Methods for diagnosis, differentiation and monitoring using urine proteins as markers in IgA nephropathy

Abstract

A method for diagnosis of IgA nephropathy is provided using a combination of alpha-1B-glycoprotein (A1BG) or a truncated fragment thereof having a molecular weight of 13-60 kDa, orosomucoid 1 (ORMI), and Ig lambda-2 chain C regions (IGLC2) as protein markers in a urine sample from a subject.

Claims

1. A method of diagnosis of IgA nephropathy in a human subject, comprising: (a) a step of detecting a combination of alpha-1B-glycoprotein (A1BG), orosomucoid 1 (ORMI), and Ig lambda-2 chain C regions (IGLC2) as protein markers in a urine sample from said human subject, wherein the markers are detected as peptide fragments using mass spectrometry; (b) a step of quantitative or semi-quantitative comparison of the markers detected in step (a) with the markers detected in a urine sample from a healthy human individual; and (c) a step of correlating results obtained in step (b) with the presence of IgA nephropathy in the human subject if the levels of all markers in the examined sample from the human subject identified in step (a) are higher than the levels of the same markers present the sample from the healthy human individual.

2. The method of claim 1, wherein a serotransferrin (TF) or platelet glycoprotein VI (GP6) as a further marker is detected and compared.

3. The method of claim 1, wherein the markers are detected using mass spectrometry in combination with an antibody-based test.

4. The method of claim 3, wherein the antibody-based test is Western blot.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) The invention was described in relation to the following figures of drawings in which

(2) FIG. 1 present sample results of Western blot analysis of A1BG, ORM1, IGLC2 and TF content in urine samples derived from patients with renal diseases versus healthy controls;

(3) FIG. 2A shows evaluation by Western blotting of the A1BG protein in selected urine samples of study participants with renal diseases (left-hand side of the blots) as compared to healthy subjects (right hand side of the blots). The presence of several subforms of the protein within various molecular weight can be noticed. Densitometry measurements were done for the upper, middle and bottom ranges separately. As shown in FIG. 2B, each of the molecular ranges correlated differently with the clinical diagnosis and also as compared to the cumulative assessment of all ranges (FIG. 2C);

(4) FIG. 3 shows a summary of densitometry readings from Western blotting of the ORM1 protein in selected urine samples of study participants with renal diseases as compared to healthy subjects.

(5) FIG. 4 shows a summary of densitometry readings from Western blotting of the IGLC2 protein (˜30 kDa) in selected urine samples of study participants with renal diseases as compared to healthy subjects.

(6) FIG. 5 shows a summary of densitometry readings from Western blotting of the TF protein in selected urine samples of study participants with renal diseases as compared to healthy subjects.

(7) FIG. 6(A) present sample results of Western blot analysis of GP6 content in urine samples derived from patients with renal diseases versus healthy controls, and (B) shows a summary of densitometry readings from Western blotting of the GP6 protein in selected urine samples of study participants with renal diseases as compared to healthy subjects.

(8) FIG. 7 presents a list of possible combinations of the markers for use according to the invention.

EXAMPLES

Example 1

(9) Discovery Phase

(10) The methodological approach of the discovery phase has been described in Mucha et al. Below, the most pertinent information is listed.

(11) Patients Characteristics

(12) Groups of Patients

(13) The study included 30 patients with IgAN and 30 healthy age- and sex-matched volunteers serving as controls. Demographic and clinical data of both groups are presented in Mucha, et al. (Supplementary material online, Table S1). Briefly, patients with biopsy-proven IgAN at different stages of chronic kidney disease (CKD) and older than 18 years were included. The inclusion criteria for the control group were as follows: age older than 18 years and absence of any kidney disease or other chronic diseases requiring treatment. The exclusion criteria for both groups included: active infection, history of malignancy, previous organ transplantation, or current pregnancy. To estimate GFR, we used the Chronic Kidney Disease Epidemiology Collaboration equations, which are the most accurate, have been evaluated in large diverse populations, and are applicable for clinical use. The study protocol was approved by the local ethics committee and informed consent was obtained from all participants. The study was performed in accordance with the Declaration of Helsinki.

(14) Urine Collection

(15) Samples were collected from all individuals according to a uniform study protocol, following the recommendations on urine proteomic sample collection. The second- or third-morning midstream urine was collected to sterile urine containers 1 to 3 h after previous urination. The pH of each sample was stabilized at 7.2 by addition of 1/10th vol. of 1M HEPES pH 7.2 immediately after collection. Then, samples were vortexed for 2 min, centrifuged at 3000×g at room temperature for 10 min to clear the debris, filtered (0.4-μm filter, Rotilabo-Spritzenfilter, Roth, Karlsruhe, Germany), and portioned into 1-ml aliquots that were stored at −80° C. before further use.

(16) Sample Filtration

(17) Membrane filters of the 10 kDa cut-off (Amicon Ultra-0.5, UFC501096, Millipore, Billerica, United States) were washed twice with MilliQ (MQ) water prior to use. Urine was centrifuged through the membrane at 14,000×g for 15 min. Next, 500 μl MQ was added to the retentate and centrifugation step was repeated. To recover the concentrated and desalted sample, the filter was placed upside down and centrifuged in a clean microcentrifuge tube for 2 min at 1000×g. The protein concentration was measured by the Bradford method. Aliquots of samples were stored at −80° C.

(18) Sample Preparation

(19) 30 IgAN samples were divided into 3 disease pooled samples (DPSs I, II, and III), and similarly, 30 control samples were divided into 3 control pooled samples (CPSs I, II, and III). Age and sex matching was preserved within the 3 pairs of pooled sample groups. All DPSs and CPSs were obtained in 2 technical replicates (marked A and B) each, making a set of 12 pooled samples to be compared after isobaric tags for relative and absolute quantitation (iTRAQ) labeling. As 4-plex iTRAQ was used, 2 technical replications of DPSs and CPSs were compared in 1 isoelectric focusing/liquid chromatography-mass spectrometry/mass spectrometry (IEF/LC-MS/MS) experiment. To analyze 12 samples, we conducted a set of 3 independent IEF/LC-MS/MS experiments. Aliquots with extracted peptides were stored at −80° C. for the IEF/LC-MS/MS analysis.

(20) Mass Spectrometry

(21) Qualitative MS/MS data processing The MS/MS data were pre-processed with Mascot Distiller (v. 2.3.2.0, Matrix Science, London, United Kingdom). Data search using the MASCOT search engine was conducted on the Swiss-Prot database with the taxonomy restricted to Homo sapiens (20,236 sequences) in a 3-step procedure described elsewhere to calculate MS and MS/MS measurement errors and to recalibrate the data for the repeated MASCOT to remove systematic bias. Protein ratios were calculated as the median ratio of their peptide's ratios. The statistical significance of a single protein ratio was assessed by an in-house program, Diffprot. Calculated P values were adjusted for multiple testing using a false discovery rate-con-trolling procedure, yielding protein ratio q values.

(22) Results of the Discovery Phase

(23) As a result of qualitative analysis (peptide and protein identification) in each of the 3 IEF/LC-MS-MS/MS experiments, 761, 951, and 956 proteins were identified, respectively, each represented by 2 or more peptides. The results of this observations were partially presented in Mucha et al. The discovery of alpha-1B-glycoprotein (A1BG), alpha-1-acid glycoprotein 1 (AGP1, ORM1), Ig lambda-2 chain C regions (IGLC2) and serotransferrin (TF) as markers for IgAN is being reported in the current invention (Table 2).

(24) TABLE-US-00002 TABLE 2 Peptide read-outs of urine proteomics specific for alpha-1B-glycoprotein (A1BG), alpha-1-acid glycoprotein 1 (AGP1, ORM1), Ig lambda-2 chain C regions (IGLC2) and serotransferrin (TF) obtained in a discovery phase of the invention. Number of ID 1 Protein name Gene peptides P04217 Alpha-1B-glycoprotein A1BG 51 Peptides q-value SEQ ID NO: 1 SLPAPW 4.26E−03 SEQ ID NO: 2 ITPGLK 4.66E−03 SEQ ID NO: 3 GVTFLLR 2.84E−03 SEQ ID NO: 4 SWVPHTF 5.71E−03 SEQ ID NO: 5 LLELTGPK 0.00E+00 SEQ ID NO: 6 SWITPGLK 4.57E−04 SEQ ID NO: 7 ATWSGAVLAGR 0.00E+00 SEQ ID NO: 8 LELHVDGPPPR 7.51E−04 SEQ ID NO: 9 SLPAPWLSMAPV 0.00E+00 SEQ ID NO: 10 VAPLSGVDFQLR 0.00E+00 SEQ ID NO: 11 IFVGPQHAGNYR 6.31E−05 SEQ ID NO: 12 HQFLLTGDTQGR 0.00E+00 SEQ ID NO: 13 LETPDFQLFK 0.00E+00 SEQ ID NO: 14 SGLSTGWTQLSK 0.00E+00 SEQ ID NO: 15 SMAPVSWITPGLK 1.67E−03 SEQ ID NO: 16 HGESSQVLHPGNK 0.00E+00 SEQ ID NO: 17 SLPAPWLSMAPVSW 6.55E−03 SEQ ID NO: 18 LELHVDGPPPRPQL 3.58E−04 SEQ ID NO: 19 HHGESSQVLHPGNK 7.56E−05 SEQ ID NO: 20 HQFLLTGDTQGRYR 7.25E−03 SEQ ID NO: 21 GVAQEPVHLDSPAIK 0.00E+00 SEQ ID NO: 22 LELIFVGPQHAGNYR 0.00E+00 SEQ ID NO: 23 LELHVDGPPPRPQLR 2.78E−04 SEQ ID NO: 24 IFFHLNAVALGDGGHY 0.00E+00 SEQ ID NO: 25 NLELIFVGPQHAGNYR 0.00E+00 SEQ ID NO: 26 TFESELSDPVELLVAES 7.70E−04 SEQ ID NO: 27 GAAANLELIFVGPQHAGNYR 0.00E+00 SEQ ID NO: 28 SLPAPWLSMAPVSWITPGLK 0.00E+00 SEQ ID NO: 29 TPGAAANLELIFVGPQHAGNYR 0.00E+00 SEQ ID NO: 30 SWVPHTFESELSDPVELLVAES 0.00E+00 SEQ ID NO: 31 TVRTPGAAANLELIFVGPQHAGNYR 0.00E+00 SEQ ID NO: 32 LHDNQNGWSGDSAPVELILSDETLPAPEFSPEPESGR 1.74E−03 SEQ ID NO: 33 TDGEGALSEPSATVTIEELAAPPPPVLMHHGESSQVLHPGNK 0.00E+00 SEQ ID NO: 34 SWVPHTFE 6.44E−03 SEQ ID NO: 35 VGPQHAGNYR 6.31E−05 SEQ ID NO: 36 STGWTQLSK 9.51E−03 SEQ ID NO: 37 HQFLLTGDTQ 0.00E+00 SEQ ID NO: 38 PVSWITPGLK 0.00E+00 SEQ ID NO: 39 HVDGPPPRPQLR 1.02E−03 SEQ ID NO: 40 LSMAPVSWITPGLK 6.31E−05 SEQ ID NO: 41 MHHGESSQVLHPGNK 3.73E−04 SEQ ID NO: 42 SGLSTGWTQLSKLLELTGPK 3.87E−05 SEQ ID NO: 43 GPPPRPQLR 7.28E−03 SEQ ID NO: 44 SLPAPWLSMA 5.85E−04 SEQ ID NO: 45 LELHVDGPPPRPQ 4.34E−04 SEQ ID NO: 46 IFFHLNAVALGDGGH 0.00E+00 SEQ ID NO: 47 NGVAQEPVHLDSPAIK 3.87E−05 SEQ ID NO: 48 TPGAAANLELIFVGPQHAGN 0.00E+00 SEQ ID NO: 49 LPAPWLSMAPVSWITPGLK 3.87E−05 Number of ID 2 Protein name Gene peptides P02763 Alpha-1-acid glycoprotein 1 ORM1 29 Peptides q-value SEQ ID NO: 50 TYMLAF 1.964E−03 SEQ ID NO: 51 AHLLILR 0.000E+00 SEQ ID NO: 52 NWGLSVY 6.310E−05 SEQ ID NO: 53 TYLNVQR 5.215E−03 SEQ ID NO: 54 YVGGQEHF 6.264E−03 SEQ ID NO: 55 FAHLLILR 1.789E−04 SEQ ID NO: 56 TTYLNVQR 4.565E−04 SEQ ID NO: 57 YVGGQEHFA 5.451E−04 SEQ ID NO: 58 YVGGQEHFAH 0.000E+00 SEQ ID NO: 59 EHFAHLLILR 6.704E−03 SEQ ID NO: 60 SDVVYTDWK 0.000E+00 SEQ ID NO: 61 YVGGQEHFAHL 0.000E+00 SEQ ID NO: 62 MLAFDVNDEK 0.000E+00 SEQ ID NO: 63 YVGGQEHFAHLL 3.866E−05 SEQ ID NO: 64 GQEHFAHLLILR 0.000E+00 SEQ ID NO: 65 SVYADKPETTK 0.000E+00 SEQ ID NO: 66 TYMLAFDVNDEK 0.000E+00 SEQ ID NO: 67 GLSVYADKPETTK 3.866E−05 SEQ ID NO: 68 EQLGEFYEALDCLR 0.000E+00 SEQ ID NO: 69 YVGGQEHFAHLLILR 0.000E+00 SEQ ID NO: 70 WGLSVYADKPETTK 3.866E−05 SEQ ID NO: 71 NWGLSVYADKPETTK 0.000E+00 SEQ ID NO: 72 DVNDEKNWGLSVYADKPETTK 0.000E+00 SEQ ID NO: 73 TYMLAFDVNDEKNWGLSVYADKPETTK 0.000E+00 SEQ ID NO: 74 VVYTDWK 7.322E−03 SEQ ID NO: 75 VYADKPETTK 1.877E−03 SEQ ID NO: 76 VGGQEHFAHLLILR 2.390E−04 SEQ ID NO: 77 IPKSDVVYTDWK 6.846E−03 SEQ ID NO: 78 GGQEHFAHLLILR 3.901E−03 Number of ID 3 Protein name Gene peptides P0CG05 Ig lambda-2 chain C regions IGLC2 4 Peptides q-value SEQ ID NO: 79 ADSSPVK 5.85E−04 SEQ ID NO: 80 GVETTTPSK 1.61E−04 SEQ ID NO: 81 AGVETTTPSK 0.00E+00 SEQ ID NO: 82 KAGVETTTPSK 8.03E−04 Number of ID 4 Protein name Gene peptides P02787 Serotransferrin TF 76 Peptides q-value SEQ ID NO: 83 VYIAGK 4.49E−03 SEQ ID NO: 84 DSGFQMN 1.79E−04 SEQ ID NO: 85 HSTIFEN 1.05E−03 SEQ ID NO: 86 GLLYNK 3.85E−03 SEQ ID NO: 87 SAGWNIPI 9.31E−03 SEQ ID NO: 88 PDPWAK 5.33E−03 SEQ ID NO: 89 MYLGYEY 4.17E−04 SEQ ID NO: 90 NPDPWAK 1.79E−04 SEQ ID NO: 91 DSAHGFLK 0.00E+00 SEQ ID NO: 92 FGYSGAFK 2.78E−04 SEQ ID NO: 93 VAEFYGSK 0.00E+00 SEQ ID NO: 94 KDSGFQMN 9.23E−04 SEQ ID NO: 95 EFQLFSSPH 2.43E−04 SEQ ID NO: 96 KPVEEYAN 6.13E−04 SEQ ID NO: 97 DGAGDVAFVK 0.00E+00 SEQ ID NO: 98 SAGWNIPIGLL 0.00E+00 SEQ ID NO: 99 EDLIWELLN 3.73E−04 SEQ ID NO: 100 YLGEEYVK 3.87E−05 SEQ ID NO: 101 HSTIFENLAN 0.00E+00 SEQ ID NO: 102 GYYGYTGAFR 0.00E+00 SEQ ID NO: 103 KPVDEYK 4.57E−04 SEQ ID NO: 104 IPMGLLYNK 3.87E−05 SEQ ID NO: 105 DSGFQMNQLR 0.00E+00 SEQ ID NO: 106 PVVAEFYGSK 0.00E+00 SEQ ID NO: 107 LAQVPSHTVVAR 0.00E+00 SEQ ID NO: 108 KPVDEYKD 8.27E−04 SEQ ID NO: 109 EGYYGYTGAFR 3.87E−05 SEQ ID NO: 110 SAGWNIPIGLLY 6.31E−05 SEQ ID NO: 111 NIPMGLLYNK 2.78E−04 SEQ ID NO: 112 HQTVPQNTGGK 0.00E+00 SEQ ID NO: 113 QYFGYSGAFK 6.31E−05 SEQ ID NO: 114 TAGWNIPMGLLY 6.31E−05 SEQ ID NO: 115 SLDGGFVYIAGK 0.00E+00 SEQ ID NO: 116 SASDLTWDNLK 0.00E+00 SEQ ID NO: 117 HSTIFENLANK 0.00E+00 SEQ ID NO: 118 EFQLFSSPHGK 0.00E+00 SEQ ID NO: 119 TAGWNIPMGLLYN 3.87E−05 SEQ ID NO: 120 SKEFQLFSSPH 2.29E−03 SEQ ID NO: 121 KDSGFQMNQLR 0.00E+00 SEQ ID NO: 122 EDLIWELLNQAQ 1.61E−04 SEQ ID NO: 123 MYLGYEYVTAIR 0.00E+00 SEQ ID NO: 124 KEGYYGYTGAFR 0.00E+00 SEQ ID NO: 125 LKPVVAEFYGSK 0.00E+00 SEQ ID NO: 126 KSASDLTWDNLK 0.00E+00 SEQ ID NO: 127 TAGWNIPMGLLYNK 0.00E+00 SEQ ID NO: 128 STLNQYFGYSGAFK 6.31E−05 SEQ ID NO: 129 NLKPVVAEFYGSK 0.00E+00 SEQ ID NO: 130 EDPQTFYYAVAVVK 0.00E+00 SEQ ID NO: 131 SKEFQLFSSPHGK 0.00E+00 SEQ ID NO: 132 AIAANEADAVTLDAGLVYDAY 0.00E+00 SEQ ID NO: 133 LAPNNLKPVVAEFYGSK 0.00E+00 SEQ ID NO: 134 EDLIWELLNQAQEHFGK 0.00E+00 SEQ ID NO: 135 IMNGEADAMSLDGGFVYIAGK 3.87E−05 SEQ ID NO: 136 AIAANEADAVTLDAGLVYDAYLAPN 0.00E+00 SEQ ID NO: 137 GKEDLIWELLNQAQEHFGK 0.00E+00 SEQ ID NO: 138 GGKEDLIWELLNQAQEHFGK 0.00E+00 SEQ ID NO: 139 EDLIWELLNQAQEHFGKDK 0.00E+00 SEQ ID NO: 140 SMGGKEDLIWELLNQAQEHFGK 0.00E+00 SEQ ID NO: 141 AIAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSK 0.00E+00 SEQ ID NO: 142 APNHAVVT 8.70E−03 SEQ ID NO: 143 APNHAVVTR 6.31E−05 SEQ ID NO: 144 SAGWNIPIGL 8.24E−03 SEQ ID NO: 145 QVPSHTVVAR 1.06E−03 SEQ ID NO: 146 STIFENLANK 0.00E+00 SEQ ID NO: 147 HLAQVPSHTVVAR 0.00E+00 SEQ ID NO: 148 GWNIPMGLLYNK 6.31E−05 SEQ ID NO: 149 MYLGYEYVTAIRNLR 3.86E−03 SEQ ID NO: 150 PNNLKPVVAEFYGSK 5.33E−03 SEQ ID NO: 151 HSTIFENLA 5.85E−04 SEQ ID NO: 152 ADRDQYELL 1.81E−03 SEQ ID NO: 153 QLFSSPHGK 1.75E−03 SEQ ID NO: 154 LGYEYVTAIR 2.69E−03 SEQ ID NO: 155 HSTIFENLANKADR 5.85E−04 SEQ ID NO: 156 HQTVPQNTGGKNPDPWAK 3.87E−05 SEQ ID NO: 157 KEDLIWELLNQAQEHFGK 1.52E−03 SEQ ID NO: 158 GLVYDAYLAPNNLKPVVAEFYGSK 3.87E−05

Example 2

(25) Validation Phase

(26) The primary difference between the discovery and validation phases is the transition from the assessment of the pooled urine samples (i.e. the discovery phase) to the individual evaluation of each protein in a given patient or a healthy person and a direct correlation of these results with the known clinical parameters in each case (i.e. the validation phase).

(27) Patient Characteristics

(28) The study included 133 renal disease patients and 19 healthy controls. Renal disease included IgAN (77 cases), ADPKD (29) and LN (27).

(29) Sample Collection

(30) Urinary samples were collected according to the protocol standardized in the Transplantation Institute, Medical University of Warsaw.

(31) SDS-PAGE

(32) Samples were defrozen to room temperature (˜23° C.), then suspended by intensive pipetting or mixing using a vortex. Leammli buffer was added to urine samples to achieve final concentrations as follows: 2% SDS; 10% glycerol; 5% β-mercaptoethanol; 0.002% bromophenol blue; 0.125 M Tris-HCl; pH 6.8. Samples were boiled at 95° C. for 2 min. 10 μl of each sample was loaded on the Mini-PROTEAN TGX 4-15% gradient gel.

(33) Western Blotting Analysis

(34) The method developed by the Department of Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences (patent application no P.415033) allows to assay all the proteins of interest in the urine samples. It permits the analysis of all the selected protein biomarkers with the accuracy not reachable by classical methods. To date, the proteome analysis of urine in medicine starts from centrifugation of the sample (in line with the European Confederacy of Laboratory Medicine guidelines), which result losing of protein which are insoluble, and exist as aggregates or degradants. This solid fraction is crucial because proteins progress into insoluble forms randomly, depending of protein, state of patient, his diet and properties of urine. For that reason, in the current study we used the whole urine in form of suspension, which next were analyzed by Western blotting technique. That gives an opportunity to examine all of proteins present in a given in urine sample. Advantages of this method are important for medicinal diagnostics. Method is noninvasive for patients, it allows collecting the samples from the patients even few times per day, and it is relatively little time consuming. For Western blotting analysis, the urine protein samples were separated on SDS-PAGE gels, as described above, and transferred to a nitrocellulose membrane. Membranes were incubated in appropriate blocking buffer (either 5% low-fat dry milk or bovine serum albumin in TBS-TWEEN® 20 (polysorbate 20) (TBST)). After an incubation in the primary antibody (A1BG (F-9); catalog number sc-374415; Santa Cruz) the cells were washed in TBST, and incubated with a horseradish peroxidase-conjugated secondary antibody. The chemiluminescence reaction for HRP was developed using SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific) and visualized with Stella 8300 bioimager. Densitometry read-outs were carried out for each of the bands in the blots. Eight randomly chosen patients samples were pooled together and used on each Western blot as a benchmark. Densitometry read-outs from other band on a given Western blot membrane were divided by the read-out of the respective benchmark. The results were referred to as a “relative band density”.

(35) List of antibodies used for Western blotting: A1BG (cat #sc-374415, Santa Cruz), ORM1 (sc-69753, Santa Cruz), IGLC2 (sc-33134, Santa Cruz), TF (sc-21011, Santa Crus), GP6 (sc-20149, Santa Cruz).

(36) Results

(37) The results of representative Western blotting analysis for A1BG, ORM1, IGLC2 and TF presence in urine samples are presented in FIG. 1.

(38) A1BG Based on the Western blotting analysis, it becomes evident that study participants with kidney diseases exhibit the presence of different forms of the protein (for the purpose of this invention segregated into within the high [˜80 kDa], medium [˜45 kDa] and low [˜15 kDa] molecular weight range), occurring in different proportions. A direct comparison of selected samples from kidney disease versus heathy patients is presented in FIG. 2A. Mutual relations between the visible forms may be important in the pathophysiology of the given disease. Indeed, as presented in FIG. 2B, various subforms of A1BG correlate differently with the type of kidney disease and also differently as compared to the cumulative assessment of the protein (FIG. 2C). Notably, the bottom-range bands tend to be most prominently elevated in IgAN patients.

(39) Validation phase as described above was also performed for the other markers, in particular ORM1, IGLC2 and TF and the results are presented in FIGS. 3-5. The results are also presented for GP6 (FIG. 6). Although GP6 is on average expressed at higher levels in LN than IgA, on the basis of our results and data, the compilation of 4 proteins reported in the GP6 compilation may increase the sensitivity and specificity of the test.