Laboratory Serologic Problems Associated with Administration of Intravenous IgG
By Benjamin Lichtiger, M.D., Ph.D.
Copyright 1993-2010 The University of Texas MD Anderson Cancer Center, Houston, Texas. All rights reserved.
Over the last several years, intravenous IgG (IVIgG) has acquired great importance in the treatment of immunodeficiency states and autoimmune disorders, in the prophylaxis of Rh isoimmunization, and in the prophylaxis of bone marrow transplant patients to decrease infections (1-3). It is important to realize though that there are clinical laboratory testing problems associated with the use of IVIgG. These problems include those caused by viral and red blood cell (RBC) alloantibody contamination of IVIgG preparations (Table 1).
Table 1. Antibodies That May Be Present in IVIgG Preparations
|Tetanus||Anti-A and anti-B|
|Hepatitis B core||Anti-B|
|hepatitis B surface antigen||Antibody (antibodies) that react with all RBC tested|
|Hepatitis C||Platelet antibodies (?)|
|Many other antibodies to bacteria, virus, etc.||Lymphocytotoxic antibodies (?)|
Preparations of IVIgG are extracted from large pools of volunteer plasma donors (greater than 10,000 donors/pool) under strict U.S. Food and Drug Administration (FDA) regulations and World Health Organization (WHO) standards and guidelines. IVIgG is extracted by a process of cold alcohol fractionation; the final product has 96-98% of the electrophoretic characteristics of gammaglobulin. Manufacturers add various amounts of biochemical compounds to achieve isotonicity, specific pH ranges, stability, and solubility (4-16). IVIgG also contains variable amounts of IgA (from 0.4 to 720 mg/mL, depending on the manufacturer). This is important to keep in mind when treating patients who may be IgA deficient (3).
After infusion, IVIgG becomes available in the circulation almost immediately. It is estimated that after 6 d, the extravascular and intravascular compartment concentrations of IVIgG are equalized. Although the physiologic half-life of IVIgG is approximately 22 d, it has been observed to extend to over 30 d in immunodeficient patients (13). Again, IVIgG prepared by different manufacturers must comply with strict WHO and FDA guidelines, production standards, and quality controls, which include having expected minimum levels of antibodies to hepatitis B surface antigen (HBsAg) and hepatitis A virus (4).
IVIgG preparations contain the various IgG subclasses in proportions similar to those in normal plasma. The importance of the IgG subclasses in replacement therapy is significant, as many disease and infection processes seem to be associated with deficiencies in specific IgG subclasses (17-23). However, IVIgG preparations derived from the large plasma donor pools described above also contain a broad spectrum of antibodies to viral, bacterial, and other microorganisms. In addition, they unavoidably contain antibodies against RBC antigens, which may produce false-positive results during workup of transfusion-dependent patients (24) (Table 1).
Consequently, the presence of alloantibodies in IVIgG preparations is a concern because of the havoc it can wreak on serologic and compatibility test results. One of the most obvious concerns is contamination with antibodies to human immunodeficiency virus (HIV) and hepatitis C virus. We here at M. D. Anderson Cancer Center's Transfusion Service recently studied IVIgG preparations used by our service. In a study reported in 1991, we tested 165 lots of IVIgG from different manufacturers (24) (Table 2). In a more recent study, we analyzed 51 lots of IVIgG (unpublished data). Definite decreases in contamination levels could be seen when comparing our two sets of data. For instance, unlike the earlier lots, none of the current lots was reactive to human immunodeficiency virus 1/2 (HIV-1/2) or rapid plasma reagin. Forty-seven of the lots in the more recent study were reactive for hepatitis C antibody, but in the future we expect to see less hepatitis C reactivity in light of a new FDA requirement for donor screening and plasma processing. It is important then neither to overlook nor to overreact to hepatitis C antibody reactivity in a patient receiving IVIgG. Obviously, testing for hepatitis and cytomegalovirus (CMV) could also produce uninterpretable results.
Table 2. Partial List of Antibodies for Infectious Disease Markers Tested for and Identified in Various IVIgG Lots
|No. lots positive|
|Hepatitis B surface||165||100%||51||100%|
|Hepatitis B core||160||97%||21||41%|
|Hepatitis A total||165||100%||Not tested|
|Rapid plasma reagin||48||20%||0||0%|
|Hepatitis C||Not tested||47||92%|
Another concern, especially in bone marrow transplant patients, is the presence of RBC alloantibodies (Tables 3 and 4). Since the recent introduction of IVIgG for CMV prophylaxis in bone marrow transplant recipients, a number of reports have described increased incidences of positive direct and indirect antiglobulin tests, confusing serologic results in pretransfusion testing, and rare cases of hemolysis (25-29). In a study by Robertson et al. of the serologic findings in 47 bone marrow transplant patients who received high doses of IVIgG (25), the frequency of positive direct antiglobulin results was 48.9% (P less than 0.001) versus 12.8% for a control group not receiving IVIgG. The frequency of positive indirect antiglobulin test results was 25.5% (P less than 0.001) in the IVIgG group versus 4.3% in the non-IVIgG group. The most frequently identified antibody specificities in the serum and the eluate were anti-A or anti-B, followed by anti-D and anti-K. In a test of 46 lots of IVIgG for antibodies against blood group antigens by Garcia et al. (26), anti-A or anti-B was detected in 88% of the lots, and indirect anti-human globulin was detected in 63% of the lots. Because IVIgG preparations, as we have pointed out, are manufactured from large donor plasma pools, they may contain varying amounts of isoagglutinins and other common alloantibodies such as anti-D and anti-K (5,30). The reported titers of isoagglutinins or anti-D in IVIgG preparations range from 0 to 128 (5,31,32) (Table 5).
Table 3. Isohemagglutinins in IVIgG Lots
|No. lots positive|
|Anti-A and anti-B||117||71%||29||59%|
*Two lots of 51 not tested.
Table 4. RBC Alloantibodies in IVIgG Lots
|No evidence of RBC alloantibodies||84||51%||8||17%|
|RBC alloantibodies present||81||49%||39||83%|
*Four lots of 51 not tested.
Table 5. RBC Alloantibodies Tested for and Identified in IVIgG Lots, 1986-1990 and 1991-1992
|No. lots positive|
|Anti-K, anti-D, and anti-Leb||No data||1||3%|
|Anti-K, anti-D, anti-Leb, and anti-C||No data||1||3%|
|Anti-C and anti-D||No data||1||3%|
|Anti-K and unidentifiable||No data||2||5%|
|Anti-D and anti-K||2||2%||6||15%|
|Anti-D and unidentifiable||12||15%||8||21%|
|Anti-D, anti-K, and unidentifiable||No data||12||31%|
|With specific RBC alloantibodies||48||59%||No data|
|Reactive with all cells tested||28||35%||No data|
|Unidentifiable RBC alloantibodies||13||16%||No data|
In addition to IVIgG, other therapeutic immunosuppressive agents that have effects similar to those of IVIgG and are used in bone marrow transplant patients, such as anti-lymphocyte globulin and anti-thymocyte globulin, may create problems during compatibility testing. For instance, in a series of 37 patients studied by Swanson et al., 8 had positive direct antiglobulin tests and positive RBC antibody screens, 15 developed RBC antibodies detectable at room temperature, 4 developed RBC antibodies detectable at 37 degrees Celsisus in albumin, and 33 developed RBC antibodies detectable in the anti-human globulin phase (33). The compatibility problems were resolved by adsorbing the anti-human globulin with RBCs coated with anti-lymphocyte globulin, which allowed the substance in the anti-lymphocyte globulin reacting with the anti-human globulin to be removed.
Hemolytic episodes resulting from passively acquired IgG antibodies are another problem in IVIgG administration, although such episodes are usually mild and self-limiting. For instance, Robertson et al., in their study of 47 bone marrow transplant recipients after IVIgG therapy, observed no significant hemolysis associated with passive transfer of antibodies (25). On the other hand, a recent report by Copelan et al. described two patients who had clinically significant hemolysis after receiving large doses of IVIgG (27). One of these two patients had an acute episode of hemolysis after receiving two lots of IVIgG. Anti-A and anti-D were found in the patient's RBC eluate; and anti-A and anti-D titers of 32 and 2, respectively, were demonstrated in the IVIgG lots. Although low titers of antibodies in IVIgG preparations have been assumed not to cause clinically significant hemolysis in patients (32), the two case reports by Copelan et al. illustrate rare exceptions (27).
Isohemagglutinins pose another problem. We and others have reported previously on the not unexpected presence of isohemagglutinins in IVIgG preparations (24,34). The presence of these antibodies, however, places a heavy burden on the logistics of the blood supply, especially in the case of IVIgG-dependent patients. If such patients are also transfusion dependent, then in most cases they must be given O, Rh- blood. In this regard then, it would be most helpful and logical for the manufacturers of IVIgG preparations to adsorb out these antibodies. However, although the technology to do this is available, such antibody removal is apparently not required by the FDA, according to the manufacturers' representatives.
It is interesting to note that in our 1991 study, roughly 50% of the IVIgG preparations showed no evidence of RBC alloantibodies (24). However, in our more recent review, only 17% of the IVIgG preparations were free of such antibodies. The reason for this is as yet unclear to us.
The list of RBC alloantibodies discussed here is not exhaustive and represents only the extent of our testing. Yet, all the alloantibodies we have discussed cloud the immunohematologic picture and further strain the process of ensuring a safe supply of blood components for transfusion-dependent patients (Tables 4-6).
Table 6. Impact of IVIgG on the Clinical Laboratory and the Transfusion Service
|Patients on IVIgG may test false-positive in IgG-based assays.|
Transfusion service is forced to use elaborate methods to identify RBC alloantibodies.
Transfusion-dependent patients on IVIgG deplete the O, Rh-negative blood inventory.
Our more recent analysis of IVIgG lots showed that other antibodies, such as anti-C, anti-c, and anti-Leb, also further complicate the workup and support of patients dependent simultaneously on IVIgG therapy and transfusions. To handle such a situation effectively, a hospital's transfusion service should be aware of patients receiving IVIgG. In hospitals in which the pharmacy dispenses IVIgG, this is not so easy; information links must be established between the pharmacy and the transfusion service to provide the names of each patient receiving IVIgG, the IVIgG dosage, the lot number, and the date of infusion. However, in other settings in which the transfusion service itself dispenses IVIgG, it is very easy and convenient for the service to keep track of who gets it.
At our institution, empty IVIgG vials are saved by the pharmacy and delivered immediately to the transfusion service each time a patient receives an IVIgG infusion. Our transfusion service then records the lot number and date of infusion of IVIgG in the patient's file. The transfusion service also is usually able to retrieve enough IVIgG from the empty vials to screen the lots for the presence of RBC alloantibodies. Subsequently, when a request for transfusion for any patient on record is received, a rapid cross-check of the transfusion file by the transfusion service staff can alert them to potential serologic problems. Often, the antibody profiles detected in the lots of IVIgG resemble those found in the serum and eluate of patients receiving IVIgG. Therefore, the transfusion service must thoroughly investigate the blood serum antibody profiles of all transplant patients to determine what type of IVIgG solutions should be administered. These steps are valuable in resolving complex serologic problems.
Unavoidably, the clinical laboratory must establish methods and procedures to avoid false-positive pitfalls triggered by IVIgG infusions. On the other hand, the transfusion service must seek out information about patients receiving IVIgG therapy so that appropriate hemotherapeutic schemes can be provided in a timely manner. Consequently, the clinical laboratory and the transfusion service should both be in the information loop regarding patients receiving IVIgG. Furthermore, each IVIgG lot being used should be analyzed to determine what contaminating antibodies may be present and may produce spurious laboratory test results, results that may in turn trigger unwarranted clinical speculation and the ordering of additional but unnecessary therapeutic and diagnostic procedures.
In dealing with IVIgG, the clinical laboratory, pharmacy, transfusion service, attending physician, and nursing staff must all have open channels of communication and information to prevent unnecessary testing and workups in such patients. As stated above, those clinical and transfusion professionals who deal with IgA-deficient patients must be particularly alert when using IVIgG preparations since such preparations may contain various amounts of IgA and since the infusion of IVIgG into an IgA-deficient patient with IgA antibodies can unleash a severe anaphylactic reaction.
Another note of caution relates to allergic transfusion reactions that may ensue during or following a posttransfusion IVIgG infusion. It may happen that a nursing staff will overlook the infusion of IVIgG in such cases and report only that the patient in question received some type of transfusion. Although the transfusion reaction should not be overlooked, knowing that IVIgG was infused would clearly help in understanding the allergic reaction.
Without a doubt, IVIgG therapy is a constantly expanding modality. Patients who receive it do experience beneficial effects, and the list of disease entities in which IVIgG can be tried and administered is currently limitless. In view of this, the clinical laboratory and transfusion service must develop a proactive strategy for dealing with the testing interferences induced by IVIgG infusions.
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