INTERNAL POLICY AND PROCEDURE

IPP

DEPARTMENT
LABORATORY AND BLOOD BANK

TITLE/DESCRIPTION POLICY NUMBER

TEST PROCEDURE (NH3) LAB-OPER-CHEM 7 Ver.1
) AMMONIA (
EFFECTIVE DATE REVIEW DUE REPLACES NUMBER NO. OF PAGES
New 7
APPROVED BY APPLIES TO
HOSPITAL DIRECTOR ALL CHEMISTRY SECTION STAFF

:INTENDED USE .1
Enzymatic in vitro test for the quantitative determination of ammonia in human plasma on Roche/cobas systems.
:CLINICAL SIGNIFICANCE .2
Ammonia is generated primarily in the gastrointestinal tract by metabolism of nitrogenous compounds. An excess
of ammonia can be toxic to the central nervous system. The Krebs-Henseleit urea cycle provides a means of
disposal of ammonia by metabolizing ammonia to urea in the liver. Hyperammonemia in infants can be caused by
inherited deficiencies of the urea cycle enzymes or acquired through acute (as in Reye’s syndrome)
or chronic (as in cirrhosis) liver disease. In adults, elevated ammonia levels can aid in diagnosis of liver failure or
hepatic encephalopathy from advanced liver diseases such as viral hepatitis or cirrhosis.
:TEST PRINCIPLE .3
Enzymatic method, with glutamate dehydrogenase.
Glutamate dehydrogenase (GLDH) catalyzes the reductive amination of 2-oxoglutarate with NH4+ and NADPH

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to form glutamate and NADP+.
NH4+ + 2-Oxoglutarate + NADPH (GLDH)→ L-Glutamate + NADP+ + H2O
The concentration of the NADP+ formed is directly proportional to the ammonia concentration. It is determined
by measuring the decrease in absorbance.
:REAGENTS &WORKING SOLUTIONS .4
R1: BICINEa buffer: 330 mmol/L, pH 8.3; GLDH (microbial): ≥234 μkat/L; 2-oxoglutarate: 50 mmol/L;
detergent; preservative; nonreactive stabilizer.
R3: NADPH: ≥1.0 mmol/L; detergent; preservative; nonreactive buffer.
a) BICINE = N,N-bis(2-hydroxyethyl)-glycine
Storage and stability
Shelf life at 2-8°C: See expiration date
On-board in use and refrigerated on the analyzer: 12 weeks
:SPECIMEN COLLECTION AND PREPARATION .5
Only the specimens listed below were tested and found acceptable.
K2-EDTA plasma (free from hemolysis and lipemia)
Do not use plasma prepared with other anticoagulants. Do not use serum since ammonia can be generated during
clotting. Collect blood from stasis-free vein of fasting patient. Smoking should be avoided prior to sampling.
Tubes should be filled completely and kept tightly stoppered at all times. Place immediately on ice and
centrifuge, preferably at 4°C. Perform analysis within 20 to 30 minutes of venipuncture or freeze separated
plasma immediately. Avoid contamination of samples by ammonia from smoking or traffic in laboratory or
patient’s room, glassware, or water. Do not use hemolyzed samples since red blood cells contain three times the
ammonia content of plasma. Ammonia concentrations can increase in vitro due to breakdown of nitrogen-
containing plasma components. One known source of spontaneous ammonia formation is an increased γ-
glutamyltransferase activity leading to decomposition of glutamine.
Centrifuge samples containing precipitates before performing the assay.
Stability:

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Several days at (-60)-(-80)°C

6. ASSAY:
For optimum performance of the assay, follow the directions given in this document for the analyzer concerned.
Refer to the appropriate operator manual for analyzer-specific assay instructions.
Test definition
Assay type 2 Point End
Reaction time / Assay points 10 / 25-57
Wavelength (sub/main) 700/340 nm
Reaction direction Decrease
Units μmol/L
Reagent pipetting Diluent (H2O)
R1 40 μL 32 μL
R2 20 μL 20
Sample volumes Sample Sample dilution
Sample Diluent (H2O)
Normal 20 μL – –
Decreased 10 μL - -

Increased 20 μL – –

:CALIBRATION .7
Calibrators S1: H2O
S2: Ammonia/Ethanol/CO2 Calibrator
Calibration mode Linear
Calibration frequency 2-point calibration
- after reagent lot change
- and as required following quality control procedures

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Traceability: This method has been standardized against SRM 194b.

:QUALITY CONTROL .8
For quality control, use control materials as listed in the “Order information” section.
Other suitable control material can be used in addition. The control intervals and limits should be adapted to each
laboratory’s individual requirements. Values obtained should fall within the defined limits. Each laboratory
should establish corrective measures to be taken if values fall outside the limits
:CALCULATION .9
Roche/ cobas systems automatically calculate the analyte concentration of each sample.
Conversion factor: μmol/L x 1.703 = μg/dL
:INTERFERENCES .10
Criterion: Recovery within ± 10% of initial values at an ammonia concentration of 50 μmol/L (85 μg/dL).
Icterus: No significant interference up to an I index of 10 for conjugated and 30 for unconjugated bilirubin
(approximate conjugated bilirubin concentration: 171 μmol/L (10 mg/dL) and approximate unconjugated
bilirubin concentration: 513 μmol/L (30 mg/dL)).
Hemolysis: No significant interference up to an H index of 200 (approximate hemoglobin concentration: 124.2
μmol/L (200 mg/dL)). Contamination with erythrocytes will elevate results, because the analyte level in
erythrocytes is higher than in normal plasma. The level of interference may be variable depending on the content
of analyte in the lysed erythrocytes.
Lipemia (native): No significant interference up to an L index of 50. There is a poor correlation between the L
index (corresponds to turbidity) and triglycerides concentration.
γ-Globulin: γ-Globulin significantly increases the apparent ammonia concentration when 3 g/dL are added to a
human plasma pool.
Drugs: No interference was found using common drug panels. Exception: Cefoxitin and Intralipid cause
artificially high and low ammonia results respectively at the therapeutic drug level. In very rare cases
gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause unreliable results.

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For diagnostic purposes, the results should always be assessed in conjunction with the patient’s medical history,
clinical examination and other findings.

:EXPECTED VALUES .11

Females 11-51 μmol/L
Males 16-60 μmol/L
Each laboratory should investigate the transferability of the expected values to its own patient population and if
necessary determine its own reference ranges.
:PRECISION .12
Reproducibility was determined using human samples and controls in an internal protocol (within-run n = 21,
total n = 63). The following results were obtained
Within-run Mean μmol/L SD μmol/L CV%
AEC Control N 60.7 1.4 2.3
AEC Control A 202 2.0 0.8
Human Plasma 1 28.6 2.5 8.8
Human Plasma 2 585 1.0 0.2
Total Mean μmol/L SD μmol/L CV %
AEC Control N 56.9 2.8 4.9
AEC Control A 203 4.0 1.8
AEC Control N 1:2 Dil. 28.1 2.6 9.4
AEC Calibrator 317 5.0 1.5
Measuring range 10-700 μmol/L
Determine samples having higher concentations via the rerun function. Dilution of samples via the rerun function
is a 1:2 dilution. Results from samples diluted by the rerun function are automatically multiplied by a factor of 2.
Lower detection limit 10 μmol/L
The lower detection limit represents the lowest measurable analyte level that can be distinguished from zero. It is

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calculated as the value lying three standard deviations above that of the lowest standard (standard 1 + 3 SD,
within-run precision, n = 21).
13. REFERENCES :

1. Balistreri WF, Rej R. Liver function. In: Burtis CA, Ashwood ER, eds. Tietz Fundamentals of Clinical
Chemistry. 4th ed. Philadelphia: WB Saunders 1996:539-568.
2. VanAnken HC, Schiphorst ME. A kinetic determination of ammonia in plasma. Clin Chim Acta 1974;56:151-
157.
3. Da Fonseca-Wollheim F. Deamidation of glutamine by increased plasma γ-glutamyltransferase is a source of
rapid ammonia formation in blood and plasma specimens. Clin Chem 1990;36:1479-1482.
4. Howanitz JH, Howanitz PJ, Skrodzki CA, Iwanski JA. Influences of specimen processing and storage
conditions on results for plasma ammonia. Clin Chem 1984;30:906-908.
5. Da Fonseca-Wollheim F. Preanalytical increase of ammonia in blood specimens from healthy subjects. Clin
Chem 1990;36:1483-1487.
6. Glick MR, Ryder KW, Jackson SA. Graphical Comparisons of Interferences in Clinical Chemistry
Instrumentation. Clin Chem 1986;32:470-474.
7. Report on the Symposium “Drug effects in clinical chemistry methods”, Breuer J, Eur J Clin Chem Clin
Biochem 1996;34:385-386.
8. Da Fonseca-Wollheim F. Direkte Plasmaammoniakbestimmung ohne Enteiweissung. Z Klin Chem Klin
Biochem 1973;11:426-431.
9. Bablok W et al. A General Regression Procedure for Method Transformation. J Clin Chem Clin Biochem
1988;26:783-790.

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14.APPROVAL PAGE:

NAME TITLE SIGNATURE DATE

PREPARED Dr/ Nagah Mohammed LAB. CONSULTANT

BY

/ .Mr LAB. DIRECTOR

REVIEWED
/Dr TQM DIRECTOR

BY

APPROVED /.Mr HOSPITAL DIRECTOR

BY

IPP HISTORY

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