Piccolo Xpress Chemistry Analyser SOP

Document POCT-SOP7 Version No.
1
No. Issue Date 09/03/2018
Piccolo Xpress Chemistry Analyser Operation
The Piccolo Xpress chemistry analyser provides quantatative determinations

of Alanine aminotransferase (ALT), Albumin, Alkaline phosphatase (ALP),
Amylase, Aspartate aminotransferase (AST), C-reactive protein (CRP),Calcium,
Creatinine,Gamma glutamyltransferase (GGT), glucose, total protein,urea and
urric acid in lithium heparin whole blood or serum
Authority For Issue: Jennifer Brown Page 1 of 16

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1. TABLE OF CONTENTS
1. TABLE OF CONTENTS..................................................................................2
2. HAZARDS AND PRECAUTIONS....................................................................3
2.1. Procedure Risk Assessment.................................................................3
2.2. Chemical...............................................................................................3
2.3. Biological Hazard..................................................................................3
2.4. Physical Hazard....................................................................................4
2.5. Room Risk Assessment (if required).....................................................4
3. CONTENT.......................................................................................................5
A) PURPOSE OF THE EXAMINATION......................................................................5
B) PRINCIPLE AND METHOD OF THE PROCEDURE USED FOR EXAMINATIONS
................................................................................................................................... 5
C) PERFORMANCE CHARACTERISTICS (SEE 5.5.1.2 AND 5.5.1.3)......................5
D) TYPE OF SAMPLE (E.G. PLASMA, SERUM, URINE)..........................................5
E) PATIENT PREPARATION.....................................................................................5
F) TYPE OF CONTAINER AND ADDITIVES..............................................................5
G) REQUIRED EQUIPMENT AND REAGENTS.........................................................5
H) ENVIRONMENTAL AND SAFETY CONTROLS....................................................5
I) CALIBRATION PROCEDURES (METROLOGICAL TRACEABILITY).....................5
J) PROCEDURAL STEPS..........................................................................................5
K) QUALITY CONTROL PROCEDURES...................................................................5
L) INTERFERENCES (E.G. LIPAEMIA, HAEMOLYSIS, BILIRUBINEMIA, DRUGS)
AND CROSS REACTIONS.........................................................................................5
M) PRINCIPLE OF PROCEDURE FOR CALCULATING RESULTS INCLUDING,
WHERE RELEVANT, THE MEASUREMENT UNCERTAINTY OF MEASURED
QUANTITY VALUES..................................................................................................6
N) BIOLOGICAL REFERENCE INTERVALS OR CLINICAL DECISION VALUES.....6
O) REPORTABLE INTERVAL OF EXAMINATION RESULTS....................................6
P) INSTRUCTIONS FOR DETERMINING QUANTITATIVE RESULTS WHEN A
RESULT IS NOT WITHIN THE MEASUREMENT INTERVAL....................................6
Q) ALERT/CRITICAL VALUES, WHERE APPROPRIATE........................................6
R) LABORATORY CLINICAL INTERPRETATION.....................................................6
S) POTENTIAL SOURCES OF VARIATION...............................................................6
T) REFERENCES.......................................................................................................6

2. HAZARDS AND PRECAUTIONS.
2.1. Procedure Risk Assessment
Hazard Risk Assessment of Overall Procedural

Risk Risk
Chemical Minor injury Possible Medium

Biological Death Rare Low
Physical Minor Injury Unlikely Low Medium
Room Death Rare Low
Mechanical N/A N/A N/A
2.2. Chemical
Please note – only chemicals which present a risk when used in this procedure need to be
included in this table.
Chemical Risk Precautions Storage and Discard 1st Aid Measures MSDS reference
Requirements (Q-pulse)
Wear gloves when Store in fridge and Fush exposed skin POCT-SDS4
handling rotor discard into a with copious amounts
sharpsafe™ of water for at least 15
Skin irritant minutes
lyophylised chemical
beads enclosed in a
plastic rotor
Chemicals used in
rotor:
D-Mannitol
Polyethylene glycol,
8000
Dextran, 70 USP
Tris(hydroxymethyl)
amino
3400
2000
Sodium Chloride
POPSO, Disodium
salt
Sodium
Thiocyanate
L-Aspartic Acid
Lithium Hydroxide,
Tris, HCL

2.3. Biological Hazard
Biological Risk Precautions Precautions
Wear Gloves when handling

There is a risk of contact with samples and quality control
blood borne viruses and other
infective agents when handling
patient samples and quality
control
2.4. Physical Hazard
Please note – this includes any manual handling and VDU risk assessments required and
need to be included in this table.
Physical Risk Precautions 1st Aid Measures Any pertinent other

Reference material
Electrical Place the analyzer on a
level surface that is free
of hair, dust, and other
contaminants. Do not
place the analyzer near
a sunny window or any
other heat source.
2.5. Room Risk Assessment (if required)
Please note – this includes any room which had special requirements for example dark
rooms, CL3 laboratories.
Room Risk Precautions 1st Aid Measures Any pertinent other

Reference material

3. CONTENT
A) PURPOSE OF THE EXAMINATION

The Piccolo Xpress chemistry system consists of a portable analyzer and
disposable single-use reagent discs. Each reagent disc contains all the
reagents needed to perform a panel of tests on a single sample. The Scottish
Organ Retrieval Team transports the analyser to the donor hospital and the
results are used to detemine the suitability of organs for transplant by
analysing blood samples every 30 minutes during Normothermic Regional
Perfusion.
B) PRINCIPLE AND METHOD OF THE PROCEDURE USED FOR

EXAMINATIONS
The operator introduces a heparinized whole blood sample (or heparinized
plasma, serum, or control) into the reagent disc. The reagent disc contains a
diluent and test-specific reagent beads.
The operator then places the disc in the Piccolo Xpress chemistry analyser
and enters the appropriate identification numbers.
The analyzer automatically performs the remainder of the testing protocol.
The reagent disc spins and whole blood is separated into plasma and blood
cells. During this time, the disc is heated to 37 ºC (98.6 ºF). Precisely
measured quantities of plasma and diluent enter the mixing chamber and are
mixed together. Through centrifugal and capillary forces, the diluted plasma is
distributed to cuvettes on the perimeter of the disc. Reagent beads in the
cuvettes are dissolved by the diluted plasma. This solution is thoroughly
mixed and the resulting chemical reactions are monitored photometrically by
the analyser. Optical signals generated by the chemical reactions are used to
calculate analyte concentrations. Calibration data specific for the chemistries
in each disc are provided to the analyzer by the bar code printed on the
barcode ring
1. Alanine Aminotransferase (ALT)

ALT catalyses the transfer of an amino group from L-alanine to α-ketoglutarate to
form L-glutamate and pyruvate. Lactate dehydrogenase catalyses the conversion
of pyruvate to lactate. Concomitantly NADH is oxidised to NAD+
L-Alanine + α-ketoglutarate → L-Glutamate + Pyruvate

Pyruvate + NADH + H+ →
Lactate + NAD+
The rate of change of the absorbance difference between 340nm and 405nm is
due to the conversion of NADH to NAD+ and is directly proportional to the
amount of ALT present in the sample
2. Albumin
Bromocresol purple (BCP) when bound with albumin, changes colour from a
yellow to blue colour. The absorbance maximum changes with the colour shift.
BCP + Albumin → BCP-Albumin Complex

Bound albumin is proportional to the concentration of albumin in the sample. This

is an endpoint reaction that is measured as absorbance at 600nm.
3. Alkaline Phosphatase (ALP)

Alkaline phosphatase hydrolyses p-Nitrophenyl phosphate in a metal – ion buffer
and forms p-nitrophenol and phosphate
p-Nitrophenyl Phosphate → p-Nitrophenol + phosphate

The amount of ALP in the sample is proportional to the rate of increase in
absorbance between 405nm and 500nm
4. Amylase (AMY)
The substrate, 2-chloro-p-nitrophenly-α-D-maltotrioside (CNPG3), reacts with α-
amylase in the patient sample, releasing 2-chloro-p-nitrophenol (CNP). The
release of CNP creates a change in colour.
CNPG3 → CNP + D- Maltotrioside
The reaction is measured bichromatically at 405nm and 500nm. The change in

absorbance due to the formation of CNP is directly proportional to α-amylase
activity in the sample
5. Aspartate Aminotransferase (AST)

AST catalyses the reaction of L-aspartate and α-ketoglutarate into oxaloacetate
and L-glutamate. Oxaloacetate is converted to malate and NADH is oxidised to
NAD+ by the catalyst MDH
L-aspartate + α-ketoglutarate → Oxaloacetate + L-glutamate
Oxaloacetate + NADH + H+ → Malate + NAD+
The rate of absorbance change at 340nm / 405mn caused by the conversion of

NADH to NAD+ is directly proportional to the amount of AST present in the
sample
6. Calcium (Ca)
Calcium in the patient sample binds with arsenaso 111 to form a calcium dye
complex
Ca + arsenazo 111 → Ca arsenazo 111 complex
The endpoint reaction is monitored at 405nm, 467nm and 600nm. The amount of
calcium in the sample is proportional to the absorbence
7. Creatinine (CRE)
Creatinine amidohydrolase hydrolyses creatinine to creatine. A second enzyme,
creatine amidinohydrolsae, catalyses the formation of sarcosine from creatine.
Sarcosine oxidase causes the oxidation of sarcosine to glycine, formaldehyde
and hydrogen peroxide (H2O2). Peroxidase catalyses the reaction among
hydrogen peroxide, 2,4,6-tribromo-3-hydroxybenzoic acid (TBHBA) and 4-
aminoantipyrine (4-AAP) into a red quinoneimine dye. Sodium ferrocyanide and
ascorbate oxidase are added to the reaction mixture to minimise the potential
interference of bilirubin and ascorbic acid, respectively.
Creatinine + H2O → Creatine

Creatine + H2O → Sarcosine + Urea

Sarcosine + H2O + O2 → Glycine + Formaldehyde + H2O2
H2O2 + TBHBA + 4-AAP → Red Quinoneimine dye + H2O
Two cuvettes are used to determine the concentration of creatinine in the sample.
Endogenous creatine is measured in the blank cuvette which is subtracted from
the combined endogenous creatine and the creatine formed from the enzyme
reactions in the test cuvette. Once the endogenous creatine is eliminated from
the calculations, the concentration of creatinine is proportional to the intensity of
the red colour produces. The endpoint reaction is measured as the difference in
absorbance between 550nm and 600 nm.
8. Gamma Glutamyltransferase (GGT)

The addition of sample containing GGT to the substrates L-ˠ-glutamyl-3-carboxy-
4-nitroaniline and glycylglycine (gly-gly) causes the formation of L-ˠ-glutamyl-
glycylglicine (glu-gly-gly) and 3-carboxy-4-nitroaniline
L-ˠ-glutamyl-3-carboxy-4-nitroanilide + gly-gly → glu-gly-gly + 3-carboxy-4-

nitroaniline
The absorbance of this rate reaction is measured at 405nm. The production of 3-

carboxy-4-nitroaniline is directly proportional to the GGT activity in the sample.
9. Glucose (GLU)
The reaction of glucose with adenosine triphosphate (ATP), catalysed br
hexokinase (HK) produces glucose-6-phosphate (G-6-P) and adenosine
diphosphate (ADP). Glucose-6-phosphate dehydrogenase (G-6-PDH) catalyses
the reaction of 6-G-P into 6-phosphogluconate and the reduction of nicotinamide
adenine dinucleotide (NAD+) to NADH.
Glucose + ATP → G-6-P + ADP
G-6-P + NAD+ → 6-phosphogluconate + NADH + H+
The absorbance is measured bichromatically at 340nm and 850nm. The

production of NADH is directly proportional to the amount of glucose present in
the sample.
10. Total Bilirubin (TBIL)

Bilirubin is oxidised by bilirubin oxidase into biliverdin
Bilirubin + O2 → Biliverdin + H2O
Bilirubin is quantitated as the difference in absorbance between 467nm and

550nm. The initial absorbance of this endpoint reaction is determined from the
bilirubin blank cuvette and the final absorbance is obtained from the bilirubin test
cuvette. The amount of bilirubin in the sample is proportional to the difference
between the initial and final absorbance measurement.
11. Total protein (TP)

The protein solution is treated with cupric (Cu11) ions in a strong alkaline
medium. Sodium potassium tartrate and potassium iodide are added to prevent
the precipitation of copper hydroxide and the auto- reduction of copper
respectively. The Cu11 ions react with peptide bonds between the carbonyl
oxygen and amide nitrogen atoms to form a coloured Cu-protein complex
Total protein + Cu11 → Cu-protein complex
The amount of total protein in the sample is directly proportional to the

absorbance of the Cu-protein complex. The total protein test is an endpoint
reaction and the absorbance is measured as the difference in absorbance
between 550nm and 850nm.
12. Blood Urea Nitrogen (BUN)

In the coupled-enzyme reaction, urease hydrolyses urea into ammonia and
carbon dioxide. Upon combining ammonia with α-ketoglutarate and reduced
nicotinamide adenine dinucleotide (NADH), the enzyme glutamate
dehydrogenase (GLDH) oxidises NADH to NAD+
Urea + H20 → 2NH3 + CO2
NH3 + α-ketoglutarate + NADH → L-Glutamate + H20 + NAD+
The rate of change of the absorbance difference between 340nm and 405nm is
caused by the conversion of NADH to NAD+ and is directly proportional to the
amount of urea present in the sample.
13. Uric Acid (UA)

Uricase catalyses the oxidation of uric acid to allantoin and hydrogen peroxide.
Peroxidase catalyses the reaction among the hydrogen peroxide (H2O2), 4-
aminoantipyrine (4-AAP) and 3,5-dichloro-2-hydroxybenzenesulphonic acid
(DHBSA) into a red quinoneimine dye. Sodium ferrocyanide and ascorbate
oxidase are added to the reaction to minimise the potential interference of
bilirubin and ascorbic acid.
Uric acid + O2 + H2O → Allantoin + CO2 + H2O2
H2O2 + 4-AAP + DHBSA → Quinoneimine dye + H2O
C) PERFORMANCE CHARACTERISTICS (SEE 5.5.1.2 AND 5.5.1.3)

C) TYPE OF SAMPLE (E.G. PLASMA, SERUM, URINE)

The Piccolo Xpress chemistry analyzer accepts lithium-heparinised whole blood,
plasma, or serum samples
E) PATIENT PREPARATION
F) TYPE OF CONTAINER AND ADDITIVES

Withdraw blood from sampling line into a plain plastic sterile syringe. Decant into a sterile
container and follow the steps for running a sample below.
.
G) REQUIRED EQUIPMENT AND REAGENTS

Operational Materials Catalog Number
Pipette tips, disposable, pack of 96 Abaxis #500-9007
Mini pipette, 100 ml, gray Abaxis #500-9006
Internal printer paper rolls, box of 6 Abaxis #1100-4410
Fan filter, covered Abaxis #1987-0009
Piccolo® Reagent Disc – General Chemistry 13
400-1029 (single); 400-0029 (10 pack); 400-0029-4 (4 pack)
H) ENVIRONMENTAL AND SAFETY CONTROLS

Refer to the various MSDS and kit inserts for instructions on the storage, handling and
disposal of the material.
POCT-EXT6
POCT-SDS4
POCT-SDS5
I) CALIBRATION PROCEDURES (METROLOGICAL TRACEABILITY)

Refer to Piccolo traceability: ref POCT-EXT6

J) PROCEDURAL STEPS
Running a patient sample
Turn on the analyser by pressing the Power button on the front of the analyser
The analyser turns on then performs a self test
If the analyser needs time to warm the disc chamber to operating temperature the
display shows ‘warming’
When the analyser reaches operating temperature it displays ‘analyse’
The reagent discs can be used straight from the fridge. The discs can remain in the
sealed pouch at room temperature for a cummulative period of up to 48 hours
Check for tears and punctures in unopened foil pouch, use disk within 20 minutes of
opening

Fill the sample chamber.

1. Using the Piccolo 100 μl volume pipette, firmly attach a new tip to the end of the
pipette.
2. With your index finger or thumb, push the pipette button to the stop position and
hold it down for sample pickup.
3. Immerse the tip 2–3 mm below the surface of the sample as shown below
4. Slowly release the button to pick up the sample, pause then remove the
pipette from the sample tube.
5. Make sure there are no air bubbles or air gaps in the pipette tip
6. Place the pipette tip into the discs sample chamber at 45° so that the
entire sample flows into the sample chamber, the tip should touch the
sample chamber
7. Push the plunger down with a slow, continuous motion. Take care not to
overfill the sample chamber
8. Keep the the pipette plunger pressed down until the pipette tip is removed
from the sample chamber
9. Discard the pipette tip into a sharpsafe™
10. Carry the prepared disc to the analyser. Hold the disc by it’s edges and keep
level to avoid spills
11. Place the disc in the recessed area in the drawer
12. Select close. The analyser closes the drawer
13. Select the sample type from patient or control
14. Enter an ID number for the sample
15. Then select done
16. The analyser checks the disc type and begins processing the sample
17. When the sample processing is complete the results will be printed
automatically
18. Select open to open the disc drawer
19. Remove the disc from the drawer and discard into a sharpsafe™
20. When finished select close to close the drawer and return the analyser to
standby mode
.
K) QUALITY CONTROL PROCEDURES
The control material used is Randox Abaxis chemistry controls level 1 and 2, which
are supplied lyophilised.
To reconstitute:
1. Carefully pipette 1ml diluent into the serum vial

Push the pipette button to the first stop and hold down. Release slowly to pick up
diluent. Push the pipette slowly to the second stop to dispense diluent into the
serum vial.
2. Close the serum vial and invert gently several times

3. Allow to stand for 30 minutes before use. Ensure contents are completely
dissolved by swirling gently. Avoid formation of foam. Do not shake.
STORAGE AND STABILITY

OPENED: Store refrigerated (+2ºC to +8ºC). Reconstituted serum is stable for 8
hours at +15ºC to +25ºC or 7 days at +2ºC to +8ºC and 1 month when frozen once at
-20ºC . Only the required amount of product should be removed. After use, any
residual product should NOT BE RETURNED to the original vial.
UNOPENED: Store refrigerated (+2ºC to +8ºC). Stable to expiration date printed on
individual vials.
Quality controls (level 1 and 2) are run before the instrument is used at the donor
hospital
To run a QC
1. In the home screen, select ‘analyse’ to open the disc drawer

2. Select the control type to use by using the up and down arrow keys
Using the reconstituted QC material analyse following steps 1 – 20 in running a

patient sample
Once complete check the printout to make sure that the results are within the target
range
L) INTERFERENCES (E.G. LIPAEMIA, HAEMOLYSIS, BILIRUBINEMIA, DRUGS)

AND CROSS REACTIONS
Any results that are affected by >10% interference from haemolysis, lipaemia or
icterus are suppressed and HEM, LIP or ICT respectively are printed on the printout
in place of the result.

For a complete list of interfering substances, please refer to the General Chemistry
13 package insert POCT-EXT6
M) PRINCIPLE OF PROCEDURE FOR CALCULATING RESULTS INCLUDING,

WHERE RELEVANT, THE MEASUREMENT UNCERTAINTY OF MEASURED
QUANTITY VALUES
Not applicable
N) BIOLOGICAL REFERENCE INTERVALS OR CLINICAL DECISION VALUES
Albumin 35 – 55 g/L
Alkaline Phosphatase 42 – 141 U/L
Alanine Aminotransferase 10 – 47 U/L
Aspartate Aminotransferase 11- 38 U/L
Amylase 14 – 97 U/L
Urea 2.5 – 7.9 mmol/L
Calcium 2.00 – 2.58 mmol/L
Creatinine 53 – 106 µmol/L
Gamma Glutamyltransferase 5 – 65 U/L
Glucose 4.1 – 6.6
Bilirubin 3 – 27 µmol/L
Total Protein 64 – 81 g/L
Uric Acid 2.2 – 8.0 mg/DL
O) REPORTABLE INTERVAL OF EXAMINATION RESULTS
Albumin 10 – 65 g/L
Alkaline Phosphatase 5 – 2400 U/L
Alanine Aminotransferase 5 – 2000 U/L
Aspartate Aminotransferase 5 – 2000 U/L
Amylase 5 – 4000 U/L
Urea 0.7 – 64.3 mmol/L
Calcium 1 – 4 mmol/L
Creatinine 18 – 1768 µmol/L
Gamma Glutamyltransferase 5 – 3000 U/L
Glucose 0.56 – 38.9 mmol/L
Bilirubin 1.7 – 513 µmol/L
Total Protein 20 – 140 g/L
Uric Acid 1-15 mg/dL
P) INSTRUCTIONS FOR DETERMINING QUANTITATIVE RESULTS WHEN A

RESULT IS NOT WITHIN THE MEASUREMENT INTERVAL
When a result is outwith the measurement interval, a sample must be sent to the
laboratory for confirmation

Q) ALERT/CRITICAL VALUES, WHERE APPROPRIATE
R) LABORATORY CLINICAL INTERPRETATION
Not applicable
S) POTENTIAL SOURCES OF VARIATION
Incorrect sample collection or collection into a tube with EDTA, fluoride oxalate or
citrate as an anticoagulant will interfere with test results
T) REFERENCES
Piccolo Xpress User Operator’s Manual POCT-EXT7

General Chemistry 13 Package insert POCT-EXT6
Piccolo Chemistry 13 SDS POCT-SDS4
Piccolo QC SDS POCT-SDS5


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Document POCT-SOP7 Version No.

Piccolo Xpress Chemistry Analyser Operation

The Piccolo Xpress chemistry analyser provides quantatative determinations

Authority For Issue: Jennifer Brown Page 1 of 16

Authority For Issue: Jennifer Brown Page 2 of 16

2. HAZARDS AND PRECAUTIONS.

2.1. Procedure Risk Assessment

Hazard Risk Assessment of Overall Procedural

Chemical Minor injury Possible Medium

Authority For Issue: Jennifer Brown Page 3 of 16

2.3. Biological Hazard

Biological Risk Precautions Precautions

Wear Gloves when handling

2.4. Physical Hazard

Physical Risk Precautions 1st Aid Measures Any pertinent other

2.5. Room Risk Assessment (if required)

Room Risk Precautions 1st Aid Measures Any pertinent other

Authority For Issue: Jennifer Brown Page 4 of 16

A) PURPOSE OF THE EXAMINATION

B) PRINCIPLE AND METHOD OF THE PROCEDURE USED FOR

1. Alanine Aminotransferase (ALT)

L-Alanine + α-ketoglutarate → L-Glutamate + Pyruvate

BCP + Albumin → BCP-Albumin Complex

Authority For Issue: Jennifer Brown Page 5 of 16

Bound albumin is proportional to the concentration of albumin in the sample. This

3. Alkaline Phosphatase (ALP)

p-Nitrophenyl Phosphate → p-Nitrophenol + phosphate

CNPG3 → CNP + D- Maltotrioside

The reaction is measured bichromatically at 405nm and 500nm. The change in

5. Aspartate Aminotransferase (AST)

L-aspartate + α-ketoglutarate → Oxaloacetate + L-glutamate

Oxaloacetate + NADH + H+ → Malate + NAD+

The rate of absorbance change at 340nm / 405mn caused by the conversion of

Ca + arsenazo 111 → Ca arsenazo 111 complex

Creatinine + H2O → Creatine

Authority For Issue: Jennifer Brown Page 6 of 16

Sarcosine + H2O + O2 → Glycine + Formaldehyde + H2O2

H2O2 + TBHBA + 4-AAP → Red Quinoneimine dye + H2O

8. Gamma Glutamyltransferase (GGT)

L-ˠ-glutamyl-3-carboxy-4-nitroanilide + gly-gly → glu-gly-gly + 3-carboxy-4-

The absorbance of this rate reaction is measured at 405nm. The production of 3-

Glucose + ATP → G-6-P + ADP

G-6-P + NAD+ → 6-phosphogluconate + NADH + H+

The absorbance is measured bichromatically at 340nm and 850nm. The

10. Total Bilirubin (TBIL)

Bilirubin + O2 → Biliverdin + H2O

Bilirubin is quantitated as the difference in absorbance between 467nm and

11. Total protein (TP)

Total protein + Cu11 → Cu-protein complex

The amount of total protein in the sample is directly proportional to the

12. Blood Urea Nitrogen (BUN)

Urea + H20 → 2NH3 + CO2

NH3 + α-ketoglutarate + NADH → L-Glutamate + H20 + NAD+

13. Uric Acid (UA)

Uric acid + O2 + H2O → Allantoin + CO2 + H2O2

H2O2 + 4-AAP + DHBSA → Quinoneimine dye + H2O

C) PERFORMANCE CHARACTERISTICS (SEE 5.5.1.2 AND 5.5.1.3)

Authority For Issue: Jennifer Brown Page 8 of 16

C) TYPE OF SAMPLE (E.G. PLASMA, SERUM, URINE)

F) TYPE OF CONTAINER AND ADDITIVES