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When is elevated testosterone not testosterone? When it is an immunoassay interfering antibody
When is elevated testosterone not testosterone? When it is an immunoassay interfering antibody A discrepancy between clinical findings and a markedly elevated testosterone (T) level stimulated search to explain this inconsistency. The cause of the falsely elevated T level was determined to be heterophile antibodies from a polyclonal gammopathy in a subject with acute myelogenous leukemia. (Fertil Steril 2008;90:886–8. 2008 by American Society for Reproductive Medicine.)
Falsely elevated hormone levels have resulted in erroneous medical decisions. The classic falsely elevated hormone is serum hCG. False-positive hCG levels have resulted in a false assumption of persistent trophoblastic tissue, which resulted in aggressive medical and surgical therapies (1, 2). Laboratory data supplements one’s clinical findings, and when there is significant discrepancy between the two, the physician needs to carefully reassess the entire scenario. A patient with no hirsuitism or virilization, but a markedly elevated testosterone (T) level is described in this report. This falsely elevated T resulted from production of immunoassay, interfering antibodies from polyclonal lymphocyte proliferation with acute myelogenous leukemia. A 31-year-old African-American woman, gravida 1, para 1002, presented with inability to conceive for 3 years and no menses for the past 2 months. Before 2 months ago her menses were regular. The patient denied vasomotor symptoms, fevers, chills, weight change, easy bruising, epistaxis, hemoptysis, hemtochezia, increased hair growth, hair loss, galactorrhea, decreased appetite, chest pain, shortness of breath, or palpitations. She reported mild epigastric discomfort after eating and general complaints of occasional fatigue, weakness, and intermittent low back pain. The patient had no history of exposure to therapeutic or diagnostic immunoglobulins and was not exposed occupationally to animal antigens. Physical examination revealed a normal woman with no evidence of hirsuitism or virilization. The patient’s external genitalia were scarred from a previous infundibulation, with an otherwise normal pelvic examination. Prolactin and thyroid function tests were normal. Total T level was elevated at 1,235 ng/dL with no significant change with dilution. A subsequent T level 3 days later remained elevated at 1,458 ng/dL. One week later a T sample was sent to a different laboratory as well as repeated in our laboratory. In the other laboratory the T result was 77 ng/dL, but in our laboratory
Received June 29, 2007. Reprint requests: Keith Hansen, M.D., Department of Obstetrics and Gynecology, Sanford School of Medicine of the University of South Dakota, 1500 West 22 Street, Sioux Falls, SD 57105 (FAX: 605-357-1528; E-mail: [email protected]).
886
the result remained elevated at 1,472 ng/dL. Estradiol was also elevated at 1,858 pg/mL. Serum protein electrophoresis (immunofixation electrophoresis) demonstrated a pattern consistent with polyclonal proliferation, with an elevated gamma globulin of 2.0 g/dL (0.6–1.7 g/dL) but a normal albumin, a and b globulin. Immunofixation electrophoresis further revealed elevated IgG at 1,893 mg/dL (700–1,600 mg/dL), elevated k of 52.2 mg/dL (3.3–19.4 mg/dL), and elevated k/l ratio of 2.46 (0.26–2.00). A complete blood count revealed an elevated white count of 45.6 K/mL with a hemoglobin of 13.3 g/dL and platelet count of 251 K/mL. The white blood cell differential demonstrated 31% blasts, which suggested an acute leukemia. The lymphoproliferative panel as well as the bone marrow aspiration revealed 50% blasts, B-cell precursor ALL, blasts positive for CD19, CD13, CD33, CD34, TDT, CD10, and CD45. Further laboratory testing revealed that the patient had acute myelocytic leukemia, which was Philadelphia chromosome positive. It was postulated that the patient’s elevated Tand E2 levels were due to antibodies produced by polyclonal proliferation of lymphocytes that interfered with the immunoassays (3, 4). To investigate this possibility, patient and control sera were immunoprecipitated with protein G–sepharose and reassayed by the same laboratory. Removal of IgG from sera was confirmed by Western blot. Based on densitometry of immunoblots, it was estimated that protein G immunoprecipitation removed greater than 80% of IgG from the patient and control sera. Samples of the patient’s blood were taken before and after chemotherapy and treated in the same manner. Immunoprecipitation did not affect immunoassay results for control serum. In contrast, removal of IgG from the patient’s sera reduced total T values by >90%, and E2 values by >75% (Table 1). These results indicated that IgG in the patient’s sera caused immunoassay interference leading to spurious laboratory results.
IMMUNOASSAY Immunoassays for E2 and total T were run on the DPC Immulite 2000 system (Diagnostic Products Corporation, Los
Fertility and Sterility Vol. 90, No. 3, September 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.07.1295
TABLE 1 Immunoassay results of patient and pooled control sera before and after protein G immunoprecipitation. Neat sera
Patient Before chemo After chemo Control
Protein G immunoprecipitated sera
E2
T
E2
T
1,309 pg/mL 1,474 pg/mL 54 pg/mL
1,232 ng/dL 1,055 ng/dL <20 ng/dL
312 pg/mL 185 pg/mL 50 pg/mL
80 ng/dL 65 ng/dL <20 ng/dL
Ramaeker. False-positive elevated testosterone. Fertil Steril 2008.
Angeles, CA). Neat sera and protein G-stripped sera from patient and control were assayed in a single run. This assay is a solid phase chemiluminescence assay. The solid phase is composed of polystyrene beads coated with polyclonal rabbit antibodies specific for E2 and T. The test sample is added to the solid phase with alkaline phosphatase-conjugated E2 or T reagents. The E2 or T in the samples than compete with the alkaline phosphatase-conjugated E2 or T for the limited number of specific antibody-binding sites on the solid phase. The reaction is then terminated by washing followed by the addition of adamantyl dioxetane. The adamantyl dioxetane is hydrolyzed by alkaline phosphatase, which results in the production of light, which is indirectly proportional to the amount of hormone in the sample. For the T assay, analytical sensitivity is 15 ng/dL. Crossreactivity with other androgens is %2.0%, and negligible for other steroids. Interassay and intra-assay coefficients of variation are <15% within the linear range of the assay. For the E2 assay, analytical sensitivity is 15 pg/mL. Crossreactivity with other naturally occurring steroids is %1.0%, and interassay and intra-assay coefficients of variation are <10%.
IMMUNOPRECIPITATION Protein G-coated sepharose (1.0 mL; GE Healthcare/Amersham, Piscataway, NJ) was washed three times in Dulbecco’s phosphate-buffered saline (PBS) containing 10% (v/v) serum protein supplement (Sage IVF, Trumbull, CT). Patient serum (0.75 mL) and control sera (pooled sera from three random subjects) were incubated with the washed protein G–sepharose for 1 hour at 4 C with gentle rocking, then centrifuged at 104 g for 2 minutes. The supernates were collected and filtered through 0.8 mm Acrodisc syringe filters (Pall Life Sciences, Ann Arbor, MI) to remove any suspended sepharose. The sepharose pellets were washed twice in PBS þ serum protein supplement and then resuspended in 2 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) reducing buffer (0.3 mL) to elute bound IgG. Fertility and Sterility
IMMUNOBLOTTING Western blotting was used to confirm that the immunoprecipitation had removed IgG from sera. Eluates (0.2 ng protein/10 mL) were electrophoresed on 12% Tris-HCl gels (BioRad, Hercules, CA) and then transferred to PVDF membranes (Biotrace, Bridgend, United Kingdom). Membranes were blocked with 5% ECL Blocking Reagent (GE Healthcare/Amersham) in TBS þ 0.5% Tween-20, then incubated with peroxidase-conjugated mouse monoclonal antihuman IgG (1:500; Zymed Laboratories, San Francisco, CA) for 2 hours at room temperature with gentle agitation, and then overnight at 4 C without agitation. After three washes, blots were incubated with Lumi-Light PLUS Blotting Substrate (Roche Diagnostics, Indianapolis, IN). Chemiluminescence was visualized on a BioRad ChemiDoc XRS Gel Documentation system. This case demonstrated the importance of further inquiry when a discrepancy is discovered between clinical and laboratory findings. The markedly elevated T and E2 levels without clinical signs consistent with these levels prompted suspicion of a laboratory error or antibody interference in the immunoassay. ‘‘Heterophile antibodies’’ are ‘‘a group of antibodies exhibiting multispecificity that react with heterogenous antigens.’’(5) Many investigators believe that these antibodies arise from the natural formation of multispecific antibodies or by immunization with a foreign animal antigen. In the latter case the foreign antigen results in an immune response creating human anti-animal antibodies. These antibodies can then cross-react in immunoassays where the assay antibodies were made in the same animal to which the individual was immunized. Human anti-animal antibodies can occur in individuals with exposure to therapeutic or diagnostic immunoglobulins as well as exposure to occupational animal antigens. It is estimated that heterophilic antibodies can interfere with 0.01%–30% of immunoassays depending on the type of assay (6). Methods that can be used to prescreen for interference include serial dilution, pretreatment of the sample with
887
heterophile blocking reagent, or mouse antibody-negative control reaction. Prescreening is often not used and not believed to be warranted because of the low frequency of abnormalities and inability of a single method to be affective for all immunoassays (3). It is suggested that screening for immunoassay interference should occur when there is an inconsistency between clinical and laboratory data. With recognition of immunoassay interference it is crucial to determine the cause of the interference. There are multiple ways that anti-immunoglobulin antibodies can develop including infectious mononucleosis, autoantibodies such as rheumatoid factor, monoclonal gammopathies, and exposure to animal antigens in the form of diagnostic or therapeutic immunoglobulins (4, 7). In this case, the patient’s discrepant clinical findings and laboratory data stimulated a search for the unexplained inconsistency. Subsequent laboratory data revealed a polyclonal gammopathy and a strikingly elevated white blood cell count, which suggested a potential source of interference. The patient’s acute myelogenous leukemia resulted in a polyclonal lymphocyte expansion with the production of multispecific immunoglobulins. These multispecific immunoglobulins presumably blocked the capture antibody in the assay, which resulted in less alkaline phosphatase-conjugated antigen bound to the capture antibody and artificially elevated the assay readings for E2 and T. This case is an example of a rare but serious and potentially fatal cause of interference in immunoassays. Devon Ramaeker, M.D.a John Brannian, Ph.D.b
888
Ramaeker et al.
Correspondence
Kristi Egland, Ph.D.b Kelly McCaul, M.D.c Keith Hansen, M.D.b a Department of Obstetrics and Gynecology, Magee-Women’s Hospital, University of Pittsburgh, Pittsburgh, Pennsylvania; b Department of Obstetrics and Gynecology, Sanford School of Medicine of the University of South Dakota and Sanford Research/USD, Sioux Falls, South Dakota; and c Avera Hematology and Transplant, Avera Cancer Institute, Avera McKennan Hospital and University Health Center, Sioux Falls, South Dakota
REFERENCES 1. Rotmensch S, Cole LA. False diagnosis and needless therapy of presumed malignant disease in women with false-positive human chorionic gonadotropin concentrations. Lancet 2000;355:712–5. 2. Bjerner J, Bormer O, Nustad K. The war on heterophilic antibody interference. Clin Chem 2005;51:9–11. 3. Emerson J, Ngo G, Emerson S. Screening for interference in immunoassays. Clin Chem 2003;49:1163–9. 4. Hennig C, Rink L, Fagin U, Wolfram J, Kirchner H. The influence of naturally occurring heterophilic anti-immunoglobulin antibodies on direct measurement of serum proteins using sandwich ELISAs. J Immunol Methods 2000;253:71–80. 5. Kaplan RV, Levinson SS. When is a heterophilic antibody not a heterophile antibody? When it is an antibody against a specific immunogen. Clin Chem 1999;45:616–8. 6. Stesterig D. Understanding and overcoming heterophilic antibody interference in immunoassays. Diagnostic Products Corporation, 2000. 7. Kairemo K, Kahn J, Taipale P. Monoclonal gammopathy may disturb oestradiol measurement in the treatment and monitoring of in-vitro fertilization. Hum Reprod 1999;14:2724–6.
Vol. 90, No. 3, September 2008
Falsely elevated hormone levels have resulted in erroneous medical decisions. The classic falsely elevated hormone is serum hCG. False-positive hCG levels have resulted in a false assumption of persistent trophoblastic tissue, which resulted in aggressive medical and surgical therapies (1, 2). Laboratory data supplements one’s clinical findings, and when there is significant discrepancy between the two, the physician needs to carefully reassess the entire scenario. A patient with no hirsuitism or virilization, but a markedly elevated testosterone (T) level is described in this report. This falsely elevated T resulted from production of immunoassay, interfering antibodies from polyclonal lymphocyte proliferation with acute myelogenous leukemia. A 31-year-old African-American woman, gravida 1, para 1002, presented with inability to conceive for 3 years and no menses for the past 2 months. Before 2 months ago her menses were regular. The patient denied vasomotor symptoms, fevers, chills, weight change, easy bruising, epistaxis, hemoptysis, hemtochezia, increased hair growth, hair loss, galactorrhea, decreased appetite, chest pain, shortness of breath, or palpitations. She reported mild epigastric discomfort after eating and general complaints of occasional fatigue, weakness, and intermittent low back pain. The patient had no history of exposure to therapeutic or diagnostic immunoglobulins and was not exposed occupationally to animal antigens. Physical examination revealed a normal woman with no evidence of hirsuitism or virilization. The patient’s external genitalia were scarred from a previous infundibulation, with an otherwise normal pelvic examination. Prolactin and thyroid function tests were normal. Total T level was elevated at 1,235 ng/dL with no significant change with dilution. A subsequent T level 3 days later remained elevated at 1,458 ng/dL. One week later a T sample was sent to a different laboratory as well as repeated in our laboratory. In the other laboratory the T result was 77 ng/dL, but in our laboratory
Received June 29, 2007. Reprint requests: Keith Hansen, M.D., Department of Obstetrics and Gynecology, Sanford School of Medicine of the University of South Dakota, 1500 West 22 Street, Sioux Falls, SD 57105 (FAX: 605-357-1528; E-mail: [email protected]).
886
the result remained elevated at 1,472 ng/dL. Estradiol was also elevated at 1,858 pg/mL. Serum protein electrophoresis (immunofixation electrophoresis) demonstrated a pattern consistent with polyclonal proliferation, with an elevated gamma globulin of 2.0 g/dL (0.6–1.7 g/dL) but a normal albumin, a and b globulin. Immunofixation electrophoresis further revealed elevated IgG at 1,893 mg/dL (700–1,600 mg/dL), elevated k of 52.2 mg/dL (3.3–19.4 mg/dL), and elevated k/l ratio of 2.46 (0.26–2.00). A complete blood count revealed an elevated white count of 45.6 K/mL with a hemoglobin of 13.3 g/dL and platelet count of 251 K/mL. The white blood cell differential demonstrated 31% blasts, which suggested an acute leukemia. The lymphoproliferative panel as well as the bone marrow aspiration revealed 50% blasts, B-cell precursor ALL, blasts positive for CD19, CD13, CD33, CD34, TDT, CD10, and CD45. Further laboratory testing revealed that the patient had acute myelocytic leukemia, which was Philadelphia chromosome positive. It was postulated that the patient’s elevated Tand E2 levels were due to antibodies produced by polyclonal proliferation of lymphocytes that interfered with the immunoassays (3, 4). To investigate this possibility, patient and control sera were immunoprecipitated with protein G–sepharose and reassayed by the same laboratory. Removal of IgG from sera was confirmed by Western blot. Based on densitometry of immunoblots, it was estimated that protein G immunoprecipitation removed greater than 80% of IgG from the patient and control sera. Samples of the patient’s blood were taken before and after chemotherapy and treated in the same manner. Immunoprecipitation did not affect immunoassay results for control serum. In contrast, removal of IgG from the patient’s sera reduced total T values by >90%, and E2 values by >75% (Table 1). These results indicated that IgG in the patient’s sera caused immunoassay interference leading to spurious laboratory results.
IMMUNOASSAY Immunoassays for E2 and total T were run on the DPC Immulite 2000 system (Diagnostic Products Corporation, Los
Fertility and Sterility Vol. 90, No. 3, September 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.07.1295
TABLE 1 Immunoassay results of patient and pooled control sera before and after protein G immunoprecipitation. Neat sera
Patient Before chemo After chemo Control
Protein G immunoprecipitated sera
E2
T
E2
T
1,309 pg/mL 1,474 pg/mL 54 pg/mL
1,232 ng/dL 1,055 ng/dL <20 ng/dL
312 pg/mL 185 pg/mL 50 pg/mL
80 ng/dL 65 ng/dL <20 ng/dL
Ramaeker. False-positive elevated testosterone. Fertil Steril 2008.
Angeles, CA). Neat sera and protein G-stripped sera from patient and control were assayed in a single run. This assay is a solid phase chemiluminescence assay. The solid phase is composed of polystyrene beads coated with polyclonal rabbit antibodies specific for E2 and T. The test sample is added to the solid phase with alkaline phosphatase-conjugated E2 or T reagents. The E2 or T in the samples than compete with the alkaline phosphatase-conjugated E2 or T for the limited number of specific antibody-binding sites on the solid phase. The reaction is then terminated by washing followed by the addition of adamantyl dioxetane. The adamantyl dioxetane is hydrolyzed by alkaline phosphatase, which results in the production of light, which is indirectly proportional to the amount of hormone in the sample. For the T assay, analytical sensitivity is 15 ng/dL. Crossreactivity with other androgens is %2.0%, and negligible for other steroids. Interassay and intra-assay coefficients of variation are <15% within the linear range of the assay. For the E2 assay, analytical sensitivity is 15 pg/mL. Crossreactivity with other naturally occurring steroids is %1.0%, and interassay and intra-assay coefficients of variation are <10%.
IMMUNOPRECIPITATION Protein G-coated sepharose (1.0 mL; GE Healthcare/Amersham, Piscataway, NJ) was washed three times in Dulbecco’s phosphate-buffered saline (PBS) containing 10% (v/v) serum protein supplement (Sage IVF, Trumbull, CT). Patient serum (0.75 mL) and control sera (pooled sera from three random subjects) were incubated with the washed protein G–sepharose for 1 hour at 4 C with gentle rocking, then centrifuged at 104 g for 2 minutes. The supernates were collected and filtered through 0.8 mm Acrodisc syringe filters (Pall Life Sciences, Ann Arbor, MI) to remove any suspended sepharose. The sepharose pellets were washed twice in PBS þ serum protein supplement and then resuspended in 2 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) reducing buffer (0.3 mL) to elute bound IgG. Fertility and Sterility
IMMUNOBLOTTING Western blotting was used to confirm that the immunoprecipitation had removed IgG from sera. Eluates (0.2 ng protein/10 mL) were electrophoresed on 12% Tris-HCl gels (BioRad, Hercules, CA) and then transferred to PVDF membranes (Biotrace, Bridgend, United Kingdom). Membranes were blocked with 5% ECL Blocking Reagent (GE Healthcare/Amersham) in TBS þ 0.5% Tween-20, then incubated with peroxidase-conjugated mouse monoclonal antihuman IgG (1:500; Zymed Laboratories, San Francisco, CA) for 2 hours at room temperature with gentle agitation, and then overnight at 4 C without agitation. After three washes, blots were incubated with Lumi-Light PLUS Blotting Substrate (Roche Diagnostics, Indianapolis, IN). Chemiluminescence was visualized on a BioRad ChemiDoc XRS Gel Documentation system. This case demonstrated the importance of further inquiry when a discrepancy is discovered between clinical and laboratory findings. The markedly elevated T and E2 levels without clinical signs consistent with these levels prompted suspicion of a laboratory error or antibody interference in the immunoassay. ‘‘Heterophile antibodies’’ are ‘‘a group of antibodies exhibiting multispecificity that react with heterogenous antigens.’’(5) Many investigators believe that these antibodies arise from the natural formation of multispecific antibodies or by immunization with a foreign animal antigen. In the latter case the foreign antigen results in an immune response creating human anti-animal antibodies. These antibodies can then cross-react in immunoassays where the assay antibodies were made in the same animal to which the individual was immunized. Human anti-animal antibodies can occur in individuals with exposure to therapeutic or diagnostic immunoglobulins as well as exposure to occupational animal antigens. It is estimated that heterophilic antibodies can interfere with 0.01%–30% of immunoassays depending on the type of assay (6). Methods that can be used to prescreen for interference include serial dilution, pretreatment of the sample with
887
heterophile blocking reagent, or mouse antibody-negative control reaction. Prescreening is often not used and not believed to be warranted because of the low frequency of abnormalities and inability of a single method to be affective for all immunoassays (3). It is suggested that screening for immunoassay interference should occur when there is an inconsistency between clinical and laboratory data. With recognition of immunoassay interference it is crucial to determine the cause of the interference. There are multiple ways that anti-immunoglobulin antibodies can develop including infectious mononucleosis, autoantibodies such as rheumatoid factor, monoclonal gammopathies, and exposure to animal antigens in the form of diagnostic or therapeutic immunoglobulins (4, 7). In this case, the patient’s discrepant clinical findings and laboratory data stimulated a search for the unexplained inconsistency. Subsequent laboratory data revealed a polyclonal gammopathy and a strikingly elevated white blood cell count, which suggested a potential source of interference. The patient’s acute myelogenous leukemia resulted in a polyclonal lymphocyte expansion with the production of multispecific immunoglobulins. These multispecific immunoglobulins presumably blocked the capture antibody in the assay, which resulted in less alkaline phosphatase-conjugated antigen bound to the capture antibody and artificially elevated the assay readings for E2 and T. This case is an example of a rare but serious and potentially fatal cause of interference in immunoassays. Devon Ramaeker, M.D.a John Brannian, Ph.D.b
888
Ramaeker et al.
Correspondence
Kristi Egland, Ph.D.b Kelly McCaul, M.D.c Keith Hansen, M.D.b a Department of Obstetrics and Gynecology, Magee-Women’s Hospital, University of Pittsburgh, Pittsburgh, Pennsylvania; b Department of Obstetrics and Gynecology, Sanford School of Medicine of the University of South Dakota and Sanford Research/USD, Sioux Falls, South Dakota; and c Avera Hematology and Transplant, Avera Cancer Institute, Avera McKennan Hospital and University Health Center, Sioux Falls, South Dakota
REFERENCES 1. Rotmensch S, Cole LA. False diagnosis and needless therapy of presumed malignant disease in women with false-positive human chorionic gonadotropin concentrations. Lancet 2000;355:712–5. 2. Bjerner J, Bormer O, Nustad K. The war on heterophilic antibody interference. Clin Chem 2005;51:9–11. 3. Emerson J, Ngo G, Emerson S. Screening for interference in immunoassays. Clin Chem 2003;49:1163–9. 4. Hennig C, Rink L, Fagin U, Wolfram J, Kirchner H. The influence of naturally occurring heterophilic anti-immunoglobulin antibodies on direct measurement of serum proteins using sandwich ELISAs. J Immunol Methods 2000;253:71–80. 5. Kaplan RV, Levinson SS. When is a heterophilic antibody not a heterophile antibody? When it is an antibody against a specific immunogen. Clin Chem 1999;45:616–8. 6. Stesterig D. Understanding and overcoming heterophilic antibody interference in immunoassays. Diagnostic Products Corporation, 2000. 7. Kairemo K, Kahn J, Taipale P. Monoclonal gammopathy may disturb oestradiol measurement in the treatment and monitoring of in-vitro fertilization. Hum Reprod 1999;14:2724–6.
Vol. 90, No. 3, September 2008