At a time when it is most needed, technologies have improved and made transfusion a safer process.

By Mary Ann Sharpe, BS, MT(ASCP)SBB

From the earliest in vivo crossmatch to the in vitro testing of the last four decades, the goal of transfusion is to provide safe, effective blood and components to recipients. Until recently, the process of determining compatibility has been a manual method, the success of the transfusion resting heavily on an individual's technique and judgment in selecting the right donor product for the patient. With the current shortage of staffing in laboratories, finding qualified, unstressed techs to perform successful crossmatching becomes quite a challenge. (Note: The distinction between crossmatching and compatibility testing is often blurred. The compatibility test is the process of records check, patient identification, sample qualification, production selection and all testing to assure proper matching between donor and recipient. The crossmatch is a single test used to test the donor against the recipient for serological compatibility. While some techniques and instruments detailed herein are not used for true crossmatching, they are important to compatibility testing and are thus considered as "crossmatch" options.) At a time when it is most needed, technologies have improved and made transfusion a safer process.

Solid phase technology is the terminology used to describe two unique systems that are taking the technique-dependent aspects and stress out of blood bank testing. This technology uses methods and materials unique from the traditional red cells to test for atypical antibodies and incompatibilities. The gel system uses plastic gel cards with acrylamide beads; the microplate system uses microplates and "fixed" antigens. While both originated and are used as manual methods, the progression to automation is a foregone conclusion.

Gel Technology

In 1994, the Food and Drug Administration (FDA) approved the use of "gel technology" for blood banking procedures in the United States. In 1995, Ortho-Clinical Diagnostics (Raritan, NJ) and Micro Typing Systems Inc. (Pompano Beach, FL), through an exclusive distribution agreement, introduced gel technology into North America. Rather than using traditional test tubes as reaction vessels, a "gel card" is used. Each card contains six microtubes that serve as individual reaction chambers, replacing test tubes. Each microtube contains dextran acrylamide gel particles and a reagent specific for the type of test to be performed.

Following addition of serum or cells, the cards are incubated, if necessary, and then centrifuged. If agglutination has occurred, the agglutinated complexes are trapped in the gel, while non-agglutinated cells are free to accumulate in the bottom of the microtube. The endpoints are read and scored as 0 (no agglutination) through 4+, similar to the grading used in traditional tube testing, depending on the disbursement of the red cell layer through the microtube. Both gel card preparation and reading can be done manually or semi-automatically.

Several blood banking procedures can be performed using the technology, including antibody screens, ABO blood grouping/Rh typing, antibody identifications, direct antiglobulin testing and crossmatches. To perform a Coombs crossmatch, pipette 50 µL of the 0.8 percent donor red cell suspension into the microtubes of an ID-MTS Anti-IgG Card™ (Fig. 1). Add 25 µL of patient serum or plasma, incubate at 37º C for 15 minutes, centrifuge for 10 minutes and read results. Immediate spin crossmatches can also be performed using gel technology.

Some of the benefits of gel technology in the crossmatch procedure include standardization, increased sensitivity and clearer endpoint determinations. Traditional steps utilized in the antiglobulin procedure, such as tube shaking, cell washing, red cell resuspension and antiglobulin controls are no longer required. Gel cards are able to preserve the reactions for 24 hours should supervisory review be warranted.

To semi-automate gel testing, Ortho-Clinical Diagnostics also markets an automated pipetting station with bar code tracking, and two types of readers, one of which centrifuges, reads and displays digital images of the card reactions for technologist review (Fig. 2).

The Capture-R System Using Microplate Technology

The microplate test uses hemagglutination, just as in tube testing and the gel technology, but has not been widely accepted for crossmatching by transfusion services. Even though long acknowledged for its sensitivity, this system has traditionally been reserved for antigen typing or non-crossmatch testing. Immucor Inc. (Norcross, GA) modified the technology to develop a system of screening for IgG antibodies. The Capture-R System is comprised of microplates, low ionic strength solution (LISS), indicator red cells and control sera. The microplate wells are precoated with dried Group O red cells from single donors and indicator cells are coated with murine monoclonal anti-IgG. The available assays test patient serum against individual screening cells or full antibody identification. Unknown serum and special LISS reagent are added to the coated microwells, incubated, washed, indicator cells added, centrifuged and reactions interpreted. The indicator cells are either dispersed and uniformly cover the bottom of the microwell (positive reaction) or form a central button of cells on the bottom of the well (negative reaction).

The manual Capture Microplate System can be used for the antiglobulin phase crossmatch but is routinely used for antibody screening, as is the ID-MTS System. Special equipment (Fig. 3) for the Capture test system includes an ImmuSPIN centrifuge and CSW100, a semi-automated microplate cell. Reading is performed manually.

Both systems test specifically for IgG antibodies and related compatibilities. The advantages of both over the routine tube tests are standardization and greater objectivity, thus saving time and costs of testing.

Automated Crossmatches

The new technologies described represent improvements in testing but continue to be used by many facilities as manual methods. An additional gain from the new technology is the ability to automate the transfusion service of both small and large volume users. Currently, the Capture® System has partnered with automation technology and now provides laboratories with several automated blood bank testing systems, including the ABS2000 (Fig. 4), ROSYS Plato, ABSHV and Dynex Dias Plus Systems, all distributed by Immucor Inc. Each system has varying degrees of automation, but all incorporate positive identification of samples, consistency of performance and objectivity of testing. These systems are designed to perform ABO/Rh, antibody screens and crossmatches. Fully automated, they accommodate pipetting of samples and reagents, incubation, cell washing, centrifugation, electronic reading of microplate results and data management.

Generally speaking, workload volumes are used to determine the appropriate system for testing. Low- to medium-volume workloads are readily handled by the ABS2000. This system performs ABO/Rh, antibody screen and donor crossmatch testing, and is truly considered walk-away. Medium- to high-volume workloads are handled by the ROSYS Plato and ABSHV microplate processing system. These instruments perform, like the ABS2000, positive identification of samples, pipetting of reagent and samples, incubation, washing and reading functions in volumes as great as 1,800 tests per shift.

A composite instrument distributed by Immucor utilizes several individual components to process 300 tests per hour. This semi-automated system uses the Hamilton MicroLab AT Plus sample handling system, DIAS PLUS system for incubation and washing, Sorvall T 6000D centrifuge and IBG MultiReader Plus for microplate scanning, interpretation and documentation. The operator of this instrument is responsible for transfer of microplates from one device to another rather than individual process performance.

The advantages of automated crossmatching are numerous. The pipette systems can assure proper sampling of each sample or reagent consistently. These devices can assure that no cross-contamination occurs and that sampling is appropriate. Incubation is performed under controlled conditions with continual monitoring and documentation of temperatures. The advantage of the automated systems with regards to centrifugation is calibration, monitoring and documentation of temperatures, rotor speeds, timing, etc. with each quality control performed and, in some cases, as tests are performed. Readers accumulate multiple data points for each well read, compile the data and formulate mathematically results of positive or negative, removing much of the subjectivity of crossmatching.

Electronic Crossmatch

The electronic or computer crossmatch is the most radical change in the blood banker's world of technology. As mentioned, few changes have truly been made since the serological crossmatch of the 1950s and 1960s. The electronic crossmatch represents a conceptual change in the provision of compatible blood components. Over time and with the input of sophisticated computer technology, the blood bank has recognized that crossmatching can be almost a matter of simply choosing the correct ABO/Rh component. Certain truisms have been discerned from our increased knowledge of antigens, antibodies and transfusion.

Approximately 0.3 percent to 3 percent of all patients tested have positive antibody screens or incompatible crossmatches.

• Many of the antibodies are not considered clinically significant.

• Individuals who have already developed antibodies as a result of allogeneic sensitization are likely to produce additional antibodies with additional exposure.

• Many individuals, regardless of the number of exposures, do not form antibodies, either because of their antigenic makeup or individual response mechanism.

• The ABO system is the most significant system in the blood bank and can cause immediate hemolytic transfusion reactions and death.

• Non-ABO blood group systems may cause delayed, rather than immediate, hemolytic reactions, thus making these reactions less dramatic or life-threatening.

Certain criteria must be met to perform a computer crossmatch. The electronic crossmatch is predicated on the use of a quality computer system and adherence to established guidelines. In accordance with 21 CFR 606.151 and 21 CFR 640.120, FDA approval is needed to implement this crossmatch process. The FDA will review and approve processes and procedures to assure all the following aspects are met.

1. The 510K-approved computer system is used.

2. Two determinations of the recipient's ABO must be performed.

3.The computer system must contain donor and recipient information.

4. A method must exist to verify correct entry of data.

5. The system must contain logic to alert the user if ABO incompatibilities exist between recipient and donor unit.

With all criteria met, the electronic crossmatch involves selecting a tentative red cell product for the patient and allowing the computer to determine if the truly appropriate product has been chosen.

The true advantages of the computer crossmatch are time and cost savings with no added risk to the patient's care. With the resulting free time, techs can focus on those recipients with alloantibodies or other serological problems requiring more attention. This technology has not been universally accepted for two reasons. Many smaller facilities do not utilize computers for blood banking because of the cost of the systems and staff proficiencies. The large facilities whose computer systems would qualify for the electronic crossmatch prefer to retain the serological crossmatch as the ultimate decision of compatibility.

The Future?

Our knowledge of blood group systems and certain immunological processes have allowed us to change the crossmatch test dramatically in recent years. Is there potential to change it further? As we gain sophistication in recombinant, cloning and other technologies, the opportunity to make transfusions safer and more effective is limited only by our imagination.

For instance, what if one could:

• create artificial antigens and develop the "two-minute crossmatch spot test" in place of antibody screening tests?

• attach artificial antigens on sepharose beads or acrylamide gel and create standardized non-red cell screening beads?

• develop a potentiator or mechanism that distinguishes clinically significant antibodies from "nuisance antibodies?"

• prepare recombinant antigens in soluble form that neutralize an individual's antibody in vivo?

• prepare stem cell stock solutions from individuals or animals that can be used to produce endless supplies of neutral transfusion fluid?

• enzyme treat red cells so the ABO antigens are destroyed and all cellular products are ABO-neutral?

While these thoughts among many others may seem like fantasy today, the potential for the future is endless. In truth, the enzyme treatment of cells to create ABO-neutral cells is not fantasy. The technology exists, albeit in a research mode. A variety of crossmatching options exist today to a far greater extent than ever imagined 10 to 15 years ago. New technologies are available to make the crossmatch more sensitive and defining. Automation is no longer just feasible, but an affordable reality in many transfusion services today. The electronic crossmatch is a standard that has been in place for many years by a handful of imaginative blood banks. What lies ahead as crossmatch options? Time, technology and the imagination only know. . .

Mary Ann Sharpe is CEO of Quarterly Connection, LLC, a blood bank-transfusion services consultation company in Savannah, GA.

Related Web Sites

Blood Banking

http://medlib.med.utah.edu/WebPath/TUTORIAL/BLDBANK/BLDBANK.html

(Scroll down for Blood Compatibility and Blood Components Available For Transfusion Tables)

http://www.slucare.edu/clinical/pathlab/bloodbank/index.shtml

Blood Substitutes

http://www.itxm.org/TMU2000/tmu12-2000.htm

Indirect and Direct Antiglobulin (Coombs) Testing and the Crossmatch

http://www.itxm.org/TMU2000/tmu10-2000.htm