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Bacterial interference with urine osmolality measurements
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Bacterial Interference with Urine Osmolality Measurements E R I K K. M I T C H E L L , 1 J O A N H. H O W A N I T Z , 2 a n d P E T E R J. H O W A N I T Z D i v i s i o n of Clinical P a t h o l o g y , D e p a r t m e n t of P a t h o l o g y , S t a t e U n i v e r s i t y of N e w Y o r k , U p s t a t e Medical Center, Syracuse, New York 13210 We report bacterial interference with urine osmolality measurements using an instrument based on the principle of freezing point depression. Although the exact nature of the interfering activity has not been defined, the phenomenon is associated with a bacterium, identified as Pseudomonas putida, and is removed from the specimens by filtration at 0.45 p.m. The bacteria led to osmometer dysfunction presumably by acting as a nidus for crystallization and preventing proper supercooling of specimens.
O
smolality of biologic fluids can be determined using one of the colligative properties of aqueous solutions. In the clinical laboratory, instruments based on either freezing point or vapor pressure depression are used to determine osmolality. Few interferences with osmolality measurements have been reported. From two renal transplant patients, we received several urine specimens in which osmolality could not be determined directly using a freezing point depression osmometer. In addition, these specimens interfered with osmometer function for as long as several hours producing error codes or erroneous values in samples measured after these urine specimens. Materials and methods
Four urine specimens from one patient and two from a second patient were studied. All measurements were performed on a Model 3CII Cryomatic Osmometer (Advanced Instruments Inc., Needham Heights, MA 02194). I n s t r u m e n t performance was monitored by standards with nominal osmolal concentrations of 14, 100, 500, and 900 m m o l / k g and a 290 m m o l / k g control (Advanced Instruments, Inc.). Specimens were centrifuged in a TJ-6 centrifuge and an L5-65 preparative ultracentrifuge with an SW-60 swinging bucket rotor (Beckman Instruments, Inc., Palo Alto, CA 94304). Type I water used for dilutions was generated from a Milli-Q water system (Worthington Diagnostics, Division of Millipore Corp., Freehold, NJ 07728). Urine filtrates were prepared using sterile filters with a nominal filter measure of 0.45 ~m (Acrodisc, Gelman Sciences Inc., Ann Arbor, MI 48106) with 8 10 mL of urine passed through by syringe and the filtrate collected, or 5 - 1 0 mL aliquots of urine passed through W h a t m a n #1 filter paper, 11 ~Lm pore size
'Current address: Dade County Medical Examiner, 1050 North West 19th Street, Miami, Florida 33136. 2To whom correspondence should be addressed. Manuscript received June 29, 1982; revised manuscript received January 24, 1983; accepted for publication January 24, 1983.
(Whatman Limited, Springfield Mill, England). Specimens were treated by 1) dilution with saline, 2) dilution with deionized water, 3) hydrochloric acid, 4) sulphuric acid, 5) ammonia hydroxide, 6) sodium hydroxide, 7) freezing and thawing, 8) heating for 120 minutes at 56°C, 9) ultracentrifugation at 60,000 rpm (369,000 x g) for 60 minutes. Urine cultures were grown in tryptic soy broth at 20°C and 37°C. Isolation and identification of organisms were accomplished by the method of Shayegani et al. (1). Two lyophilized strains of Pseudomonas putida, grown at 26°C before lyophilization, were obtained from the American Type Culture Collection (Rockville, MD 20852); they were cultured in tryptic soy broth at 20°C and 37°C. All solutions used for culture and dilutions were tested for interference with the freezing point depression osmometer. Results
With four specimens (three from first patient, one from second) the digital display of the osmometer showed no supercooling or plateau region of the temperature curve as indicated by the display readings, and samples measured subsequently gave error codes or inaccurate results even after vigorous cleaning of the probe. Dilution of the specimens with normal saline or deionized water did not attenuate the effect until dilutions of greater than 1 to 100 were reached. Centrifugation at 1500 x g, passage through the filter paper of 11 ~m pore size, or ultracentrifugation at 369,000 x g did not remove the interference. T r e a t m e n t of the urine with hydrochloric or sulphuric acid to less t h a n pH 3 and with ammonia or sodium hydroxide to greater t h a n pH 10 abolished the interference only on an erratic basis. Freezing and thawing did not destroy the activity. Heating of the urines to 56 ° for two hours as well as passage through a 0.45 ~Lm filter removed all interference. Sediment left on the 0.45 ~Lm filters retained activity. A Gram-negative bacillus with flagella could be visualized and was identified as Pseudomonas putida. The interfering activity could be reproduced by subcultures of the organism grown at 20°C, but those grown at 37°C lost the activity, which could not be restored by further subcultures at 20°C. Cultures of two strains ofPseudomonas putida purchased from American Type Culture Collection showed no interference. Discussion
Freezing point is based on indirect determination of osmolality through measurement of colligative proper-
260
MITCHELL, HOWANITZ, HOWANITZ
ti~es of solutions (2). In freezing point depression osmometers, the sample is cooled below the freezing t e m p e r a t u r e without solidifying. When a physical stimulus such as vibration is applied, crystallization is started but freezing is not complete. A slush is formed in which a small percentage of the sample t u r n s solid (3). The sample then continues to freeze liberating its latent h e a t of fusion as crystallization occurs. The t e m p e r a t u r e rises to a plateau which is related to the osmolality of the sample. Interferences with osmolality m e a s u r e m e n t s in freezing point depression i n s t r u m e n t s can be related to improper m a i n t e n a n c e or to particulate m a t t e r such as dust or similar particles which act as nuclei for crystallization (4). Hyperviscosity can interfere with freezing point depression osmometry by causing nonuniform freezing of the sample (5). I n s t r u m e n t error codes can result from problems with t e m p e r a t u r e , the level or concentration of ethylene glycol in the refrigerated b a t h of the osmometer, or difficulties with adj u s t m e n t of the stir/freeze wire. We have isolated a bacterium which we have identified as the organism Pseudomonas putida. We speculate t h a t this organism is associated with the interference we report in m e a s u r i n g osmolality using freezing point instrumentation. Pseudomonas species usually are motile with polar multitrichous flagella and m a y produce slime. This raises the hypothesis t h a t flagella, flagellar fragments or slime m a y be i m p o r t a n t in the interference by perhaps adhering to the osm o m e t e r probe and acting as a nucleus for crystallization. Two strains of Pseudornonas putida from the American Type Culture Association did not lead to the interference in freezing point depression os-
mometry. It is possible these strains did not produce the interfering factor, or the organism isolated from the urine specimens was misidentified as Pseudomonas putida. We propose the bacterium or its by-products provide a source causing crystal formation to commence i m m e d i a t e l y on cooling the specimen below 0°C. With progressive freezing, supercooling cannot take place in these urine specimens and hence, osmolality cannot be measured. The most significant aspect of this phenomenon is its prolonged nature which simulates a situation t h a t requires i n s t r u m e n t repair. The difficulty can be easily overcome by filtering the specimen at 0.45 p.m before d e t e r m i n i n g the osmolality. Acknowledgements We are grateful to Ms. F. Morgenstern for help with the bacterial identification. References 1. Shayegani, M., Lee, A. M., and Parsons, L. M. A scheme for identification of nonfermentative gram-negative bacteria. Health Lab. Sci. 14, 83-94 (1977}. 2. Barlow, W. K., Schneider, P. G., Schult, S. L., and Viebell, M. Clinical osmometry. Med. Electronics 9, 64-69 (1978). 3. Weisberg, H. F., Osmolality. Check Sample Program. Clinical Chemistry, No. CC-71 American Society of Clinical Pathologists ( 1971 ). 4. User's Guide. Advanced cryomatic osmometer. Advanced Instruments, Inc., Needham Heights, MA, 1977, pp. 21-22. 5. Barlow, W. K., Volatiles and osmometry (cont.}. Clin. Chem. 22, 1230-1232 {1976}.
Bacterial Interference with Urine Osmolality Measurements E R I K K. M I T C H E L L , 1 J O A N H. H O W A N I T Z , 2 a n d P E T E R J. H O W A N I T Z D i v i s i o n of Clinical P a t h o l o g y , D e p a r t m e n t of P a t h o l o g y , S t a t e U n i v e r s i t y of N e w Y o r k , U p s t a t e Medical Center, Syracuse, New York 13210 We report bacterial interference with urine osmolality measurements using an instrument based on the principle of freezing point depression. Although the exact nature of the interfering activity has not been defined, the phenomenon is associated with a bacterium, identified as Pseudomonas putida, and is removed from the specimens by filtration at 0.45 p.m. The bacteria led to osmometer dysfunction presumably by acting as a nidus for crystallization and preventing proper supercooling of specimens.
O
smolality of biologic fluids can be determined using one of the colligative properties of aqueous solutions. In the clinical laboratory, instruments based on either freezing point or vapor pressure depression are used to determine osmolality. Few interferences with osmolality measurements have been reported. From two renal transplant patients, we received several urine specimens in which osmolality could not be determined directly using a freezing point depression osmometer. In addition, these specimens interfered with osmometer function for as long as several hours producing error codes or erroneous values in samples measured after these urine specimens. Materials and methods
Four urine specimens from one patient and two from a second patient were studied. All measurements were performed on a Model 3CII Cryomatic Osmometer (Advanced Instruments Inc., Needham Heights, MA 02194). I n s t r u m e n t performance was monitored by standards with nominal osmolal concentrations of 14, 100, 500, and 900 m m o l / k g and a 290 m m o l / k g control (Advanced Instruments, Inc.). Specimens were centrifuged in a TJ-6 centrifuge and an L5-65 preparative ultracentrifuge with an SW-60 swinging bucket rotor (Beckman Instruments, Inc., Palo Alto, CA 94304). Type I water used for dilutions was generated from a Milli-Q water system (Worthington Diagnostics, Division of Millipore Corp., Freehold, NJ 07728). Urine filtrates were prepared using sterile filters with a nominal filter measure of 0.45 ~m (Acrodisc, Gelman Sciences Inc., Ann Arbor, MI 48106) with 8 10 mL of urine passed through by syringe and the filtrate collected, or 5 - 1 0 mL aliquots of urine passed through W h a t m a n #1 filter paper, 11 ~Lm pore size
'Current address: Dade County Medical Examiner, 1050 North West 19th Street, Miami, Florida 33136. 2To whom correspondence should be addressed. Manuscript received June 29, 1982; revised manuscript received January 24, 1983; accepted for publication January 24, 1983.
(Whatman Limited, Springfield Mill, England). Specimens were treated by 1) dilution with saline, 2) dilution with deionized water, 3) hydrochloric acid, 4) sulphuric acid, 5) ammonia hydroxide, 6) sodium hydroxide, 7) freezing and thawing, 8) heating for 120 minutes at 56°C, 9) ultracentrifugation at 60,000 rpm (369,000 x g) for 60 minutes. Urine cultures were grown in tryptic soy broth at 20°C and 37°C. Isolation and identification of organisms were accomplished by the method of Shayegani et al. (1). Two lyophilized strains of Pseudomonas putida, grown at 26°C before lyophilization, were obtained from the American Type Culture Collection (Rockville, MD 20852); they were cultured in tryptic soy broth at 20°C and 37°C. All solutions used for culture and dilutions were tested for interference with the freezing point depression osmometer. Results
With four specimens (three from first patient, one from second) the digital display of the osmometer showed no supercooling or plateau region of the temperature curve as indicated by the display readings, and samples measured subsequently gave error codes or inaccurate results even after vigorous cleaning of the probe. Dilution of the specimens with normal saline or deionized water did not attenuate the effect until dilutions of greater than 1 to 100 were reached. Centrifugation at 1500 x g, passage through the filter paper of 11 ~m pore size, or ultracentrifugation at 369,000 x g did not remove the interference. T r e a t m e n t of the urine with hydrochloric or sulphuric acid to less t h a n pH 3 and with ammonia or sodium hydroxide to greater t h a n pH 10 abolished the interference only on an erratic basis. Freezing and thawing did not destroy the activity. Heating of the urines to 56 ° for two hours as well as passage through a 0.45 ~Lm filter removed all interference. Sediment left on the 0.45 ~Lm filters retained activity. A Gram-negative bacillus with flagella could be visualized and was identified as Pseudomonas putida. The interfering activity could be reproduced by subcultures of the organism grown at 20°C, but those grown at 37°C lost the activity, which could not be restored by further subcultures at 20°C. Cultures of two strains ofPseudomonas putida purchased from American Type Culture Collection showed no interference. Discussion
Freezing point is based on indirect determination of osmolality through measurement of colligative proper-
260
MITCHELL, HOWANITZ, HOWANITZ
ti~es of solutions (2). In freezing point depression osmometers, the sample is cooled below the freezing t e m p e r a t u r e without solidifying. When a physical stimulus such as vibration is applied, crystallization is started but freezing is not complete. A slush is formed in which a small percentage of the sample t u r n s solid (3). The sample then continues to freeze liberating its latent h e a t of fusion as crystallization occurs. The t e m p e r a t u r e rises to a plateau which is related to the osmolality of the sample. Interferences with osmolality m e a s u r e m e n t s in freezing point depression i n s t r u m e n t s can be related to improper m a i n t e n a n c e or to particulate m a t t e r such as dust or similar particles which act as nuclei for crystallization (4). Hyperviscosity can interfere with freezing point depression osmometry by causing nonuniform freezing of the sample (5). I n s t r u m e n t error codes can result from problems with t e m p e r a t u r e , the level or concentration of ethylene glycol in the refrigerated b a t h of the osmometer, or difficulties with adj u s t m e n t of the stir/freeze wire. We have isolated a bacterium which we have identified as the organism Pseudomonas putida. We speculate t h a t this organism is associated with the interference we report in m e a s u r i n g osmolality using freezing point instrumentation. Pseudomonas species usually are motile with polar multitrichous flagella and m a y produce slime. This raises the hypothesis t h a t flagella, flagellar fragments or slime m a y be i m p o r t a n t in the interference by perhaps adhering to the osm o m e t e r probe and acting as a nucleus for crystallization. Two strains of Pseudornonas putida from the American Type Culture Association did not lead to the interference in freezing point depression os-
mometry. It is possible these strains did not produce the interfering factor, or the organism isolated from the urine specimens was misidentified as Pseudomonas putida. We propose the bacterium or its by-products provide a source causing crystal formation to commence i m m e d i a t e l y on cooling the specimen below 0°C. With progressive freezing, supercooling cannot take place in these urine specimens and hence, osmolality cannot be measured. The most significant aspect of this phenomenon is its prolonged nature which simulates a situation t h a t requires i n s t r u m e n t repair. The difficulty can be easily overcome by filtering the specimen at 0.45 p.m before d e t e r m i n i n g the osmolality. Acknowledgements We are grateful to Ms. F. Morgenstern for help with the bacterial identification. References 1. Shayegani, M., Lee, A. M., and Parsons, L. M. A scheme for identification of nonfermentative gram-negative bacteria. Health Lab. Sci. 14, 83-94 (1977}. 2. Barlow, W. K., Schneider, P. G., Schult, S. L., and Viebell, M. Clinical osmometry. Med. Electronics 9, 64-69 (1978). 3. Weisberg, H. F., Osmolality. Check Sample Program. Clinical Chemistry, No. CC-71 American Society of Clinical Pathologists ( 1971 ). 4. User's Guide. Advanced cryomatic osmometer. Advanced Instruments, Inc., Needham Heights, MA, 1977, pp. 21-22. 5. Barlow, W. K., Volatiles and osmometry (cont.}. Clin. Chem. 22, 1230-1232 {1976}.