13 Prolactin

STANDARD OPERATING PROCEDURE
PROLACTIN (PRL)
1. PURPOSE OF EXAMINATION:
a. To measure Prolactin levels quantitatively in the specimen by Automated

Method.
b. To use Prolactin Measurements as an aid in the diagnosis, management and
follow up of prolactin secreting tumours accompanied by secondary
amenorrhea or galactorrhoea, hyperprolactenemia and infertility
2. PRINCIPLE OF TEST METHOD:

The VITROS Prolactin assay is performed using the VITROS Prolactin reagent Pack
and VITROS Immunodiagnostic Products Prolactin Calibrators on the VITROS
Immunodiagnostic System. An immunometric technique is used, this involves the
simultaneous reaction of prolactin present in the sample with a biotinylated
antibody(sheep polyclonalanti-prolactin) and a horseradish peroxidase (HRP)-labeled
antibody conjugate (mouse monoclonal anti-prolactin). The antigen-antibody
complex is captured by streptavidin on the wells, unbound materials are removed by
washing.
The bound HRP conjugate is measured bya luminescent reaction 8. A reagent
containing luminogenic substrates (a luminol derivative and a per acid salt) and an
electron transfer agent, is added to the wells. The HRP in the bound conjugate
catalyzes the oxidation of the luminol derivative, producing light. The electron
transfer agent (a substituted acetanilide) increases the level of light produced and
prolongs its emission. The light signals are read by the VITROS System. The amount
of HRP conjugate bound is directly proportional to the concentration of prolactin
present
3. SPECIMEN COLLECTION AND HANDLING:

a. Primary sample/Patient preparation:Whole blood collected by
venipuncture; Vacutainer containing blood up to the mark, is gently inverted
5 times to ensure proper mixing of specimen with clot activator.Collect
sample 3-4 hrs after patient has awakened.
Laboratory Medicine & Pathology

Doc No.:LMP/IA 13 Document Name: Prolactin
Issue No.: Effective Date: Next Review Date: 18/09/2018 Review by: Dev Shankar Yadav
17/09/2017 Designation: Technical Manager
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Amendment No.: Amendment Date: Prepared by: Dil Bahadur Saru Approved by: Dr. Gopi Aryal Issued by: Nageshwar Chaudhary
Designation: Section Incharge Designation: Laboratory Director Designation: Quality Manager
b. Specimen type required (secondary sample): Serum.

c. Other specimens analysed by this method: NA
d. Type of container and additives for
i. Serum: Red capped vacutainer containing Clot activator/SST.
e. Sample separation: Serum should be separated from cells within 2 hrs of
collection. Blood Samples are allowed to clot completely at room
temperature (RT) for 15 to 30 minutes. Blood samples are centrifuged
at 3000 rpm for 10 minutes.
f. Rejection criteria: Unlabeled, wrongly labeled, contaminated specimens;
Serum exceeding HIL interference limits (refer 5g).
g. Sample stability and storage:
 Serum/plasma free from evaporation effects is stable at room temperature

(RT) for 8 hrs, at 2-8°C for 48 hrs, at -20°C for longer storage.
Samples may be thawed only once.
 Post test sample storage by the lab: Sample portions at -20°C, for three
days.
4. SPECIFIC PERFORMANCE CHARACTERISTICS:

a. Assay Range/AMR : 1.4- 329 ng/mL [30.8-7000 µIU/mL]
b. Analytical Sensitivity: 1.4 ng/mL
c. Calibration and Its Verification :The details of calibration carried out during
validation are available in the validation Report.
Acceptable CV for Low Cal : 10.3
Acceptable CV for High Cal : 7.9
Master Curve Data : Critical Precision is 0.995
d. Measurement Imprecision:
Measurement repeatability( Within-run/Intra run) :
Measurement reproducibility (Inter assay) :
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Level 1 is 3.16, Level 2 is 5.51 and Level 3 is 4.95.

e. Measurement accuracy : 2.1 %
f. Method comparison:
Comparison with Unicel DxI 600 from Beckman Coulter and Centaur XP from
Siemens : Correlation (r) = 0.9947
g. Interferences / Analytical specificity: HIL (hemolysis, icterus & lipemia)
interference.
No significant interference in serum with Hemolysis (Hemoglobin upto 500
mg/dL), Icterus (Bilirubin upto 20 mg/dL) and Lipaemia (upto 1000 mg/dL of
Triglycerides)
5. REQUIRED EQUIPMENTS AND REAGENTS:

Equipments: vitros 5600 and vitros 4600, micro-pipettes, refrigerators, centrifuges.
Reagents (Refer to pack insert):
VITROS Immunodiagnostic Products Reagent Packs contain homogeneous liquid
reagents that do not require shaking or mixing prior to loading onto the system. As
with all immunoassay protein-based solutions, inappropriate handling of the reagent
pack can cause foam to occur on the surface of the reagent.
The reagent pack is supplied ready for use. Store unopened at2-8 °C, do not freeze.
Use opened reagent packs within 8 weeks of first loading onto a System; do not use
beyond the expiration date. Store opened reagent packs on board the System, or at
2-8 °C in a sealed reagent pack storage box containing dry desiccant.
VITROS Prolactin Calibrators are supplied freeze-dried. Store unopened at 2-8 °C. Do
not use beyond the expiration date. Reconstitute with 1 mL distilled water. After
reconstitution store for up to 5 weeks at 2-8 °C or 10 weeks at –20 °C (with no more
than 1 freeze-thaw cycle
Onboard Stability: 28 days.
6. CALIBRATION PROCEDURES & metrological traceability:

Calibration and Traceability of Calibration
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The calibration range of the VITROS Prolactin assay is 0-7 000 mIU prolactin/L
(traceable to in-house reference calibrators, which have been value-assigned to
correlate to another commercially available assay,with reference to the Third
International Standard of prolactin 84/500). Prolactin concentrations are quoted in
mIU/L or ng/mL. Conversion between units is made using
the formula: Result in ng/mL = Result in mIU (3rd IS 84/500)/L x 0.047
Accuracy
A correlation between the VITROS Prolactin assay and a commercially available
assay(X) has been obtained by measuring a panel of 137 patient samples from a
variety of clinical categories. The range of samples used covered 39.0-6 549 mIU/L in
the VITROS Prolactin assay and 52.0-7 527 mIU/L in the commercially available assay:
VITROS Prolactin assay= 1.048 x X - 94 (mIU/L), with a correlation coefficient of
0.982.
Precision
Precision was evaluated according to the National Committee for Clinical Laboratory
Standards protocol EP5-T2 12. Four freeze-dried Control sera were used. One
replicate of Control 1 and two replicates of Controls 2, 3 and 4 were assayed on at
least 2 separate occasions per day on atleast 20 different days. The experiment was
performed using 2 reagent lots on different Systems. The data presented are
representative of the product performance.
7. PROCEDURAL STEPS: Refer to Point 7 on Common page

Test Conditions:
Sample Volume: 25 µL;
Test Temperature: 37°C;
Type of measurement: Two-step sandwich immunoassay
Dilution:
 Auto dilution-1:2, 1:5 as required for samples with result in excess of 200
ng/mL.
 Results obtained may be reported directly when the test value is within 1000
ng/mL.

Page | 4 of 7
 In case the test value obtained after dilution (1: 2), exceeds 200 ng/mL on the
result monitor, then report the results as > 1000 ng/mL.
Sample Test value Action Test value

displayed displayed Action to
Dilution volume to be
be taken
(µL) (ng/mL) taken (ng/mL)
Proceed to
1:2 100 Upto 400 > 200 the next
Report dilution
directly
Report as
1:5 40 Upto 1000 > 200
> 1000
8. QUALITY CONTROL PROCEDURES:

a. Internal Quality Control (IQC): IQC material with known PRL concentrations are
analyzed on the days of use and data is reviewed.
QC product material:
Serum: LYPHOCHECK Immunoassay Plus Control (IPC), Level I, II and III from
Bio-Rad Laboratories.
Frequency: Any two levels IPC once a day.
 Lab mean, SD and CV% is established for the IQC. If the value lies
within the cut off limits as specified by the lab or pack insert for
Quality Control (before establishing the lab mean), the test results
are released. Levey-Jennings control charts are maintained.
9. PRINCIPLE OF PROCEDURE FOR CALCULATING RESULTS

Chemiluminometric principle employs the calculation based on the measurement of
light emitted when an electron returns from an excited state to ground state
(Excitation event is by a chemical reaction).

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The instrument automatically calculates results from the absorbance and prints the
concentration of PRL in ng/mL.
The system reports serum prolactin results in ng/mL (mass units) or μIU/mL (SI units),
depending on the units defined when setting up the assay. The conversion formula is
1ng/mL = 21.2 μIU/mL.
10. REPORTABLE INTERVAL OF PATIENT EXAMINATION RESULTS:
Reportable range : 1.4-329 ng/mL
Lowest value reported : <1.4 ng/mL
Reporting of test values above the reportable range (Clinical reportable range)
Serum : >1000 ng/mL
11.BIOLOGICALREFERENCEINTERVALS:
Male 3.7-17.9
Unit:
ng/mL Female Non – pregnant 3.0-18.6
12. ALERT/ CRITICAL VALUES, WHERE APPROPRIATE: NA
13. CLINICAL SIGNIFICANCE:

Increase (Hyperprolactinemia) Decrease (Hypoprolactinemia)
PRL secreting pituitary adenomas (prolactinomas); L-Dopa, Bromocryptine; Decreased
Functional and organic diseases of the pituitary function (Sheehan’s
hypothalamus; Hypothyroidism; renal failure; syndrome)
ectopic tumors; psychotropic drugs
(phenothiazines), anti-hypertensive drugs
(reserpine) and TRH, Estrogen therapy.
14. LABORATORY INTERPRETATION: Done, where appropriate in consultation with

clinician.
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15. SAFETY PRECAUTIONS: Refer to Advia centaur user manual for PRL .
16. POTENTIAL SOURCES OF VARIABILITY:

1. Refer to Pack insert for interferences from various substances and drugs
2. Refer to Point 4 on Common page for interferences from pre analytical,
analytical and post analytical phases
3. Elevations in fetus and newborn
4. Elevations during sleep (peak in early morning hrs), stress, exercise
5. Elevations in females during pregnancy and nursing.
6. Limitations
Lactation and the administration of oral contraceptives can increase prolactin
concentrations.
7. High Dose Hook Effect
Patient samples with high prolactin levels can cause a paradoxical decrease
in the RLUs (high dose hook effect). In this assay, patient samples with
prolactin levels as high as 30,000 ng/mL (636,000μIU/mL) will assay greater
than 200 ng/mL (4240 μIU/mL).
17. REFERENCES:
 Vitros 5600® PRL Reagent & calibrator pack insert sheets
 Operator’s guide – vitros 5600.
 TEITZ Textbook of Clinical Chemistry- Burtis. C.A. Ashwood E. R. 3rd edition.

TEITZ Textbook of Clinical Chemistry & Molecular Diagnostics- Burtis. C.A.
Ashwood E. R. 4th editions.
 Clinical Guide to Laboratory Tests, 3rd edition- Norbert W. Teitz, 4th edition-
Alan H.B WY.
 A manual of laboratory and diagnostic tests – Francis Fischbach 6th edition
 Medical Laboratory technology 6th edition by Ramnik Sood.

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STANDARD OPERATING PROCEDURE

a. To measure Prolactin levels quantitatively in the specimen by Automated

2. PRINCIPLE OF TEST METHOD:

3. SPECIMEN COLLECTION AND HANDLING:

Laboratory Medicine & Pathology

b. Specimen type required (secondary sample): Serum.

 Serum/plasma free from evaporation effects is stable at room temperature

4. SPECIFIC PERFORMANCE CHARACTERISTICS:

Level 1 is 3.16, Level 2 is 5.51 and Level 3 is 4.95.

5. REQUIRED EQUIPMENTS AND REAGENTS:

6. CALIBRATION PROCEDURES & metrological traceability:

7. PROCEDURAL STEPS: Refer to Point 7 on Common page

Laboratory Medicine & Pathology

Sample Test value Action Test value

8. QUALITY CONTROL PROCEDURES:

9. PRINCIPLE OF PROCEDURE FOR CALCULATING RESULTS

Laboratory Medicine & Pathology

12. ALERT/ CRITICAL VALUES, WHERE APPROPRIATE: NA

13. CLINICAL SIGNIFICANCE:

14. LABORATORY INTERPRETATION: Done, where appropriate in consultation with

16. POTENTIAL SOURCES OF VARIABILITY:

 Operator’s guide – vitros 5600.

 TEITZ Textbook of Clinical Chemistry- Burtis. C.A. Ashwood E. R. 3rd edition.

 A manual of laboratory and diagnostic tests – Francis Fischbach 6th edition

 Medical Laboratory technology 6th edition by Ramnik Sood.

Laboratory Medicine & Pathology

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