Particle-counting immunoassay (PACIA) of pregnancy-specific beta 1-glycoprotein, a possible marker of various malignancies and Crohn's ileitis

Pregnancy-specific beta 1-glycoprotein (SP1) was assayed by particle-counting immunoassay (PACIA) with a sensitivity of 1 microgram/L. In serum from 50 men, the SP1 concentration was less than 1 microgram/L, whereas three of the specimens from 46 nonpregnant women had values exceeding 1 microgram/L. In 29% of 950 consecutive patients' sera, SP1 concentrations exceeded 1 microgram/L--in sarcoma (six of six), in malignant hemopathies (101/127, 80%) such as myeloma (20/26, 92%) and acute myeloblastic leukemia (23/27, 90%), and in various other types of cancer (11/19, 58%) except for bronchial epithelioma, which did not lead to any significant increase of SP1 in the five patients examined. The concentration of SP1 was also frequently increased in patients with Crohn's ileitis (28/43, 65%) but not in patients with other inflammatory disorders.     

Elimination of the need for urine studies in the screening algorithm for monoclonal gammopathies by using serum immunofixation and free light chain assays

OBJECTIVE: To determine the relative diagnostic contribution of urine assays as part of the screening algorithm for monoclonal gammopathies. PATIENTS AND METHODS: We identified 428 patients with a monoclonal gammopathy and monoclonal urinary protein at initial diagnosis of plasma cell dyscrasia who had also undergone serum immunofixation and serum free light chain quantitation within 30 days of diagnosis. The laboratory results for serum protein electrophoresis, serum immunofixation, serum free light chain, urine protein electrophoresis, and urine immunofixation were reviewed. RESULTS: The patients had diagnoses of multiple myeloma, primary amyloid, monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, solitary plasmacytomas, and other less frequently detected monoclonal gammopathies. All 428 had a monoclonal urine protein, 85.7% had an abnormal serum free light chain kappa/lambda ratio, 80.8% had an abnormal serum protein electrophoresis, and 93.5% had an abnormal serum immunofixation result. All 3 serum assays were normal in only 2 patients, 1 of whom had monoclonal gammopathy of undetermined significance (idiopathic Bence Jones proteinuria) and 1 whose urine sample contained an intact monoclonal immunoglobulin but whose serum and subsequent urine samples showed no evidence of a monoclonal gammopathy. CONCLUSION: Discontinuation of urine studies and reliance on a diagnostic algorithm using only serum studies (protein electrophoresis, immunofixation, and free light chain quantitation) missed 2 (0.5%) of the 428 monoclonal gammopathies with urinary monoclonal proteins, and these 2 cases required no medical intervention.     

Practical considerations for the measurement of free light chains in serum

BACKGROUND: A new immunoassay for free light chain measurements has been reported to be useful for the diagnosis and monitoring of monoclonal light chain diseases and nonsecretory myeloma. We describe experience with and some potential pitfalls of the assay. METHODS: The assay was assessed for precision, sample type and stability, recovery, and harmonization of results between two analyzers on which the reagents are used. Free-light-chain concentrations were measured in healthy individuals (to determine biological variation), patients with monoclonal gammopathy of undetermined significance, myeloma patients after autologous stem cell transplants, and patients with renal disease. RESULTS: Analytical imprecision (CV) was 6-11% for kappa and lambda free-light-chain measurement and 16% for the calculated kappa/lambda ratio. Biological variation was generally insignificant compared with analytical variation. Despite the same reagent source, values were not completely harmonized between assay systems and may produce discordant free-light-chain ratios. In some patients with clinically stable myeloma, or post transplantation, or with monoclonal gammopathy of undetermined significance, free-light-chain concentration and ratio were within the population reference interval despite the presence of monoclonal intact immunoglobulin in serum. In other patients with monoclonal gammopathy of undetermined significance, values were abnormal although there was no clinical evidence of progression to multiple myeloma. CONCLUSIONS: The use of free-light-chain measurements alone cannot differentiate some groups of patients with monoclonal gammopathy from healthy individuals. As with the introduction of any new test, it is essential that more scientific data about use of this assay in different subject groups are available so that results can be interpreted with clinical certainty.     

The predictive power of serum kappa/lambda ratios for discrimination between monoclonal gammopathy of undetermined significance and multiple myeloma.

The predictive power of serum kappa/lambda ratios on initial presentation of immunoglobulin G (IgG) or IgA monoclonal component was studied to differentiate between monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients. The retrospective study involved 145 patients clinically diagnosed with monoclonal gammopathy of undetermined significance or multiple myeloma, who had serum M-protein IgG <35 g/L or IgA <20 g/L at M-protein detection. Serum light chains kappa and lambda were measured by fixed-time nephelometry. Test performance indices, predictive values and likelihood ratios were calculated according to the Weissler recommendation. MM patients were considered as diseased and MGUS patients as non-diseased in order to estimate the performance characteristics of serum kappa/lambda ratios. There was a statistically significant difference in kappa/lambda ratios distribution between both groups of patients, in both M-protein kappa-type (Mann-Whitney U=168, p<0.001) and in M-protein lambda-type (Mann-Whitney U=143, p<0.001). Negative likelihood ratios at threshold levels of 0.6 and 4.2 were 2.17- and 3.32-fold greater, respectively, than positive likelihood ratios, so that the predictive power of a serum kappa/lambda ratio within these limits is better in ruling out (negative predictive power) than ruling in disease (positive predictive power). The post-test characteristics of a serum kappa/lambda ratio interval between 0.6 and 4.2 in discriminating MGUS from MM in our geographic population were: sensitivity 0.96 (0.93-0.99 95% CI); specificity 0.70 (0.63-0.77); positive predictive value 0.68 (0.64-0.73); negative predictive value 0.96 (0.94-0.99); likelihood ratios (+)LR 3.23 (2.68-4.04); and (-)LR 17.16 (11.00-63.00). Thus, serum M-protein with a kappa/lambda ratio between 0.6 and 4.2 increases the posterior probability of MGUS from 0.60 to 0.96 in asymptomatic patients, for whom only monitoring may be suggested when the serum kappa/lambda ratio is within these limits.     

A case of hook effect in the serum free light chain assay using the Olympus AU400e

We describe a case where a woman with an IgG lambda monoclonal gammopathy had an undetectable serum free lambda light chain due to antigen excess. The patient had a three year history of multiple myeloma and initially presented with an elevated serum free lambda light chain. The serum free lambda light chain concentration increased over the course of several months to >4000 mg/L (reference interval 5.7-26.3 mg/L) and then suddenly dropped to <3.0 mg/L. Paradoxically, during this time the monoclonal IgG lambda concentration measured using serum protein electrophoresis increased from 44 to 59 g/dL. Serial dilution of the patient's serum specimen revealed that the serum free lambda light chain was actually 3130 mg/L. This case represents an example of antigen excess or 'hook effect' using the serum free light chain assay.     

Two-site monoclonal antibody-based immunoradiometric assay for measuring prostate secretory protein in serum.

We developed a double-determinant immunoradiometric assay for measuring serum prostate secretory protein (PSP), using monoclonal antibodies (MAb) against two different epitopes: MAb PSP-19 was the capture antibody and MAb PSP-6 was the tracer antibody. Assay sensitivity was 0.1 microgram/L. Analytical recovery of PSP was 93.5-104.6%, whereas the intra- and interassay mean CVs were 4.2% and 6.9%, respectively. In 92 normal men, ages greater than 50 years, the mean PSP concentration was 5.7 micrograms/L, with 10 (10.9%) men having concentrations greater than 10 micrograms/L. In contrast, 20 of 49 (40.8%) patients with benign prostate hyperplasia (BPH; mean PSP concentration 9.4 micrograms/L) and 46 of 100 (46%) patients with prostate cancer (mean PSP concentration 22.2 micrograms/L) had PSP concentrations greater than 10 micrograms/L. Mean serum PSP concentrations of the BPH (P less than 0.05) and prostate cancer (P less than 0.01) groups were significantly different from those of age-matched normal men. In a small group of patients, serial PSP concentrations correlated with the clinical course during therapy. Thus, PSP may be a useful marker for evaluating patients with prostate cancer.     

Elevation of serum beta 2 microglobulin in liver diseases

beta 2 Microglobulin levels were measured by radioimmunoassay in the serum of 160 patients with liver disease and compared to 63 normal controls and 75 asymptomatic HBs-Ag carriers. All the latter subjects had normal values. Elevated serum beta 2 microglobulin levels were found in most of the other categories: acute viral hepatitis (35/45); chronic persistent (8/26) or active (35/41) hepatitis and liver cirrhosis (27/38). beta 2 Microglobulin values were significantly lower in chronic persistent hepatitis than in the three other groups (p less than 0.05). Steroid therapy was followed by reduction of serum beta 2m levels in 11/11 cases of chronic active hepatitis, eight of whom returned to normal value. Although linked to the course of the disease, variations of beta 2 microglobulin were independent of transaminases, bilirubin and gamma globulins. Elevated serum beta 2 microglobulin correlated with demonstration of rheumatoid factor but not with detection of circulating immune complexes, hepatitis B virus markers or autoantibodies. The results suggest that elevation of serum beta w microglobulin is encountered mostly in the active forms of inflammatory liver diseases.     

Monoclonal protein reference change value as determined by gel-based serum protein electrophoresis

IntroductionThe International Myeloma Working Group recommendations for monitoring disease progression or response include quantitation of the involved monoclonal immunoglobulin. They have defined the minimum change criteria of ≧ 25% with an absolute change of no < 5 g/L for either minimal response or progression. Limited evidence is available to accurately determine the magnitude of change in a monoclonal protein to reflect a true change in clinical status. Here we determined the analytical and biological variability of monoclonal proteins in stable monoclonal gammopathy of undetermined significance (MGUS) patients.MethodAnalytical variability (CVa) of normal protein fractions and monoclonal proteins were assessed agarose gel-based serum protein electrophoresis. Sixteen clinically stable MGUS patients were identified from our clinical hematology database. Individual biological variability (CVi) was determined and used to calculate a monoclonal protein reference change value (RCV).ResultAnalytical variability of the normal protein fractions (albumin, alpha-1, alpha-2, beta, total gamma) ranged from 1.3% for albumin to 5.8% for the alpha-1 globulins. CVa of low (5.6 g/L) and high (32.2 g/L) concentration monoclonal proteins were 3.1% and 22.2%, respectively. Individual CVi of stable patients ranged from 3.5% to 24.5% with a CVi of 12.9%. The reference change value (RCV) at a 95% probability was determined to be 36.7% (low) 39.6% (high) using our CVa and CVi.ConclusionsSerial monitoring of monoclonal protein concentration is important for MGUS and multiple myeloma patients. Accurate criteria for interpreting a change in monoclonal protein concentration are required for appropriate decision making. We used QC results and real-world conditions to assess imprecision of serum protein fractions including low and high monoclonal protein fractions and clinically stable MGUS patients to determine CVi and RCV. The calculated RCVs of 36.7% (low) and 39.6% (high) in this study were greater that reported previously and greater than the established criteria for relapse. Response criteria may be reassessed to increase sensitivity and specificity for detection of response.     

A radioimmunoassay of the pregnancy-specific beta1-glycoprotein (SP1).

A radioimmunological method for determination of the pregnancy specific beta1-glycoprotein (SP1) is described. Antigen specificity of rabbit anti-human SP1 serum (Behringwerke) was investigated against human placental and pituitary hormones. The sensitivity was about 10 microgram/l. The concentration ranges of SP1 in umbilical cord blood and amniotic fluid were 0.10--0.60 mg/l and 0.3--3.8 mg/l, respectively. A good correlation (r = +0.95) was found between the radioimmunological and electroimmunological methods for determination of SP1 in maternal blood.     

Understanding and interpreting serum protein electrophoresis

Serum protein electrophoresis is used to identify patients with multiple myeloma and other serum protein disorders. Electrophoresis separates proteins based on their physical properties, and the subsets of these proteins are used in interpreting the results. Plasma protein levels display reasonably predictable changes in response to acute inflammation, malignancy, trauma, necrosis, infarction, burns, and chemical injury. A homogeneous spike-like peak in a focal region of the gamma-globulin zone indicates a monoclonal gammopathy. Monoclonal gammopathies are associated with a clonal process that is malignant or potentially malignant, including multiple myeloma, Waldenstrom's macroglobulinemia, solitary plasmacytoma, smoldering multiple myeloma, monoclonal gammopathy of undetermined significance, plasma cell leukemia, heavy chain disease, and amyloidosis. The quantity of M protein, the results of bone marrow biopsy, and other characteristics can help differentiate multiple myeloma from the other causes of monoclonal gammopathy. In contrast, polyclonal gammopathies may be caused by any reactive or inflammatory process.     

Underestimation of monoclonal proteins by serum protein electrophoresis on cellulose acetate

Our study of 95 serum samples from 37 patients with monoclonal gammopathy revealed distorted irregular monoclonal (M) protein bands after serum protein electrophoresis (SPE) on cellulose acetate membrane. In 71 (75%) of the 95 sera, the M-protein was underestimated and the albumin concentration overestimated. Dilution of the serum sample before SPE eliminated the abnormality of the M-protein bands. By SPE, the mean albumin concentration in these 71 undiluted sera was 45.8 (SD 7.4) g/L vs 37.9 (SD 5.8) g/L for the diluted sera; moreover, this was true of individual samples: measured albumin concentration in each diluted serum sample was always less than in the undiluted serum. As measured by the bromcresol green dye-binding method, the albumin concentration was 32.8 (SD 5.9) g/L. Similarly, the M-protein concentration in SPE was 49.5 (SD 12.3) g/L for the undiluted sera vs 61.8 (SD 15.1) g/L for the diluted sera, and the M-protein concentration in each diluted serum sample always exceeded that in the undiluted serum. Underestimation of M-protein limits the usefulness of M-protein measurement in evaluating the patient's response to therapy and for early detection of disease progression. SPE strips should be carefully inspected visually, and sera with M-protein band abnormalities should be diluted and re-assayed if SPE is to quantify concentrations of M-protein and albumin accurately.     

Radioimmunoassays of human myoglobin in serum and urine.

Two solid-phase radioimmunoassays have been developed for the detection of myoglobin in serum and urine. The sensitivity of the methods is 0.1 and 0.5 microgram/l, respectively, with a coefficient of variation of the respective method of 7-8%. The mean serum concentration of myoglobin in ninety-nine healthy blood donors was 44.3 microgram/l +/- 18.0 microgram/l (SD) with a significant difference (P less than 0.001) between men (50.6 +/- 19.8) and women (35.7 +/- 10.4). Serum myoglobin was positively correlated to age (P less than 0.05), body weight (P less than 0.02), serum creatine kinase (P less than 0.001), and serum creatinine (P less than 0.001) to galactose elimination rate. Serum myoglobin levels were not influenced by exhaustive short time dynamic exercise. The mean urinary excretion of myoglobin in twenty-four healthy students was 1.2 microgram/24 h (range 0.1-4 microgram/24 h). Myoglobin excretion was correlated to excretion of beta 2-microglobulin (P less than 0.02) but not to serum levels of myoglobin. No indications of circulating antibodies to myoglobin were obtained when assaying sixty-seven rheumatoid arthritis and thirteen myastenia gravis sera. Presence of other myoglobin binding substances in serum, which would interfere with the assays also seemed unlikely. Determination of myoglobin in serum by sensitive and specific method might be of clinical value in the diagnosis of diseases involving muscle tissues.     

Multiple myeloma: recognition and management

Multiple myeloma is the malignant proliferation of plasma cells involving more than 10 percent of the bone marrow. The multiple myeloma cell produces monoclonal immunoglobulins that may be identified on serum or urine protein electrophoresis. Bone pain related to multiple lytic lesions is the most common clinical presentation. However, up to 30 percent of patients are diagnosed incidentally while being evaluated for unrelated problems, and one third of patients are diagnosed after a pathologic fracture, commonly of the axial skeleton. Multiple myeloma must be differentiated from other causes of monoclonal gammopathy, including monoclonal gammopathy of undetermined significance, heavy chain disease, plasmacytoma and Waldenstrom macroglobulinemia. Chemotherapy with melphalan-prednisone is the standard treatment for multiple myeloma. Other treatment modalities include polychemotherapy and bone marrow transplantation. Only 50 to 60 percent of patients respond to therapy. The aggregate median survival for all stages of multiple myeloma is three years.     

Ultrasensitive radioimmunoassay of prostate-specific antigen.

We describe a modification of the Yang Pros-check radioimmunoassay for prostate-specific antigen (PSA) that increases the analytical sensitivity of the assay approximately threefold (from a working range of 0.3-50 to 0.1-1.2 micrograms/L). It can detect PSA added to zero-concentration diluent (bovine serum albumin solution) at 0.10 microgram/L or added to zero-concentration control female serum at 0.20 microgram/L (P less than 0.05). In 26 patients tested after cystoprostatectomy for bladder cancer (who had normal prostates without cancer on histologic examination), PSA values by this ultrasensitive assay were all less than 0.10 microgram/L. Therefore, we propose this value as the upper limit of the 95% reference interval. In a retrospective study of two patients who developed recurrent prostate cancer, serum PSA values increased above the 0.1 microgram/L detection limit 175 and 581 days before increasing above the 0.3 microgram/L detection limit of the standard Yang assay. This ultrasensitive radioimmunoassay of PSA should prove more useful than current methods for detecting early recurrence of prostate cancer.     

Diagnostic performance of quantitative kappa and lambda free light chain assays in clinical practice

BACKGROUND: The quantitative assay for free light chains (FLCs) is a recently introduced commercial test reported to be sensitive and specific for detecting FLC diseases such as primary systemic amyloidosis (AL), light chain deposition disease (LCDD), nonsecretory multiple myeloma (NSMM), and light chain multiple myeloma. We evaluated its diagnostic performance in clinical practice. METHODS: All FLC clinical test results generated in 2003 were abstracted from the Laboratory Information System. Diagnoses were obtained from the Dysproteinemia database and the patient medical history. RESULTS: In 2003, we received samples for FLC assays from 1020 Mayo Clinic patients. The majority of these patients (88%) had bone marrow-derived monoclonal plasma cell disorders (PCDs). The 121 patients who did not have monoclonal gammopathy all had FLC kappa/lambda ratios within the range of values obtained for a reference population in our laboratory. Among the patients with monoclonal gammopathies were patients with multiple myeloma (330), AL (269), monoclonal gammopathy of undetermined significance (114), smoldering multiple myeloma (72), plasmacytoma (22), NSMM (20), macroglobulinemia (9), LCDD (7), and a variety of other PCDs. Among the 110 AL patients who had not been previously treated and who had a FLC assay performed within 120 days of diagnosis, the FLC kappa/lambda ratio was positive in 91% compared with 69% for serum immunofixation electrophoresis (IFE) and 83% for urine IFE. The combination of serum IFE and serum FLC assay detected an abnormal result in 99% (109 of 110) of patients with AL. CONCLUSION: The performance of the FLC assay in this analysis of clinical laboratory data is consistent with results from published retrospective validation studies.     

Activity concentration and mass concentration (monoclonal antibody immunoenzymometric method) compared for creatine kinase MB isoenzyme in serum

Results of the "Tandem-E CKMB" immunoenzymometric procedure (y) for creatine kinase (CK; EC 2.9.3.2) were compared with electrophoresis (x) for 160 serum samples from patients suspected of having sustained myocardial infarctions. The results correlated well: y, microgram/L (Tandem assay) = 1.3x-6.3 U/L(electrophoresis) (r = 0.95). CK-MB mass measurement was more stable than enzyme activity after storage and appeared to be more sensitive. Sera from 86 other people, which had no detectable CK-MB upon electrophoresis, gave a mean CK-MB value of 1.1 microgram/L (SD 1.3, range 0-8) with the Tandem assay. To determine whether these low values represented actual isoenzyme, we tested for possible interference by heterophile antibodies in the patients' sera by preincubating the samples with mouse serum before the Tandem assay. The mouse serum did not interfere with the assay of sera that had substantial quantities of CK-MB by electrophoresis. However, in five of six samples that were negative by electrophoresis, the CK-MB values were substantially smaller, indicating that the values measured were false-positives caused by the presence of heterophile antibodies directed against mouse proteins, an interference that could be eliminated by pretreatment with mouse serum.     

Radioimmunoassay of "pregnancy-specific"-beta 1-glycoprotein (SP1).

A radioimmunoassay for SP1 ("pregnancy-specific" beta 1-glycoprotein) is described in which a sensitivity of 0.1 microgram/L was achieved by use of an antiserum with Ka approximately equal to 1.7 x 10(11) L/mol and delayed addition of a 125I-SP1 tracer (specific acty, 25 Ci/g) that was stable for use in the assay for at least six to seven weeks. We also report an additional anti-SP1 serum, from an early bleeding, that displayed "apparent positive cooperativity" in the IgG but not the IgM fraction.     

Predictive values of thyroxine, thyrotropin, and triiodothyronine concentrations in serum.

The predictive values of total thyroxine, thyrotropin, and total triiodothyronine concentrations in serum for clinically apparent and subclinical hypothyroidism and hyperthyroidism were determined by retrospective analysis of clinical cases chosen from the frequency distributions of hormone test results. Very low total thyroxine concentrations, less than 25 microgram/L, were specific for hypothyroidism. Most subclinical hypothyroid cases had intermediate or low total thyroxine concentrations, 35-60 microgram/L, with moderately increased thyrotropin concentrations, greater than 12 milli-int. units/L. Thyrotropin cut-off concentrations were identified that had predictive values greater than 90% for classifying untreated and hormone-treated hypothyroid cases. Above-normal total thyroxine concentrations, regardless of absolute value, were not specific for hyperthyroidism, because binding-protein alterations related to estrogen taking were prevalent and produced occasional marked increases in serum total thyroxine. Nevertheless, cut-off concentrations for serum total thyroxine and total triiodothyronine were identified that had a predictive value greater than 90% for hyperthyroidism, without necessitating measurement of the capacities or concentrations of hormone-binding proteins.     

Monoclonal antibody–based immunoassays for serum myoglobin quantification in acute myocardial infarction

We have developed a monoclonal antibody–based enzyme immunoassay and a solid-phase radioimmunoassay for human myoglobin. Both assays are based on competition for the monoclonal antibody between the free myoglobin present in the standards or serum samples and the myoglobin coated to the wells of microtiter plates. Consequently, the absorbance at 630 nm and the radioactivity are inversely related to the concentrations of free myoglobin. The sensitivity of both assays was 10 μg/L with linearity up to 1,000 μg/L. There was no interference with other serum proteins, as judged from analysis of specimens with high concentrations of lactate dehydrogenase, creatine kinase, or hemoglobin. The average serum myoglobin concentration in 30 normal individuals was 67 μg/L. Five patients with cardiac arrhythmias had normal values (average, 63 μg/L) while four patients with myocardial infarction had abnormally high concentrations of myoglobin (300–1,000+ μg/L). In a typical case of myocardial infarction, serum myoglobin rose 21 hr earlier and peaked 12 hr earlier than creatine kinase and its cardiac isoenzyme. These rapid imrnunoassays appear to be useful for the early detection of increased serum myoglobin indicative of myocardial infarction.     

Combined liquid chromatography/radioimmunoassay with improved specificity for serum digoxin

This method for assaying digoxin in serum with improved specificity combines small-column extraction of serum, "high-performance" liquid chromatography, and RIA of the eluted fractions. Analytical recoveries of 1.0, 0.5, and 0.1 microgram/L standards were 95%, 93%, and 84%, respectively. The CVs for duplicates and replicates of sera with values of 0.5 to 1 microgram/L were 4 to 6%. Fifty-nine sera from 50 patients receiving digoxin were so studied. All digoxin metabolites appear to cross react with antibody to digoxin to various degrees. The most polar metabolites were quantitatively the most important, their average cross reactivity being 33%. For eight patients the value for digoxin by the present method was less than 60% of the RIA value. Sera from nine patients not taking digoxin but with falsely high digoxin values were also studied by the present method. The digoxin peak was well resolved from those for (a) digoxin metabolites (except dihydrodigoxin), (b) digitalis-like factors in neonates and in patients with renal failure or combined hepatic and renal failure, and (c) two cross reacting drugs and their metabolites.