Sop of Interleukin-6(IL-6) testing

a.PURPOSE:

Immunoassay for the in vitro quantitative determination of Interleukin-6

(IL-6) in serum and plasma. This assay can be used to aid in the
management of critically ill patients as early indicator for acute
inflammation using
The electrochemiluminescence immunoassay “ECLIA” on the Hitachi cobas e-411 immunoassay
analyzers
b. PRINCIPLE OF TEST:
Sandwich principle. Total duration of assay: 18 minutes.
▪ 1st incubation: 30 µL of sample are incubated with a biotinylated
monoclonal IL-6-specific antibody.
▪ 2nd incubation: After addition of a monoclonal IL-6-specific antibody
labeled with a ruthenium complexa) and streptavidin-coated
microparticles, the antibodies form a sandwich complex with the antigen
of the sample.
▪ The reaction mixture is aspirated into the measuring cell where the
microparticles are magnetically captured onto the surface of the
electrode. Unbound substances are then removed with
ProCell/ProCell M. Application of a voltage to the electrode then induces
chemiluminescent emission which is measured by a photomultiplier.
▪ Results are determined via a calibration curve which is instrumentspecifically generated by 2-point
calibration and a master curve provided
via the reagent barcode or e-barcode.
C.PERFORMANCE CHARECTERISTICS:

Linearity:

1.5-5000 pg/mL (defined by the lower detection limit and the maximum of

the master curve).

Values below the lower detection limit are reported as < 1.5 pg/mL.

Values above the measuring range are reported as > 5000 pg/mL (or up to 50000 pg/mL for 10-fold
diluted samples).

Precision:

Precision was determined using Elecsys reagents, samples and controls in

a protocol (EP5-A2) of the CLSI (Clinical and Laboratory Standards
Institute): 2 runs per day in duplicate each for 21 days (n = 84). The
following results were obtained:
Serum and plasma
cobas e 411 analyzer

Repeatabil Intermedia
 ity te
precision

Samp Mea SD C SD CV
le n pg/ V pg/ %
pg/ mL % mL
mL
Huma 6. 1.46 8.
n 17.3 1.03 0 5
seru
m1

Huma 117 2.90 2. 3.71 3.

n 5 2
seru
m2

Huma 891 22.8 2. 25.5 2.

n 6 9
seru
m3

PC 38.3 0.540 1. 1.04 2.

MMb 4 7
)1

PC 229 3.29 1. 6.71 2.

MM 2 4 9

PC MM = PreciControl Multimarker

Method comparison
Inorganic phosphate values obtained on a COBAS INTEGRA 700 analyzer with the COBAS INTEGRA
Phosphate (Inorganic) ver.2 reagent (y) were compared to those determined with the same reagent on a
Roche/Hitachi 917 analyzer (x) and to the previous reagent (PHOS) on a COBAS INTEGRA 700 analyzer
(x).

Seum and plasma

Roche/Hitachi 917 analyzer Sample size (n) = 100

Passing/Bablok Linear regression

y = 1.043x + 0.022 mmol/L y = 1.040x + 0.025 mmol/L
τ = 0.955 r = 1.000
SD (md 95) = 0.040 Sy.x = 0.018

The sample concentrations were between 0.572 to 5.69 mmol/L (1.77 to17.7 mg/dL).
COBAS INTEGRA 700 analyzer Sample size (n) = 96

Passing/Bablok Linear regression

y = 1.029x - 0.047 mmol/L y = 1.040x - 0.067 mmol/L
τ = 0.942 r = 0.999
SD (md 95) = 0.077 Sy.x = 0.032
The sample concentrations were between 0.619 to 4.76 mmol/L (1.92 to
14.9 mg/dL).

Urine
Roche/Hitachi 917 analyzer Sample size (n) = 86

Passing/Bablok Linear regression

y = 1.052x - 0.0235 mmol/L y = 1.044x - 0.028 mmol/L
τ = 0.983 r = 1.000
SD (md 95) = 0.743 Sy.x = 0.349

The sample concentrations were between 6.08 to 89.4 mmol/L (18.9 to277 mg/dL).

COBAS INTEGRA 700 analyzer Sample size (n) = 68

Passing/Bablok Linear regression
y = 1.000x - 0.399 mmol/L y = 1.002x - 0.405 mmol/L
τ = 0.989 r = 1.000
SD (md 95) = 0.396 Sy.x = 0.180

The sample concentrations were between 6.08 to 44.8 mmol/L (18.9 to 139 mg/dL).

d. SAMPLES:

Serum: Collect serum using standard sampling tubes with yellow capped tube containing gel+BCA(CAT)
or red capped tube containing silica gel.

Plasma: Green capped Heparin (Li-, Na-, NH4+-) or lavender capped EDTA (K2-, K3-) tube.

Urine: Urine should be collected in an acid-washed, detergent-free container. Acidify with hydrochloric
acid after collection (pH < 3).

Sample stability: Sample stability has been validated in the lab for a period of 24 hours at 2-8◦C.the
storage period of 1 day has been decided as the routine protocol and NABL 112.

Sample requirement: Minimum sample volume required is 5.0 ml in yellow capped tube containing
gel+BCA(CAT) or red capped tube containing silica gel.

Sample rejection: Haemolysed, inadequate sample volume, unlabeled sample.

e. PATIENT PREPARATION:

Random sample can be collected.

f. TYPE OF CONTAINER:

Blood sample should be collected in yellow capped tube containing gel+BCA(CAT) or red capped tube
containing silica gel.Spot urine sample can be collected in a 50ml plastic container Acidified with
hydrochloric acid after collection (pH < 3).

g. EQUIPMENTS AND REAGENT REQUIREMENT:

EQUIPMENTS:

Cobas Integra 400 Plus Biochemistry analyzer.

REAGENTS:

working solutions

R1 Sulfuric acid 0.36 mol/L, detergent

SR SR Ammonium molybdate 3.5 mmol/L, Sulfuric acid 0.36 mol/L,
Sodium chloride 150 mmol/L

R1 is in position B and SR is in position C.

Reagent handling :

Ready for use.

Pipetting parameters

Serum, plasma, and urine

Diluent (H2O)
R1 90 µL 11 µL
Sample 2.5 µL 27.5 μL
SR 38 µL
Total volume 158 µL

STORAGE AND STABILITY:

Shelf life at 2-8 °C See expiration date on cobas c pack label

On-board in use at 10-15 °C 12 weeks

h. SAFTEY:

Exercise the normal precautions required for handling all laboratory reagents.

To avoid the possible build-up of azide compounds,flush waste -pipes with water after the disposal of
undiluted reagent.

Dispose all waste material in accordance with BMW guidelines.

The materials used as a calibrator or quality control should be handled as infectious and universal
precautions to be followed.

i. CALIBRATION:

CALIBRATOR REQUIRED:

Lyophilized serum : Calibrator f.a.s. ref. 10759350 190

Use deionized water as zero calibrator.

CALIBRATION FREQUENCY:

AS per manufacture’s instruction recalibration lot /lot recovery 30days or whenthe following occurs:

 Change in reagent lot or significant shift in control values.

 Major preventive maintenance was performed on the analyzer or a critical part was
replaced.

However daily or bottle to bottle calibration may be done as per the discretion of the lab manager.

CALIBRATOR PREPARATION:

CALIBRATOR STABILITY:

TRACEABILITY:

This method has been standardized against NERL primary reference material.

j. TESTING PROCEDURE:

K.QUALITY CONTROL:

INTERNAL CONTROL;

EXTERNAL CONTROL:
l. INTERFERENCE AND LIMITATIONS:

Substance Limits
Icterus No significant interference up to an I index of 51 (approximate conjugated bilirubin
concentration: 872 μmol/L or 51 mg/dL). No significant interference with
unconjugated bilirubin.
Hemolysis No significant interference up to an H index of 420 (approximate hemoglobin
concentration: 261 μmol/L or 420 mg/dL).
Lipemic No significant interference up to an Intralipid level of 1000 mg/dL. There is poor
correlation between turbidity and triglycerides concentration.
Drugs Phospholipids contained in liposomal drug formulations (e.g. AmBisome) may be
hydrolyzed in the test due to the acidic reaction pH and thus lead to elevated
phosphate results.

m. CALCULATING RESULTS TO INCLUDE MEASURMENT UNCERTANITY:

n. BIOLOGICAL TEST INTERVALS:

Serum, plasma

Adults14
0.81-1.45 mmol/L (2.5-4.5 mg/dL)

Children15
Age Male mmol/L (mg/dL) Female mmol/L (mg/dL)
1-30 days 1.25-2.25 (3.9-6.9 ) 1.40-2.50 (4.3-7.7)
1-12 months 1.15-2.15 (3.5-6.6) 1.20-2.10 (3.7-6.5)
1-3 years 1.00-1.95 (3.1-6.0) 1.10-1.95 (3.4-6.0)
4-6 years 1.05-1.80 (3.3-5.6) 1.05-1.80 (3.2-5.5)
7-9 years 0.95-1.75 (3.0-5.4) 1.00-1.80 (3.1-5.5)
10-12 years 1.05-1.85 (3.2-5.7) 1.05-1.70 (3.3-5.3)
13-15 years 0.95-1.65 (2.9-5.1) 0.90-1.55 (2.8-4.8)
16-18 years 0.85-1.60 (2.7-4.9) 0.80-1.55 (2.5-4.8)

Urine
1st morning urine16 13-44 mmol/L (40-136 mg/dL)
24h urine6 13-42 mmol/d (0.4-1.3 g/d)

o.REPORTABLE RANGE: 1.1-92 mmol/L (3.41-285 mg/dL)

p.DETERMINING RESULTS WHEN THE RESULT IS NOT WITHIN MEASURABLE RANGE:

LINEARITY:The test is linear within a concentration range of 1.1-92 mmol/L (3.41-285 mg/dL) for
serum,plasma and Urine

Limits of detection (LOD) verified in the lab: 1.1-92 mmol/L (3.41-285 mg/dL)

POTENTIAL SOURCE OF VARIATION:

In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may cause
unreliable results.11 For diagnostic purposes, the results should always be assessed in conjunction with
the patient’s medical history, clinical examination and other findings.

INTERPRETATION:

88 % of the phosphorus contained in the body is localized in bone in the form of calcium phosphate as
the apatite Ca2+[Ca3(PO4)2]3 . The remainder is involved in intermediary carbohydrate metabolism and
in physiologically important substances such as phospholipids, nucleic acids and ATP. Phosphorus occurs
in blood in the form of inorganic phosphate and in
organically bound phosphoric acid. The small amount of extracellular organic phosphorus is found
almost exclusively in the form of phospholipids. The ratio of phosphate to calcium in the blood is
approximately 6:10.
LOW:

HIGH:

An increase in the level of phosphorus causes a decrease in the calcium level. The mechanism is
influenced by interactions between parathormone and vitamin D. Hypoparathyroidism, vitamin D
intoxication and renal failure with decreased glomerular phosphate filtration give rise to
hyperphosphatemia. Hypophosphatemia occurs in rickets, hyperparathyroidism and Fanconi’s
syndrome.
REFERENCE: