Chapter 11-12 Overview of the Routine Blood Bank Laboratory & Other Technologies and Automation

• Study notes on alternative technologies and automation in the routine blood bank

Gel Technology

• Gel tests are performed in a specially designed microtube
• The test is based on controlled centrifugation of RBCs through a dextran-acrylamide gel
• Gel particles act as a reaction medium and filter
• They sieve RBC agglutinates according to size during centrifugation
• Agglutinated RBCs remain fixed or suspended in the gel
• Unagglutinated RBCs travel unimpeded through the microtube, forming a pellet
• Agglutination reactions are graded from 1+ to 4+, negative, or mixed field
• Standardization is a major advantage of gel technology
• It is stable where washing and control cells for AHG is unnecessary
• Gel tests use small sample amounts
• Disadvantages include the need for a special incubator, centrifuge, specific pipettes, and long centrifugation time
• The principle of SPRCA is solid-phase technology

Solid-Phase Technology

• Solid-phase immunoassay is used for red blood cell typing and antibody screening
• Solid-phase technology is approved for antibody screening, identification, and compatibility testing
• Microplate test wells have intact reagent RBCs bound to microwells
• Patient's serum or plasma and low ionic strength saline (LISS) are added to the RBC-coated microwells
• After incubation and washing unbound Ig, anti-IgG coated RBCs are added
• Centrifugation is performed
• Positive tests show indicator RBC adherence to part or all of the well bottom, which depends on reaction strength
• Solid-phase advantages include standard, stable, and well-defined endpoints
• It is easy to use, requires no reagent pre-dilution
• Hemolyzed, lipemic, or icteric samples can be processed
• There is an indicator for adding any sample
• The disadvantages of solid-phase technique is the need for special equipment like a centrifuge, incubator, and light source
• Higher sensitivity may detect weak autoantibodies that are missed by other antibodies

Comparison of Technologies

• Traditional tube tests involve agglutination in a tube, whereas gel tests use microtube cards and solid-phase tests use microplate wells
• Traditional tests have no reaction matrix whereas gel tests use immunologically inert dextran-acrylamide gels but solid-phase tests use chemically modified polystyrene microplate wells
• Traditional and gel tests use AHG (antihuman globulin sera) for testing detection
• Solid-phase tests use anti-IgG coated red cells
• Washing and centrifugation is required for traditional and solid-phase testing, but not for gel testing
• Reaction readings are quantitative (1+ to 4+, MF) for traditional and gel tests
• Reaction readings are semi quantitative for solid phase
• Traditional tests have no stable reactions
• Gel tests are stable for 2–3 days whereas solid-phase testing are stable for 2 days
• Quality control includes positive and negative controls/Coomb's check cells for traditional tests
• Gel tests use lot numbers of cards, and diluent on the day of use
• LISS color change, positive and negative controls are used for solid phase testing
• Special equipment and automation is not FDA-approved for traditional but it is for gel and solid phase

Automation

• Automated equipment partially addresses personnel shortages and turnaround times
• They also ensure the level of quality standards by new regulatory standards
• Barcoding and standardized techniques reduce testing errors

Summary Chart

• All blood bank laboratory operations are regulated by law and must meet requirements for education, training or experience
• Apheresis machines selectively remove components from donor blood for hemapheresis
• All blood and blood components must be stored properly to ensure viability
• Every unit must be tested for ABO group and Rh type including weak D when indicated and screened for specific viral markers
• Gel and solid-phase technologies have the following advantages:
• Standardization (no more tube resuspension of RBCs)
• Stability because there are well defined reaction endpoints
• Decreased sample volume requirements
• Enhanced sensitivity and specificity

Gel Test Key Points

• The principle is hemagglutination
• RBCs and serum or plasma are allowed to incubate together in a reaction chamber
• After incubation, controlled centrifugation drives the RBCs through a specially designed microtube filled with beads of dextran-acrylamide gel
• Agglutinated cells remain at the top of the tube, or are trapped where unagglutinated will move through to the bottom
• Gel reagents are stable for 2-3 days
• Gel is approved for ABO forward and reverse grouping, Rh typing, DAT, antibody screening, antibody identification, and compatibility testing
• A major disadvantage is the equipment required

SPRCA Key Points

• The principle is solid-phase technology
• In SPRCA tests, the target antigen is affixed to the bottom of the microplate wells
• If the test plasma contains antibodies to the antigen, indicator cells detect the antibodies attached to the fixed antigen by forming a monolayer of RBCs
• If the test plasma contains no antibodies, indicator cells form a clearly delineated button as attachment is not present
• Solid-phase reactions are stable for two days
• Solid-phase testing is approved for testing antibody screening, antibody identification, and compatibility testing
• The advantages are ease of use, no predilution, and ability to test hemolyzed, lipemic or icteric samples but a major disadvantage is the need to purchase specific equipment

Solid-phase protein A technology

• IgG antibodies are captured in microwells that are coated with protein A.
• Solidscreen II, which uses solid-phase protein A technology, uses an assay that uses traditional antiglobulin technique
• It is available only in the United States as an automated technology on the TANGO optimo instrument

ELISA Key Points

• The MACE products provide well-characterized monoclonal antibodies immobilized in microwells that are used to capture glycoproteins from platelets and are used primarily for compatibility testing
• The PAK products either provide immobilized platelet glycoproteins, or captures by monoclonal antibodies in microplate wells
• PAK is used to detect and differentiate between antibodies that bind to platelet-specific glycoproteins (IIb/IIIa, Ib/IX, Ia/IIa, and IV) and class I HLA, primarily for compatibility testing

Luminex-based assay

• The Lumienx-based assay is a bead-based assay that uses fluorescence and flow cytometry to test for platelet/HLA antibodies.
• Luminex assay uses a mixture of 100 different colored beads each coated with a different protein
• The flow cytometer determines between the 100 beads by the internal dyes