Nephrolithiasis is a common disease, occurring in both industrialized and developing countries and mainly affecting adults aged 20–60 years [
A recent survey in Italy has shown a prevalence of 7.5% in an urban population [
It is worldwide underlined that the determination of crystalline and molecular composition and the quantification of all stone components are helpful to establish the etiology of stones disease. Different methodologies exist for the analysis of renal stones. These include qualitative “dry” chemical spot tests and quantitative X-ray crystallography, infrared spectroscopy (FT-IR), and “wet” chemistry tests [
Chemical spot tests are relatively inaccurate because of false-positive and false-negative results and do not allow distinguishing between the crystalline phases. Among physical methods, X-ray diffraction is appropriate for quantification of mineral samples, but it cannot adequately detect amorphous species such as carbapatite or struvite. FT-IR spectroscopy is the most appropriate technique for stones analysis and is becoming the gold standard for stone analysis [
The aim of this paper is to compare a semiquantitative method (DiaSys) with a quantitative method (FT-IR spectroscopy technique) for urinary stone analysis, in order to introduce in our laboratory a more reliable technique.
We analyzed 48 urinary stones, from 48 patients (28 men and 20 women, age range 21–75) from our Divisions of Nephrology and Urology. Stones were analyzed by both spot test and FT-IR method as described below. Shape, colour, size and weight were registered for each stone at the time of delivery. The stones submitted to analysis were washed with deionized water and dried at room temperature for 24 h; subsequently the stones were powdered in a mortar and aliquoted in 2 vials before being subjected to the following analysis.
Spot test analysis for the qualitative tests of urinary calculi composition was performed according to kit instructions (Urinary Calculi Analysis kit, DiaSys, Diagnostic System GmbH, Holzheim, Germany). This method allows detecting the presence of cystine and following ions usually present in urinary calculi: carbonate, calcium, oxalate, ammonium, phosphate, magnesium, and urate. The assay consists of the addition of chemical reagents labeled R1 to R15 dropwise to the finely pulverized sample and placed into a vessel with 50 mL of distilled water. Then the appearance of certain colors, precipitates, or air bubbles would indicate positive results for one of the ions and cystine [
The second aliquot of the pulverized stone was mixed with an inert powdered support (dried potassium bromide) in a proportion of 0.5 to 2% in agate mortar. This mixture was transferred into an appropriate die and pressed at 10 t/cm2 to form a transparent pellet 13 mm in diameter. The pellet assembled in a holder was placed in the IR beam of the spectrometer. The spectral region investigated was from 4000 to 400 cm−1; 32 scans were averaged with a 4 cm−1 resolution for each spectrum. A background spectrum was collected before every analysis, for the sample blank.
Again a background spectrum was measured to provide a relative scale for the absorption intensity. Background spectra were performed at air or pure KBr pellet. Spectra were recorded by means of a Perkin Elmer Spectrum One [
Spectra were then computer-matched with the Euclidean search application, a tool of SPECTRA NICODOM IR Library (obtained from Nicodom s.r.o., Hlavni 2727 CZ-14100 Praha 4, Czech Republic, EU) that compares the unknown spectrum with reference spectra contained in the library between 4000 and 400 cm−1. A report is then generated for the various stone components. The results of the automatic comparison for a spectrum identification were provided as a list of the best-fitting spectra with their score. The score value can range from 0.000 to 1.000. Score 1.000 indicates a perfect likeness between the unknown spectrum and the reference one. In each case, a visual inspection of the spectra was performed to check the results.
On the basis of FT-IR analysis, the 48 calculi were divided into three groups: pure stone (
Classification criteria were established by comparing results obtained with the two methods in reference to the identification of cystine and the ionic species as follows: agreement: when FT-IR and the chemical spot test identify the same components, partial agreement: when the chemical spot test identifies the main component detected by FT-IR in addition to other ions not attributable to a particular crystalline species, disagreement: when the spot test does not identify the main substance or when it identifies the main component but also other ions referable to particular crystalline species which were not detected by FT-IR.
The percentage values of major constituents for the chemical spot test and FT-IR method are shown in Tables
Chemical constituents of urinary stone (%) obtained using chemical spot test (a) and FT-IR analysis (b).
Chemical spot test analysis
Components detected | Number | Frequency (%) |
---|---|---|
Oxalate | 45/48 | 93.7 |
Calcium | 39/48 | 81.2 |
Magnesium | 16/48 | 33.3 |
Phosphate | 14/48 | 29.1 |
Urate | 9/48 | 18.7 |
Ammonium | 7/48 | 14.5 |
Cystine | 5/48 | 10.4 |
Carbonate | 3/48 | 6.2 |
FT-IR analysis
Components detected | Number | Frequency (%) |
---|---|---|
Calcium oxalate monohydrate | 32/48 | 66.6 |
Carbonate apatite | 16/48 | 33.3 |
Anhydrous uric acid | 12/48 | 25.0 |
Calcium oxalate dihydrate | 8/48 | 16.6 |
Dihydrate uric acid | 2/48 | 4.1 |
Magnesium ammonium phosphate | 2/48 | 4.1 |
Cystine | 2/48 | 4.1 |
Atazanavir | 1/48 | 1.0 |
Comparison between urinary stone composition (frequency %) obtained with chemical spot test (extrapolated associating the single chemical constituents) and FT-IR analysis.
Chemical spot test versus FT-IR in components identification | ||
---|---|---|
Components detected | Frequency (%) by chemical spot test | Frequency (%) by FT-IR |
Oxalate | 93.7 | 75.0 |
Calcium | 81.2 | 77.1 |
Magnesium | 33.3 | 4.1 |
Phosphate | 29.1 | 37.5 |
Urate | 18.7 | 25.0 |
Ammonium | 14.5 | 4.1 |
Cystine | 10.4 | 4.1 |
Carbonate | 6.2 | 37.5 |
In the pure stone group (Table
Agreement between results obtained with chemical spot test and FT-IR in the pure stone group.
Pure stones | |||
---|---|---|---|
Substances detected |
Agreement ( |
Partial agreement ( |
Disagreement ( |
Calcium oxalate monohydrate (14) | 10 | 3 | 1 |
Anhydrous uric acid (6) | 0 | 0 | 6 |
Cystine (2) | 1 | 1 | 0 |
Atazanavir (1) | 0 | 0 | 1 |
|
|||
Total (23) | 11 (47.8%) | 4 (17.4%) | 8 (34.8%) |
Tables
Agreement between results obtained with chemical spot test and FT-IR in the pure stone with substances in trace group.
Pure stones with substance in trace | |||
---|---|---|---|
Components detected ( |
Agreement ( |
Partial agreement ( |
Disagreement ( |
Calcium oxalate monohydrate + carbonate apatite trace (4) | 0 | 3 | 1 |
Calcium oxalate dihydrate + carbonate apatite trace (1) | 0 | 1 | 0 |
Calcium oxalate monohydrate + calcium oxalate dihydrate trace (1) | 0 | 1 | 0 |
|
|||
Total (6) | 0 (0.0%) | 5 (83.3%) | 1 (16.7%) |
Agreement between results obtained with chemical spot test and FT-IR in the mixed stone group.
Mixed stones | |||
---|---|---|---|
Substances detected ( |
Agreement ( |
Partial agreement ( |
Disagreement ( |
Anhydrous uric acid + calcium oxalate monohydrate (3) | 2 | 0 | 1 |
Calcium oxalate monohydrate + calcium oxalate dihydrate + carbonate apatite (4) | 0 | 2 | 2 |
Magnesium ammonium phosphate + carbonate apatite + calcium oxalate monohydrate (1) | 0 | 0 | 1 |
Carbonate apatite + magnesium ammonium phosphate + protein (1) | 0 | 0 | 1 |
Anhydrous uric acid + calcium oxalate monohydrate + carbonate apatite (1) | 0 | 0 | 1 |
Anhydrous uric acid + dihydrate uric acid (2) | 1 | 0 | 1 |
Carbonate apatite + calcium oxalate monohydrate + calcium oxalate dihydrate (3) | 0 | 0 | 3 |
Calcium oxalate monohydrate + carbonate apatite (2) | 0 | 1 | 1 |
Calcium oxalate monohydrate + calcium oxalate dihydrate (1) | 0 | 1 | 0 |
Calcium oxalate dihydrate + carbonate apatite + protein (1) | 0 | 0 | 1 |
|
|||
Total (19) | 3 (15.8%) | 4 (21.0%) | 12 (63.2%) |
For the pure stone with substances in trace group the two methods were in agreement in none (0%), in partial agreement in 5/6 (83.3%), and in disagreement in 1/6 (16.7%) cases.
Thus, the mixed stone groups show the major disagreement.
Urolithiasis is a frequent disease whose incidence is progressively increased in the last years in both men and women.
Urinary stone composition is important both for correct diagnosis and for patient follow-up. Among the methods available for urinary stone analysis, chemical analysis has been traditionally used most widely due to its ease and low cost even if this technique is time consuming and necessitates large stone samples.
Guidelines on Urolithiasis of European Association of Urology 2013 underlines the obsolescence of chemical analysis and recommends the use of FT-IR for urinary stone analysis [
Yet, we think that there is one stronger reason to discourage the use of chemical spot tests, that is, the many relevant drawbacks of these techniques even for the very frequent forms of nephrolithiasis.
In this study, 48 urinary stones were analyzed using both the chemical spot test and the FT-IR method and results were compared. As expected, the most common components detected by both methods were calcium and oxalate. However we observed oxalate in 75.0% and calcium in 77.1% using the FT-IR method, while we observed oxalate in 93,7% and calcium in 81.2% using the chemical spot test. The difference between the two methods in detecting oxalate is remarkable since it was incorrectly recognized in 25.0% more stones.
However, the insufficient discrimination by the chemical spot method of uric acid stones is even more amazing. In fact, the chemical method does not or, only partially, recognize the presence of uric acid in stone samples (number 6) detected by FT-IR analysis (Figure
FT-IR spectrum of a uric acid anhydrous stone (top) matched with reference spectra contained in the NICODOM library. The reference spectrum of uric acid anhydrous stone with the best fit value was showed in the bottom.
Also critical is the number of false positive ammonium or magnesium containing stones by the chemical spot method. In fact this finding may suggest to clinicians (despite absence of phosphates) that the stone is constituted by PAM, which mistakenly would lead to the recognition of an infective pathogenesis. Similarly, with this method five cystine containing stones were detected while the true number was two; this finding would lead to specific diagnostic and therapeutic measures which may even worsen the renal stone disease.
Another example can be found in calcium phosphates, which constitute a very heterogeneous group with multiple etiology including infections (in the case of carbonated calcium phosphate and whitlockite), hypercalciuric mechanism (in the case of brushite and octacalcium phosphate), and disorders related to tubular acidification function (in the case of carbonate apatite).
Moreover, there are significant differences on identification of substances present in trace in mixed stones, in fact FT-IR technique shows a high sensitivity and allows an accurate identification of stone composition.
The elaborated treatment of samples and the subjective interpretation of results are the major disadvantages of the spot test, adding a variability out of control to structural method limits. Chemical methods have repeatedly proved to be unreliable in numerous quality control programs, with error rates in identifying certain components above 90% [
For these reasons, according to these results, the introduction of the FT-IR technique in our clinical laboratory may be more responsive to clinicians’ expectations.
Calcium oxalate monohydrate
Calcium oxalate dihydrate
Carbonate apatite
Magnesium ammonium phosphate
Uric acid anhydrous
Uric acid dihydrate
Fourier transform infrared spectroscopy.
The authors declare that there is no conflict of interests regarding the publication of this paper.