-code tracking system and 3 cameras that record sample, reagent, and card identification
• Instrument incorporates a camera for image analysis with a mathematical algorithm to interpret the results

Solid-Phase Technology

• Used for years in immunology and chemistry laboratories with one of the test reactants (either antigen or antibody) bound to a solid support (usually a microtiter well)
• Plastic's (like polystyrene) ability to absorb proteins from solution and to bind them irreversibly makes plastic microplate wells ideal
• Solid-phase serologic assays were first applied to RBC typing/antibody screening tests in 1978
• Solid-phase technologies relevant to serologic testing includes: solid-phase red cell adherence (SPRCA), solid-phase protein A, and solid-phase enzyme-linked immunosorbent assay (ELISA)

Solid-Phase Red Cell Adherence

• In 1984, Plapp and coworkers reported using solid-phase red cell adherence (SPRCA) for detecting RBC antigens and antibodies
• Immucor manufactured the first SPRCA assays commercially under the trade name of Capture for the detection of RBC and platelet antibodies.
• Capture immunoassays detect antibodies to RBCs, platelets, and cytomegalovirus
• SPRCA assays are either first- or second-generation tests
• First-generation tests require the user to add the target antigen (RBCs, platelets, or platelet proteins, etc.) to the microplate wells before starting the test
• Second-generation tests are pre-bound to the target antigen (RBC, platelet glycoproteins, or CMV) to the microplate test wells during manufacturing
• First- and second-generation SPRCA assays are FDA approved for antibody screening/identification, weak-D testing, IgG autologous control, and compatibility testing.
• Second-generation assays use microwells that are preloaded with antibody screening cells
• Two- or four-cell sets are are recommended for antibody detection in transfusion recipients
• Pooled cells are used for donor antibody detection when increased sensitivity is undesirable
• Reagent panels of preloaded cells are also available for RBC antibody identification
• Intact reagent RBCs are added by the user to chemically modified microwells (first-generation test), when it is desirable to test selected cells
• SPRCA assays can performed with either serum/plasma, but plasma is preferable
• Incompletely clotted samples causes difficulties during the wash cycle and create unreadable tests due to serum clotting
• The manufacturer recommends adding a pH-stabilizing buffer to the isotonic saline used to wash the microplates (available from many manufacturers)
• Capture technology tests performed in microplate wells like 96-well U-bottomed plates or in1 x 8 or 2 x 8 U-bottomed strips
• Both serum/plasma and low ionic strength saline (LISS) are added to microwells that are coated with the target antigen during second-generation

Solid-Phase Protein A Technology

• In 1992, Biotest AG developed a test in Europe for detecting IgG antibodies, using microplate wells that are coated with protein A
• Protein A is a component of the cell wall of Staphylococcus aureus with a very high affinity for the Fc portion of most immunoglobulin classes
• In 2005, the FDA approved the distribution of the Solidscreen II assay in the United States, and in 2010, Bio-Rad Laboratories acquired this test from Biotest AG
• Solidscreen II uses antiglobulin technique to detect and identify red cell antibodies, perform compatibility tests, detect IgG on patients' red blood cells, and type red cells for weak D and partial D antigens (DVI and DVII).
• The Solidscreen II assay is performed in the automated TANGO™™ optimo
• During the indirect test: 50 µL of patient serum/plasma, typing serum, or control reagents is combined in a microwell with a 1% suspension of reagent red blood cells (antibody screen and identification), patient/ donor cells (antigen typing), or donor red cells (crossmatch), depending on which test is being performed
• A low ionic strength solution (MLB 2) is also added to the microwells
• Contents of the microwell are mixed and incubated at 37°C
• A 1% suspension of the unknown red blood cells is prepared for the direct tests, no incubation is required
• Both mixtures are centrifuged, supernatant is aspirated, and the microwell is washed with phosphate buffered saline (PBS)
• Next, 100 µL of anti-IgG anti-human globulin (AHG) is added
• Microwell is remixed and centrifuged
• Antibodies attached to red blood cells are captured by protein A on the microwell's surface and form a homogeneous layer on the microwell bottom (positive reaction)
• Compact red blood cell button is formed in the center of the well if no antibodies are attached (negative reaction)
• The reaction patterns are recorded as a digital image and interpreted by the TANGO™™ optimo
• Demonstrates a standardized method for performing antiglobulin testing using a solid-phase capture method
• Solid-phase protein A provides a standardized method for performing antiglobulin testing using a unique solid-phase capture method

Solid-Phase Enzyme-Linked Immunosorbent Assay (ELISA)

• In 1994, GTi Diagnostics (acquired by Gen-Probe in 2011 now known as Gen-Probe GTi Diagnostics) developed an ELISA assay
• The assay tests for antibodies to platelet glycoproteins using separate microwells for each glycoprotein
• PAK1 detects antibodies such as anti-HPA-1a that react with platelet antigens that occur on glycoprotein IIb/IIIa
• PAK2 detects antibodies to antigens on platelet glycoproteins IIb/IIIa, Ia/IIa, Ib/IX, IV, and class I HLA
• PAK1 and PAK2 were combined into a single assay called PAKPLUS®
• Other ELISA-based variations followed, including PAK12 and PAK2-LE
• In addition, Gen-Probe GTi Diagnostics has developed ELISA assays to detect and identify HLA (human leukocyte antigen) antibodies (both class I and class II)
• A bead-based Luminex assay detects platelet /HLA antibodies and it uses fluorescence and flow cytometry, and Luminex assay uses a mixture of up to 100 different colored beads, each bead coated with a different protein and tests by different antigens
• PAK® tests may be used to test for alloantibody in patient's serum or autoantibody on patent's autologous platelets
• Requires specialized laboratory equipment like adjustable micropipettes with disposable tips.
• No dedicated automation is available for the Gen-Probe GTi solid-phase ELISA assays, but some labs have automation for portions of the assay, like sample handling and plate washing

Gel and SPRCA Technology Comparison

• Both provide stable, reproducible endpoints vs tube testing
• The gel test uses a special pipette for cell/sera measurement
• SPRCA has more sensitivity and specificity