Measurement of beta 2-microglobulin in serum by a particle-enhanced nephelometric immunoassay

A particle-enhanced immunoassay of beta 2-microglobulin in serum is described. It is based on the agglutination of complexes formed between the serum beta 2-microglobulin and latex particles coated with F(ab')2 fragments of polyclonal anti-beta 2-microglobulin antibodies. The analytical range of the method is 0.50 to 16 mg/l; it can be extended by appropriate dilution to 0.12 to 80 mg/l with good precision (CV less than 5% over the whole range). The accuracy and the precision are confirmed by a good correlation with radioimmunoassay (n = 123, r = 0.993). No error due to antigen excess was observed, even up to 292 mg/l. The main advantages of the method are its simplicity, its low cost per test and its high sensitivity (final dilution of the sample at 1/1200) with no known interference. The calibration curve is stable for at least 2 weeks.     

Beta 2-microglobulin levels in cerebrospinal fluid of children with leukemia and lymphoma

Beta 2-microglobulin levels were determined in the cerebrospinal fluid (CSF) of 119 children (ages 1 1/2 to 18 years) with malignant conditions; 87 with acute lymphoblastic leukemia, 9 with acute myeloblastic leukemia, 15 with lymphoma, and 8 with solid tumours. A total of 491 CSF specimens and 202 serum specimens were analyzed over a 12-month period. The mean CSF beta 2-microglobulin and serum beta 2-microglobulin were 1.11 +/- 0.58 mg/L and 1.5 +/- 0.64 mg/L respectively and were not different from the mean CSF (1.20 +/- 0.45 mg/L) and serum beta 2-microglobulin levels (1.70 +/- 0.45 mg/L) found in control patients. Meningeal leukemia was diagnosed on the basis of cytology in 7 patients. No elevation of CSF beta 2-microglobulin was found in any specimen at the time of CNS disease. Eleven other patients showed a transient rise in CSF beta 2-microglobulin above the reference range (greater than 2.1 mg/L). No evidence of CNS involvement was found in any of these patients. Five of these patients had received a combination of intrathecal methotrexate and irradiation therapy within the previous 4 months. A transient rise in CSF beta 2-microglobulin (2-3-fold increase over baseline CSF levels), which did not exceed the upper limit of the reference range was seen in 5 of 7 other children receiving the above therapy. Our study fails to demonstrate the usefulness of CSF beta 2-microglobulin for the diagnosis of CNS metastases but suggests that a transient elevation of CSF beta 2-microglobulin may occur after intrathecal methotrexate and irradiation therapy.     

Immunonephelometric determination of retinol-binding protein in serum and urine

An immunonephelometric method developed for measurement of retinol-binding protein (RBP) in serum and urine can detect it in concentrations of about 30 micrograms/L, which is in the lower limit of its normal concentration in urine (range 0-0.56 mg/L; mean +/- SD 0.19 +/- 0.15; n = 44). Urinary RBP was increased (range 0.93-29.5 mg/L) in all of 25 urine specimens from 13 subjects being treated with aminoglycoside (tobramycin). Urinary excretion of RBP was correlated (r = 0.83) with the excretion of beta 2-microglobulin. The within-assay and day-to-day precision (CV) was determined over the detection range of 0.03-8 mg/L. Within these limits the corresponding CVs varied from 4 to 27% and from 8 to 30%, respectively. The method had fairly good precision within the optimal measuring range of approximately 0.4 to 4.5 mg/L for both urine and 20-fold diluted serum samples. For various RBP concentrations our analytical recovery was 89-114% of added RBP. Results by this method correlated well (r = 0.96, n = 24) with those by a radial immunodiffusion method.     

Beta 2 microglobulin in serum--a "tumor marker" in malignant lymphomas?

Serum beta 2-microglobulin concentration was investigated in 221 patients with malignant lymphoma (Hodgkin type and non-Hodgkin type). In 55% of the unselected group of patients increased serum concentrations of beta 2-microglobulin were found. There was a close correlation between tumor extension and beta 2-microglobulin levels. In the tumor stage I the beta 2-microglobulin concentration was 1.65 +/- 0.45 mg/l, in the stage II 2.41 +/- 0.65 mg/l. The levels in the stage III (3.48 +/- 1.15 mg/l) and in the stage IV (5.49 +/- 1.99 mg/l) were significant higher than normal. Using longitudinal studies, measurement of beta 2-microglobulin was followed up during the course of the disease. During tumor regression by an effective antineoplastic therapy initially elevated beta 2-microglobulin concentrations decreased. In patients who achieved a complete remission a normalization of beta 2-microglobulin concentration was seen. We conclude from our findings that beta 2-microglobulin has the characteristics of a tumor associated marker in patients with malignant lymphomas. The low specificity and sensitivity limits the clinical use of this parameter.     

Establishment of a reference interval for beta 2-microglobulin in cerebrospinal fluid with use of two commercial assays

Increased concentrations of beta 2-microglobulin in cerebrospinal fluid have been used to detect central nervous system involvement with metastatic cancer and with neurological complications of AIDS. However, no adequate reference interval study has been reported for beta 2-microglobulin in cerebrospinal fluid. We established a reference interval with both the Abbott IMx microparticle enzyme-linked immunoassay (EIA) (0.6-2.0 mg/L) and the Pharmacia beta 2-micro EIA 96 method (0.8-2.2 mg/L) for beta 2-microglobulin in cerebrospinal fluid. The two methods correlate well, with the latter method giving slightly higher values. beta 2-Microglobulin increases with age in adults by about 0.1 mg/L every 7.5 years, with no significant difference between genders.     

Serum cystatin C level as a potentially good marker for impaired kidney function

OBJECTIVES: To evaluate the diagnostic significance of serum cystatin C levels in clinical practice. DESIGN AND METHODS: Serum (99m)Tc-DTPA clearance was compared with serum cystatin C, creatinine, beta(2)-microglobulin levels and creatinine clearance in a group of patients aged 42.61 +/- 7.55 years with glomerular filtration rates of 10-60 mL/min/1.73 m(2) (n = 52) and healthy controls aged 43.90 +/- 12.06 years (n = 52). RESULTS: No effect of sex on serum cystatin C levels was observed, but average levels increased with age. No significant difference was evident between the mean cystatin C levels of three blood samples taken at 1 month intervals from healthy subjects. Reference clearance was correlated with creatinine clearance (r = 0.957), cystatin C (r = 0.828), beta(2)-microglobulin (r = 0.767) and creatinine (r = 0.682). 60 mL/min/1.73 m(2) was chosen as the borderline for receiver-operating characteristics analysis. The values for the cut-off point, sensitivity, specificity and the area under curve were determined for cystatin C as 1.36 mg/L, 98%, 99% and 0.99 +/- 00.1, respectively; for creatinine, the values were 103 micromol/L, 80%, 100% and 0.97 +/- 0.01, respectively, and for beta(2)-microglobulin, the values were 2.51 mg/L, 86%, 92% and 0.94 +/- 0.02, respectively. CONCLUSION: Serum cystatin C level can be used as a marker for renal damage.     

Turnover in humans of β2-microglobulin: the constant chain of HLA-antigens

The turnover of beta 2-microglobulin, the common subunit of the HLA antigens, has been examined in normal subjects and in some patients with kidney disorders, multiple myeloma and rheumatoid arthritis. All patients displayed elevated serum levels of beta 2-microglobulin. The plasma disappearance curve of 125I-beta 2-microglobulin demonstrated that the protein has a rapid turnover (t 1/2 = 2.1 h; range 1.1-2.8 h) in normal persons and in patients with a normal glomerular filtration rate. In patients with kidney disorders the impaired renal filtration prolonged the turnover time and led to elevated serum levels of beta 2-microglobulin. Simultaneous measurements of 125I-beta 2-microglobulin in serum and urine allowed estimations of the beta 2-microglobulin net reabsorption in the renal tubuli. Two patients with renal disease reabsorbed 84% and 89%, respectively, of the beta 2-microglobulin filtered in the glomeruli. In normal persons the net reabsorption is close to 100%. In patients with normal kidney function increased serum levels of beta 2-microglobulin seem to be due to an increased synthetic rate of the protein as the elimination rate is normal. HLA antigen heavy chains in serum are present in smaller amounts than beta 2-microglobulin. The present data, therefore, suggest an imbalanced synthesis of the two chains.     

Clearance and synthesis rates of beta 2-microglobulin in patients undergoing hemodialysis and in normal subjects.

Retention of beta 2-microglobulin in patients undergoing hemodialysis is associated with a beta 2-microglobulin-derived amyloidosis. Removal of beta 2-microglobulin by renal replacement therapy has been proposed for the prevention of this amyloidosis. Currently, however, data on the beta 2-microglobulin synthesis rate in patients undergoing hemodialysis are scarce, and consequently it remains speculative how much removal would be necessary to counterbalance synthesis. The plasma kinetics of iodine 131-labeled beta 2-microglobulin were therefore examined in 11 patients with anuria who were undergoing long-term hemodialysis. Five healthy persons served as controls. Kinetic modeling of the plasma curves showed that the data fitted a two-pool model (r2 greater than 0.96) consisting of a rapid 2 to 4 hour distribution phase followed by a less steep curve, described by the plasma (metabolic) clearance (Clp). Synthetic rates were calculated from Clp and the beta 2-microglobulin steady state plasma concentration (plus beta 2-microglobulin removal during hemodialysis in the case of high flux hemodialysis). The results showed a significantly higher Clp in normal controls as compared with patients undergoing hemodialysis (65.5 +/- 12.8 ml/min (mean +/- SD) versus 3.4 +/- 0.7 ml/min). In contrast, the beta 2-microglobulin synthesis rate in the patient group (3.10 +/- 0.79 mg/kg/day) was not significantly different from that of normal controls (2.40 +/- 0.67 mg/kg/day), which was due to markedly elevated beta 2-microglobulin plasma concentrations in the patients (37.6 +/- 14.1 mg/L vs 1.92 +/- 0.27 mg/L). These findings suggest that the presence of end-stage renal disease does not have a significant impact on the beta 2-microglobulin generation rate. The degree of accumulation of beta 2-microglobulin in patients undergoing hemodialysis seems to depend on the loss of renal excretory function.     

Beta-trace protein--a marker of kidney function in children: "Original research communication-clinical investigation"

OBJECTIVES: To determine the pediatric reference interval for serum beta-trace protein (beta-TP) and to compare beta-TP with established LMW markers of GFR, i.e., cystatin C (CysC) and beta(2)-microglobulin (beta(2)-M). DESIGN AND METHODS: All three LMW markers were measured immunonephelometrically. In 106 children above the age of 2 years without evidence of kidney disease, non-parametric reference intervals were calculated. The relative rise of the GFR marker concentrations above the upper reference was studied in 107 samples from 96 patients covering the entire GFR range. RESULTS: Above 2 years, the reference range of beta-TP was constant at 0.43-1.04 mg/L. With decreasing Schwartz-GFR, there was a comparable rise in beta-TP and beta(2)-M, while CysC rose less in the group with GFR below 30 mL/min/1.73 m(2) (278+/-49% [CysC] versus 336+/-65% [beta-TP] and 342+/-76% [beta(2)-M]; p=0.043 and 0.027, respectively). CONCLUSIONS: These data confirm the potential of ss-TP as an endogenous GFR marker in children.     

Determination of beta 2-microglobulin in human urine and serum by latex immunoassay

This highly sensitive method for determination of beta 2-microglobulin (beta 2-m) in human urine or serum is based on direct agglutination by beta 2-m of latex particles on which an antibody against beta 2-m is adsorbed. The agglutination is quantified by counting the remaining unagglutinated particles, or by turbidimetry. A novel aspect of this method is the capability to prevent nonspecific agglutination of the antibody-coated particles by diluting them with an albumin solution of well-defined characteristics (pH, freshness, concentration) just before the assay. The assayable concentration range is 1--32 micrograms/L, the detection limit 0.5 micrograms/L. Within-assay CV, based on 10 determinations of beta 2-m in urine and serum at two different dilutions, ranged from 4.6 to 8.7%. Between-assay CV, calculated from 10 determinations of beta 2-m in urine and serum, was 10 and 8.4%, respectively. Analytical recovery of beta 2-m in urine averaged 97% and in serum 104% (n = 10). No component of urine or serum interfered. Coefficients of correlation for beta 2-m in urine or serum as measured by radioimmunoassay and latex immunoassay were 0.97 and 0.93, respectively. Concentrations of beta 2-m in serum and urine from 33 healthy men (ages 20 to 67 years) averaged 1.5 mg/l and 54 micrograms/g of creatine, respectively.     

Limitations of conventional laser nephelometry for the measurement of beta 2-microglobulin, lysozyme, alpha 1 fetoprotein and myoglobin in serum and urine

We have tested some assay procedures for the measurement of beta 2-microglobulin, lysozyme, alpha 1-fetoprotein and myoglobin in serum and/or urine with the use of a manual Behring laser nephelometer. The assay working ranges were: beta 2-microglobulin: 0.0038-0.038 g/L; lysozyme: 0.005-0.325 g/L. We have studied the effect of different antiserum dilution ratios and of different concentrations of polyethylene glycol 6000 on the calibration curves. The best standard curves were obtained with the use of the following antiserum dilutions: anti-beta 2-microglobulin: 1:3 with saline, 40 g/L PEG; anti-lysozyme: 1:5 with saline, 40 g/L PEG; anti alpha 1-fetoprotein: concentrated; anti-myoglobin: concentrated with added 40 g/L PEG. In the case of beta 2-microglobulin and lysozyme, laser nephelometry, could be a fast and simple procedure if a 10 times increase in sensitivity can be achieved. For the measurement of alpha 1-fetoprotein and myoglobin, the sensitivity of laser nephelometry was disappointing when compared to those reported for radioimmunoassay and enzyme immunoassay.     

Comparison of gas-liquid chromatography and EMIT assay for serum valproic acid

We describe a simple direct extraction method for the gas-liquid chromatography determination of serum valproic acid. The working range for the assay is 2-180 mg/L and our within-run precision was 5.8 and 4.3% at the 40 and 90 mg/L concentrations respectively. Hemolyzed and lipemic sera as well as samples from patients with hyperbilirubinemia and from patients with decreased renal function were put through the assay and no interfering peaks were noted. Interference occurred when teflon-lined screw caps were used during the extraction step. The method was proven to be accurate by linear regression analysis of samples containing weighed-in amounts of valproic acid. The above assay was compared to an enzyme immunoassay technique (EMIT). The working range for the latter is 10-150 mg/L and the with-run precision was 10.8 and 5.9% and 90 mg/L concentration respectively. Samples were run by both the gas-liquid chromatograph and enzyme immunoassay methods and gave very similar results over the range 16-139 mg/L.     

Beta 2-microglobulin in the diagnosis of specific involvement of the kidneys in acute leukemias]

Measurements of the serum and urinary beta 2-microglobulin (beta 2-Mg) concentrations in 138 adult patients with various forms of acute leukemia, which were carried out in various periods of the disease, have revealed that serum beta 2-Mg levels were two times higher in the patients with leukemic involvement of the kidneys (5.54 +/- 3.50 mg/l) than in those without renal involvement (2.77 +/- 1.07 mg/l). Consequently, urinary beta 2-Mg excretion was much higher in the patients with renal involvement than in those with intact kidneys (1.512 +/- 3.222 mg/l vs. 0.388 +/- 1.175 mg/l). Mathematical analysis has shown that the risk of leukemic involvement of the kidneys makes up 93.3 percent in acute leukemia patients with serum beta 2-Mg levels surpassing 6.0 mg/l. This permitted a conclusion on the diagnostic value of beta 2-Mg measurements for the detection of specific involvement of the kidneys in acute leukemia.     

A comparative study of serum alpha 1-microglobulin and beta 2-microglobulin levels in cancerous and other diseases

The levels of serum alpha 1-microglobulin in 60 normal persons and in 191 patients suffering from a variety of benign and malignant disorders were determined by an enzyme immunoassay, and these values were compared with the levels of beta 2-microglobulin. A discrepancy between the serum levels of these proteins was found in hepatobiliary disorders; that is, an increased serum level of beta 2-microglobulin was observed in 73.9%, while in only 4.3% was there an elevation of alpha 1-microglobulin. In particular, alpha 1-microglobulin levels in patients with liver cirrhosis were well below the normal range, while beta 2-microglobulin levels were elevated. Elevated levels of both proteins were noted in patients with some impairments of renal function, particularly in chronic renal failure, and in immunological diseases. In 81 patients with neoplastic diseases, a high alpha 1-microglobulin value was found in only 15 patients (16.4%), while a high beta 2-microglobulin value in 62 patients (76.5%). The serum levels of both alpha 1-microglobulin and beta 2-microglobulin were especially high in plasma cell dyscrasia with Bence Jones protein, but other neoplastic diseases were mostly associated with beta 2-microglobulin elevation alone.     

Heterogeneous enzyme immunoassay of alpha-fetoprotein in maternal serum by flow-injection amperometric detection of 4-aminophenol.

A sandwich-type heterogeneous enzyme immunoassay with flow-injection analysis for alpha-fetoprotein (AFP) in human serum has been developed. 4-Aminophenol, the product of enzymatic reaction, is detected amperometrically. The interassay CV for this electrochemical enzyme immunoassay was less than 8.2%, with a minimum detection limit for AFP of 0.163 micrograms/L. The calibration curve had a linear range of 0.316-100 micrograms/L. Studies with 48 human maternal serum samples, comparing results by this method with those by a commercial kit, showed a good correlation (r = 0.961). This procedure provides an alternative method for determining low concentrations of AFP in human maternal serum.     

Measurement of urinary retinol-binding protein by enzyme-linked immunosorbent assay, and its application to detection of tubular proteinuria

An enzyme-linked immunosorbent assay (ELISA) for urinary retinol-binding protein (RBP) has been developed and compared with urinary beta 2-microglobulin for the detection of tubular proteinuria. The assay has a working range of 10 to 250 micrograms of RBP per liter of urine. The within-assay CV was 3.2-7.1%, the between-assay CV 12.5%. A control population of 118 male subjects gave a geometric mean urinary RBP concentration of 7.7 micrograms per millimole of creatinine and a 95th centile of 22 micrograms per millimole of creatinine. Comparison of urinary RBP and beta 2-microglobulin concentrations in 80 control subjects and 117 subjects exposed to cadmium fumes gave correlations of r = 0.59 and 0.91, respectively. Of the 117 subjects exposed to cadmium fumes, 103 gave both RBP and beta 2-microglobulin concentrations on the same side of the upper 95th centile values of 22 and 38 micrograms per millimole of creatinine for RBP and beta 2-microglobulin respectively (Chi-square analysis p less than 0.001), demonstrating that RBP and beta 2-microglobulin detect tubular proteinuria with equal sensitivity and specificity. ELISA and an established latex immunoassay gave well-correlated results.     

Method of quantitative analysis of beta2-microglobulin in human blood

A simple and economic method of radial immunodiffusion is developed for diagnostic measurements of beta 2-microglobulin in human serum. Human serum and plasma concentrations of beta 2-microglobulin can be measured sufficiently accurately; the results are reproducible. The reproducibility and addictiveness of the method were evaluated. The error of the method is no more than 15% (3-36 mg/liter), which is permissible for methods of this type.     

Immunoassay of low concentrations of albumin in urine by latex particle counting

We describe here a nonisotopic immunoassay, based on particle-counting technology, for the determination of urinary albumin. The assay takes only 35 min and has been fully automated on the IMPACT (Acade Diagnostic Systems, Brussels, Belgium) machine. The system measures albumin within a linear range between 6.25 and 50 mg/L and has a detection limit of 0.4 mg/L. Analytical recoveries at three concentrations ranged between 96% and 102%. Within-run precision ranged from 1.6% to 9.5%. The method was compared with a commercial nephelometric immunoassay system and a correlation coefficient of 0.996 was found for 216 urine samples. No antigen excess affects the shape of the curve in our system, whereas in nephelometry a 3 g/L solution of albumin starts to decrease the dose-response curve.     

A simple radial immunodiffusion method for assay of beta 2-microglobulin in serum

In this method for clinical measurement of beta 2-microglobulin, a 10 g/L agarose gel containing anti-beta 2-microglobulin is used, with subsequent staining with Coomassie Blue. Although the method is slow (requiring 30 h), it is inexpensive, reliable, and accurate over the range of 1 to 10 mg/L, and is useful in the determination of beta 2-microglobulin in serum and cerebrospinal fluid. A modified procedure in which staining with silver is used may be sufficiently sensitive for the clinical assay of urine.     

Serum beta2-microglobulin in liver disease.

The concentration of beta 2-microglobulin in serum was determined in seventy-one patients with various liver disorders. Elevated values were found in most patients with chronic active or chronic persistent hepatitis and in over 80% of patients with alcohol-induced liver cirrhosis. In contrast, patients with alcohol-induced fatty liver, the serum beta 2-microglobulin concentrations were mostly within the normal range. Significant correlation (P less than 0.001) was noted between the elimination rate of galactose from blood and the serum beta 2-microglobulin concentration in patients with alcoholic liver damage but not in patients with chronic hepatitis. The reasons for the increased S-beta 2-microglobulin concentrations in liver diseases are unknown. Several explanations including a release of beta 2-microglobulin from necrotic liver cells or an increased synthesis of beta 2-microglobulin consequent to inflammation in the liver are possible. Alternatively, raised beta 2-microglobulin levels may reflect the hepatic synthesis during reparative growth.