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Critically High Plasma Ammonia in an Adolescent Girl.

CASE DESCRIPTION

A 12-year-old female patient presented with nonspecific symptoms including fatigue, fever, and headache persisting for approximately 4 months. Plasma ammonia was 230 [micro]g/dL (135 [micro]mol/L) (critical value >187 [micro]g/dL or 110 [micro]mol/L). The patient was referred to a pediatric emergency center for urgent evaluation of hyperammonemia. Plasma and urine amino acid testing was performed to investigate the possibility ofa urea cycle disorder; however, all results were normal. Over the following 2 weeks, the patient was treated with intravenous sodium benzoate/sodium phenylacetate and L-arginine to reduce the blood ammonia concentration. However, the patient's ammonia concentration never dropped below 94 [micro]g/dL (55 [micro]mol/L) (upper limit of reference interval, 80 [micro]g/dL or 47 [micro]mol/L), with concentrations as high as 296 [micro]g/dL (174 [micro]mol/L) with inconsistent associations between ammonia concentrations and headaches. During the course of treatment, the patient suffered substantial side effects of intravenous therapy and underwent numerous costly clinical and laboratory investigations. Amino acid investigations were repeated but results remained normal. Given the large fluctuations in blood ammonia concentrations with inconsistent response to intravenous medications, the metabolic service contacted the laboratory to inquire about a possible preanalytical or analytical cause. One week before this query, a community physician also contacted the laboratory regarding hyperammonemia in several patients. However, no obvious preanalytical or analytical errors were discovered despite numerous audits of preanalytical conditions and review of quality control data.

QUESTIONS TO CONSIDER

1. What is plasma ammonia and why is it important?

2. What are the physiologic causes of hyperammonemia?

3. What is the appropriate treatment for hyperammonemia?

4. What are the preanalytical causes of hyperammonemia?

Discussion

Ammonia is a metabolic waste product produced mainly in the gut, kidney, and skeletal muscle during exercise (1). It is generated from microbial metabolism, deamination ofamino acids, nucleic acid degradation, and protein catabolism. Ammonia is normally removed from circulation by incorporation into the urea cycle after condensation with bicarbonate and phosphorylation to form carbamoyl phosphate in the liver. The urea cycle includes 5 enzymes and 2 transporters that remove ammonia by generating urea and fumarate. Fumarate is incorporated into the Krebs cycle, whereas urea is excreted in urine. At physiological pH, ammonia exists mainly as ammonium ions (N[H.sub.4.sup.+]), with 1%--2% as ammonia gas (N[H.sub.3]). Ammonia readily crosses the blood--brain barrier where it combines with [H.sup.+] to form ammonium, which competes with potassium for transport across neuronal membranes (2). Symptoms of hyperammonemia include lethargy, irritability, vomiting, and poor feeding in children, as well as hyperventilation, seizures, coma, and eventually death.

Common causes of hyperammonemia include liver failure (hepatocyte destruction and reduced urea cycle enzymes), drug reactions (e.g., inhibition of the urea cycle by valproic acid), hemolytic disease (release of ammonia from red blood cells), gastrointestinal bleeds (increased ammonia generation due to microbial catabolism of hemoglobin), or urea cycle disorders (defective or decreased urea cycle enzymes) (1). Urea cycle disorders are inborn errors of metabolism that normally present with severe hyperammonemia in the first few weeks of life, although less severe forms can manifest at any age (3). Early diagnosis and treatment is important to avoid delays in mental development.

Ammonia is frequently measured in the clinical laboratory with a one-step enzymatic assay using glutamate dehydrogenase (GLDH), [4] [alpha]-ketoglutarate, and NADPH. When ammonia is added to this mixture, glutamate and NADP+ are generated by GLDH activity, with a decrease in NADPH absorbance at 340 nm proportional to ammonia concentration. In this case, the ammonia assay (Randox GLDH) also included a background subtraction at 700 nm.

Ammonia concentrations can be falsely increased by numerous preanalytical conditions. Delays in blood centrifugation and room temperature storage can lead to increased ammonia due to in vitro generation by [gamma]-glutamyltransferase (GGT) activity (4, 5). Traumatic blood collection and hemolysis can increase ammonia, as red blood cells contain a high concentration of ammonia compared to plasma (6). Excess muscle activity (e.g., seizures) or muscle activity under ischemia (e.g., fist clenching with tourniquet) increases ammonia generation via AMP-deaminase activity (7). To limit the influence of preanalytical error, blood collection is frequently conducted without a tourniquet using prechilled tubes transported on ice for separation in a refrigerated centrifuge. Dipotassium EDTA is the anticoagulant of choice as heparin interferes with several ammonia assays (8).

In this case, the patient presented with vague symptoms and a critical (>187 [micro]g/dL or >110 [micro]mol/L) plasma ammonia which raised suspicion for a urea cycle disorder (Fig. 1, day--13). Ammonia results over the next few days remained high despite the use of intravenous ammonia scavengers, ranging from 94 to 296 [micro]g/dL (55-174 [micro]mol/L) (Fig. 1, days--13 to--9). There were also significant fluctuations in ammonia concentrations taken hours apart with no apparent change in clinical symptoms. On day 0 (Fig. 1), a community physician contacted the laboratory regarding hyperammonemia in several patients; however, no obvious preanalytical or analytical causes were discovered at that time. In these patients, ammonia results were either disregarded or patients received limited follow-up. On day 7, the metabolics service contacted the laboratory regarding their patient, prompting a second investigation.

The second investigation by laboratory staff found that the centrifuge (Eppendorf 5810 R with swinging bucket, rotor radius = 173 mm) used to separate samples for ammonia testing was set at 1200 rpm instead of 1200g, resulting in a relative centrifugal force (RCF) of only 280g (Eq. 1). This centrifuge's display distinguished between rpm and g with an asterisk (i.e., "1200" = 1200 rpm; "1200 *" = 1200g), which was missed during the initial investigation. The centrifuge speed was immediately corrected and the patient ammonia results quickly normalized (Fig. 1, day 7):

RCF = 1.12 X Radius(mm) X [(rpm/1000).sup.2] (1)

To confirm that incorrect centrifugation speed was the cause, 2 prechilled 3-mL tubes of [K.sub.2]-EDTA anticoagulated whole blood were collected from 10 healthy volunteers. Tubes were stored on ice before separation in a refrigerated (4[degrees]C) centrifuge. One tube from each volunteer was spun at low speed (1200 rpm, 280g), and the other at normal speed (1200g), each for 10 min. Plasma was aliquoted on ice and tested for ammonia, GGT, complete blood count, and Roche serum indices (hemolysis, icterus, lipemia). Differences were assessed using the Wilcoxon signed rank test.

Plasma from samples centrifuged at low speed were visibly more turbid and had significantly (P < 0.01) higher ammonia and platelets than those spun at normal speed (Fig. 2, A and B). There were no detectable red or white blood cells in any of the plasma samples and no significant difference in hemolysis or icterus indices (data not shown). Consistent with the visual assessment of turbidity, the lipemia index was significantly increased (data not shown; P <0.01) in samples centrifuged at low speed. There was no significant difference in GGT (data not shown; P = 0.56). Linear regression was used to determine the relationship between platelet count and ammonia, yielding a coefficient of determination ([R.sup.2]) of 0.97, a slope of 0.19 [micro]mol/L/109 platelets/L, and an intercept of 20.9 [micro]mol/L ammonia (Fig. 2C). Differences were less pronounced when the study was repeated using 1.0-mL microcollection containers, likely due to the smaller number ofplatelets available to concentrate in an incompletely separated sample (data not shown).

As these results suggested that the patient's high ammonia concentrations were from residual platelets, a second experiment was performed with pooled [K.sub.2]-EDTA whole blood. Blood was centrifuged at 1200 rpm, 1200g, and 2000g, and plasma from each condition was split into 2 aliquots--one of which was additionally centrifuged at 2500g for 15 min to completely remove residual platelets. Ammonia and platelets were significantly decreased with increasing centrifugation speed and the additional centrifugation at 2500gremoved nearly all residual platelets, consistently yielding the lowest ammonia concentration (data not shown). This experiment confirmed that increased ammonia in samples spun at low speed was due to residual platelets in plasma. Interestingly, this phenomenon was first described by Cowley et al. over 30 years ago (9); however, to our knowledge this is the first report of a significant patient impact resulting from a falsely high ammonia result owing to incomplete specimen separation.

Other causes of increased ammonia were investigated; however, these were quickly ruled out. For example, while samples spun at low speed were visibly turbid, the degree of turbidity assessed by the Roche lipemia index (dichromatic absorbance at 660 and 700 nm) was too low to cause a significant interference, which interestingly would have been a negative interference (false low) for this assay. Interestingly, GGT concentrations were within the reference interval and did not significantly differ according to centrifugation speed.

As there was a clear relationship between centrifugation speed, residual platelets, and ammonia concentration, several engineering controls and operational changes were made to reduce the chances of centrifuges being set incorrectly in the future: (a) centrifuge speeds would be required to be verified at the start of each shift, and (b) a brightly colored stop sign would be attached to centrifuges to prevent accidental use when speeds were changed. In this case, the speed of the centrifuge in question was changed to 1200 rpm to process a bone marrow sample; however, it was not changed back to 1200g. Finally, (c) samples collected for ammonia testing would be considered STAT priority to reduce processing and analysis time.

While these changes combined with staff education will reduce preanalytical errors in ammonia testing, they cannot eliminate them. Communication with phlebotomists, clinician teams, and biochemical genetics laboratories is essential to determine whether an increased ammonia result is truly real. This is because review of QC results (i.e., Levey--Jennings charts) cannot uncover errors that occur in the preanalytical phase of testing. Furthermore, patient [delta] checks appear to be seldom used in ammonia testing. Tracking mean patient ammonia results over time could help detect large-scale preanalytical errors; however, this would require a large ammonia testing workload.

Regardless of the test, preanalytical quality must be maintained for physicians to make appropriate clinical decisions. In this case, an incorrectly set centrifuge resulted in costly additional treatment that turned out to be unnecessary, repeated testing, and significant physical and psychological harm to a patient. This caused several quality improvements to be made regarding the control and monitoring of centrifuge use. Because of their central importance in clinical laboratories, centrifuges and their operating procedures should be carefully evaluated to minimize preanalytical errors (10).

POINTS TO REMEMBER

* Ammonia is a toxic nitrogenous waste product that is normally processed by the urea cycle into urea.

* Urea cycle disorders can be responsible for hyperammonemia and usually appear in childhood.

* Hyperammonemia is a serious condition that must be urgently treated.

* Ammonia testing is confounded by numerous preanalytical conditions. Residual platelets in plasma can cause large false increases of ammonia.

* Control of preanalytical error is paramount to prevent treatment based on an incorrect laboratory result, as this can cause patient harm.

Author Contributions: All authors confirmed they have contributed to the intellectual content ofthispaper and have met the following 3 requirements: (a) significant contribution to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval ofthe published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: L. de Koning, Calgary Laboratory Services.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: None declared.

Expert Testimony: None declared.

Patents: None declared.

References

(1.) Adeva MM, Souto G, Blanco N, Donapetry C. Ammonium metabolism in humans. Metabolism 2012; 61: 1495-511.

(2.) Thrane VR, Thrane, AS, Wang F, Cotrina ML, Smith NA, Chen M, et al.Ammonia triggers neuronal disinhibition and seizures by impairing astrocyte potassium buffering. Nat Med 2013; 19:1643-8.

(3.) Summar ML, Dobbelaere D, Brusilow S, Lee B. Diagnosis, symptoms, frequency and mortality of 260 patients with urea cycle disorders from a 21-year, multicentre study of acute hyperammonaemic episodes. Acta Paediatr 2008; 97:1420-5.

(4.) da Fonseca-Wollheim F, Deamidation of glutamine by increased plasma gamma-glutamyltransferase is a source of rapid ammonia formation in blood and plasma specimens. Clin Chem 1990; 36:1479-82.

(5.) Nikolac N, OmazicJ, Simundic AM. The evidence based practice for optimal sample quality for ammonia measurement. Clin Biochem 2014; 47:991-5.

(6.) Apushkin M, Das A, Joseph C, Leung EK, Yeo KT, Baron JM, Baron BW. Reducing the risk of hyperammonemia from transfusion of stored red blood cells. Transfus ApherSci 2013; 49:459-62.

(7.) Valen PA, Nakayama DA, Veum J, Sulaiman AR, Wortmann RL. Myoadenylate deaminase deficiency and forearm ischemic exercise testing. Arthritis Rheum 1987; 30:661-8.

(8.) Wannasilp N,SribhenK,Pussara N,HwanpuchT,Wangchaijaroenkit S, Opartkiattikul N. Heparin is an unsuitable anticoagulant for the detection of plasma ammonia.ClinChimActa2006; 371:196-7.

(9.) CowleyDM, Nagle BA, ChalmersAH,Sinton TJ. Effects of plateletsoncollectionofspecimensforassayofammonia in plasma. Clin Chem 1985; 31:332-3.

(10.) Pyle -Eilola,A.Centrifuges:labsunsunghighperformance instruments. Clin Lab News; 2015. https://www.aacc.org/ publications/cln/articles/2015/july/centrifuges-labsunsung-high-performance-instruments (Accessed April 2016).

Commentary

D. Robert Dufour, MD *

The case described by Orton et al. is of a young girl who presented with nonspecific complaints of headache and fatigue. One possible explanation for such symptoms is an inborn error in urea cycle enzymes, a group of rare disorders that more commonly presents acutely and in early childhood (1). Less commonly, chronic presentations with nonspecific symptoms can occur later in life (sometimes even in adults) and can be difficult to diagnose. Most cases also have various neuropsychiatric symptoms in addition to headache and fatigue, and the lack of these being described in this child make this diagnosis less likely. Plasma ammonia concentrations are the most common screening test for this condition, as well as for the similar hepatic encephalopathy that occurs in adults (2).

An experienced clinician often recognizes that laboratory results are inconsistent with the clinical picture, as happened in this case. Despite another clinician contacting the laboratory about apparently incorrect ammonia concentrations, it took a second inquiry from the clinicians caring for this child for the laboratory to complete a full investigation, leading to further complications of treatment in the patient and, presumably, large medical bills.

A good working relationship between clinicians and laboratorians is critical to optimal laboratory practice. The National Academies report on Improving Diagnosis in Health Care recommends such improved communication between clinicians and laboratorians (3). Clinicians expect that laboratory results accurately reflect the status of their patients but recognize that no test is perfect. Inquiries from clinicians should be looked on as a form of "quality control" and a chance for both the provider and the laboratory to improve their understanding of the issues that affect laboratory results. This can only result in better patient care and improved patient outcomes.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 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; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts ofinterest.

References

(1.) Haberle J, Boddaert N, Burlina A, Chakrapani A, Dixon M, Huemer M, et al. Suggested guidelines for the diagnosis and management of urea cycle disorders. Orphanet J Rare Dis2012; 7:32.

(2.) Prakash R, Mullen KD. Mechanisms, diagnosis, and management of hepatic encephalopathy. Nat Rev Gastroenterol Hepatol 2010; 7:515-25.

(3.) National Academies of Sciences, Engineering, and Medicine. Improving diagnosis in health care. Washington (DC): The National Academies Press; 2015.

D. Robert Dufour, MD *

George Washington University Medical Center, Washington, DC.

* Address correspondence to the author at: 3825 N. Kenmore Ave.#1S, Chicago, IL 60613.

Fax 240-432-7760; e-mail chemdoctorbob@earthlink.net.

Received September 7, 2016; accepted September 14, 2016.

DOI: 10.1373/clinchem.2016.263566

Commentary

Sarah M. Brown *

When troubleshooting potential laboratory errors, the entire test system must be considered, including pre-analytical steps. In this case, the authors had previously investigated analytical error associated with their ammonia assay after a community physician questioned results. The initial investigation, including review of QC, did not indicate analytical error. Although QCs are fantastic tools for evaluating the analytical performance of a test, they are not robust at identifying preanalytical errors. Alternatively, [delta] checks can be used to identify preanalytical errors. The [delta] checks are comparisons of results to previous results of the same analyte from the same patient. A [delta], or significant change from a previous result, may indicate preanalytical errors. The [delta] checks are good for analytes that show little day-to-day variation, have low critical difference values, and have low intraindividual variation. Alternatively, tracers can be used to evaluate the entire testing system. A laboratory tracer undergoes all of the pre- and postanalytical steps. Tracers can uncover errors that standard quality controls and [delta] checks cannot, and are good for analytes that are not candidates for [delta] checks.

Ammonia is in analyte that is particularly susceptible to preanalytical error such as environmental contamination, improper tourniquet use, and hemolysis. Improper sample handling can also cause falsely increased ammonia. To prevent in vitro formation of ammonia from cellular metabolism, samples should be drawn on ice and plasma should be separated from cells immediately. Ammonia has wide intraindividual variation; concentrations can change with exercise and the amount of dietary protein. Ammonia can also show dramatic changes during the treatment of hyperammonemia.

This case highlights (a) the need to remove cells from plasma quickly and thoroughly before measuring ammonia and (b) how tracing back through an entire testing system can identify problems that would not be uncovered with routine quality assurance measures.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following3 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; and (c) final approval ofthe published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

Sarah M. Brown *

Department of Pediatrics, Washington University School of Medicine, St. Louis, MO.

* Address correspondence to the author at: Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8116, St. Louis, MO 63104. Fax3l4-454-2274; e-mail brown_sa@kids.wustl.edu.

Received September 18,2016; accepted September 20,2016.

DOI: 10.1373/clinchem.2016.263574

Dennis J. Orton, [1,2] Jessica L. Gifford, [1,2] Isolde Seiden-Long, [1,2] Aneal Khan, [3] and Lawrence de Koning [1,2] *

[1] Calgary Laboratory Services, Calgary, Alberta, Canada, and Departments of2 Pathology and Laboratory Medicine and3 Medical Genetics and Paediatrics, University of Calgary, Calgary, Alberta, Canada.

* Address correspondence to this author at: Alberta Children's Hospital, Rm. B3-724, 2888 Shaganappi Trail Northwest, Calgary, AB T3B 6A8, Canada. Fax 403-955-2321; e-mail Lawrence.DeKoning@cls.ab.ca.

This study was previously presented at the Joint Conference of the Canadian Society of Clinical Chemists (CSCC)and Canadian College of Medical Geneticists (CCMG) in Edmonton, Alberta, Canada, on June 20,2016.

Received April 18, 2016; accepted June 10, 2016.

DOI: 10.1373/clinchem.2016.259473

[4] Nonstandard abbreviations: GLDH, glutamate dehydrogenase; GGT, y-glutamyltransferase; RCF, relative centrifugal force.

Caption: Fig.1. Patient ammonia results overtime. Dashed line: critical value for pediatric patients (187 [micro]g/dL or 110 [micro]mol/L). Dotted line: upper limit of reference interval (80 [micro]g/dL or 47 [micro]mol/L). Correction of the centrifuge speed occurred at day 7, indicated by the arrow.To convert ammonia results in [micro]mol/Lto [micro]g/dL, multiply by 1.703.

Caption: Fig.2. Results from volunteers. Light gray: samples spun at low speed. Dark gray: samples spun at normal speed. There was significantly (P <0.05) higher ammonia (A) and platelets (B) in plasma spun at low vs normal speed. Ammonia versus platelet count (C). Dotted lines: ammonia upper limit of normal (80 [micro]g/dL or 47 [micro]mol/L).To convert ammonia results in [micro]mol/L to [micro]g/dL, multiply by 1.703.
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Title Annotation:Clinical Case Study
Author:Orton, Dennis J.; Gifford, Jessica L.; Seiden-Long, Isolde; Khan, Aneal; de Koning, Lawrence
Publication:Clinical Chemistry
Date:Dec 1, 2016
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