Prolactin A

Prolactin

For use on the IMMULITE® and IMMULITE® 1000 systems

IMMULITE /IMMULITE 1000 Prolactin ®

®

CDC Analyte Identifier Code: 4915 CDC Test System Identifier Code: 10159 CLIA Complexity Category: Moderate

In assessing the significance of moderate elevations, it is important to keep in mind that prolactin is a stress hormone. Not only surgery, but events no more distressing than venipuncture or a clinical interview have been reported to occasion a transient rise. Moreover, the release of prolactin is inherently episodic, and dayto-day fluctuations with CVs as high as 30% have been encountered. Finally, there is a sleep-related diurnal variation: prolactin levels increase during sleep and reach their lowest a few hours after waking. The advice sometimes given to draw samples "between nine and noon" is based on the assumption that subjects observe reasonably normal waking hours.

Summary and Explanation

Principle of the Procedure

Human prolactin is a polypeptide hormone of the anterior pituitary with a molecular mass of about 22,800. It plays an essential role in the secretion of milk and has the ability to suppress gonadal function. The very existence of prolactin as a substance distinct from human growth hormone was established only in 1970. Since that time, determination of prolactin has become an important tool in the investigation of amenorrhea, galactorrhea and hypothalamic-pituitary disorders.

IMMULITE/IMMULITE 1000 Prolactin is a solid-phase, two-site chemiluminescent immunometric assay.

As a reference range for circulating prolactin, the literature suggests concentrations up to approximately 20 ng/mL. Values are distinctly elevated at birth but decline to adult levels in less than three months.

EDTA plasma should not be used as a sample type.

English Intended Use: For in vitro diagnostic use with the IMMULITE and IMMULITE 1000 Analyzers — for the quantitative measurement of prolactin in serum, as an aid in the diagnosis and treatment of pituitary disorders. Catalog Number: LKPR1 (100 tests), LKPR5 (500 tests) Test Code: PRL Color: Dark Blue

Women are reported to have slightly higher mean levels than men, with a slight rise at puberty — apparently estrogen related — and a corresponding fall at menopause. During pregnancy, the prolactin level climbs steadily to ten or twenty times its former value, then drops back down to normal after delivery — within three weeks in nonnursing mothers. In those who breast-feed, the decline to normal is more gradual because of the prompt and dramatic surges in prolactin release induced by suckling. Women taking oral contraceptives or under estrogen treatment may have prolactin levels higher than normal. 2

Incubation Cycles: 1 × 30 minutes.

Specimen Collection The use of an ultracentrifuge is recommended to clear lipemic samples. Hemolyzed samples may indicate mistreatment of a specimen before receipt by the laboratory; hence the results should be interpreted with caution.

Centrifuging serum samples before a complete clot forms may result in the presence of fibrin. To prevent erroneous results due to the presence of fibrin, ensure that complete clot formation has taken place prior to centrifugation of samples. Some samples, particularly those from patients receiving anticoagulant therapy, may require increased clotting time. Blood collection tubes from different manufacturers may yield differing values, depending on materials and additives, including gel or physical barriers, clot activators and/or anticoagulants. IMMULITE/IMMULITE 1000 Prolactin has not been tested with all possible variations of tube types. Consult the section on

IMMULITE/IMMULITE 1000 Prolactin (PILKPR-5, 2005-05-16)

Alternate Sample Types for details on tubes that have been tested. Volume Required: 25 µL serum. (Sample cup must contain at least 100 µL more than the total volume required.) Storage: 7 days at 2–8°C or 3 months at –20°C.

Warnings and Precautions For in vitro diagnostic use. Reagents: Store at 2–8°C. Dispose of in accordance with applicable laws. Follow universal precautions, and handle all components as if capable of transmitting infectious agents. Source materials derived from human blood were tested and found nonreactive for syphilis; for antibodies to HIV 1 and 2; for hepatitis B surface antigen; and for antibodies to hepatitis C. Sodium azide, at concentrations less than 0.1 g/dL, has been added as a preservative. On disposal, flush with large volumes of water to prevent the buildup of potentially explosive metal azides in lead and copper plumbing.

refrigerated: stable at 2–8°C until expiration date. Recommended usage is within 30 days after opening when stored as indicated. LKPR1: 1 wedge. LKPR5: 5 wedges. Prolactin Adjustors (LPRL, LPRH) Two vials (Low and High), of lyophilized human prolactin in a serum/buffer matrix. Reconstitute each vial with 2.0 mL distilled or deionized water. Mix by gentle swirling or inversion until the lyophilized material is fully dissolved. Aliquot and freeze. Stable at –20°C for 60 days after reconstitution. LKPR1: 1 set. LKPR5: 2 sets.

Kit Components Supplied Separately Prolactin Sample Diluent (LPRZ) For the manual dilution of patient samples. 25 mL of a prolactin-free serum/buffer matrix, with preservative. Stable at 2–8°C for 30 days or for 6 months (aliquotted) at –20°C.

Chemiluminescent Substrate: Avoid contamination and exposure to direct sunlight. (See insert.)

LSUBX: Chemiluminescent Substrate LPWS2: Probe Wash Module LKPM : Probe Cleaning Kit LCHx-y: Sample Cup Holders (barcoded) LSCP: Sample Cups (disposable) LSCC: Sample Cup Caps (optional)

Water: Use distilled or deionized water.

CON6: Tri-level, multi-constituent control

Materials Supplied

Also Required Sample transfer pipets, distilled or deionized water, controls.

Components are a matched set. The barcode labels are needed for the assay. Prolactin Test Units (LPR1) Each barcode-labeled unit contains one bead coated with monoclonal murine antiprolactin antibody. Stable at 2–8°C until expiration date. LKPR1: 100 units. LKPR5: 500 units. Allow the Test Unit bags to come to room temperature before opening. Open by cutting along the top edge, leaving the ziplock ridge intact. Reseal the bags to protect from moisture. Prolactin Reagent Wedge (LPR2) With barcode. 7.5 mL mL alkaline phosphatase (bovine calf intestine) conjugated to polyclonal goat antiprolactin antibody in buffer, with preservative. Store capped and IMMULITE/IMMULITE 1000 Prolactin (PILKPR-5, 2005-05-16)

Assay Procedure Note that for optimal performance, it is important to perform all routine maintenance procedures as defined in the IMMULITE or IMMULITE 1000 Operator's Manual. See the IMMULITE or IMMULITE 1000 Operator's Manual for: preparation, setup, dilutions, adjustment, assay and quality control procedures. Visually inspect each Test Unit for the presence of a bead before loading it onto the system. Recommended Adjustment Interval: 4 weeks. Quality Control Samples: Use controls or sample pools with at least two levels (low and high) of prolactin. 3

Expected Values

Prolactin, mIU/L

Based on its relationship to DPC's Coat-ACount Prolactin IRMA, the assay can be expected to have the following preliminary reference range for men. Group

n

Median

Adult Males

19

95% Range

6.2 ng/mL

2.5 – 17

131 mIU/L

53 – 360

Group Females

17

Group

Median

n

Adult Females 115

95% Range

9.4 ng/mL

1.9 – 25

199 mIU/L

40 – 530

Cord

n Median

0.6 – 9 Males

Cord

55

Cord

318

21 – 2,968

233

42 – 912

27 6,254 3,180 – 11,978

0.1 – 0.5 36 0.6 – 9

55

403

85 – 1,378

170

13 – 615

55 7,208 3,392 – 14,098

0.1 – 0.5 64 2,480 0.6 – 9

Central 95%

28 8,056 4,240 – 14,310

0.1 – 0.5 28

Combined

A study performed with IMMULITE Prolactin yielded the following reference range for women.

Age (yr)

110

191

42 – 2,650 21 – 848

Consider these limits as guidelines only. Each laboratory should establish its own reference ranges.

Limitation As summarized in DPC's technical report 17 ZB157, in a study which followed normal ovulating women on a daily basis throughout one complete cycle, somewhat higher values were obtained—an outcome consistent with the increasingly stressful sample collection process entailed by the study design. A cross-sectional study of pediatric fertility values performed with IMMULITE Prolactin at a "wellness" clinic in the southwestern United States yielded the following results. Prolactin, ng/mL Group Females

Males

Combined

Age (yr)

n Median Central 95%

Cord

28

380

200 – 675

0.1 – 0.5

28

15

1 – 140

0.6 – 9

55

11

2 – 43

Cord

27

295

150 – 565

0.1 – 0.5

36

19

4 – 65

0.6 – 9

55

8

0.6 – 29

Cord

55

340

160 – 665

0.1 – 0.5

64

117

2 – 125

0.6 – 9

110

9

1 – 40

Heterophilic antibodies in human serum can react with the immunoglobulins included in the assay components causing interference with in vitro immunoassays. [See Boscato LM, Stuart MC. Heterophilic antibodies: a problem for all immunoassays. Clin Chem 1988:34:2733.] Samples from patients routinely exposed to animals or animal serum products can demonstrate this type of interference potentially causing an anomalous result. These reagents have been formulated to minimize the risk of interference; however, potential interactions between rare sera and test components can occur. For diagnostic purposes, the results obtained from this assay should always be used in combination with the clinical examination, patient medical history, and other findings.

Performance Data See Tables and Graphs for data representative of the assay's performance. Results are expressed in ng/mL. (Unless otherwise noted, all were generated on serum samples collected in tubes without gel barriers or clot-promoting additives.) Conversion Factor: ng/mL × 21.2 → mIU/L Calibration Range: Up to 150 ng/mL rd (3,180 mIU/L) [WHO 3 IS 84/500].

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IMMULITE/IMMULITE 1000 Prolactin (PILKPR-5, 2005-05-16)

Analytical Sensitivity: 0.5 ng/mL (11 mIU/L). High-dose Hook Effect: None up to 22,000 ng/mL. Intraassay Precision (Within-Run): Statistics were calculated for samples from the results of 20 replicates in a single run. (See “Intraassay Precision” table.) Interassay Precision (Run-to-Run): Statistics were calculated for samples assayed in 26 different runs. (See “Interassay Precision” table.) Linearity: Samples were assayed under various dilutions. (See "Linearity" table for representative data.) Recovery: Samples spiked 1 to 19 with three prolactin solutions (129, 270 and 610 ng/mL) were assayed. (See "Recovery" table for representative data.) Specificity: The antibody is highly specific for prolactin. (See "Specificity" table.) Bilirubin: Presence of bilirubin in concentrations up to 200 mg/L has no effect on results, within the precision of the assay. Hemolysis: Presence of packed red blood cells in concentrations up to 30 µL/mL has no effect on results, within the precision of the assay. Alternate Sample Type: To assess the effect of alternate sample types, blood was collected from 36 volunteers into plain, heparinized and Becton Dickinson ® SST vacutainer tubes. Equal volumes of the matched samples were spiked with various concentrations of prolactin, to obtain values throughout the calibration range of the assay, and then assayed by the IMMULITE Prolactin procedure. (Heparin) = 0.97 (Serum) + 0.24 ng/mL r = 0.98 (SST) = 0.93 (Plain Tubes) + 0.39 ng/mL r = 0.96 Means: 12.1 ng/mL (Serum) 12.0 ng/mL (Heparin) 11.7 ng/mL (SST)

EDTA: Because EDTA would have a significant effect on results, it should not be used as an anticoagulant.

Prolactin on 148 samples. (Concentration range: approximately 1 to 125 ng/mL. See graph.) By linear regression: (IML) = 0.98 (IML 2000) + 1.97 ng/mL r = 0.988 Means: 45 ng/mL (IMMULITE) 44 ng/mL (IMMULITE 2000)

References 1) Cowden E, et al. Laboratory assessment of prolactin status. Ann Clin Biochem 1979;16:113–21. 2) Cowden E, et al. Tests of prolactin secretion. Lancet 1979;1155–8. 3) Djursing H. Short- and long-term fluctuations in plasma prolactin concentration in normal subjects. Acta Endocrinol 1981; 97:1–6. 4) Greer ME, et al. Prevalence of hyperprolactinemia in anovulatory women. Obstet Gynecol 1980;56:65–9. 5) Harrington RA, et al. Metoclopramide: an updated review. Drugs 1983;25:451–94. 6) Healy DL, et al. Pituitary autonomy in hyperprolactinemia secondary amenorrhea: results of hypothalamicpituitary testing. J Clin Endocrinol Metab 1977;44:809–18. 7) Kleinberg DL, Noel GL, Frantz AG. Galactorrhea: a study of 235 cases. N Engl J Med 1977;296:589–600. 8) Knuth UA, Friesen HG. Prolactin and pregnancy. Curr Top Exp Endocrinol 1983;4:69–96. 9) Maxson WS, Hammond CB. Hyperprolactinemia. Contemp Ob/Gyn 1982 Jan;19(1):49–63, 19:67–83. 10) Pepperell RJ. Prolactin and reproduction. Fertil Steril 1981;35:267–74. 11) Babson, AL. The IMMULITE Automated Immunoassay System. J Clin Immunoassay 1991;14:83-8. 12) Thorner MO. Prolactin: clinical physiology and management of hyperprolactinemia. In: Martini L, Besser GM, et al, editors. Clinical endocrinology. New York: Academic Press, 1977;319–61. 13) Tolis G, Franks S. Physiology and pathology of prolactin secretion. In: Tolis G, et al, editors. Clinical endocrinology: a pathophysiological approach. New York: Raven Press, 1979;291–317. 14) Tolis G, et al. Aspects of prolactin pathophysiology. Periodicum Biologorum 1983;85 Suppl 1:29–36. 15) Yen SSC. Neuroendocrine regulation of gonadotropin and prolactin secretion in women: disorders in reproduction. In: Vaitukaitis J, editor. Clinical reproductive endocrinology. New York: Elsevier Biomedical, 1982;137–76. 16) Zacur HA. Use of the human prolactin immunoassay. J Clin Immunoassay 1983 Spring;6(1):63–67. 17) Vankrieken L. IMMULITE reproductive hormone assays: multicenter reference range data. Los Angeles: Diagnostic Products Corporation, 2000. Document No. ZB157-D.

Method Comparison: The assay was compared to DPC's IMMULITE 2000 IMMULITE/IMMULITE 1000 Prolactin (PILKPR-5, 2005-05-16)

5

Recovery (ng/mL)

Technical Assistance In the United States, contact DPC's Technical Services department. Tel: 800.372.1782 or 973.927.2828 Fax: 973.927.4101. Outside the United States, contact your National Distributor. Manufactured by EURO/DPC Ltd. under a Quality System registered to ISO 13485:2003.

Solution1 1

2

Tables and Graphs 3

Intraassay Precision (ng/mL)

Observed2 Expected3 %O/E4

—

16.6

—

—

A

24.3

22.2

110%

B

29.6

29.3

101%

C

45.0

46.3

97%

—

18.9

—

—

A

26.3

24.4

108%

B

34.1

31.5

108%

C

58.6

48.5

121%

—

37.1

—

—

A

42.4

41.7

102%

Mean1

SD2

CV3

B

51.9

48.8

106%

1

8.0

0.54

6.8%

C

76.5

65.8

116%

2

16.3

1.00

6.1%

3

36.6

2.07

5.7%

Specificity

Mean1 1

SD2

14.1

CV3

1.36

ng/mL Added2

Apparent ng/mL 3

% Crossreactivity4

FSH

1,000

ND

ND

HCG

35,000

ND

ND

HGH

1,000

ND

ND

HPL

200,000

ND

ND

500

ND

ND

1,000

ND

ND

Compound1

Interassay Precision (ng/mL) 9.6%

2

22.2

2.10

9.5%

3

51.0

3.26

6.4%

LH TSH

ND: not detectable.5

Dilution1 1

2

3

Observed2 Expected3 %O/E4

5

8 in 8

30.3

—

—

4 in 8

15.1

15.1

100%

2 in 8

6.6

7.6

87%

1 in 8

3.5

3.8

92%

8 in 8

39.9

—

—

4 in 8

21.3

20.0

107%

2 in 8

10.3

10.0

103%

1 in 8

4.3

5.0

86%

8 in 8

139.3

—

—

4 in 8

72.6

69.7

104%

2 in 8

36.8

34.8

106%

1 in 8

20.7

17.4

119%

Method Comparison 150

120

IMMULITE Prolactin

Linearity (ng/mL)

90

60

30

0 0

30

60

90

120

150

IMMULITE 2000 Prolactin, ng/mL

(IML) = 0.98 (IML 2000) + 1.97 ng/mL r = 0.988 Deutsch. Intraassay Precision: 1Mittelwert, 2S (Standardbereich), 3CV (Variationskoeffizient). Interassay Precision: 1Mittelwert, 2S 6

IMMULITE/IMMULITE 1000 Prolactin (PILKPR-5, 2005-05-16)

(Standardbereich), 3CV (Variationskoeffizient). Linearity: 1Verdünnung, 2Beobachten (B), 3 Erwarten (E), 4% B/E, 58 in 8. Recovery: 1 Lösung, 2Beobachten (B), 3Erwarten (E), 4% B/E. Specificity: 1Verbindung, 2zugesetzte Menge, 3 Ausgewiesene Konzentration, 4% Kreuzreaktivität, 5NN: Nicht nachweisbar. Method Comparison: Prolactin: Prolaktin. Español. Intraassay Precision: 1Media, 2DS, 3 CV. Interassay Precision: 1Media, 2DS, 3CV. Linearity: 1Dilución, 2Observado (O), 3Esperado (E), 4%O/E, 58 en 8. Recovery: 1Solución, 2 Observado (O), 3Esperado (E), 4%O/E. Specificity: 1Compuesto, 2Cantidad añadida, 3 Concentración aparente, 4% Reacción cruzada, 5 ND: no detectable. Method Comparison: Prolactin: Prolactina. Français. Intrraassay Precision: 1Moyenne, 2 SD, 3CV. Interassay Precision: 1Moyenne, 2 SD, 3CV. Linearity: 1Dilution, 2Observé (O), 3 Attendu (A), 4%O/A, 58 dans 8. Recovery: 1 Solution, 2Observé (O), 3Attendu (A), 4%O/A. Specificity: 1Composé, 2ajouté, 3 Concentration apparente, 4Réaction croisée%. 5ND: non détectable. Method Comparison: Prolactin: Prolactine. Italiano. intraassay Precision: 1Media, 2SD (Deviazione Standard), 3CV (Coefficiente di Variazione). Interassay Precision: 1Media, 2SD (Deviazione Standard), 3CV (Coefficiente di Variazione). Linearity: 1Diluizione, 2Osservato (O), 3Atteso (A), 4%O/A, 58 in 8. Recovery: 1 Soluzione, 2Osservato (O), 3Atteso (A), 4%O/A. Specificity: 1Composto, 2quantità aggiunta, 3 Concentrazione apparente, 4Percentuale di Crossreattività, 5ND: non determinabile. Method Comparison: Prolactin: Prolattina. Português. Intraassay Precision: 1Média, 2 Desvio padrão, 3Coeficiente de variação. Interassay Precision: 1Média, 2Desvio padrão, 3 Coeficiente de variação. Linearity: 1Diluição, 2 Observado (O), 3Esperado (E), 4%O/E, 58 em 8. Recovery: 1Solução, 2Observado (O), 3 Esperado (E), 4%O/E. Specificity: 1Composto, 2 Quantidade adicionada, 3Apparent Concentration, 4Percentagem de reacção cruzada, 5ND: não detectável. Method Comparison: Prolactin: Prolactina.

Deutsch Prolaktin Anwendung: Zur in vitro-Diagnostik unter Verwendung der IMMULITE und IMMULITE 1000 Systeme – zur quantitativen Messung von Prolaktin im Serum, zur Untersuchung und Therapiekontrolle von pituitären Störungen. IMMULITE/IMMULITE 1000 Prolactin (PILKPR-5, 2005-05-16)

Artikelnummern: LKPR1 (100 Tests), LKPR5 (500 Tests) Testcode: PRL Farbe: dunkelblau

Klinische Relevanz Humanes Prolaktin ist ein PolypeptidHormon der vorderen Hypophyse mit einem Molekulargewicht von ca. 22 800. Es ist von essentieller Wichtigkeit für die Produktion von Muttermilch und kann gonadale Funktionen unterdrücken. Die genaue Definition von Prolaktin, als eine vom menschlichen Wachstumhormon zu unterscheidende Substanz, gelang erst 1970. Seit diesem Zeitpunkt, wurde die Bestimmung von Prolaktin eine wichtige Methode in der Diagnostik der Amenorrhoe, Galactorrhoe und Hypothalamus-, bzw. Hypophysendysfunktionen. Als Referenzwertebereich für zirkulierendes Prolaktin werden in der Literatur Konzentrationen bis annähernd 20 ng/ml angegeben. Die Prolaktinspiegel sind zum Zeitpunkt der Geburt deutlich erhöht, sinken aber auf normale Erwachsenenwerte in weniger als 3 Monaten. Für Frauen werden geringfügig höhere Durchschnittswerte angegeben, mit einem leichten Anstieg während der Pubertät und ein entsprechender Abfall nach der Menopause, als für Männer. Während der Schwangerschaft steigen die Prolaktinwerte beständig bis zum 10- oder 20-fachen der Ausgangswerte an. Nach der Geburt fallen die Werte für nicht stillende Mütter innerhalb 3 Wochen auf ein normales Niveau. Bei stillenden Müttern ist der Rückgang auf normale Werte verzögert, da das Saugen des Kindes zu einem schnellen und dramatischen Anstieg der Prolaktinfreisetzung führt. Frauen die Kontrazeptiva einnehmen bzw. unter Östrogenbehandlung stehen, weisen ebenfalls erhöhte Prolaktinspiegel auf. In der Beurteilung geringfügiger Konzentrationsanstiege sollte immer berücksichtigt werden, daß Prolaktin ein Streßhormon ist. Nicht allein das Stillen sondern auch geringfügige Eingriffe wie Blutentnahme oder eine Anamnesebefragung, sind als Ursache für kurzfristige Konzentrationserhöhungen registriert 7

worden. Überdies ist die Freisetzung von Prolaktin von Natur aus episodisch. Von Tag zu Tag wurden Fluktuationen mit Variationskoeffizienten höher als 30% beobachtet. Schließlich gibt es schlafabhängige tägliche Variationen: Die Prolaktinkonzentrationen steigen während des Schlafes und erreichen ihre niedrigsten Werte wenige Stunden nach dem Aufwachen. Die Empfehlung Proben zwischen 9,00 und 12,00 Uhr zu entnehmen, beruht auf der Annahme „normaler“ Aufwachzeiten.

dem Kapitel "Alternative Probenarten" zu entnehmen.

Methodik

Reagenzien: Bei 2–8°C lagern. Unter Einhaltung der geltenden gesetzlichen Vorschriften entsorgen.

IMMULITE/IMMULITE 1000 Prolaktin ist ein Festphasen, Zwei-phasen Chemilumineszenz immunometrischer Assay. Inkubationszyklen: 1 × 30 minuten.

Probengewinnung Der Einsatz einer Ultrazentrifuge wird zur Klärung von lipämischen Proben empfohlen. Bei hämolysierten Proben besteht die Möglichkeit einer unsachgemäßen Handhabung vor Eintreffen im Labor, daher sind die Ergebnisse zurückhaltend zu interpretieren. EDTA-Plasma ist als Probenart nicht geeignet. Die Zentrifugation der Serumproben vor dem völligen Abschluss der Gerinnung kann zu Fibringerinnseln führen. Um fehlerhaften Analysenergebnissen infolge von Gerinnseln vorzubeugen, ist sicherzustellen, dass die Gerinnung vor der Zentrifugation der Proben vollständig abgeschlossen ist. Insbesondere Proben von Patienten unter Antikoagulantientherapie können eine verlängerte Gerinnungszeit aufweisen. Blutentnahmeröhrchen von verschiedenen Herstellern können differierende Werte verursachen. Dies hängt von den verwendeten Materialien und Additiven (Gel oder physische Trennbarrieren, Gerinnungsaktivatoren und /oder Antikoagulantien) ab. IMMULITE/IMMULITE 1000 Prolaktin sind nicht mit allen möglichen Röhrchenvariationen ausgetestet worden. Details der getesteten Röhrchenarten sind

8

Erforderliche Menge: 25 µl Serum. (Inhalt der Probenschale muss mindestens 100 µl über der erforderlichen Gesamtmenge liegen.) Lagerung: 7 Tage bei 2–8°C oder 3 Monate bei –20°C.

Hinweise und Vorsichtsmaßnahmen Zur In-vitro-Diagnostik.

Die generell geltenden Vorsichtsmaßnahmen sind einzuhalten und alle Komponenten als potenziell infektiös zu behandeln. Alle aus menschlichem Blut gewonnenen Materialien wurden auf Syphilis, Antikörper gegen HIV-1 und HIV-2, Hepatitis-B-Oberflächenantigen und Hepatitis-C-Antikörper untersucht und negativ befundet. Bestimmten Komponenten wurde Natriumazid (