Immunoturbidimetric in urine - jcp.bmj.com · assayed in serum and urine using goat anti-humanIgA,IgG,andIgM(Guildhay, Surrey, England) in an immunoturbidimetric assay after the method

J Clin Pathol 1991;44:466-471

Immunoturbidimetric assay for estimating freelight chains of immunoglobulins in urine andserum

C R Tillyer, J Iqbal, J Raymond, M Gore, T J McIlwain

AbstractAn immunoturbidimetric assay for theassessment of free K and A. light chains ofimmunoglobulins was developed using acommercial polyclonal antiserum withreactivity towards epitopes on the lightchains, which are not expressed whenthey are bound to heavy chains. Theassay, on a centrifugal analyser, issimple and rapid. The limit of detectionis 5 mg/l of free light chain, with anassay range of 5-120 mg/l, intrabatchprecisions from 1-5-64%, and interbatchprecisions from 6-5-8-9%. The assay wasonly slightly less sensitive than colloidalgold staining of cellulose acetate electro-phoreses for the detection of Bence-Jones protein in urine. For the serialmonitoring of response to chemotherapyin patients with myeloma, the assaycorrelated well with serum paraproteinestimates obtained by densitometricscanning of Ponceau stained celluloseacetate electrophoreses, but not withserum ,B-2 microglobulin measurements,even after correction for the effects ofcreatinine.These assays may prove to be of use for

the monitoring of tumour response inthe treatment of Bence-Jones myeloma.

Department ofChemical Pathologyand Section ofMedicine, RoyalMarsden Hospital andInstitute of CancerResearch, LondonSW3 6JJ and DownsRoad, Sutton, SurreyC R TillyerJ IqbalJ RaymondM GoreT J McllwainCorrespondence to:Dr C R Tillyer, RoyalMarsden Hospital SW3Accepted for publication9 January 1991

The monoclonal immunoglobulins secreted inmyeloma have many of the propertiesrequired of the ideal tumour marker and thevalue of quantifying the concentration ofparaprotein in serum as a guide to the detec-tion, treatment, and monitoring of this condi-tion is undisputed.' About 10-20% ofmyelomas, however, secrete only the free lightchains of immunoglobulins, historicallyknown as Bence-Jones protein,2 and untilrecently there has been relatively little effortroutinely to quantitate these paraproteins inurine as an aid to disease monitoring andtreatment. There are several possible reasonsfor this. The method used for quantifyingserum paraproteins (densitometric scanning ofstained cellulose acetate or agarose electro-phoreses) has not been routinely applied todetection of urinary Bence-Jones protein.This has probably been due to therequirement to concentrate urines because ofthe insensitivity of the protein stains used,which renders the estimation far more

laborious. A further problem is the highlyvariable protein composition of urine, par-ticularly in patients with myeloma. This canlead to inaccuracies in the scan, due to thedifferent dye binding properties ofthe differentproteins, and also in the reference proteinassay used to calibrate the scan. The accuracyand imprecision of this assay is also verydependent on the degree of homology andinvariability of the proteins in the sample andstandard.Immunochemical assays have been

developed,3 but as the antisera were raised tobound light chains any immunoglobulin in theassay had to be removed by gel or membranefiltration techniques which were not suitablefor routine analysis.45 Antisera to epitopeswhich are only expressed by the free lightchain and not on bound light chains willovercome this problem, but the developmentof free light chains assays has been hinderedbecause antisera with sufficient specificity andavidity have been commercially available onlyrecently, even though they were first reported25 years ago.6 Immunochemical assays alsohave a protein calibration problem due to thedifferential reactivity of antisera to standardscontaining a heterogenous collection ofproteins and samples containing a homogen-ous or different heterogenous set of proteins.Furthermore, different antisera can react in acompletely different manner to the same stan-dard or sample. In practice, however,immunochemical assays are reliably consistentfor monitoring paraproteins in patients whenthe same batch of antiserum is used.7

In the past eight years several free lightchain assays have appeared using radio-immunoassay' and enzyme immunoassaytechniques"' with commercial polyclonal ormonoclonal) antisera, which have been used tomonitor treatment in patients with Bence-Jones proteinuria and myeloma. We usedcommercially available polyclonal anti-freelight chain antisera to develop a simple,direct, and rapid immunoturbidimetric assayson a centrifugal analyser for free K and A lightchains in serum and urine.

MethodsFREE LIGHT CHAIN ASSAYSReagents: sodium phosphate buffer pH 7 0(0-067 mol/l). Polyethylene glycol (PEG) 6000(BDH, Poole, Dorset, England) 6% (w/v) inphosphate buffer (6% PEG).

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Immunoturbidimetric assay for estimating free light chains in urine and serum

Antiserum: rabbit anti-human free K(A100:Lot 088) and anti-human free i(A101:Lot 079) light chains (Dako Ltd, HighWycombe, Buckinghamshire, England).Preparation of light chain standards: urinesamples from patients with Bence-Jonesprotein myeloma were pooled, precipitatedwith 70% ammonium sulphate, dialysedagainst phosphate buffered saline (PBS), pH7 0, and light chain fractions obtained afterpassage over an Ultrogel AcA 54 columnequilibrated with PBS; fractions were pooledand adjusted to a final concentration of 0 5 g/l.Purity was more than 99% as assessed bypolyacrylamide and cellulose acetate electro-phoresis. Working standards were preparedfrom this solution by dilution in 6% PEG.Assay procedure: standards and samples wereassayed on a Cobas Bio centrifugal analyser(Roche, Welwyn Garden City, Hertfordshire)using a 1 in 20 dilution of antiserum in 6%PEG. The Cobas Bio variable listing was asfollows: (1) units mg/l; (2) calculation factor1000; (3) standard 1 concentration 0; (4) stan-dard 2 concentration 0; (5) standard 3 concen-tration 0; (6) limit 0; (7) temperature °C 25;(8) type of analysis 5; (9) wavelength (nm)340; (10) sample volume (p1) 20; (11) diluentvolume (,ul) 45; (12) reagent volume (MI) 250;(13) incubation time (seconds) 0; (14) startreagent volume (pl) 0; (15) time of first reading(seconds) 0 5; (16) time interval (seconds) 30;(17) number of readings 30; (18) blankingmode 1; (19) printout mode 1.

CELLULOSE ACETATE ELECTROPHORESIS AND

PARAPROTEIN QUANTITATIONThe method of Kohn,'2 with the addition ofTween-20 in the wetting buffer, was used.Strips were stained with Ponceau S for den-sitometric scanning, and with nigrosine andcolloidal gold stain'3 for detection of urinaryBence-Jones protein. Immunofixation wascarried out using a modification of the methodof Kohn and Riches.'4/3-2 microglobulin assay: the immunotur-bidimetric assay of Tillyer and Rawal wasused.'5 Calibration standards were from theProtein Reference Unit, Sheffield, England.The working range of this assay was 0-5-16 mg/l, with samples going into antigen excessabove 30 mg/l.Serum and urine immunoglobulins: These wereassayed in serum and urine using goat anti-human IgA, IgG, and IgM (Guildhay, Surrey,England) in an immunoturbidimetric assayafter the method of Deverill (personal com-munication), using6% PEG reaction buffer andSPS-01 calibration standard (ProteinReference Unit, Sheffield).

Calibration curves were fitted using a thirdorder polynomial least-squares algorithm.Imprecisions were estimated using an ANOVAtechnique for duplicate control samples in eachbatch. Parallelism was assessed by the regres-sion of the diluted sample on the standard.Spearman's rank correlation (p) was used toassess the correlation between estimation of

serum paraprotein, urinary free light chains,and /B-2 microglobulin concentration.

CLINICAL EVALUATIONA random sample of 12 patients attending themyeloma unit at the Royal Marsden hospital,all with Bence-Jones protein in the urine,determined by cellulose acetate electro-phoresis, were monitored for about 100 daysfor serum paraprotein concentrations, /3-2microglobulin and immunoglobulins; andurine total protein, immunoglobulins, and freelight chains. The paraprotein types wereGK,BJK (n = 3); GA,BJB (n = 4); AK,BJK(n = 1); A),BJ) (n = 1); DK,BJK (n = 1);and BJK (n = 2). Nine of the patients under-went various combinations of high dosechemotherapy (including cyclophosphamide/vincristine, adriamycin, methylprednisolone/melphalan/busulphan, with or without bonemarrow autograft).

ResultsFREE LIGHT CHAIN ASSAYSCalibration curve: calibration curves for both Kand A light chain assays were non-linear overthe chosen concentration range (5-120 mg/l),but showed a high degree of reproducibilityover the range for each batch of antiserum (figs1 and 2).Recovery: recovery of light chain using spikedurines was 90-103% (mean 96%) for the Kassay and 95-101% (mean 99%) for the A assay.Detection limit: the detection limit (three timesthe standard deviation of the blank responsefrom zero) was 5 mg/l for both assays.Cross reactivity: using free light chain stan-dards the cross reactivity of free K light chainswith anti-free A antiserum and free A lightchains with anti-free K antiserum were both lessthan 3 2%, using the free light chains standard.The true cross reactivity between light chains isless than this according to others using anti-serum from the same source8 9; the sensitivity ofour assay was limiting in this respect. The crossreactivities of the free light chain antisera tosera of patients with myeloma containing nofree light chains (as determined by electro-phoresis) are shown in table 1. As expected thedegree of cross reactivity varied considerablyamong the patients, the maximum being 2 2%.This is much higher than others have shownusing polyclonal IgG,89 but is not sufficient tocause any major problems in the urine free lightchain assay.Imprecision: imprecision estimates for pooledurines are shown in table 2. These are compar-able with immunoturbidimetric assays forother proteins.Parallelism: data are presented in table 3.Although sample and standard response curveswere apparently parallel, regression analysis ofthe observed and expected concentrationsshowed evidence for non-parallelism in one outof three A urines and one of the K urines. Again,this may be expected in the assay ofmonoclonalfree light chains. The deviations from parallel-ism were not very high, however, and we did

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0 50 100 150 200Free light chain (mg/1)

250

Figure I Calibration curvesfor Kfree light chainassays. Points show mean SDfor nine different assaycalibration curves using the same batch of antiserum.

not consider that they would seriously affect theaccuracy of the assay.

CLINICAL STUDIESReference ranges: the detection limit ofthe assaywas not low enough to determine a true normalrange. The working detection limit of the assaywas 8 mg/l, the concentration of the lowest

Table 1 Cross reactivity offree light chain antisera tobound light chains

Cross reactivity (%)Paraprotein(no of samples) Anti-K Anti-A

GK (8) <0 24-1 96 <004-<04GA (1) <0-8 0-16AK(1) 1-6 <1AA (2) <0-06, <0 45 0-02, <0 45MK (2) <0 37,2 1 <0 37,2-24MA (2) < 0-06, 1 25 0-023,0 35

Table 2 Imprecision estimates for light chain assays

K A

mg/l CV, CVb n mg/L CV, CV,b n

12 7 2.1 - 10 14-1 2-7 - 10956 26 - 10 723 1-5 - 10187-6 6-4 8-9 8 177.3 4-6 6-5 10

CV, = intrabatch CV (%).CVb = interbatch CV (%).

Table 3 Parallelism of samples and standards

Regression of observed sample concentration on expected

Sample Slope (95% CI) Intercept (95% CI)

A 0-98 (0-94-103) -1 30 (-299- 0 39)A 1-03 (0-83-1-23) 7-86 (-8 40-24-1)A 093 (090-096)* 297 (-1-50- 7-4)K 0-88 (0-76-0-98)* -8-70 (-23-50-6 13)K 1-01 (0-93-1-10) 4-45 ( -0-39-9-31)

*Confidence interval (CI) for slope does not encompassexpected value of unity.

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,100-

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0 50 100 150 200 250Free Light chain (mg/I)

Figure 2 Calibration curvesfor A free light chainassays. Points show mean SDfor seven different assaycalibration curves using the same batch of antiserum.

standard, which is very close to the upper limitof the normal range for absolute concentrationof free light chains as determined by otherauthors (8 mg/l for K9 10, and 2-1 mg/l for '9). Ina sample of seven 24 hour urine collectionsfrom normal hospital staff absolute concentra-tions of free K and A chains were all less than8 mg/l. During chemotherapy, most of thepatients showed extremely high variation inglomerular filtration rates due to factors such asthe toxic effect,s of chemotherapy, administra-tion ofintravenous fluids, diuretics and varyingfluid intake. Results were therefore correctedfor the effects ofcreatinine to permit a meaning-ful comparison of spot urine samples.Patient monitoring: the nine treated patientshad initial urine free light chain concentrationsranging from 615 to 3 mg/mmol creatinine; fiveof these responded biochemically by droppingtheir urinary free light chains to undetectableconcentrations in at least one sample duringtreatment. Two typical cases are shown in figs3A and B. The patient in fig 3B showed atransient rise in ,B-2 microglobulin in conjunc-tion with the development of a transientparaprotein (GK) in the serum. This seemed tobe part of the general bone marrow responseobserved after high dose chemotherapy as thepatient achieved clinical remission soon after.Two cases showed a partial response tochemotherapy; one (fig 3C) showing a rapid fallto low concentrations of urinary free lightchain but still remained positive in the urinaryfree light chain assay and on electrophoresis;urinary free light chain concentrations in thesecond case fell from 615 to 62 mg/mmolcreatinine but rose thereafter. Two showed nobiochemical response. These both had very lowinitial concentrations of urinary free lightchains (3 mg/mmol creatinine); one showedvery little change after chemotherapy and the

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Immunoturbidimetric assayfor estimatingfree light chains in urine and serum

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Figure 3 Comparison of the response of urine free light chain, serum paraprotein (ifpresent), and serum p-2 microglobulin in four patients withmyeloma receiving high dose chemotherapy. The qualitative result ( + positive, - negative) of a colloidal gold stain on the unconcentrated urinesample is shown at each assay point (BJP). Points on the urine free light chain assay graph, which were below the absolute sensitivity of the assay(8 mg/l), are shown in parentheses.

other (fig 3D) died shortly after a large rise inurinary free light chains and fl-2microglobulin. Correction of serum ,B-2microglobulin valves for creatinine did notsignificantly affect the trend in each figure.The urinary free light chain assay results

correlated well with detection of Bence-Jonesprotein by cellulose acetate electrophoresis.Out of 75 samples, 64 (85%) were positive andthree (4%) were negative on both assays. Fivesamples (7%) were positive on electrophoresisand negative on free light chain assay. The limitof detection for gold stained cellulose acetateelectrophoresis of unconcentrated urines hasbeen estimated to be 2 mg/I,"6 and this iscorroborated by this study. It is noteworthythat two of these samples came from the twopatients with paraprotein leaks in the urine.Although the presence of separate Bence-Jones

bands was confirmed by immunofixation,paraprotein leaks might have been the cause offalse positive results on electrophoresis. Threesamples (4%), all in one patient, were"negative" on electrophoresis but positive inthe free light chain assay. There are severalpossible reasons for this and for false positiveresults generally in the free light chain assay.The free light chain assay does not distinguishbetween monoclonal and polyclonal lightchain, so although a particular monoclonalBence-Jones protein may have disappearedfrom the urine on electrophoresis, the free lightchain assay will still detect polyclonal free lightchain which may be present in increasedamounts, particularly if the patient has atubular proteinuria or renal failure.'7 In thispatient there was not only a mild tubularproteinuria, but several transient paraprotein

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bands had appeared in the urine and serumafter chemotherapy. These could have beenlight chain fragments or transient Bence-Jonesprotein secreting clones, again as part of anoligoclonal response after chemotherapy. Theoriginal Bence-Jones protein band had goneand so they were reported as negative onelectrophoresis, but the free light chain assayhad presumably measured them. It would alsobe possible for a urinary "leak" of serumparaprotein to be of sufficient size to cause across reaction of urinary bound light chain inthe free light chain assay and hence a falsepositive result. This was not apparently thecase in the two patients with serum paraproteinleaks here, because the urinary immuno-globulins were too low.Comparison with other markers: urinary freelight chain concentrations with serumparaproteins in the nine cases which also had aserum monoclonal immunoglobulin(p = 0542; t = 4-2for42 df; p < 0001)anditwas our impression that significant falls in theserum paraprotein were generally accompaniedby falls of a similar magnitude in urinary freelight chain. In contrast, serum /3-2microglobulin did not correlate with urine freelight chain concentrations (p = -0 15;t = -1-04 for 46 df; p > 0-05) or with theserum paraprotein concentration (p = 0 057;t = 0 43 for 56 df; p > 0 05). Correcting the/3-2 microglobulin for the serum creatinineusing the regression formula of Cuzick et al'8and comparing the difference between theobserved /3-2 microglobulin and that predictedfrom the serum creatinine again showed nocorrelation with either urine concentrations(p = 0 13; t = 0 95 for 53 df; p > 0-05) orserum paraprotein (p = -0- 19; t = -1 -28 for42 df; p > 0 05). This would suggest that /3-2microglobulin may not be a suitable marker formonitoring Bence-Jones myeloma, as has beenrecommended"9; the serum /3-2 microglobulinconcentration in the patients studied here didnot change as rapidly or by as much aftertreatment as the other markers.

ConclusionsThe immunoturbidimetric assay for free lightchains described here is much simpler andmore rapid than any previously described freelight chain radioenzyme immunoassay. It doesnot require very prolonged incubation timesand multiple washing steps, and a result can bemade available within 30 minutes ofreceiving asample. Although it requires more antiserumthan that used in the more sensitive assays,where high antiserum dilutions and prolongedincubations are used to achieve high sensitivity,the raw material cost is offset by the largereduction in time required to perform theassay. Furthermore, the shelf-life of theimmunoturbidimetric assay is determined onlyby the antiserum, which is considerably longerthan that of I"25 and at least equivalent to an-enzyme immunoassay.The sensitivity of the immunoturbidimetric

(5 mg/l) is not as great as some of the enzymeimmunoassays (0 5 to 60 ,g/1), but the utility of

such sensitive assays for Bence-Jones proteinassays is highly questionable when the upperlimit of normal for free light chains in urines isof the order of 2-8 mg/l. The only method fordetermining the presence of Bence-Jonesprotein below this level of polyclonal back-ground is electrophoresis, which is alwaysrequired to establish the clonal state of anyabnormal immunoglobulin or free light chainmeasurement.

It could be argued that there is little role atpresent for Bence-Jones protein quantitationin myeloma. Urinary Bence-Jones protein,particularly A Bence-Jones protein, is not asgood a marker of tumour burden as serumparaprotein, probably because of the con-siderable metabolism of light chains by thekidney and their adverse effect on renal func-tion.'o Serum free light chains measurementsmay need to be considered in renal failure andfor those Bence-Jones proteins whichpolymerise and are not filtered at theglomerulus. For Bence-Jones myeloma,however, other quantitative assays for monitor-ing response to treatment are quite unsatisfac-tory: urine total protein assays are notoriouslyinaccurate and imprecise and they only trulyreflect Bence-Jones protein concentrations inthe urine when Bence-Jones protein is themajor protein present. Patients with myelomacommonly have considerable degrees ofproteinuria which will seriously decrease thesensitivity of the total protein assay. Serum /3-2microglobulin has an undoubtedly importantrole for assessment of prognosis in myeloma,'8but its role in monitoring disease is less clear. Ithas been reported as showing a good correla-tion with disease stage, total myeloma cellmass, chemotherapy response and diseaseactivity, 21-3 but good21 22 or poor2126 correlationwith paraprotein concentrations. Its mainproblem as a tumour marker is that the serumconcentrations are affected by the glomerularfiltration rate, requiring creatinine correction toremove the effect of diminished renal function,and urine concentrations are unreliable due tothe effect of urine acid. It can show a rise afterchemotherapy as in one of the patients studiedhere, and as a preterminal event.'8 It has beensuggested that /3-2 microglobulin should beregarded as reflecting plasma cell proliferativeactivity and mass whereas the paraproteinreflects myeloma cell mass alone.26 The resultspresented here suggest that the magnitude ofthe response to treatment does not seem to be asgreat as that of the paraprotein or free lightchains and that it is less specific and sensitivethan either of these assays for serial monitoringof tumour cell mass.The adoption of an immunoturbidimetric

free light chain assay for long term monitoringof patients will require not only a supply ofantiserum raised against a sufficiently largelight chain pool, but also a systematic approachto the preparation of suitable multiclonal orpolyclonal standards and cross calibration ofsuch standards. These problems are not insur-mountable and not much more difficult thanthose encountered in the immunochemicalassay of serum immunoglobulins and

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Immunoturbidimetric assay for estimating free light chains in urine and serum

paraproteins. The simplicity and convenienceofthis assay may lead to greater use ofurine andserum free light chain measurements in Bence-Jones myeloma as a parameter on which to basepossible response, outcome, and adoption ofalternative therapeutic options.

We are most grateful to Mary Meldrum, clinical data manager,for collating the therapeutic information on the patients. MaryMeldrum and TJMcE are supported by the Cancer ResearchCampaign and Medical Research Council.

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