Clinical Chemistry

CLINICAL CHEMISTRY
PROCEDURE MANUAL
PREPARED BY:
NFHSI MGMT
2023
TITLE: CLINICAL CHEMISTRY PROCEDURE MANUAL DOCUMENT NUMBER: 1
AUTHOR: JOANNE TERAMOTO REVIEWED BY:
LABORATORY NAME: NFHSI, INC. APPROVED BY:
DATE CREATED: 3/1/2023 EFFECTIVE DATE:
I. GLUCOSE
SUMMARY. Measurement of glucose concentration in serum or plasma is mainly used in the
diagnosis and monitoring of treatment in diabetes mellitus. Other applications are the detection
of neonatal hypoglycemia, the exclusion of pancreatic islet cell carcinoma as well as the
evaluation of carbohydrate metabolism in various diseases.
METHOD. “GOD-PAP”: enzymatic photometric test
PRINCIPLE. Determination of glucose after enzymatic oxidation by glucose oxidase. The
colorimetric indicator is quinoneimine, which is generated from 4-aminoantipyrine and phenol
by hydrogen peroxide under the catalytic action of peroxidase (Trinder’s reaction).
SPECIMEN. See below.
Serum, heparin plasma or EDTA plasma
Separate at the latest 1h after blood collection from cellular contents.
Stability in plasma after addition of a glycolytic inhibitor (Flouride, monoiodacetate, mannose):
2 days at 20-25℃
7 days at 4-8℃
1 day at -20℃
Stability in serum (separated from cellular contents, hemlysis free) without adding a glycolytic
inhibitor:
8h at 25℃
72h at 4℃
REAGENTS. See below.
Components and Concentrations
Phosphate buffer pH7.5 250mmol/L
Phenol 5 mmol/L
4-Aminoantipyrine 0.5 mmol/L
Glucose oxidase (GOD) ≥ 10 kU/L
Peroxidase (POD) ≥ 1 kU/L
Storage Instructions and Reagent Stability
Reagent is stable up to the end of the indicated month of expiry, if stored at 2-8℃, protected
from light and contamination is avoided. Don not freeze the reagents.
NOTE: It has to be mentioned that the measurement is not influenced by occasionally ocuring
color changes, as long as the absorbance of the reagent is <0.3 at 546nm.
Reagent Preparation
Reagent is ready to use.
ASSAY PROCEDURE. Application sheets for automated systems re available on request.
Wavelength 500 nm, Hg 546 nm
Optical path 1 cm
Temperature 20-25℃/37℃
Measurement Against reagent blank
Blank Sample/Calibrator
Sample/Calibrator - 10uL
Dist. Water 10uL -
Reagent 1000uL 1000uL
Mix, incubate 20 min at 20-25℃ or 10 min at 37℃. Read absorbance against the blank within 60
min.
CALCULATION. See below.
With calibrator
Glucose (mg/dL) = A Sample x Conc. Cal. (mg/dL)
A Cal.
Conversion factor
Glucose (mg/dL) x 0.05551 = Glucose (mmol/L)
CALIBRATORS AND CONTROLS. For the calibration of automated photometric systems, DiaSys
TruCal U Calibrator is recommended. The assigned values of this calibrator have been made
traceable to the reference metho gas chromatography – isotope dilution mass spectrometry
(GC-IDMS). Glucose Standard FS may be used alternatively for calibration. For internal quality
control, DiaSys Trulab N and P controls should be assayed. Each laboratory should establish
corrective action in case of deviations in control recovery.
PERFORMANCE CHARACTERISTICS. See below.
Measuring range
The test have been developed to determine glucose concentrations within a measuring range
from 1-400 mg/dL (0.06-22.2 mmol/L). When values exceed this range samples should be
diluted 1 + 4 with NaCl solution (9g/L) and the result multiplied by 5.
Specificity/Interferences
No interference was observed by ascorbic acid up to 15 mg/dL, bilirubin up to 40 mg/dL,
hemoglobin up to 200 mg/dL and lipemia up to 2000 mg/dL triglycerides.
Sensitivity/Limit of Detection
The lower limit of detection is 1 mg/dL.
WARNINGS AND PRECAUTIONS. The reagent contains sodium azide (0.95 g/L) as preservative.
Do not swallow. Avoid contact with skin and mucous membranes. Please refer to the safety data
sheets and take necessary purposes, the results should always be assessed with the patients
medical history, clinical examinations and other findings.
REFERENCE RANGE AND CLINICAL INTERPRETATION. See below.
Adult : FBS 70-115 mg/dL
RBS
Elevated plasma glucose levels are expected in a variety of clinical conditions especially diabetes
mellitus, Cushing’s syndrome and hyperadrenalism. Decreased plasma glucose levels are
observed in hyper-insulinism, anti-diabetic treatment and hypoadrenalism.
II. UREA
SUMMARY. Urea contributes most of the body’s non-protein nitrogen, accounting 45% of the
total. Urea is the nitrogen-containing end product of protein catabolism. It is synthesized in the
liver, released into blood circulation and excreted by the kidneys. States associated with
elevated levels of urea in blood are referred to as hyperuremia or azotemia. Parallel
determination of urea and creatinine is performed to differentiate between pre-renal and post-
renal azotemia. Furthermore, measurement of urea in blood is a useful indicator of renal and
hepatic integrity.
Pre-renal azotemia, caused by e.g. dehydration, increased protein catabolism, cortisol
treatment or decreased renal perfusion, leads to increased urea levels, while creatinine values
remain within the reference range. In post-renal azotemias, caused by the obstruction of the
urinary tract, both urea and creatinine levels rise, but creatinine in a smaller extent. In renal
diseases urea concentrations are elevated when the glomerular filtration rate is markedly
reduced and when the protein intake is higher than 200g/day.
METHOD. “Urease-GLDH”: enzymatic UV test
PRINCIPLE. Urea reacts directly with diacetyl monoxime under strong acidic conditions to give a
yellow condensation product. The reaction is intensified by the presence of ferric ions and
thiosemicarbazide. The intense red colour formed is measured at 540nm/ yellow green filter.
Serum, plasma (no ammonium heparin), fresh urine
Dilute urine 1 + 0 with dist. water and multiply results by 51.
TruLab Urine controls must be prediluted the same way as patient samples.
Stability in serum or plasma:
7 days at 20-25℃
7 days at 4-8℃
1 year at -20℃
in urine:
2 days at 20-25℃
7 days at 4-8℃
1 month at -20℃
Freeze only once. Discard contaminated specimens.
R1: TRIS pH7.8 150 mmol/L
2-Oxoglutarate 9 mmol/L
ADP 0.75 mmol/L
Urease ≥7 kU/L
GLDH (bovine) ≥1 kU/L
R2: NADH 1.3 mmol/L
Reagents are stable up to the end of the indicated month of expiry, if stored at 2-8℃, protected
from light and contamination is avoided. Do not freeze the reagents.
Reagent Preparation
Reagents are ready to use.
ASSAY PROCEDURE. Application sheets for automated systems are available on request.
Wavelength 340 nm, Hg 334 nm, Hg 365 nm
Optical path 1 cm
Temperature 25℃/30℃/37℃
Measurement Against reagent blank; 2-point kinetic
Reagent 1 1000uL 1000uL
Mix, incubate 0-5 min., then
add:
Mix, incubate for approx. 60
sec at 25℃/30℃ r approx.
30-40 sec at 37℃, then read
absorbance A1. Read
absorbance A2 exactly after
another 60 seconds.
△A=(A2-A1) Sample/Calibrator
NOTES:
1. The method is optimized for 2-point kinetic measurement. It is recommended to perform the
method only on mechanized equipment because it is difficult to incubate all samples ad the
reagent blank strictly for the same time intervals. The assay scheme may be used for adaptation
purposes or instruments with no specific adaptation sheet. The volumes may be proportionally
smaller.
2. The statement “approx. 60 sec or approx. 30-40 sec” means that the time period chosen does
not need to be exactly 60 resp. 30-40 sec. A time once chosen has to be respected exactly for all
samples, standards and the reagent blanc.
With calibrator
Urea (mg/dL) = ∆A Sample x Conc. Cal. (mg/dL)
∆A Cal.
Conversion factor
Urea (mg/dL) x 0.1665 = Urea (mmol/L)
Urea (mg/dL) x 0.467 = BUN (mg/dL)
BUN (mg/dL) x 2.14 = Urea (mg/dL)
TruCal U calibrator is recommended. The assigned values of the calibrators have been made
traceable to NIST SRM-909 Level 1. Urea standard FS may be used alternatively for calibration.
Diasys Trulab N, P and Trulab Urine controls should be assayed for internal quality control. Each
laboratory should establish corrective action in case of deviations in control recovery.
Measuring range
The test has been developed to determine urea concentrations within a measuring range from
2-300 mg/dL (0.3-50 mmol/L) in serum/plasma respectively up to 30 g/dL (5mol/L) in urine.
When values exceed this range, the samples should be diluted 1+ 2 with NaCl solution (9g/L)
and the result multiplied by 3.
hemoglobin up to 500 g/dL and lipemia up to 2000 mg/dL triglycerides. Ammonium ions
interfere; therefore, do not use ammonium heparin as anticoagulant for collection of plasma.
Sensitivity/Detection Limit
The lower limit of detection is 2 mg/dL.
WARNING AND PRECAUTIONS. The reagents contain sodium azide (0.95 g/L) as preservative.
Do not swallow. Avoid contact with skin and mucous membranes. Reagent 1 contains animal
material. Handle the product as potentially infectious according to universal precautions and
good laboratory practices. Please refer to the safety data sheets and take necessary purposes,
the results should always be assessed with the patients medical history, clinical examinations
and other findings.
Adult : BUN serum/plasma
Women <50 7.94 – 20.1 mg/dL
Women >50 7.01- 18.7 mg/dL
Men <50 8.87-20.5 mg/dL
Men>50 8.41-25.7mg/dL
Urea/Creatinine ratio in serum:
25-40 (mmol/L)
20-35 (mg/dL)
Elevated serum urea levels may be due to pre-renal, renal or post-renal etiology. Pre-renal
causes could be cardiac related or due to increased protein catabolism, and dehydration. Renal
causes include glomerulonephritis, chronic nephritis, nephrotic syndrome and other kidney
disease. Post-renal causes include obstruction of the urinary tract.
Decreased serum urea levels could be due to pregnancy, intravenous infusion, low antidiuretic
hormone secretion hormone secretion, low protein intake, severe liver disease, inborn errors of
urea cycle and SIADH (Syndrome of inappropriate ADH secretion).
III. CREATININE
SUMMARY. Creatinine is a waste product excreted by the kidneys mainly by glomerular
filtration. The concentration of creatinine in plasma of a healthy individual is fairly constant,
independent from water intake, exercise and rate f urine production. Therefore increased
plasma creatinine values always indicate decreased excretion. i.e. impaired kidney function.
Creatinine clearance is a good indicator for the glomerular filtration rate (GFR) which allows
better detection of kidney diseases and monitoring of renal function. For this purpose,
creatinine is measured simultaneously in serum and urine collected over a defined time period.
METHOD. Kinetic test without depolarization according to the Jaffe method
PRINCIPLE. Creatinine forms a colored orange-red complex in an alkaline picrate solution. The
difference at fixed times during conversion is proportional to the concentration of creatinine in
the sample.
Human serum, heparin plasma or urine.
Only use suitable tubes or collection containers for specimen collection and preparation.
When using primary tubes, follow the manufacturer’s instructions.
Stability in serum/plasma:
7 days at 4-25℃
3 months at -20℃
Stability in urine:
2 days at 20-25℃
6 days at 4-8℃
6 months at -20℃
Dilute urine 1 + 49 with dist. water, multiply the result by 50. TruLab Urine controls must be
prediluted the same way as patient samples.
Only freeze once. Discard contaminated specimens.
R1: Sodium hydroxide 0.2 mol/L
R2: Picric acid 20 mmol/L
Storage and Stability
Reagents are stable up to the date of expiry indicated on the kit, if stored at 2-25℃ and
contamination is avoided. Do not freeze and protect from light.
The in-use stability of the reagent is 18 months.
Reagent Preparation
The reagents are ready to use.
ASSAY PROCEDURE. See below.
Basic settings for BioMajesty
Wavelength 505/571 nm
Temperature 37℃
Measurement Kinetic
Sample/Calibrator 5.0uL
Reagent 1 80uL
Reagent 2 20uL
Addition reagent 2 Cycle19 (286 s)
Absorbance Cycle 24/32 (354 s/464 s)
Calibration Linear
With calibrator
Serum/Plasma
Creatinine [mg/dL] = ∆A Sample x Conc. Cal. [mg/dL]
∆A Cal.
Urine
Creatinine [mg/dL] = ∆A Sample x Conc. Cal. [mg/dL] x 50
∆A Cal.
Creatinine Clearance [mL/min/1.73 m2] [5]
= mg Creatinine/ 100 mL Urine x mL Urine
mg Creatinine/ 100 mL Serum x min Urine collection time
The calculated creatinine clearance refers to the average body surface of an adult (1.73 m 2).
Conversion Factor
Creatinine [mg/dL] x 88.4 = Creatinine [umol/L]
Creatinine [mg/dL] x 0.0884 = Creatinine [mmol/L]
CALIBRATORS AND CONTROLS. DiaSys TruCal U is recommended for calibration. Calibrator
values for the compensated method have been made traceable to the NIST Standard Reference
Material SRM 967 using level 1 and 2 and, therefore, to GC-IDMS. Creatinine Standard FS may
be used alternatively for calibration. Use DiaSys TruLab N and P or TruLab Urine Level 1 and 2
controls for internal quality control. Quality control must be performed after calibration. Control
intervals and limits have to be adapted to the individual requirements of each laboratory.
Results must be within the defined ranges. Follow the relevant legal requirements and
guidelines. Each laboratory should establish corrective action in case of deviations in control
recovery.
Data evaluated on BioMajesty
Measuring range up to 14mg/dL
When values exceed this range samples should be diluted 1 + 1 with NaCl solution (9g/L) and
the result multiplied by 2.
Interfering substance Interferences ≤ 10% up to

Ascorbic acid 30 mg/dL
Bilirubin conjugated 3 mg/dL
Bilirubin unconjugated 1.5 mg/dL
Hemoglobin 600 mg/dL
Lipemia 1800 mg/dL
WARNING AND PRECAUTIONS. See below.

1. Components contained in Creatinine FS are classified according to EC regulation 1272/2008
(CLP) as follows:
Reagent 1: Warning. H290 May be corrosive to metals. H315 Causes skin irritation. H319 Causes
serious eye irritation. P234 Keep only in original packaging. P264 Wash hands and face
thoroughly after handling. P280 Wear protective gloves/protective clothing/eye protection. If
on skin: Wash with plenty of water/soap. If in eyes: Rinse cautiously with water for several
minutes. Remove contact lenses, if present and easy to do. Continue rinsing. If skin irritation
occurs: Get medical advice/attention. P390 Absorb spillage to prevent material damage.
Reagent 2: Warning. H290 May be corrosive to metals. P234 Keep only in original packaging.
P280 Wear protective gloves/protective clothing/eye protection. P390 Absorb spillage to
prevent material damage.
2. High homogentisic acid concentrations in urine samples lead to false results.
3. In very rare cases, samples of patients with gammopathy might give falsified results.
4. Eltrombopag medication leads to falsely low or high results in patient samples/
5. In case of product malfunction or altered appearance that could affect the performance,
contact the manufacturer.
6. Any serious incident related to the product must be reported to the manufacturer and the
competent authority of the Member State where the user and/or patient is located.
7. Please refer to MSDS and take the necessary precautions for the use of laboratory reagents.
8. For professional use only.
Serum Creatinine: Male 0.9 – 1.3 mg/dL
Female 0.8 – 1.1 g/dL
Serum creatinine concentration is related to muscle mass and the values are lower in children.
Increased serum creatinine is associated with decrease in glomerular filtration rate (GFR),
whether the cause is pre-renal, renal or post-renal. Pre-renal factors include conditions such as
congestive heart failure, shock, diarrhea, uncontrolled diabetes mellitus, se of diuretics, etc.
Renal factors involve mainly damage to the glomeruli. Post-renal factors may be prostatic
hypertrophy, calculi blocking the ureters or neoplasms compressing the ureters. The serum
creatinine concentration is monitored closely after a renal transplantation because a rising
concentration, even though small, may be an indication of graft rejection.
IV. CHOLESTEROL
SUMMARY. Cholesterol is a component of cell membranes and a precursor for steroid
hormones and bile acids synthesized by body cells and absorbed with food. Cholesterol is
transported in plasma via lipoproteins, namely complexes between lipids and apolipoproteins.
There are four classes of lipoproteins: HDL, LDL, VLDL and chylomicrons. While LDL is involved in
the cholesterol transport to the peripheral cells, HDL is responsible for the cholesterol uptake
from cells. The four different lipoprotein classes show distinct relationship to coronary
atherosclerosis. LDL-C contributes to atherosclerotic plaque formation within the arterial intima
and is strongly associated with coronary heart disease (CHD) and related mortality. Even with
total cholesterol within the normal range and increased concentration of LDL-C indicates high
risk. HDL-C has a protective effect impeding plaque formation and shows an inverse relationship
to CHD prevalence. In fact, low HDL-C values constitute an independent risk factor. The
determination of the individual total cholesterol level is used for screening purposes while for a
better risk assessment it is necessary to measure additionally HDL-C and LDL-C.
In the past few years, several controlled clinical trials using diet, life style changes and/or
different drugs have demonstrated that lowering total cholesterol and LDL-C levels reduce
drastically CHD risk.
METHOD. “CHOD-PAP”: enzymatic photometric test
PRINCIPLE. Determination f cholesterol after enzymatic hydrolysis and oxidation. The
colorimetric indicator is quinoneimine which is generated from 4-aminoantipyrine and phenol
by hydrogen peroxide under the catalytic action of peroxidase (Trinder’s reaction).
Serum, heparin plasma or EDTA plasma
Stability:
7 days at 20-25℃
7 days at 4-8℃
3 months at -20℃
Discard contaminated specimens. Freeze only once.
Reagent
Good’s buffer pH6.7 50 mmol/L
Phenol 5 mmol/L
4-Aminoantipyrine 0.3 mmol/L
Cholesterol esterase (CHE) ≥200 U/L
Cholesterol oxidase (CHO) ≥50 U/L
Peroxidase (POD) ≥3 kU/L
Reagent is stable up to the end of the indicated month of expiry. If stored at 2-8℃, protected
from light and contamination is avoided. Do not freeze the reagents.
Note: It has to be mentioned that the measurement is not influenced by occasionally occurring
changes, as long as the absorbance of the reagent is <0.3 at 546 nm.
Reagent Preparation
The reagent is ready to use.
ASSAY PROCEDURE. Application sheets for automated systems are available on request.
Wavelength 500 nm, Hg 546 nm
Optical path 1 cm
Temperature 20-25℃
Dist. water 10uL -
Mix, incubate for 20 min. at 20-25℃ or for 10min. at 37℃. Read absorbance within 660 min
against reagent blank.
With calibrator
Cholesterol [mg/dL] = A Sample x Conc. Cal. [mg/dL]
A Cal.
Conversion factor
Cholesterol [mg/dL] x 0.02586 = Cholesterol [mmol/L]
TruCal U calibrator is recommended. The assigned values of the calibrators have been made
traceable to the reference method GC-IDMS. Cholesterol Standard FS may be used alternatively
for calibration. For internal quality control, DiaSys TruLab N and P or TruLab L controls should be
assayed. Each laboratory should establish corrective action in case of deviations in control
recovery.
Measuring range
The test has been developed to determine cholesterol concentrations within a measuring range
from 3-750 mg/dL (0.08-19.4 mmol/L). When values exceed this range, the samples should be
diluted 1+ 4 with NaCl solution (9g/L) and the result multiplied by 5.
hemoglobin up to 200 g/dL and lipemia up to 2000 mg/dL triglycerides.
Sensitivity/Detection Limit
The lower limit of detection is 3 mg/dL (0.08 mmol/L).
WARNING AND PRECAUTIONS. The reagents contain sodium azide (0.95 g/L) as preservative.
Do not swallow. Avoid contact with skin and mucous membranes. Reagent 1 contains animal
material. Handle the product as potentially infectious according to universal precautions and
good laboratory practices. Please refer to the safety data sheets and take necessary purposes,
the results should always be assessed with the patients medical history, clinical examinations
and other findings.
Desirable ≤200 mg/dL
Borderline high risk 200-240 mg/dL
High risk >240 mg/dL
Serum cholesterol is increased in hypothyroidism, diabetes mellitus, nephrotic syndrome and in
various hyperlipidemias especially those causing xanthomatosis. Elevated serum cholesterol is a
serious risk factor for the development of coronary artery disease. Decreased serum cholesterol
is seen in severe hepatocellular hyperthyroidism and anemia.
The European Task Force on Coronary Prevention recommends to lower TC concentration to less
than 190 mg/dL and LDL-C to less than 115 mg/dL.
V. TOTAL PROTEIN
SUMMARY. Measurement of total protein is a useful test in a variety of disorders. Decreased
total protein concentrations can be detected in defective protein synthesis in the liver, protein
loss due to impaired kidney function, intestinal malabsorption or nutritional deficiency. Elevated
protein levels occur in chronic inflammatory disorders, liver cirrhosis and dehydration.
METHOD. Photometric test according to biuret method
PRINCIPLE. Proteins form a violet blue color complex with copper ions in alkaline solution. The
absorbance of the color is directly proportional to the concentration.
Human serum or heparin plasma
Only use suitable tubes or collection containers for specimen collection and preparation. When
using primary tubes, follow the manufacturer’s instructions.
Stability:
6 days at 20-25℃
4 weeks at 4-8℃
At least one year at -20℃
R1: Sodium hydroxide 100 mmol/L
Potassium sodium tartrate 17 mmol/L
R2: Sodium hydroxide 500 mmol/L
Potassium sodium tartrate 80 mmol/L
Potassium iodide 75 mmol/L
Copper sulphate 30 mmol/L
Reagents are stable up to date of expiry indicated on the kit, if stored at 2-25℃ and
contamination is avoided. Protect from light. The in-use stability of the reagent is 18 months.
Reagent Preparation
Wavelength 545 nm
Temperature 37℃
Measurement Endpoint
Reagent 1 80uL
Reagent 2 20 uL
Addition Reagent 2 Cycle 19 (286 s)
Absorbance 1 Cycle 17/18 (231 s/244 s)
Calibration Linear
With calibrator
Total Protein [g/dL] = A Sample x Conc. Cal. [g/dL]
A Cal.
CALIBRATORS AND CONTROLS. DiaSys TruCal U calibrator is recommended. The assigned values
of the calibrators have been made traceable to the biuret method. Total Protein Standard FS
may be used alternatively for calibration. Use DiaSys TruLab N and P for internal quality control.
Quality control must be performed after calibration. Control intervals and limits have to be
adapted to the individual requirements of each laboratory. Results must be within the defined
ranges. Each laboratory should establish corrective action in case of deviations in control
recovery.
Exemplary data mentioned below may slightly differ in case of deviating measurement
conditions.
Measuring range up to 14 g/dL.
When values exceed this range, samples should be diluted 1 + 1 with NaCl solution (9 g/L) and
Limit detection 0.05g/dL
Interfering substances Interferences ≤ 10% up to

Bilirubin (conjugated and unconjugated) 60 mg/dL
Lipemia 1000 mg/dL

1. Components contained in Total Protein FS are classified according to EC regulation 1272/2008
(CLP) as follows:
Reagent 1: Warning. H290 May be corrosive to metals. P234 Keep only in original packaging.
P390 Absorb spillage to prevent material damage.
Reagent 2: Warning. Contains Potassium iodide. H315 Causes skin irritation. H319 Causes
serious eye irritation. H373 May cause damage to organs through prolonged or repeated
exposure. H412 Harmful to aquatic life with long lasting effects. P234 Keep only in original
packaging. P273 Avoid release to the environment. P280 Wear protective gloves/protective
clothing/eye protection. If in the eyes: Rinse cautiously with water for several minutes. Remove
contact lenses, if present and easy to do. Continue rinsing. P314 Get medical advice/attention if
you feel unwell.
2. In serum or plasma of patients who have received large intravenous amount of polydextrans,
too high values can be measured with the biuret method. In such cases an alternative method
has to be used.
Adults 6.6 – 8.8 g/dL
Children Female Male
1-30 day (s) 4.2 – 6.2 4.1 – 6.3
1-6 month (s) 4.4 – 6.6 4.7 – 6.7
6 months-1 year 5.6 – 7.9 5.5 – 7.0
1-18 year(s) 5.7 – 8.0 5.7 – 8.0
In addition to dehydration and diarrhea, increased serum-total protein levels are observed n
multiple myeloma. Hypoproteinemia is generally seen in conditions associated with
hypoalbuminemia.
VI. ALBUMIN
SUMMARY. Albumin is an important binding and transport protein for various substances in
plasma and the main contributor to the plasma osmotic pressure. Measurement of albumin in
serum is used for diagnosis and monitoring of liver diseases, e.g. liver cirrhosis. Furthermore,
albumin levels indicate the health and nutritional status of an individual and, therefore are used
for detecting malnutrition and for prognosis in elderly hospitalized patients.
METHOD. Photometric test using bromocresol green
PRINCIPLE. In the presence of bromocresol green at a slightly acid pH, serum albumin produces
a color change of the indicator from yellow-green to green-blue.
Human serum or heparin plasma
Only use suitable tubes or collection containers for specimen collection and preparation. When
using primary tubes, follow the manufacturer’s instructions.
Stability:
10 weeks at 20-25℃
5 months at 4-8℃
3 months at -20℃
Citrate buffer pH4.2 30 mmol/L
Bromocresol green 0.26 mmol/L
Reagents are stable up to date of expiry indicated on the kit, if stored at 2-25℃ and
contamination is avoided. Do not freeze and protect from light. The in-use stability of the
reagent is 18 months.
Reagent Preparation
Temperature 37℃
Measurement Endpoint
Reagent 1 90uL
Addition Reagent Cycle 19 (286 s)
Absorbance Cycle 7/9 (95 s/122 s)
Calibration Linear
With calibrator
Albumin [g/dL] = A Sample x Conc. Cal. [g/dL]
A Cal.
Conversion Factor
Albumin [g/dL] x 144.9 = Albumin [umol/L]
CALIBRATORS AND CONTROLS. DiaSys TruCal U calibrator is recommended. Calibrator values
have been made traceable to the ERM-DA470. Albumin Standard FS may be used alternatively
for calibration. Use DiaSys TruLab N and P for internal quality control. Quality control must be
performed after calibration. Control intervals and limits have to be adapted to the individual
requirements of each laboratory. Results must be within the defined ranges. Each laboratory
should establish corrective action in case of deviations in control recovery.
Exemplary data mentioned below may slightly differ in case of deviating measurement
conditions.
Measuring range up to 6 g/dL.
When values exceed this range, samples should be diluted 1 + 1 with NaCl solution (9 g/L) and
Limit detection 0.1 g/dL
Interfering substances Interferences ≤ 10% up to

Bilirubin (conjugated and unconjugated) 60 mg/dL
Lipemia 1200 mg/dL

Adults 3.5 – 5.2 g/dL 507 – 756 umol/L
Serum levels of albumin are used to assess nutritional status and have important influences on
the metabolism of endogenous substances such as calcium, bilirubin and fatty acids and on the
effect of drugs and hormones. Hyperalbuminemia has little diagnostic significance except in
dehydration. Hypoalbuminemia is very common in many illnesses like impaired synthesis (liver
disease), increased catabolism, reduced amino acid absorption, protein loss in urine,
malnutrition and protein losing enteropathy, and in hospital patients with acute illness.
VII. ALKALINE PHOSPHATASE
SUMMARY. Alkaline phosphatase, a hydrolytic enzyme acting optimally at alkaline pH, exists in
blood in numerous distinct forms which originate mainly from bone and liver, but also from
other tissues as kidney, placenta, testes, thymus, lung and tumors. Physiological increases are
found during bone growth in childhood and in pregnancy, while pathological increases are
largely associated with hepatobiliary and bone disease. In hepatobiliary disease, they indicate
obstruction in bile ducts as in cholestatis caused by gallstones, tumors, or inflammation.
Elevated activities are also observed in infectious hepatitis. In bone diseases elevated AP
activities originate from increased osteoblastic activity as in Paget’s disease, osteomalacia
(rickets), bone metastases and hyperparathyroidism.
METHOD. Kinetic photometric test according to IFCC
PRINCIPLE. Paranitrophenyl phosphate, which is colorless is hydrolysed by alkaline phosphatase
at pH 10.5 and 37℃ to form free paranitrophenol which is colored yellow. The addition of NaOH
stops enzyme activity and the final color shows maximum absorbance at 410 nm.

Serum or heparin plasma
Do not use hemolytic samples.
Stability:
7 days at 20-25℃
7 days at 4-8℃
2 months at -20℃
R1: 2-Amino-2-methyl-1-propanol pH10.4 1.1 mol/L
Magnesium acetate 2 mmol/L
Zinc zulphate 0.5 mmol/L
HEDTA 2.5 mmol/L
R2: p-Nitrophenylphosphate 80 mmol/L
Reagent Preparation
Substrate Start
Sample Start
Mix 4 parts of R1 + 1 part of R2
Stability: 4 weeks at 2-c
5 days at 15-25℃
The mono reagent must be protected from light.
ASSAY PROCEDURE. Applications for automated systems are available on request.
Wavelength Hg 405nm, 400-420 nm
Optical path 1cm
Temperature 37℃
Substrate Start
Sample/calibrator - 20uL
Dist. Water 20uL -
Mix incubate for approx. 1
min then add:
Mix, read absorbance after 1
min and start stopwatch.
Read absorbance again after
1,2 and 3 min.
Sample Start
Dist. Water 20uL -
Mono reagent 1000uL 1000uL
Mix, read absorbance after 1
min and start stopwatch.
Read absorbance again after
1,2, and 3 min.

With factor
From absorbance readings calculate ∆ A/min and multiply by the corresponding factor from
table below:
∆ A/min x factor = AP activity [U/L]
Substrate start 405 nm 3433
Sample start 405 nm 2757
With calibrator
AP [U/L] = ∆A/min Sample x Conc. Cal. [U/L]
∆A Cal. Calibrator
Conversion Factor
AP [U/L] x 0.0167 = AP [ukat/L]
CALIBRATORS AND CONTROLS. DiaSys TruCal U is recommended for calibration. This method is
traceable to the molar extinction coefficient. Use TruLab N and P for internal quality control.
Each laboratory should establish corrective action in case of deviations in control recovery.
Measuring range up to 1400U/L.
In case of a manual procedure, the test is suitable for AP activities which correspond to a
maximum of ∆A/min of 0.25. If such values are exceeded the samples should be diluted 1 + 9
with NaCl solution (9g/L) and results multiplied by 10.
Limit detection 0.6 U/L
Onboard stability 6 days
Calibration stability 6 days
Interfering substance Interferences ≤ 10% up to

Bilirubin conjugated 60 mg/dL
Bilirubin unconjugated 36 mg/dL
Lipemia 2000 mg/dL

1. The reagents contain sodium azide (0.95 g/L) as preservative. Do not swallow. Avoid contact
with skin and mucous membranes.
2. During the reaction, p-nitrophenol is produced which is poisonous when inhaled, swallowed
or absorbed through skin. If the reaction mixture comes in contact with skin or mucous
membranes wash copiously with water.
Adults:
Women 35-104 U/L
Men 40-129 U/L
Levels up to 3 times may be normal in children.
Liver, bone and placenta contain very high concentrations of ALP. Therefore, increase in ALP
activity is usually related to hepatobiliary and bone disorders. Increased ALP levels are observed
in liver diseases, osteomalacia, rickets and bone disorders. Moderate elevations are sometimes
noted in congestive heart failure, intestinal disease and intra-abdominal bacterial infections.
VIII. ALAT (GPT)
SUMMARY. Alanine aminotransferase, formerly called Glutamic Pyruvic Transaminase (GPT). As
a liver specific enzyme, ALAT is only significantly elevated in hepatobiliary diseases.
METHOD. Optimized UV-test according to IFCC [modified]
PRINCIPLE. Transamination is the process in which an amino group is transferred from amino
acid to a a-keto acid. The enzymes responsible for transamination are called transaminases. The
substrated in the reaction are a-ketoglutaric acid (a KG) plus L-aspartate for AST, and aKG plus L-
alanine for ALT. The products formed by enzyme action are glutamate and oxaloacetate for AST
and glutamate and pyruvate for ALT. Addition of 2,4, dinitrophenyl hydrazine results in the
formation of hydrazone complex with the ketoacids. A red color is produced on the addition of
sodium hydroxide. The intensity of color is related to enzymatic activity.
Human serum, heparin plasma.
Stability:
3 days at 20-25℃
7 days at 4-8℃
7 days at -20℃
R1: TRIS pH7.15 0.2 mol/L
L-alanine 700 mmol/L
LDH ≥2300 U/L
R2: 2-Oxoglutarate 1 mmol/L
NADH
Pyridoxal-5-Phosphate FS
Good’s buffer pH9.6 100 mmol/L
Pyridoxal-5-phosphate 13 mmol/L
Reagent Preparation
For determination with P-5-P mix 1 part of P-5-P with 100 parts of reagent 1.
Stability after mixing:
6 days at 2-8℃
24 hours at 15-25℃
Basic settings for BioMajesty
Temperature 37℃
Measurement Kinetic
Reagent 1 80uL
Reagent 2 20uL
Addition reagent 2 Cycle19 (286 s)
Absorbance 1 -
Calibration Linear
With calibrator
ALAT [U/L] = ∆A Sample x Conc. Cal. [mg/dL]
∆A Cal.
Conversion Factor
ALAT [U/L] x 0.0167 = ALAT [ukat/L]
CALIBRATORS AND CONTROLS. DiaSys TruCal U is recommended for calibration. This method
has been standardized against the original IFCC formulation. Use TruLab N and P for internal
quality control. Each laboratory should establish corrective action in case of deviations in
control recovery.
With P-5-P
Measuring range up to 1000U/L.
When values exceed this range samples should be diluted 1 + 9 with NaCl solution (9g/L) and
Limit detection 4U/L
Interfering substance Interferences ≤ 10% up to Analyte Concentration

[U/L]
Ascorbic acid 30 mg/dL 36.0
60 mg/dL 110
Bilirubin conjugated 54 mg/dL 36.0
60 mg/dL 120
Bilirubin unconjugated 54 mg/dL 36.0
60 mg/dL 106
Hemoglobin 500 mg/dL 36.0
500 mg/dL 118
Lipemia 400 mg/dL 36.0
900 mg/dL 99.2

1. The reagents contain sodium azide (0.95 g/L) as preservative. Do not swallow. Avoid contact
2. Reagent 1 contains animal and biological material. Handle the product as potentially
infectious according to universal precautions and good clinical laboratory practice.
3. Reagent 2 contains biological material. Handle the product as potentially infectious according
to universal precautions and good clinical laboratory practice.
4. Sulfasalazine and sulfapyridine medication may cause false results in patient samples. Blood
collection must be performed prior to drug administration.
With P-5-P:
Women <34
Men <45
Children 1-30 day(s) <25
2-12 months <35
1-3 years <30
4-6 years <25
7-9 years <25
10-18 years <30
ALT is distributed mainly in the liver and to a lesser extent in the kidney and muscles. Increased
ALT activity s observed in hepatitis and cirrhosis. Values may be increased to >10 times – 100
times ULN in hepatitis.
IX. CALCIUM
SUMMARY. Calcium plays an essential role in many cell functions: intracellulary in muscle
contraction and glycogen metabolism; extracellulary, in bone mineralization, blood coagulation
and in transmission of nerve impulses. Calcium is present in plasma in three forms: free, bount
to proteins or complexed with anions as phosphate, citrate and bicarbonate. Decreased total
calcium levels can be associated with diseases of bone apparatus (especially osteoporosis),
kidney diseases (especially under dialysis), defective intestinal absorption and
hypoparathyroidism, malignant diseases with metastases and sarcoidosis. Calcium
measurements also help in monitoring calcium supplementation mainly in prevention of
osteoporosis.
METHOD. Photometric test using arsenazo III
PRINCIPLE. Calcium with arsenazo III at neutral pH yields a blue colored complex whose
intensity is proportional to the calcium concentration. Interference by magnesium is eliminated
by addition of 8-hydroxyquinoline-5-sulfonic acid.
Serum, heparin plasma or urine
Do not use EDTA plasma.
Stability:
In serum/plasma
7 days at 20-25℃
3 weeks at c
8 months at -20℃
In urine
2 days at 20-25℃
4 days at 4-8℃
3 weeks at -20℃
Add 10 mL of concentrated HCl to 24 h urine and heat the specimen to dissolve calcium
oxalate.
Reagent:
Phosphate buffer pH7.5 50mmol/L
8-Hydroxyquinoline-5-sulfonic acid 5mmol/L
Arsenazo III 120umol/L
Reagent Preparation
Wavelength 650nm, Hg 623nm (630-670nm)
Optical path 1cm
Dist. Water 10uL -
Mix incubate for 5 min and read absorbance against reagent blank.

With calibrator
Calcium [mg/dL] = A Sample x Conc. Cal. [mg/dL]
A Cal.
Conversion Factor
Calcium [mg/dL] x 0.2495 = Calcium [mmol/L]
Calcium/U [mg/24 h] x 0.025 = Calcium/U [mmol/24h]
CALIBRATORS AND CONTROLS. For calibration of automated photometric systems, the DiaSys
TruCal U calibrator is recommended. This method has been standardized against the reference
method Atomic Absorption Spectrometry (AAS). Calcium Standard FS may be used alternatively
for calibration. Each laboratory should establish corrective action in case of deviations in control
recovery.
Measuring range
The test has been developed to determine calcium concentrations within a measuring range
from 0.04-20 mg/dL. When values exceed this range, samples should be diluted 1 + 1 with NaCl
solution (9g/L) and the result multiplied by 2.
hemoglobin up to 500 mg/dL, lipemia up to 2000 mg/dL, triglycerides and magnesium up to 15
mg/dL. Strontium salts in medicine may lead to strongly increased calcium values.
Sensitivity/Limit Detection
The lower limit of detection is 0.04 mg/dL.
1. As calcium is an ubiquitous ion, essential precaution must be taken against accidental
contamination. Only use disposable materials.
2. Traces of chelating agent such as EDTA can prevent the formation of the colored complex.
3. The reagent contains sodium azide (0.95g/L) as preservative. Do not swallow. Avoid contact
Serum/plasma 8.6-10.3 mg/dL
Calcium measurements are used in the diagnosis and treatment of parathyroid diseases, a
variety of bone diseases, chronic renal failure and tetany. Increased serum calcium levels are
associated with primary hyperparathyroidism, multiple myeloma, metastatic bone lesions and
hypervitaminosis D. Hypocalcemia is associated with hypoparathyroidism, nephrotic syndrome
and rickets and renal failure.
X. PHOSPHORUS
SUMMARY. Phosphorus exists in the body almost exclusively as phosphate, mainly an inorganic
substance of the bones, but also in cells, phospholipids and nucleic acids as well as in adenosine
triphosphate, which is involved in the energy transfer. In plasma, it is present as calcium
phosphate; therefore the level of plasma phosphorus is strongly associated with that of calcium
levels. Measurement of phosphorus in serum and urine is mainly performed to detect disorders
of kidneys, bones and parathyroid glands. Increased concentrations are found in renal failure,
hypoparathyroidism, pseudo-hyperparathyroidism and loss of calcium phosphate of bones and
cells. Decreased values occur in malabsorption, hyperparathyroidism and vitamin D deficiency.
METHOD. Photometric UV test with endpoint determination
PRINCIPLE. Phosphorus in serum reacts with ammonium molybdate to form
phosphomolybdate, which is then reduced by stannous chloride and hydrazine sulphate to
molybdenum blue. The intensity of the color is measured at 640nm.
Serum, heparin plasma or urine
Stability:
In serum/plasma
1 day at 20-25℃
4 days at 4-8℃
1 year at -20℃
In urine
2 days at 20-25℃ at pH<5
Discard contaminated specimens.
For collection of 24 h urine, add 10 mL of 0g/dL HCl into the collection bottle to avoid
phosphate precipitations Dilute urine 1 + 10 with dist. water before determination and multiply
the result by 11.
R1: Glycine/sulphuric acid buffer 50mmol/L
R2: Glycine buffer 50mmol/L
Ammonium molybdate 1.75mmol/L
Reagent Preparation
Wavelength 340nm, Hg 334nm, Hg 365nm
660nm bichromatic
Optical path 1cm
Dist. Water 10uL -
Mix, incubate 5 min, read
absorbance A1, then add:
Mix and read absorbance A2
within 5-60 min
∆A=(A2-A1) Sample/Calibrator
With calibrator
Phosphorus [mg/dL] = A Sample x Conc. Cal. [mg/dL]
A Cal.
Conversion Factor
Phosphorus [mg/dL] x 0.3229 = Phosphorus [mmol/L]
Phosphorus [mg/dL] x 3.06619 = Phosphorus [mg/dL]
CALIBRATORS AND CONTROLS. For calibration of automated photometric systems, the DiaSys
TruCal U calibrator is recommended. The assigned values of calibrators have been made
traceable to a primary phophorus standard. Phosphorus Standard FS may be used alternatively
for calibration. Each laboratory should establish corrective action in case of deviations in control
recovery.
Measuring range
The test has been developed to determine phosphorus concentrations within a measuring
range from 0.2-30 mg/dL. When values exceed this range, samples should be diluted 1 + 10 with
NaCl solution (9g/L) and the result multiplied by 11.
hemoglobin up to 1000 mg/dL, lipemia up to 2000 mg/dL.
Sensitivity/Limit Detection
The lower limit of detection is 0.2 mg/dL.
Serum
Adults 2.6-4.5 md/dL
Plasma
Concentrations of inorganic phosphate are about 0.2-0.3 mg/dL lower in heparinized plasma
than in serum.
An increase in serum phosphorus is found in chronic nephritis progressing with increase renal
failure. A moderate increase is observed in hypoparathyroidism n vitamin D excess.
A decrease in serum phosphorus is observed in rickets or osteomalacia and also in
hyperparathyroidism. Hypophosphatemia may result due to disorders of renal tubular
reabsorption.

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Clinical Chemistry

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CLINICAL CHEMISTRY

Interfering substance Interferences ≤ 10% up to

WARNING AND PRECAUTIONS. See below.

Interfering substances Interferences ≤ 10% up to

WARNING AND PRECAUTIONS. See below.

Interfering substances Interferences ≤ 10% up to

WARNING AND PRECAUTIONS. See below.

SPECIMEN. See below.

CALCULATION. See below.

Interfering substance Interferences ≤ 10% up to

WARNING AND PRECAUTIONS. See below.

Interfering substance Interferences ≤ 10% up to Analyte Concentration

WARNING AND PRECAUTIONS. See below.

CALCULATION. See below.

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