Introduction. Protocol for crystalluria investigation. Mini Review. Michel Daudon* and Vincent Frochot Crystalluria

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1 Clin Chem Lab Med 2015; 53(Suppl): S1479 S1487 Mini Review Michel Daudon* and Vincent Frochot Crystalluria DOI /cclm Received May 22, 2015; accepted July 28, 2015; previously published online October 28, 2015 Abstract: Crystalluria is a marker of urine supersaturation with substances deriving from metabolic disorders, inherited diseases or drugs. The investigation of crystalluria must be done according to a protocol which includes the delivery to the laboratory of a proper urine sample, the use of a microscope equipped with polarized light, the accurate knowledge of urine ph, and a comprehensive examination of the crystals, which is based on their identification, quantification and size measurement. For unusual crystals, infrared spectroscopy may also be needed. The main urinary crystalline categories include: calcium oxalates, calcium phosphates, uric acids and urates, struvite, aminoacids (cystine), purines (2,8-dihydroxyadenine and xanthine) and drugs (e.g. sulfamethoxazole, amoxycillin, ceftriaxone, atazanavir). The investigation of crystalluria is a cheap and valuable tool for the detection and the monitoring of inherited and acquired diseases associated with urinary stone formation or renal function impairment either acute or chronic due to intrarenal crystal precipitation. Keywords: 2,8-dihydroxyadenine; calcium oxalate; calcium phosphate; crystalluria; cystine; drugs; purines. Introduction Crystalluria is a frequent finding both in normal individuals and in patients suffering from urolithiasis from various causes. The study of crystalluria is useful for: the diagnosis of lithogenic inherited diseases (e.g. primary hyperoxaluria, cystinuria, adenine phosphoribosyltransferase deficiency); the identification of crystals due to drugs, which may be responsible for acute kidney injury as well as *Corresponding author: Michel Daudon, PhD, Laboratoire des Lithiases, Service des Explorations Fonctionnelles, APHP, Hôpital Tenon, 4, rue de la Chine, Paris cedex 20, France, michel.daudon@tnn.aphp.fr Vincent Frochot: Laboratoire des Lithiases, Service des Explorations Fonctionnelles, Hôpital Tenon, APHP, Paris, France chronic kidney disease; the assessment of metabolic disorders associated with stone formation; the assessment, in stone formers, of the risk of stone recurrence. Crystals from metabolic origin are the result of an equilibrium break between two groups of substances, the promoters and the inhibitors of crystallization. Promoters are substances excreted by the kidney, whose concentration exceeds the ability of urine to maintain them as soluble molecules and ions. Calcium, oxalate, urate and phosphate ions are the main promoters of crystals [1 5]. Inhibitors, which are either filtered through glomeruli or are produced locally by tubular cells, are substances which are able to avoid or delay crystal formation, growth, aggregation and/or adhesion to the tubular epithelium [6, 7]. Among inhibitors, small ions and molecules such as magnesium, citrate and pyrophosphate, form complexes with some promoters, a fact which decreases urine supersaturation [6]. Macromolecules such as osteopontin, bikunin, matrix GLA protein, Tamm-Horsfall protein or the urinary fragment 1 of prothrombin also are able to stop crystal growth, aggregation and/or adhesion to the tubular cells, thus allowing the crystals to be eliminated from the kidney with the urine flow [6 8]. This paper describes how crystalluria should be investigated, the main categories of urinary crystals, and the utility of the evaluation of crystalluria for the diagnosis and follow-up of recurrent stone formers. Protocol for crystalluria investigation The investigation of crystalluria must be done according to a proper methodology [9 11]. It should be performed under the usual subject s life habits and alimentation, on fasting conditions, on the whole volume of the first morning urine, which is usually the best due to the reduced water intake during night, even though also the second urine of the morning, produced in fasting conditions may be considered. The container with the whole volume of urine should be released to the laboratory within 2 h from voiding, kept at room temperature and processed without delay [12].

2 S1480 Daudon and Frochot: Crystalluria Crystals must be examined using preferably a phase contrast microscope, which must also be equipped with a polarized light device, which shows the birefringence features of crystals and is mandatory for their correct identification, especially when they come with unusual morphologies. Today, automated instruments for urine sediment examination, when able to supply images of good quality, may also be used for the identification of the most common crystals [13]. Urine ph is an important feature to know when examining a sample for crystalluria. In this respect, it must be remembered that most crystalline species are ph-sensitive, with the only exception being calcium oxalate and 2,8-dihydroxyadenine crystals (Table 1), for which the main factor favoring precipitation is an excessive molar concentration. By contrast, highly ph-dependent species may form crystals in specific ph ranges even at normal or relatively low molar concentration. The best example of a ph-dependent solute is uric acid: at a urine ph of 5.0, it may crystallize at molar concentrations of about 2 mmol/l, while at a ph of a concentration of 4 mmol/l is required for crystallization. Thus, an accurate measurement of urine ph is mandatory for the investigation of crystalluria. After homogenization of the sample by gentle shaking, microscopic examination includes urine cytology and a comprehensive evaluation of crystals namely, their identification, quantitation and measurement of crystals and their aggregates size. However, the wide morphological spectrum of each crystalline category, coupled with their complex chemical composition, may make the identification of crystals difficult, a task which can be facilitated by the consultation of papers on the subject and atlases on urinary sediment [9, 11, 14 16]. For crystals which cannot be identified after their morphology coupled with their polarizing features and the knowledge of urinary ph, especially if they are suspicious for inherited diseases or for being due to drugs, infrared spectroscopy is recommended [17]. Classification of urinary crystals Calcium oxalates Calcium oxalate monohydrate, or whewellite (Figure 1A) is closely associated with high oxalate concentration in the presence of normal or low calcium [18]. Urine samples that contain only whewellite crystals without weddellite are highly suggestive for a high oxalate molar Table 1: Dependence on urinary ph of the metabolic crystalline categories described in text. Cristalline species Dependence on urine ph Average ph (ranges) Calcium oxalates Calcium oxalate monohydrate Low 5.9 ( ) Calcium oxalate dihydrate Low 5.8 ( ) Calcium phosphates Calcium ortophosphates (amorphous carbonated calcium phosphate and carbapatites) High 6.7 ( ) Brushite High 6.5 ( ) Uric acids Amorphous uric acid Moderate 5.5 ( ) Anhydrous uric acid (uricite) High 5.2 ( ) Uric acid monohydrate High 5.2 ( ) Uric acid dihydrate High 5.2 ( ) Urates Ammonium hydrogen urate Moderate 7.2 ( ) Magnesium ammonium phosphate Struvite High 7.7 ( ) Other purines Xanthine Moderate 5.6 ( ) 2,8-dihydroxyadenine Very low 6.2 ( ) Aminoacids Cystine Moderate 6.8 ( )

3 Daudon and Frochot: Crystalluria S1481 Figure 1: Calcium oxalate crystals. (A) Whewellite crystals with the typical ovoid shape. (B) Typical octahedral (bipyramidal) crystals of weddellite and a small red-cell like crystal of whewellite (arrow). (C) Elongated hexagonal crystals of whewellite as found in the urine of patients after the ingestion of ethyleneglycol. (D) Octahedral and dodecahedral (arrow) crystals of weddellite. concentration, a fact that should prompt the search for primary hyperoxaluria. Conversely, calcium oxalate dihydrate, or weddellite (Figure 1B), is usually associated with hypercalciuria. In our experience, more than 94% of urine samples containing calcium oxalate crystals with a calcium to oxalate ratio < 5 exhibit whewellite crystals, either alone or accompanied by weddellite. By contrast, more than 90% of urine samples containing calcium oxalate crystals with a calcium to oxalate ratio > 14 contain weddellite alone [16]. Whewellite crystals with oval or dumb-bell shape (Figure 1A) are commonly associated with high levels of urine oxalate, while small crystals resembling erythrocytes may be found in urine with normal or moderately increased oxalate (Figure 1B). Peculiar types of whewellite crystals, with the shape of elongated and narrow hexagons and diamonds (Figure 1C) are found in the urine after the ingestion of ethylene glycol, which results in severe hyperoxaluria. Weddellite, in most instances, appears as octahedral (bipyramidal) crystals (Figure 1B), while more rare dodecahedral crystals (Figure 1D) are associated with high concentration of urinary calcium (Figure 2). Figure 2: Correlation between the frequency of weddellite dodecahedral crystals (axis) and the urinary molar concentration of calcium (abscissa) (adapted from reference [6]). Calcium phosphates The formation of crystals of calcium orthophosphates (mainly amorphous carbonated calcium phosphate and carbapatites) (Figure 3A) and brushite (dicalcium phosphate dihydrate) (Figure 3B) depend on different

4 S1482 Daudon and Frochot: Crystalluria Figure 3: Calcium phosphate crystals. (A) Amorphous granulations of carbonated calcium phosphate. (B) Asymmetrical rod-shaped crystals of calcium hydrogen phosphate dihydrate (brushite). (C) Crystal aggregate of brushite intermingled with weddellite (arrows). biochemical conditions [19]. Calcium orthophosphate species are mainly dependent on urine ph and they form easily when urine ph is above 6.5 (Table 1). Brushite formation usually requires high concentrations of calcium and phosphate, even though it may also be favored by low citrate concentration. Brushite can also be found in aggregates with weddellite crystals (Figure 3C), being often associated with hypercalciuria. Uric acids and urates Uric acid crystals form in acidic urine (Table 1). Four types of uric acid precipitates may be found in urine: amorphous uric acid, anhydrous uric acid (uricite) and two hydrated forms, namely uric acid monohydrate and dihydrate. Uric acid dihydrate (Figure 4A) and amorphous uric acid (Figure 4B) are the most common species found in urine. As shown in Figure 4C and D, they depend on different metabolic conditions: uric acid dihydrate is mostly dependent on low urine ph while amorphous uric acid is mainly related to high urine urate concentration. By contrast, urate salts, such as ammonium hydrogen urate, form in alkaline urine (Table 1), explaining why this crystalline species may accompany struvite in the case of urinary tract infection by urea-splitting bacteria. Struvite Magnesium ammonium phosphate hexahydrate or struvite, of which several morphological variants can be observed (Figure 5), precipitates in urine only in the presence of high urine ph (Table 1) associated with high ammonium content. This condition is found only in the case of urinary tract infection by micro-organisms able to hydrolyze urea, which results in the production of high amounts of ammonia and alkaline urine. Thus, struvite may be considered virtually pathognomonic of the presence of urea-splitting bacteria in the urine specimen.

5 Daudon and Frochot: Crystalluria S1483 Figure 4: Uric acid crystals. (A) Typical lozenges of uric acid dihydrate crystals. (B) Amorphous uric acid granulations. (C) Correlation between uric acid dihydrate crystals and urinary ph. (D) Correlation between amorphous uric acid granulations and uricosuria. Infrequent crystals Cystine Cystine crystals are pathognomonic of cystinuria, which is a hereditary disease characterized by a proximal tubular dysfunction due to mutations on genes coding for the transporter of dibasic amino acids. Cystine crystals present as hexagonal plates, often irregular, either isolated or in large aggregates (Figure 6A). 2,8-Dihydroxyadenine The inherited condition of adenine phosphoribosyltransferase deficiency can be diagnosed by the identification of crystals in urine. In fact, in virtually all untreated patients, characteristic spherical crystals of 2,8-dihydroxyadenine which under polarized light appear as a black Maltese cross, are observed in urine (Figure 6B). However, as these crystals can be confused with other spherical crystals such as ammonium hydrogen urate, in suspected cases the investigation with infrared spectroscopy on a centrifuged pellet is mandatory. The same approach is advisable for patients presenting with acute kidney injury due to massive precipitation of 2,8-dihydroxyadenine crystals within the renal tubules [20], in whom only few and atypical crystals can be found in the urine [20, 21]. Xanthine Xanthine crystals, which are found in xanthinuria, a rare genetic disorder caused by the deficiency of the enzyme xanthine oxidase, present as polarizing granules or sticks without a characteristic aspect. Therefore infrared analysis is required for accurate identification. Tyrosine, leucine or potassium orotate crystals can also be found in urine. However, since they are associated with rare metabolic disease [14, 15], they are an uncommon finding. Crystals due to drugs Several drugs, mainly antimicrobial or antiviral agents, may crystallize in urine when used at high dose and/or for long periods [22]. Some sulfonamides may induce crystalluria. Among them, N-acetylsulfamethoxazole chlorhydrate, a metabolite of sulfamethoxazole which is widely used in combination with trimethoprim. Crystals, which precipitate at a ph around 5.0, may have the shape of lozenges, hexagons

6 S1484 Daudon and Frochot: Crystalluria Figure 5: Struvite crystals as seen under polarized light. (A) A coffin-shaped crystal of struvite. (B) Rod-shaped and coffin-shaped crystals of struvite. (C) A hexagonal crystal of struvite and small agglomerates of amorphous carbonated calcium phosphate grains. (D) Trapezoidal crystals of struvite. (E) A large X-shaped crystal of struvite with birefringent small aggregates of ammonium hydrogen urate crystals (black arrows). or ovoid structures, which may mimic uric acid dihydrate, cystine or whewellite, respectively (Figure 6C E). N-acetylsulfadiazine, a metabolite of sulfadiazine used in the treatment of cerebral toxoplasmosis, also crystallizes in acidic urine. For an accurate identification of these crystals, infrared spectroscopy is recommended [17, 23]. Occasionally, other antibacterial agents such as aminopenicillines, particularly amoxicilline [24], fluoroquinolones [25] and, more rarely, ceftriaxone may induce crystalluria, which at times may be associated with acute kidney injury due to the intratubular precipitation of crystals. Antiviral agents currently are a common cause of crystalluria, especially atazanavir [26], an antiprotease widely used in combination therapy in AIDS patients, which form very large bundles of drug needles ( μm or more) (Figure 6F). Acyclovir, used in the treatment of herpes virus infections, also forms crystals with an aspect of long, thin needles that may form aggregates. Other drugs which may cause crystalluria are the inhibitor of HIV-1 protease indinavir, the potassium-sparing diuretic triamterene, the barbiturate primidone, and the antiepileptic felbamate, whose crystals have most

7 Daudon and Frochot: Crystalluria S1485 Figure 6: Uncommon crystals. (A) Cystine crystals. (B) 2,8-dihydroxyadenine crystals as seen by polarized light. (C) Diamond-shaped crystal of N-acetylsulfamethoxazole hydrochloride very similar to uric acid dihydrate crystals. (D) Oval-shaped crystal of N-acetylsulfamethoxazole hydrochloride very similar to whewellite. (E) Hexagonal crystals of N-acetylsulfamethoxazole hydrochloride that may easily be misidentified as cystine crystals. (F) Needle-shaped crystals of atazanavir surrounded by leukocytes. unusual and pleomorphic morphologies, a fact which should alert the urine microscopist. Other drugs such as the vasodilator naftidrofuryl oxalate, the gastrointestinal lipase inhibitor orlistat and vitamin C, when given intravenously at high doses, may also cause urinary crystals, which are made up of calcium oxalate undistinguishable from calcium oxalate crystals from other causes [15]. Quantification and frequency of crystalluria in stone formers In stone formers, the disappearance of crystalluria indicates that lithogenic activity is under control. This result is most often attainable in patients with calcium or uric acid nephrolithiasis, while a complete disappearance of crystalluria is more difficult to achieve in inherited disorders such as primary hyperoxaluria type 1 (PH1), cystinuria or dihydroxyadeninuria, which have a very active and permanent crystallization. In these conditions, a decrease in the amount of crystals present in urine samples, expressed as global crystal volume (GCV), is often sufficient to efficiently reduce the lithogenic process [27]. By comparing clinical outcomes and variations in GCV in serial urine samples, we have determined quantitative thresholds of harmful crystalluria in PH1 and cystinuria, thus defining the desirable goals to achieve [27, 28]. In PH1, a crystal volume < 500 μm 3 /mm 3 of urine was associated with a lowered risk of tubular plugging with calcium oxalate crystals in the days following combined liver-kidney transplantation, a crucial period when

8 S1486 Daudon and Frochot: Crystalluria considerable amounts of calcium oxalate stored in bones are excreted through the kidneys after restoration of renal function, with the risk of massive tubular obstruction and loss of the kidney transplant [29]. By closely monitoring calcium oxalate GCV, early post-transplant care of PH1 children could be optimized [28], which resulted in improved long-term outcome [30]. In cystinuric patients, a cystine GCV > 3000 μm 3 /mm 3 of urine was predictive of recurrence of cystine stones, whereas a stable lower value was associated with a lack of recurrence [27]. In all stone formers, crystalluria is more frequent than in healthy subjects, although in these latter some crystals may transiently be found in urine, especially during the hours following a meal. By evaluating the presence or absence of crystals in first morning urine samples at each visit in 204 calcium oxalate stone formers followed at our institution for a median duration of 7 years (5 15 years), we observed that the presence of crystals in 50% of urine samples was associated with stone recurrence in 87% of cases, whereas stone recurrence was observed in only 9% of patients with less frequent crystalluria [31]. Thus, we proposed a crystalluria index defined as the ratio of the number of urine samples containing crystals to the total number of examined samples in a given patient, with a crystalluria index > 0.50 as the threshold value indicative of persistent lithogenic activity and risk of stone recurrence [32]. 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