Laboratory Utilization and Analytical Validation of Fecal Electrolyte Tests

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1 ARTICLES Laboratory Utilization and Analytical Validation of Fecal Electrolyte Tests Rugvedita Parakh, 1,2 Dina N. Greene, 1 Patrick C. Mathias, 1 Darci R. Block, 3 and Pratistha Ranjitkar 1 * Background: Chronic diarrhea can be categorized as fatty, watery, or inflammatory. Watery diarrhea is further divided into secretory or osmotic types and can be differentiated by measuring fecal electrolytes and osmotic gap. However, with widespread use of endoscopy, it is unclear if these measurements are being used clinically. Furthermore, because stool is not a validated specimen type for Food and Drug Administration approved electrolyte assays, utilization is a practical concern for laboratories before analytical validation. Here, we determined the clinical utility and validated the performance characteristics of stool electrolytes on the Beckman Coulter AU680. Methods: Historical results and literature review were used to determine the clinically relevant ranges for stool electrolytes (Na +,Cl,K +, phosphate, and Mg 2+ ). Additionally, medical chart review was performed (n = 44 patients) on results to evaluate their clinical utility in chronic diarrhea work-up. Linearity, precision, and stability studies were performed on the AU680. Accuracy was evaluated by comparing results to the Roche Cobas 6000 c501. Results: For all cases, stool electrolytes and osmotic gap proved valuable in chronic diarrhea work-up. The imprecision of the assays ranged from 0% to 5.9%. All assays were found to be linear within the instrument's analytical measurement range with appropriate slopes and intercepts. The bias between the AU680 and the Roche c501 ranged from 0.48 to 2.39 (mmol/l or mg/dl). Na +,Cl, and K + were stable refrigerated for 5 days and up to 1 freeze-thaw cycle. Phosphate and Mg 2+ were stable refrigerated for 48 h, but unstable to freeze-thaw cycles. Conclusions: Stool osmotic gap is valuable for evaluating chronic diarrhea and can be calculated using electrolyte concentrations measured on the AU680. IMPACT STATEMENT Utilization of fecal electrolytes and osmotic gap in the diagnosis of chronic diarrhea is unclear with the widespread use of endoscopic procedures. In addition, laboratory validation of nonstandard body fluids is a challenge because fecal fluid is not a validated specimen type for Food and Drug Administration approved electrolyte assays. Here, we investigated whether clinicians at our institution used fecal electrolytes and osmotic gap to guide clinical decisions as well as describe the validation of fecal electrolytes on the Beckman Coulter AU680 chemistry analyzer. 1 Department of Laboratory Medicine, University of Washington, Seattle, WA; 2 Department of Pathology, University of Washington, Seattle, WA; 3 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN. *Address correspondence to this author at: Medical College of Wisconsin, 9200 W. Wisconsin Ave., Department of Pathology, Froedtert/Medical College Lab Building, FMCLB 239, Milwaukee, WI Fax ; pranjitkar@mcw.edu. DOI: /jalm American Association for Clinical Chemistry 4 Nonstandard abbreviations: CRR, clinical reportable range; AMR, analytical measurement range. 668 JALM :06 May 2017

2 Fecal Electrolytes: Utilization and Analytical Validation ARTICLES Chronic diarrhea is defined as consistent liquid bowel movements that last 4 weeks or more and is typically categorized into 3 basic types: watery, fatty, or inflammatory (1, 2). The most challenging aspect of clinically assessing chronic diarrhea is determining the etiology (1). The differential diagnosis is broad, and the condition itself may be multifactorial. Therefore, the most practical approach to working up chronic diarrhea is to first categorize the type of diarrhea. The American Academy of Family Physicians and British Medical Journal Best Practices in Gastroenterology recommend that physicians categorize the condition using a detailed clinical history and by evaluating stool characteristics during the initial visit (2, 3). This approach narrows down the list of diagnostic possibilities, hence reducing unnecessary testing. The differential diagnoses within each category can then be followed up by performing category-specific tests. The clinical laboratory can aid in the differential diagnosis of watery diarrhea by further categorizing the condition as secretory or osmotic. Secretory diarrhea results from impaired fluid absorption in the intestine; there is an overall reduction in the intestinal absorption rate leading to the production of stool rich in electrolytes (3). In contrast, osmotic diarrhea results from poorly absorbed, osmolytically active substances pulling fluid into the bowel lumen leading to water retention and low electrolyte concentration in the intestinal lumen (4). As such, determination of fecal osmolal gap is beneficial in differentiating osmotic and secretory causes of chronic diarrhea where a fecal osmotic gap <50 mosm/kg suggests secretory diarrhea and >75 mosm/kg suggests osmotic diarrhea (5). The fecal osmolal gap is defined as the difference between 290 mosm/kg fecal osmolality, in which the fecal osmolality is calculated as 2 [fecal (Na + ) + fecal (K + )]. The concentrations of Na + and K + can be measured potentiometrically in liquid stool samples by the clinical laboratory. Stool osmolality should not be measured directly, since ongoing fermentation during transport or storage will falsely increase the result (4, 6). However, direct measurement of stool osmolality does aid in determining sample adulteration, contamination, or sample integrity. For instance, a liquid stool osmolality <290 mosm/kg suggests intentional addition of water, as can be the case in fictitious diarrhea (7). Alternatively, stool osmolality >330 mosm/kg in the absence of concurrently increased serum osmolality suggests improper sample storage or contamination with concentrated urine (8). Cl,Mg 2+, and phosphate measurements can be useful in the diagnosis of certain unique conditions. Quantification of fecal Cl may be helpful in the diagnosis of congenital chloride diarrhea, a condition resulting from defective intestinal absorption of Cl that leads to watery secretory diarrhea (9). Affected patients may present with fecal Cl >90 mmmol/l (9, 10). Both phosphate- and Mg 2+ -based laxatives induce osmotic diarrhea wherein a high soluble fraction of these analytes is expected in fecal output. It is important to rule out laxative-induced diarrhea during the initial evaluation of watery diarrhea, since patients may be using laxatives surreptitiously or be unaware of a potential laxative that is causing diarrheal symptoms (11, 12). Stool Mg 2+ >110 mg/dl and phosphate >102 mg/dl suggests Mg 2+ and phosphate induced diarrhea, respectively (11 14). There is a definitive but limited role for evaluating fecal electrolytes and the associated osmolal gap. However, there are still questions surrounding their utility due to the widespread use of endoscopic procedures. Stool is not a matrix cleared by the Food and Drug Administration for most chemistry assays; therefore, utilization becomes a practical concern of validation, rather than solely a clinical concern. The objective of this study was to determine if fecal electrolytes offered clinical utility to our ordering physicians and, if useful, to validate the quantification of these analytes in stool samples using the Beckman Coulter AU680. May : JALM 669

3 ARTICLES Fecal Electrolytes: Utilization and Analytical Validation MATERIALS AND METHODS Sample collection and instrumentation This project was granted exemption from the University of Washington institutional review board as part of ongoing quality improvement practices. Residual, unpreserved liquid stool samples submitted for clinical testing were sequestered. Samples were centrifuged at 12470g for 2 5 min, and the resulting supernatant was collected and stored at 20 C until further analysis. Samples were analyzed within 1 month of collection. Samples were thawed and analyzed for Na +,K +,Cl, phosphate, and Mg 2+ using the Beckman Coulter AU680. Na +,K +, and Cl were measured using indirect potentiometry, and Mg 2+ and phosphate were measured using colorimetric assays. For all assays, the urine mode of the instrument was used. This function allows the stool tests to share the instrument parameters, calibrators, reagents, and QC materials with the parallel AU680 urine tests. Osmolality was measured via freezing point depression using the Advanced Microosmometer Model Stool electrolyte utilization studies Historical laboratory results for electrolytes (Na +,K +,Cl ) and osmolality measured in stool samples between 1 January 2014 and 1 July 2015 at 2 hospital laboratories were retrieved from the laboratory information system (LIS; Sunquest, Version 7.1). Because a final diagnosis is often impossible in chronic diarrhea, the clinical utility of testing was determined by asking the following set of objective questions: (a) Was the submitted stool specimen watery? (b) Was the diarrhea chronic? (c) Was a gastrointestinal specialist consulted? (d) Was endoscopy performed? (e) Was the laboratory result taken into consideration before further work-up? Individual patient chart review (n = 44) was performed to extract the information needed to answer the above questions, including symptoms, duration of symptoms, medications, other relevant laboratory test results (biochemistry and microbiology results), specific diagnostic modalities used (endoscopy and biopsy results), treatment, and if determined, the final diagnosis. Analytical validation Clinical reportable range. Historical results (1 January to 1 July 2015) and literature review were used to determine the clinical reportable range (CRR) 4 for Na +,K +, and Cl. The CRR for phosphate and Mg 2+ were established using only literature review because these electrolytes were not previously measured in our laboratory and therefore historical results were not available (13). Appropriate AU680 onboard dilution factor (0, 2, 3, 5, or 10) for each analyte was selected to meet the necessary CRR if the analytical measuring range was deemed insufficient. To validate the onboard dilution, samples (n = 2) with analytes within the analytical measurement range (AMR) of the assay were measured undiluted (neat) and with dilution. The neat result was then compared to the autodilution result. An additional manual dilution was similarly validated for Mg 2+ (n = 3) to encompass the desired CRR. Intraday and interday imprecision. Intra- and interday imprecision were evaluated at 2 different concentrations (low and high) for each analyte. Low Na +,K +,Cl,Mg 2+, phosphate, and osmolality samples were prepared by performing appropriate dilution of processed stool samples with clinical reagent grade water to obtain desired concentrations. High Na + and phosphate samples were prepared by spiking in concentrated stocks of NaH 2 PO 4 monohydrate (<10% volume) into stool samples. Patient samples with desired high concentration of K +,Cl,Mg 2+, and osmolality were available; therefore, spiking exogenous electrolytes was not necessary. Samples were stored at 4 C until analysis could be performed. Both low and high patient samples were analyzed 20 times for electrolyte concentrations and osmolality to 670 JALM :06 May 2017

4 Fecal Electrolytes: Utilization and Analytical Validation ARTICLES determine the intraday imprecision. Specimens were further analyzed over 5 consecutive days (n = 4 replicates/day) to determine the interday imprecision. Linearity. Linearity was assessed by adding standard solutions of NaCl, KCl, NaH 2 PO 4 H 2 O, or MgPO 4 (<10% by volume) into processed stool specimens to create (n = 4) high electrolyte concentration specimens. The concentrations of analytes in these samples were as following: Na mmol/l, Cl 400 mmol/l, K mmol/l, Mg mg/dl, phosphate 193 mg/dl, and osmolality 1235 mosm/kg. The highconcentration samples were subsequently diluted with various processed stool samples determined to have a low starting concentration of the analyte being challenged to produce a series of 5 intermediateand low-concentration samples. The low analyte concentrations were as follows: Na + 19 mmol/l, Cl 21 mmol/l, K + 14 mmol/l, Mg mg/dl, phosphate 21 mg/dl, and osmolality 20 mosm/kg. Each specimen was measured in triplicate, and the average of these measures was evaluated by linear regression. Stability studies. Stability of electrolyte concentrations at 4 C was evaluated using 4 patient samples over 5 days. Results from successive days were compared to the baseline concentration (Na mmol/l, K mmol/l, Cl mmol/l, Mg mg/dl, phosphate 2 72 mg/dl), and the absolute difference was plotted as a function over time. In addition, the freeze-thaw stability was evaluated using 5 8 patient samples per electrolyte for 1 freeze-thaw cycle. Samples were stored for 4 24 h between the freeze-thaw cycles. Baseline concentrations in patient samples (Na mmol/l, K mmol/l, Cl mmol/l, phosphate mg/dl, Mg mg/dl) were determined, and the samples were frozen at 20 C. Samples were thawed at room temperature before reevaluating the electrolyte concentration and refreezing. Percent recovery was calculated compared to baseline values for each analyte. Method comparison. The accuracy of the AU680 electrolyte concentrations were evaluated by comparing results to the Roche Cobas c501 (Roche Diagnostics). Residual clinical samples were collected at 2 laboratories: samples submitted for stool electrolytes testing at a reference laboratory (n = 26; Mayo Medical Laboratories) and samples submitted to our microbiology laboratory for stool cultures (n = 17). At both institutions, samples were processed, aliquoted, and kept frozen until they could be exchanged between the two laboratories. Samples were thawed at both sites and analyzed within 12 h of each other. The following range of values was compared: Na + (6 129 mmol/l), K + ( mmol/l), Cl ( mmol/l), Mg 2+ (3 20 mg/dl), and phosphate (n = 24; mg/dl). Deming regression was used to determine the bias between instruments. Statistics. Statistical analyses, calculations, and data display were performed using Microsoft Excel (Microsoft), GraphPad Prism (Prism Software for Science), and EP evaluator (Data Innovations). RESULTS Utilization studies A total of 120 samples (108 unique patients) were submitted for stool Na +,K +, and Cl during the study period. Out of these, 60 samples were rejected because of inappropriate specimen type (i.e., formed stool). For test orders that were performed (n = 60 samples, 58 unique patients), 44 patient charts were accessible for review: 43/44 met the criteria for chronic diarrhea, 42/44 cases required consultation from a gastrointestinal specialist, and 41/44 patients received an endoscopic procedure. More importantly, in all cases, the laboratory result was used for the following purposes: categorizing the patient as having secretory or May : JALM 671

5 ARTICLES Fecal Electrolytes: Utilization and Analytical Validation Table 1. Institutional historical results and patient ranges from literature review. Analyte AU680 AMR, urine mode Historical results Dilution utilized Patient ranges from literature review Sodium mmol/l < mmol/l Not necessary mmol/l (13) mmol/l (6) Potassium mmol/l mmol/l Not necessary mmol/l (13) mmol/l (6) Chloride mmol/l < mmol/l Not necessary <90 mmol/l (9, 10) Magnesium mg/dl NA a Onboard: 2-fold Manual: 15-fold mg/dl (13) Phosphate mg/dl NA Not necessary >102 mg/dl (14) a NA, not applicable. osmotic type of diarrhea, guiding downstream diagnostic tests or procedures, and formulating treatment plans such as diet changes. Analytical validation The AU680 AMR for Na +,K +, and Cl was sufficient to provide a numerical result when simulated against historical results (Table 1). Similarly, the AU680 AMR for phosphate encompassed literature specified clinical reportable range excluding the need for performing dilutions. In contrast, the AMR for Mg 2+ ranged from 0.5 to 10 mg/dl with a 2-fold onboard dilution capability, extending the onboard range up to 20 mg/dl. However, stool Mg 2+ concentration is useful in the diagnosis of factitious diarrhea with a clinical decision limit of 110 mg/dl and reported concentrations found in the literature were between 2 and 270 mg/dl (11 13). Therefore, a 15 manual dilution was validated to have minimal variation from expected (10% difference) and further extended the clinical reportable range to 150 mg/dl. The intra- and interday CVs for each analyte are listed in Table 2. Intraday imprecision for all 5 analytes ranged from 0% to 2.7%, and interday imprecision ranged from 0.6% to 5.9%. Mixing studies to assess the linearity of the assay showed the slopes of all assays to be within 1.00 Table 2. Intra- and interday precision. Intraday Interday Units Mean SD %CV Mean SD %CV Na +, high mmol/l Na +, low K +, high mmol/l K +, low Cl, high mmol/l Cl, low Mg 2+, high mg/dl Mg 2+, low Phosphate, high mg/dl Phosphate, low Osmolality, high mosm/kg Osmolality, low JALM :06 May 2017

6 Fecal Electrolytes: Utilization and Analytical Validation ARTICLES Fig. 1. Linearity of the assays. (A), Sodium. (B), Chloride. (C), Potassium. (D), Phosphate. (E), Magnesium. (F), Osmolality. Samples determined to contain low electrolyte concentrations (Na + = 19 mmoleq/l; Cl = 21 mmoleq/l; K + = 14 mmoleq/l; phosphate = 21 mg/dl; Mg 2+ = 1.1 mg/dl; phosphate = 21 mg/dl; and osmolality = 20 mosm/kg) were admixed with samples determined to contain high electrolyte concentrations (Na + = 368 mmoleq/l; Cl = 400 mmoleq/l; K + = 193 mmoleq/l; Mg 2+ = 8.6 mg/dl; phosphate = 193 mg/dl; and osmolality = 1235 mosm/kg). Expected concentrations for the intermediate mixes were calculated based on the proportions of the high and low sample volumes mixed. to 0.98 and the y intercept to be between 4.9 and 0.19 (Fig. 1). Method comparison experiments showed an unexpected bias of results obtained on the AU680 vs the Roche Cobas comparative method for all analytes, with the AU680 consistently producing higher results (see Fig. 1 in the Data Supplement that accompanies the online version of this article at The following mean biases were observed for each analyte: Na mmol/l, K mmol/l, Cl 14.7 mmol/l, Mg mg/dl, and phosphate 4.3 mg/dl. However, when the AU680 results were compared to results obtained on the Roche analyzer before freezing and shipping the samples, the following mean biases between the methods were observed: Na mmol/l, K mmol/l, Cl 2.39 mmol/l, phosphate 0.96 mg/dl, and Mg mg/dl (Fig. 2, right panel). Deming regression analysis produced slopes ranging from 0.97 to 1.07 and intercepts ranging from 1.42 to 1.92 (Na +,K +,Cl = mmol/l; Mg 2+, phosphate = mg/dl) (Fig. 2, left panel). Stability studies Stability studies were carried out at 4 C for 5 days (Fig. 3). All electrolyte concentrations remained stable throughout the study period. Na +,K +, and Cl were stable in all specimens tested for up to 1 freeze-thaw cycle (Fig. 4). However, the freeze-thaw stability of phosphate and Mg 2+ varied between specimens. In some specimens, both phosphate and Mg 2+ decreased after a single freeze-thaw cycle, with recoveries as low as 35% phosphate or 40% Mg 2+. May : JALM 673

7 ARTICLES Fecal Electrolytes: Utilization and Analytical Validation Fig. 2. Assessment of method accuracy. Comparison of Na + (A), K + (B), Cl (C), phosphate (D), and Mg 2+ (E) measurement on the Beckman AU680 and the Roche Cobas 6000 (left panel) and Bland Altman plot showing the absolute difference and the average bias between the 2 methods. Residual clinical samples collected at the reference laboratory were initially stored at 80 C and then shipped on dry ice. Samples were stored at 20 C until analysis could be performed. Samples were subsequently thawed at room temperature. Cloudy samples or samples with visible debris were cleared at 12470g for 2 3 s. Results obtained on the AU680 were compared to clinically reported results obtained on the Roche Cobas JALM :06 May 2017

8 Fecal Electrolytes: Utilization and Analytical Validation ARTICLES Fig. 3. Stability of processed stool Na + (A), K + (B), Cl (C), Mg 2+ (D), and phosphate (E) at 4 C over 5 days. Difference plots showing the absolute difference compared to day 1. DISCUSSION Chronic diarrhea is a symptom that results from several different conditions making the differential diagnosis broad. Experts recommend categorizing the type of diarrhea to focus the diagnostic spectrum and then perform only those tests relevant to the category. Watery diarrhea can arise from secretory or osmotic reasons and can be differentiated by measuring the fecal osmotic gap. Our institution recently switched from the Beckman Coulter DxC instruments to AU680 instruments, which warranted validation of fecal electrolytes on the new instrument. However, with endoscopic procedures being easily accessible and considered to be the most definitive test, we investigated whether these tests had clinical utility to our physicians and if the tests were being used in the correct clinical setting. The challenge with determining proper utility retrospectively with chronic diarrhea is that most cases do not have a final diagnosis. In our chart review, a specific cause of chronic diarrhea was identified only in 8% of cases. In addition, the test is not definitive but merely a diagnostic clue that categorizes the type of diarrhea. Therefore, to discern utility, we designed a questionnaire to answer fundamental questions in utilization. The first question we asked was whether an appropriate sample had been submitted for testing. Fecal electrolytes can only be May : JALM 675

9 ARTICLES Fecal Electrolytes: Utilization and Analytical Validation Fig. 4. Freeze-thaw stability (cycle = 1) in patient samples (n = 8) showing percent recovery compared to baseline results. Mg 2+ was quantifiable in n=5patient samples. Baseline measurements were obtained on freshly processed stool samples. Samples were frozen at 20 C for 4 24 h and subsequently thawed at room temperature for reanalysis. measured in liquid stool. In our analysis, we found that 60 orders (50%) were cancelled because of inappropriate specimen submission such as formed stool, which essentially rules out diarrhea. This may represent a diagnostic finding unto itself that may alter the course of the work-up. Alternately, it is possible the order was placed inappropriately, or some combination of both. However, with the remaining specimens, almost all patients had chronic diarrhea with symptoms lasting longer than 4 weeks, indicating that the tests were ordered in the proper clinical context. More importantly, in these patients, the results were used for categorization, further diagnostic work-up, and/or guiding treatment decisions such as diet adjustments. Analytical validation of the 5 electrolytes demonstrated acceptable precision and linearity. The accuracy of the methods was assessed with splitsample comparisons against the Roche Cobas. However, results from the AU680 were consistently higher for all analytes, which led us to suspect preanalytical processing difference. An obvious difference in sample processing between the 2 institutions was the temperature at which samples were thawed. At the reference laboratory, samples were thawed in the refrigerator while at our institution samples were thawed at room temperature. We investigated further if thawing samples at 4 C vs room temperature affected quantification, but in controlled studies, significant differences were not observed (data not shown). We were unable to pinpoint the reason why repeat analysis on the Roche Cobas was discrepant, but these inconsistencies could be related to the complexity of stool as a matrix, where bacterial fermentation is ongoing even if samples are refrigerated. The complexity of liquid stool analysis is further illustrated by our stability studies. In controlled experiments, both phosphate and Mg 2+ were stable for up to 5 days. However, in some specimens during the validation process we noticed incomplete recovery when measured 48 h later in select specimens. Similarly, we also found that phosphate and Mg 2+ were not stable to freeze-thaw cycles in some samples, while others were unaffected. This phenomenon appeared to be sample specific because both phosphate and Mg 2+ were similarly affected or unaffected. Therefore, we recommend analyzing stool samples while still fresh or at the most, within 48 h of sample receipt. Furthermore, as evidenced by the discrepancies observed in method comparison studies, sample processing and handling could be a contributing factor to producing inaccurate results. In this regard, complying with the exact sample preparation and storage conditions described in the manuscript is recommended to laboratories intending to validate these tests in liquid stool. In conclusion, we have found that clinicians do consider stool osmolal gap in the evaluation of chronic diarrhea. The analytical validation of electrolytes in stool can be challenging due to the heterogeneity of the samples and the unpredictable stability of analytes in certain samples. 676 JALM :06 May 2017

10 Fecal Electrolytes: Utilization and Analytical Validation ARTICLES Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Authors Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest. Role of Sponsor: No sponsor was declared. Acknowledgments: We would like to thank Minh D.N. Hoang and Nickloy V. Voskoboev for technical assistance. REFERENCES 1. Fine KD, Schiller LR. AGA technical review on the evaluation and management of chronic diarrhea. Gastroenterology 1999;116: Juckett G, Trivedi R. Evaluation of chronic diarrhea. Am Fam Physician 2011;84: Schiller LR. Definitions, pathophysiology, and evaluation of chronic diarrhoea. Best Pract Res Clin Gastroenterol 2012;26: Hammer HF, Santa Ana CA, Schiller LR, Fordtran JS. Studies of osmotic diarrhea induced in normal subjects by ingestion of polyethylene glycol and lactulose. J Clin Invest 1989;84: Eherer AJ, Fordtran JS. Fecal osmotic gap and ph in experimental diarrhea of various causes. Gastroenterology 1992;103: Shiau YF, Feldman GM, Resnick MA, Coff PM. Stool electrolyte and osmolality measurements in the evaluation of diarrheal disorders. Ann Intern Med 1985; 102: Topazian M, Binder HJ. Brief report: factitious diarrhea detected by measurement of stool osmolality. N Engl J Med 1994;330: Steffer KJ, Santa Ana CA, Cole JA, Fordtran JS. The practical value of comprehensive stool analysis in detecting the cause of idiopathic chronic diarrhea. Gastroenterol Clin North Am 2012;41: Wedenoja S, Hoglund P, Holmberg C. Review article: the clinical management of congenital chloride diarrhoea. Aliment Pharmacol Ther 2010;31: Holmberg C. Congenital chloride diarrhoea. Clin Gastroenterol 1986;15: Phillips S, Donaldson L, Geisler K, Pera A, Kochar R. Stool composition in factitial diarrhea: a 6-year experience with stool analysis. Ann Intern Med 1995;123: Fine KD, Santa Ana CA, Fordtran JS. Diagnosis of magnesium-induced diarrhea. N Engl J Med 1991;324: Voskoboev NV, Cambern SJ, Hanley MM, Giesen CD, Schilling JJ, Jannetto PJ, et al. Fecal electrolyte testing for evaluation of unexplained diarrhea: validation of body fluid test accuracy in the absence of a reference method. Clin Biochem 2015;48: Fine KD, Ogunji F, Florio R, Porter J, Ana CS. Investigation and diagnosis of diarrhea caused by sodium phosphate. Dig Dis Sci 1998;43: May : JALM 677