Effects of storage in formalin on composition of canine and feline uroliths

Hasan Albasan Minnesota Urolith Center, Veterinary Clinical Sciences Department, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108.
Department of Internal Medicine, Faculty of Veterinary Medicine, Ankara University, Ankara, 06110 Turkey.

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Carl A. Osborne Minnesota Urolith Center, Veterinary Clinical Sciences Department, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108.

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Jody P. Lulich Minnesota Urolith Center, Veterinary Clinical Sciences Department, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108.

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Lisa K. Ulrich Minnesota Urolith Center, Veterinary Clinical Sciences Department, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108.

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Lori A. Koehler Minnesota Urolith Center, Veterinary Clinical Sciences Department, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108.

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Abstract

Objective—To determine whether storage in neutral-buffered 10% formalin in vitro has any effect on the composition of biogenic minerals of canine and feline uroliths.

Design—Prospective in vitro study.

Sample Population—Canine and feline uroliths submitted to the Minnesota Urolith Center from 34 dogs and 27 cats.

Procedures—Submissions from each dog or cat consisted of multiple uroliths of a single mineral type. After retrieval from the urinary tract, none of the uroliths had been placed in a preservative before submission. Evaluated uroliths were exclusively composed of the following: only struvite (uroliths from 5 dogs and 5 cats), calcium oxalate (5 dogs and 5 cats), calcium phosphate apatite (5 dogs and 5 cats), cystine (5 dogs and 5 cats), ammonium urate (5 dogs and 5 cats), or silica (5 dogs). One urolith from each dog or cat was quantitatively analyzed by polarized light microscopy, infrared spectroscopy, or both. Another urolith from the same animal was immersed in 1 mL of neutral-buffered 10% formalin for 48 hours at room temperature (22.5°C). Uroliths exposed to formalin were then air-dried for 30 minutes, and the analysis was repeated.

Results—After exposure to formalin, a portion of every struvite urolith was transformed into newberyite. This was not observed with any other urolith mineral type. Quantitative mineral analysis of nonstruvite uroliths revealed no detectable change in mineral composition. However, 3 of 10 ammonium urate uroliths dissolved when placed in formalin.

Conclusions and Clinical Relevance—To avoid misdiagnosis of mineral composition, uroliths should not be immersed in formalin prior to analysis.

Abstract

Objective—To determine whether storage in neutral-buffered 10% formalin in vitro has any effect on the composition of biogenic minerals of canine and feline uroliths.

Design—Prospective in vitro study.

Sample Population—Canine and feline uroliths submitted to the Minnesota Urolith Center from 34 dogs and 27 cats.

Procedures—Submissions from each dog or cat consisted of multiple uroliths of a single mineral type. After retrieval from the urinary tract, none of the uroliths had been placed in a preservative before submission. Evaluated uroliths were exclusively composed of the following: only struvite (uroliths from 5 dogs and 5 cats), calcium oxalate (5 dogs and 5 cats), calcium phosphate apatite (5 dogs and 5 cats), cystine (5 dogs and 5 cats), ammonium urate (5 dogs and 5 cats), or silica (5 dogs). One urolith from each dog or cat was quantitatively analyzed by polarized light microscopy, infrared spectroscopy, or both. Another urolith from the same animal was immersed in 1 mL of neutral-buffered 10% formalin for 48 hours at room temperature (22.5°C). Uroliths exposed to formalin were then air-dried for 30 minutes, and the analysis was repeated.

Results—After exposure to formalin, a portion of every struvite urolith was transformed into newberyite. This was not observed with any other urolith mineral type. Quantitative mineral analysis of nonstruvite uroliths revealed no detectable change in mineral composition. However, 3 of 10 ammonium urate uroliths dissolved when placed in formalin.

Conclusions and Clinical Relevance—To avoid misdiagnosis of mineral composition, uroliths should not be immersed in formalin prior to analysis.

In 2009, the Minnesota Urolith Center performed quantitative analysis on uroliths retrieved from 47,036 dogs and 11,697 cats.1,2 Our protocol requests that veterinarians submit uroliths dry without preservatives. However, 81 (55 from dogs; 26 from cats) uroliths were submitted in formalin. Of these 81 uroliths, 38 (47%; 27 from dogs; 11 from cats) contained newberyite (magnesium hydrogen phosphate trihydrate [MgHPO4•3H2O]). In comparison, 30 (21 from dogs; 9 from cats; < 1%) uroliths submitted without any preservatives contained newberyite. Our presumption was that these samples were not immersed in formalin; however, there was no specific information provided as to whether those samples were preserved in formalin. In 1 study3 of human uroliths, investigators concluded that newberyite was not biogenic but an in vitro artifact. In several other studies4–9 of human uroliths, investigators hypothesized that struvite decomposed to newberyite with time.

We were unable to find studies designed to determine whether exposure to neutral-buffered 10% formalin changed the composition of biogenic struvite to newberyite. We examined the hypothesis that newberyite is an in vitro artifact caused by exposure of struvite to formalin. The purpose of the in vitro study reported here was to determine whether storage in neutral-buffered 10% formalin changes the composition of biogenic minerals found in canine and feline uroliths.

Materials and Methods

Source of uroliths—Submissions containing multiple uroliths of a single mineral type (ie, exclusively composed of struvite, calcium oxalate, calcium phosphate apatite, cystine, ammonium urate, or silica) were evaluated. Uroliths from 34 dogs and 27 cats that had been submitted by veterinarians to the Minnesota Urolith Center were conveniently selected. Urolith submissions were only included if multiple uroliths from individual cases were available for quantitative analysis. Uroliths were composed of struvite; calcium oxalate monohydrate, calcium oxalate dihydrate, or both; calcium phosphate; cystine; ammonium urate; or silica. The size of feline uroliths was ≤ 5 mm in diameter. The size of canine uroliths was > 5 to ≤ 10 mm in diameter.

Analyses of uroliths—Mineral composition of all uroliths was determined by optical crystallography,a infrared spectroscopy,11 or both. The areas within a urolith were described with the following terms (Figure 1) as previously described10: nidus, area of obvious initiation of urolith growth (it was not necessarily the geometric center of the sample); stone, the main body of the urolith; shell, a complete outer concentric lamination of the urolith; and surface crystals, an incomplete outer lamination of the urolith. In the present study, the nidus and the stone were combined and designated as the inner layers of uroliths. The shell and surface crystal components were combined and designated as outer layers of uroliths.

Figure 1—
Figure 1—

Illustration of layers that may be present within a urolith. In the present study, the nidus and the stone were combined and designated as the inner layers of uroliths and the shell and surface crystal components were combined and designated as outer layers of uroliths. Nidus = Area of obvious initiation of urolith growth (it is not necessarily the geometric center). Shell = A complete outer concentric lamination of the urolith. Stone = The main body of the urolith. Surface crystals = An incomplete outer lamination of the urolith.

Citation: Journal of the American Veterinary Medical Association 241, 12; 10.2460/javma.241.12.1613

Formalin—Commercially available formalin containing 37% formaldehydec was diluted to neutral-buffered 10% formalin (4% formaldehyde) with distilled water. The formalin solution was buffered to a neutral pH with sodium phosphate.11

Short-term formalin exposure—To answer the question of whether formalin alters the mineral composition of uroliths, the following procedure was performed. Urolith submissions exclusively composed of struvite (uroliths from 5 dogs and 5 cats), calcium oxalate (5 dogs and 5 cats), calcium phosphate (5 dogs and 5 cats), cystine (5 dogs and 5 cats), ammonium urate (5 dogs and 5 cats), or silica (5 dogs), with no preservative after retrieval from the urinary tract, were selected.

One urolith from each dog or cat was quantitatively analyzed. A second urolith from the same animal was immersed in a capped container with 1 mL of neutral-buffered 10% formalin for 48 hours at room temperature (22.5°C). The formalin was decanted, and uroliths were air-dried for 30 minutes. The second urolith was quantitatively analyzed in an manner identical to the first urolith.

Effect of pH on mineral transformation—To determine the possible effects of pH on mineral transformation, uroliths exclusively composed of struvite (uroliths from 1 dog and 1 cat), calcium oxalate (1 dog and 1 cat), cystine (1 dog), or ammonium urate (1 dog) were tested. One urolith from each dog or cat was quantitatively analyzed. A subsequent urolith from the same animal was immersed in a capped container with 1 mL of neutral-buffered 10% formalin at room temperature (22.5°C) for 168 hours. The pH of formalin in each container was determined with a pH meterd at baseline and after 72 and 168 hours.

As a control, pH was also measured in 2 capped containers of neutral-buffered 10% formalin stored at room temperature but without uroliths. The pH of formalin was determined at 72 hours in one and at 168 hours in the other.

Results

Canine and feline struvite uroliths—After immersion in neutral-buffered 10% formalin for 48 hours, newberyite was detected in the outer layers of all 5 canine struvite uroliths. Newberyite was not detected in the inner layers of any canine urolith (Table 1). Similarly after 48 hours of immersion, newberyite was detected in the outer layers of all 5 feline struvite uroliths. Newberyite was not detected in the inner layers of any feline uroliths. By use of x-ray diffraction and energy-dispersive spectroscopy at the Characterization Facility of the University of Minnesota,e however, bobierritee (Mg2(PO4)2•8H2O) and hydromanganesite were observed in the inner layers of 2 of 5 feline uroliths and in the outer layers of 4 of 5 feline uroliths.

Table 1—

Effect of immersion in neutral-buffered 10% formalin for 48 hours on the composition of struvite uroliths from 5 dogs and 5 cats.

Struvite urolithInner layerOuter layer
Struvite (%)Bobierrite (%)*Struvite (%)Newberyite (%)Bobierrite (%)*
Canine100001000
Canine100001000
Canine100001000
Canine100001000
Canine100001000
Feline100001000
Feline100006040
Feline100007525
Feline95509010
Feline955255025

Bobierrite and hydromanganesite.

Canine and feline ammonium urate uroliths—After immersion of canine ammonium urate uroliths in neutral-buffered 10% formalin for 48 hours, the mineral portion of 1 of 5 canine uroliths dissolved (Table 2). The remaining material contained concentric rings of noncrystalline matrix. Unidentified matrix material was present in the center of rings of the matrix. The color of formalin changed from clear to the same green color observed in the canine ammonium urate urolith prior to immersion in formalin. After immersion of 5 feline ammonium urate uroliths in neutral-buffered 10% formalin, 2 completely dissolved, the outer layers of 2 dissolved, and the outer and inner layers of 1 were partially dissolved. The non-mineral substance remaining in feline uroliths after dissolution of ammonium urate appeared to be matrix. The color of formalin changed from clear to green.

Table 2—

Effect of immersion in neutral-buffered 10% formalin for 48 hours on the composition of ammonium urate uroliths from 5 dogs and 5 cats.

Ammonium urate urolithInner layerOuter layer
Ammonium urate (%)Matrix (%)Ammonium urate (%)Matrix (%)
Canine10001000
Canine10001000
Canine10001000
Canine10001000
Canine10000100
Feline1000595
Feline10000100
Feline50505050
Feline01000100
Feline01000100

Other types of canine and feline uroliths—After immersion of canine and feline uroliths composed solely of calcium oxalate monohydrate and calcium oxalate dihydrate, calcium phosphate, cystine, or silica in neutral-buffered 10% formalin for 48 hours, neither the outer nor the inner layers of uroliths were changed.

pH of neutral-buffered 10% formalin with immersed uroliths—Compared with baseline values, the pH of neutral-buffered 10% formalin changed after exposure to uroliths for 72 and 168 hours (Table 3). Compared with baseline values, the pH increased when formalin was stored without a urolith sample for 72 and 168 hours.

Table 3—

The pH of neutral-buffered 10% formalin after storage of feline or canine uroliths of various compositions for 72 and 168 hours.

UrolithSpeciespH of neutral-buffered 10% formalin
Before storageAfter 72 hours of storageAfter 168 hours of storage
Ammonium urateCanine7.346.365.93
CystineCanine7.347.247.13
Calcium oxalateCanine7.347.347.33
Calcium oxalateFeline7.347.357.34
StruviteFeline7.347.427.49
StruviteCanine7.347.527.71
No urolithNA7.317.347.35

NA = Not applicable.

Discussion

Results of our study confirmed the hypothesis that immersion of struvite uroliths in formalin changed portions of struvite into newberyite. The appearance of bobierrite and hydromanganesite when sterile struvite uroliths from cats were immersed in formalin was unexpected. Bobierrite and hydromanganesite were not observed in canine struvite uroliths.

Struvite crystals form in alkaline solutions. They dissolve when the pH of the solution becomes acidic.3 Formaldehyde (CH2O) is available commercially as acidic formalin.11 During storage of formaldeyhde in aqueous solutions, formaldehyde is present as methylene hydrate (H2C(OH)2). Methylene hydate dissociates into formic acid (HCOOH).11 Formic acid also forms when formaldehyde is oxidized by atmospheric oxygen (H2C(OH)2 + O2 → HCOOH + H2O).11,12 When struvite uroliths are stored in formalin, in vitro transformation of struvite (MgNH4PO4•6H2O) to newberyite (MgHPO4•3H2O) starts by the reaction of formic acid with magnesium ammonium phosphate: struvite + formic acid → newberyite + ammonium + carbon dioxide + hydrogen ion. This reaction provides a plausible explanation as to why varying quantities of struvite uroliths were transformed into newberyite.

Supersaturation of urine with struvite (Mg2+ + NH3 + HPO42−) and newberyite (Mg2+ + HPO42−) depends on initial concentrations of these minerals and the initial pH of solution. In general, dissolution of struvite uroliths results in an increased concentration of magnesium, ammonium, and hydrogen phosphate in the solution. Increasing ionic concentrations of magnesium and hydrogen phosphate and decreasing concentration of ammonia in the solution promote the transformation of struvite to newberyite.13,14

In our study, the pH of neutral-buffered 10% formalin increased after struvite uroliths were added. Dissolution of struvite uroliths in a solution results in an increase in the concentration of ammonium (NH4+). This reaction increases the pH of the solution by decreasing the concentration of hydrogen ion.15

Magnesium ammonium phosphates undergo hydrolytic alteration in water.16 In water, struvite is slowly replaced by cattiite (Mg3(PO4)2•22H2O), then cattiite is replaced by bobierrite (Mg2(PO4)2•8H2O), which has been reported to be more stable than cattiite.16

Ammonium urate occurs in vivo. Therefore, formalin-induced in vitro dissolution of ammonium urate uroliths could result in erroneous diagnoses. Because mineral composition cannot be accurately determined by visual inspection, to avoid misdiagnosis, uroliths should not be preserved in formalin prior to submission for quantitative mineral analysis.

a.

Olympus BH-2 Polarizing Microscope, Olympus America Corp, Center Valley, Pa.

b.

AVATAR350, Thermo Electron Corp, Madison, Wis.

c.

Formaldehyde solution, Mallinckrodt Baker Inc, Philipsburg, NJ.

d.

pH meter, Mallinckrodt Baker Inc, Philipsburg, NJ.

e.

The Characterization Facility, University of Minnesota, Minneapolis, Minn.

References

  • 1. Osborne CA. Canine urolith update, 2009: perspectives from the Minnesota Urolith Center. DVM Newsmagazine 2010; 41(7):1S.

  • 2. Osborne CA. Epidemiology of feline uroliths and urethral plugs: update 1981 to 2009. DVM Newsmagazine 2010; 41(6):2S.

  • 3. Abbona F, Lundager Madsen HE, Boistelle R. Crystallization of two magnesium phosphates, struvite and newberyite: effect of pH and concentration. J Crystal Growth 1982; 57:614.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Parsons J. Magnesium dibasic phosphate identified as a crystalline component of a urinary calculus. J Urol 1956; 76:228230.

  • 5. Lonsdale K, Sutor DJ. Newberyite in ancient and modern urinary calculi: Identification and space group. Science 1966; 154:13531354.

  • 6. Sutor DJ. Newberyite—its formation in human urinary calculi. Nature 1968; 218:295.

  • 7. Whitaker A. The composition of struvite. Mineral Mag 1968; 36:820824.

  • 8. Whitaker A. The decomposition of struvite: further evidence. Mineral Mag 1969; 37:290291.

  • 9. Carmona P, Bellanato J, Cifuentes-Delatte L. Trimagnesium orthophosphate in renal calculi. Invest Urol 1980; 18:151154.

  • 10. Ulrich LK, Bird KA, Koehler LA, et al. Urolith analysis: submission, methods, and interpretation. Vet Clin North Am Small Anim Pract 1996; 26:393400.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Kierman JA. Fixation. In: Histological & histochemical methods: theory & practice. 2nd ed. New York: Pergamon Press, 1990;1031.

  • 12. Fox CH, Johnson FB, Whiting J, et al. Formaldehyde fixation. J Histochem Cytochem 1985; 33:845853.

  • 13. Boistelle R, Abbona F, Lundager HE. On the transformation of struvite into newberyite in aqueous system. Phys Chem Miner 1983; 9:216222.

  • 14. Abbona F. On the decomposition of struvite, MgNH4PO4•6H2O. Miner Petrogr Acta 1991; 34:2737.

  • 15. Babic-Ivancic V, Kontrec J, Brecevic L. Formation and transformation of struvite and newberyite in aqueous solution under conditions similar to physiological. Urol Res 2004; 32:350356.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16. Frazier WA, Lehr JR, Smith JP. The magnesium phosphate hannayite, schertelite and bobierrite. Am Mineral 1963; 48:635641.

  • Figure 1—

    Illustration of layers that may be present within a urolith. In the present study, the nidus and the stone were combined and designated as the inner layers of uroliths and the shell and surface crystal components were combined and designated as outer layers of uroliths. Nidus = Area of obvious initiation of urolith growth (it is not necessarily the geometric center). Shell = A complete outer concentric lamination of the urolith. Stone = The main body of the urolith. Surface crystals = An incomplete outer lamination of the urolith.

  • 1. Osborne CA. Canine urolith update, 2009: perspectives from the Minnesota Urolith Center. DVM Newsmagazine 2010; 41(7):1S.

  • 2. Osborne CA. Epidemiology of feline uroliths and urethral plugs: update 1981 to 2009. DVM Newsmagazine 2010; 41(6):2S.

  • 3. Abbona F, Lundager Madsen HE, Boistelle R. Crystallization of two magnesium phosphates, struvite and newberyite: effect of pH and concentration. J Crystal Growth 1982; 57:614.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Parsons J. Magnesium dibasic phosphate identified as a crystalline component of a urinary calculus. J Urol 1956; 76:228230.

  • 5. Lonsdale K, Sutor DJ. Newberyite in ancient and modern urinary calculi: Identification and space group. Science 1966; 154:13531354.

  • 6. Sutor DJ. Newberyite—its formation in human urinary calculi. Nature 1968; 218:295.

  • 7. Whitaker A. The composition of struvite. Mineral Mag 1968; 36:820824.

  • 8. Whitaker A. The decomposition of struvite: further evidence. Mineral Mag 1969; 37:290291.

  • 9. Carmona P, Bellanato J, Cifuentes-Delatte L. Trimagnesium orthophosphate in renal calculi. Invest Urol 1980; 18:151154.

  • 10. Ulrich LK, Bird KA, Koehler LA, et al. Urolith analysis: submission, methods, and interpretation. Vet Clin North Am Small Anim Pract 1996; 26:393400.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Kierman JA. Fixation. In: Histological & histochemical methods: theory & practice. 2nd ed. New York: Pergamon Press, 1990;1031.

  • 12. Fox CH, Johnson FB, Whiting J, et al. Formaldehyde fixation. J Histochem Cytochem 1985; 33:845853.

  • 13. Boistelle R, Abbona F, Lundager HE. On the transformation of struvite into newberyite in aqueous system. Phys Chem Miner 1983; 9:216222.

  • 14. Abbona F. On the decomposition of struvite, MgNH4PO4•6H2O. Miner Petrogr Acta 1991; 34:2737.

  • 15. Babic-Ivancic V, Kontrec J, Brecevic L. Formation and transformation of struvite and newberyite in aqueous solution under conditions similar to physiological. Urol Res 2004; 32:350356.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16. Frazier WA, Lehr JR, Smith JP. The magnesium phosphate hannayite, schertelite and bobierrite. Am Mineral 1963; 48:635641.

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