Serum Total Prolactin and Monomeric Prolactin Reference Intervals Determined by Precipitation with Polyethylene Glycol: Evaluation and Validation on Common ImmunoAssay Platforms

Abstract Background: Macroprolactin is an important source of immunoassay interference that commonly leads to misdiagnosis and mismanagement of hyperprolactinemic patients. We used the predominant immunoassay platforms for prolactin to assay serum samples treated with polyethylene glycol (PEG) and establish and validate reference intervals for total and monomeric prolactin. Methods: We used the Architect (Abbott), ADVIA Centaur and Immulite (Siemens Diagnostics), Access (Beckman Coulter), Elecsys (Roche Diagnostics), and AIA (Tosoh) analyzers with samples from healthy males (n = 53) and females (n = 93) to derive parametric reference intervals for total and post-PEG monomeric prolactin. Concentrations of immunoreactive prolactin isoforms in serum samples from healthy individuals were established by gel filtration chromatography (GFC). We then used samples from 22 individuals whose hyperprolactinemia was entirely attributable to macroprolactin and 32 patients with true hyperprolactinemia to compare patient classifications and prolactin concentrations measured by GFC with the newly derived post-PEG reference intervals. Results: Parametric reference intervals for post-PEG prolactin in male and female serum samples, respectively, were (in mIU/L): 61–196, 66–278 (Centaur); 63–245, 75–381 (Elecsys); 70–301, 92–469 (Access); 72–229, 79–347 (Architect); 73–247, 83–383 (AIA); and 78–263, 85–394 (Immulite). Concordance between GFC and immunoassay-specific post-PEG reference intervals was observed in 311 of 324 cases and for 31 of 32 patients with true hyperprolactinemia and 17 of 22 patients with macroprolactinemia. Results leading to misclassification occurred in a few analyzers for 5 macroprolactinemia patient samples with relatively minor increases in post-PEG prolactin (mean 61 mIU/L). Conclusions: Our validated normative reference data for sera pretreated with PEG and analyzed on the most commonly used immunoassay platforms should facilitate the more widespread introduction of macroprolactin screening by clinical laboratories.

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Eġġ proteins in cod serum. Natural occurrence and induction by injections of oestradiol 3-benzoate

Biochemical Journal ◽

10.1042/bj1210847 ◽

1971 ◽

Vol 121 (5) ◽

pp. 847-856 ◽

Cited By ~ 89

Author(s):

P. A. Plack ◽

D. J. Pritchard ◽

N. W. Fraser

Keyword(s):

Molecular Weight ◽

Gel Filtration ◽

Double Diffusion ◽

Normal Serum ◽

Total Serum Protein ◽

Total Serum ◽

Natural Occurrence ◽

Serum Samples ◽

Male And Female ◽

Oestradiol Benzoate

1. Before the uptake of water that precedes spawning, eggs of cod (Gadus morhua L.) contained 30% dry matter, of which 80% was protein. Some 75% of this protein was soluble in 0.5m-sodium chloride. The major components in the extract were two similar lipoproteins, of molecular weight about 400000, containing 21% lipid, some two-thirds of which was phospholipid, and about 0.5% protein phosphorus. 2. These lipoproteins were identified by immunochemical methods in the serum of female cod with developing ovaries, but not in the serum of male or of immature female fish. 3. The concentrations of egg proteins in the serum of female cod were determined by a serial-dilution double-diffusion immunological method, and were shown to increase with development of the ovaries, reaching a value of about 32mg/ml when the weight of the ovaries was 10% of the weight of the fish. 4. Immature male and female cod were injected intramuscularly with a solution of oestradiol-17β 3-benzoate in oil and the concentration of egg proteins in their serum was measured by the immunodiffusion method. The serum contained no detectable egg proteins before injection of the fish, but 30μg of oestradiol benzoate/kg gave rise to detectable amounts of egg proteins in 10 days, and with 300μg or 1mg of oestradiol benzoate/kg the concentration of egg proteins rose to 32mg/ml. The values for male and female cod were similar and represented about one-half of the total serum protein. 5. With a dose of 1mg of oestradiol benzoate/kg, egg proteins were first detected in the serum 2 days after injection and the concentration increased up to 10 days. 6. Serum samples taken before and 10 days after an injection of 1mg of oestradiol benzoate/kg were fractionated by gel-filtration on Sephadex G-200. The difference curves obtained from fractionation curves after and before injection confirmed the values of the concentrations of egg proteins obtained from the immunodiffusion test and showed that the concentrations of the normal serum components fell by 20–50% of the initial value, the high-molecular-weight globulins showing the most marked fall. 7. Egg proteins were detected in the liver and testes of the injected fish, but not in the ovaries.

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A possible cause of the variable detectability of macroprolactin by different immunoassay systems

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2015-0484 ◽

2016 ◽

Vol 54 (4) ◽

Cited By ~ 5

Author(s):

Naoki Hattori ◽

Kohzo Aisaka ◽

Akira Shimatsu

Keyword(s):

Polyethylene Glycol ◽

Gel Filtration ◽

Protein G ◽

Normal Range ◽

Serum Samples ◽

Chemiluminescence Immunoassay ◽

Factors Influencing ◽

Group 2 ◽

Possible Cause ◽

Group 1

AbstractMacroprolactinaemia is a major cause of hyperprolactinaemia. The detectability of macroprolactin varies widely among different immunoassay systems, but the causes are not fully known. This study aimed to identify the factors influencing the detectability of macroprolactin by immunoassay systems.The study included 1544 patients who visited an obstetric and gynaecological hospital. Macroprolactinaemia was screened using the polyethylene glycol (PEG) method and confirmed using gel filtration chromatography and the protein G method. The prolactin (PRL) values determined by enzyme immunoassay (EIA) were compared with those of a chemiluminescence immunoassay system (Centaur) that is known to cross-react the least with macroprolactin.Macroprolactinaemia was found in 62 of 1544 patients (4.02%) who visited an obstetric and gynaecological hospital. The ratio of EIA-determined total PRL to free PRL in the supernatant after PEG precipitation was significantly elevated in all 62 serum samples with macroprolactin compared to those in 1482 serum samples without macroprolactin. In contrast, the ratio of Centaur-determined total PRL to free PRL was significantly elevated in 32 serum samples (group 1) and was within the normal range in 30 (group 2) of 62 serum samples with macroprolactin. The prevalence of non-IgG-type macroprolactin was significantly higher in group 1 than in group 2. Centaur diagnosed hyperprolactinaemia less frequently than EIA (n=2 vs. 16) in 62 patients with macroprolactinaemia. Those two hyperprolactinaemic patients diagnosed by Centaur had non-IgG-type macroprolactin.Macroprolactinaemia was present in 4% of patients visiting an obstetric and gynaecological hospital. The nature of macroprolactin (IgG-type or non-IgG-type) may partly explain why macroprolactin detectability varies among different immunoassay systems.

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Paediatric reference intervals for 17 Roche cobas 8000 e602 immunoassays in the CALIPER cohort of healthy children and adolescents

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2019-0707 ◽

2019 ◽

Vol 57 (12) ◽

pp. 1968-1979 ◽

Cited By ~ 2

Author(s):

Mary Kathryn Bohn ◽

Victoria Higgins ◽

Shervin Asgari ◽

Felix Leung ◽

Barry Hoffman ◽

...

Keyword(s):

Children And Adolescents ◽

Reference Values ◽

Laboratory Tests ◽

Clinical Decision Making ◽

Reference Intervals ◽

Healthy Children ◽

Serum Samples ◽

Clinical Laboratories ◽

Age And Sex ◽

Roche Cobas

Abstract Background The diagnostic utility of laboratory tests in paediatric medicine relies heavily on the availability of appropriate reference intervals (RIs). The Canadian Laboratory Initiative on Paediatric Reference Intervals (CALIPER) has established a comprehensive database of covariate-stratified RIs for many paediatric laboratory tests using a large, healthy reference population. Several automated analysers in widespread use in clinical laboratories have already been studied. Here, we extend the testing to Roche immunoassays and report, for the first time, comprehensive paediatric RIs for 17 endocrine and special chemistry markers. Methods A total of 741 healthy children and adolescents (1 day to <19 years) were recruited and serum samples were analysed for 17 immunoassays on the Roche cobas 8000 e602 Immunoassay Analyzer. Age and sex-specific RIs were established and corresponding 90% confidence intervals (CIs) were calculated in accordance with Clinical and Laboratory Standards Institute guidelines. Results Reference values for all analytes measured required age partitioning, particularly during early life and throughout adolescence. Of the 17 analytes measured, eight required sex partitioning, including ferritin, thyroid stimulating hormone (TSH), total triiodothyronine (TT3) and all fertility/sex hormones, except prolactin. Conclusions This is the first study to determine accurate paediatric RIs for Roche immunoassays. RIs were generally similar to those previously published by CALIPER on other analytical platforms, highlighting the reproducibility of age- and sex-specific trends in reference values observed across the paediatric age range. The RIs established in this study will improve the accuracy of test result interpretation and clinical decision-making in clinical laboratories utilising Roche immunoassays.

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Serum prolactin revisited: parametric reference intervals and cross platform evaluation of polyethylene glycol precipitation-based methods for discrimination between hyperprolactinemia and macroprolactinemia

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2016-0902 ◽

2017 ◽

Vol 55 (11) ◽

Cited By ~ 6

Author(s):

Martin Overgaard ◽

Susanne Møller Pedersen

Keyword(s):

Polyethylene Glycol ◽

Elevated Serum ◽

Gel Filtration ◽

Reference Interval ◽

Reference Intervals ◽

Serum Prolactin ◽

Specific Reference ◽

Screening Methods ◽

Discriminative Ability ◽

Discrimination Methods

AbstractBackground:Hyperprolactinemia diagnosis and treatment is often compromised by the presence of biologically inactive and clinically irrelevant higher-molecular-weight complexes of prolactin, macroprolactin. The objective of this study was to evaluate the performance of two macroprolactin screening regimes across commonly used automated immunoassay platforms.Methods:Parametric total and monomeric gender-specific reference intervals were determined for six immunoassay methods using female (n=96) and male sera (n=127) from healthy donors. The reference intervals were validated using 27 hyperprolactinemic and macroprolactinemic sera, whose presence of monomeric and macroforms of prolactin were determined using gel filtration chromatography (GFC).Results:Normative data for six prolactin assays included the range of values (2.5th–97.5th percentiles). Validation sera (hyperprolactinemic and macroprolactinemic; n=27) showed higher discordant classification [mean=2.8; 95% confidence interval (CI) 1.2–4.4] for the monomer reference interval method compared to the post-polyethylene glycol (PEG) recovery cutoff method (mean=1.8; 95% CI 0.8–2.8). The two monomer/macroprolactin discrimination methods did not differ significantly (p=0.089). Among macroprolactinemic sera evaluated by both discrimination methods, the Cobas and Architect/Kryptor prolactin assays showed the lowest and the highest number of misclassifications, respectively.Conclusions:Current automated immunoassays for prolactin testing require macroprolactin screening methods based on PEG precipitation in order to discriminate truly from falsely elevated serum prolactin. While the recovery cutoff and monomeric reference interval macroprolactin screening methods demonstrate similar discriminative ability, the latter method also provides the clinician with an easy interpretable monomeric prolactin concentration along with a monomeric reference interval.

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Multicenter performance evaluation of a second generation cortisol assay

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2016-0400 ◽

2017 ◽

Vol 55 (6) ◽

pp. 826-835 ◽

Cited By ~ 12

Author(s):

Michael Vogeser ◽

Jürgen Kratzsch ◽

Yoon Ju Bae ◽

Mathias Bruegel ◽

Uta Ceglarek ◽

...

Keyword(s):

Second Generation ◽

Reference Ranges ◽

Good Precision ◽

Healthy Individuals ◽

Serum Samples ◽

Intermediate Precision ◽

Coefficients Of Variation ◽

Liquid Chromatography Tandem Mass ◽

Cortisol Levels ◽

Roche Diagnostics

Abstract Background: Untreated disorders of the adrenocortical system, such as Cushing’s or Addison’s disease, can be fatal, and accurate quantification of a patient’s cortisol levels is vital for diagnosis. The objective of this study was to assess the analytical performance of a new fully-automated Elecsys® Cortisol II assay (second generation) to measure cortisol levels in serum and saliva. Methods: Four European investigational sites assessed the intermediate precision and reproducibility of the Cortisol II assay (Roche Diagnostics) under routine conditions. Method comparisons of the Cortisol II assay vs. liquid chromatography-tandem mass spectrometry (LC-MS/MS), the gold standard for cortisol measurement, were performed. Cortisol reference ranges from three US sites were determined using samples from self-reported healthy individuals. Results: The coefficients of variation (CVs) for repeatability, intermediate precision, and reproducibility for serum samples were ≤2.6%, ≤5.8%, and ≤9.5%, respectively, and for saliva were ≤4.4% and ≤10.9%, and ≤11.4%, respectively. Agreement between the Cortisol II assay and LC-MS/MS in serum samples was close, with a slope of 1.02 and an intercept of 4.473 nmol/L. Reference range samples were collected from healthy individuals (n=300) and serum morning cortisol concentrations (5–95th percentile) were 166.1–507 nmol/L and afternoon concentrations were 73.8–291 nmol/L. Morning, afternoon, and midnight saliva concentrations (95th percentile) were 20.3, 6.94, and 7.56 nmol/L, respectively. Conclusions: The Cortisol II assay had good precision over the entire measuring range and had excellent agreement with LC-MS/MS. This test was found suitable for routine diagnostic application and will be valuable for the diagnosis of adrenocortical diseases.

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Establishment of reference intervals of monomeric prolactin to identify macroprolactinemia in Chinese patients with increased total prolactin

BMC Endocrine Disorders ◽

10.1186/s12902-021-00861-z ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yao Hu ◽

Jiajin Ni ◽

Buyue Zhang ◽

Wei Cheng ◽

Huating Zhang ◽

...

Keyword(s):

Gel Filtration ◽

Reference Interval ◽

Reference Intervals ◽

Chinese Patients ◽

Clinical Feature ◽

Specific Reference ◽

Healthy Female ◽

Roche Diagnostics ◽

Improved Accuracy ◽

First Time

Abstract Background Macroprolactin is responsible for pseudohyperprolactinemia and is a common pitfall of the prolactin immunoassay. We aimed to determine the frequency of macroprolactinemia in Chinese hyperprolactinemic patients using monomeric prolactin discriminated by precipitation with polyethylene glycol (PEG). Methods Post-PEG monomeric prolactin gender-specific reference intervals were established for the Elecsys immunoassay method (Roche Diagnostics) using sera from healthy female (n = 120) and male (n = 120) donors. The reference intervals were validated using 20 macroprolactinemic (as assessed by gel filtration chromatography (GFC)) sera samples, and presence of monomeric prolactin was discriminated by GFC. Patients with high total prolactin were then screened by PEG precipitation to analyze macroprolactin. The demographic and biochemical details of patients with true hyperprolactinemia and macroprolactinemia were compared. Results Reference intervals for monomeric prolactin in females and males were 3.4–18.5 and 2.7–13.1 ng/mL, respectively. Among 1140 hyperprolactinemic patients, macroprolactinemia was identified in 261 (22.9 %) patients while the other 879 (77.1 %) patients were diagnosed with true hyperprolactinemia. Menstrual disturbances were the most common clinical feature in both groups. Galactorrhea, amenorrhea, and visual disturbances occurred more frequently in true hyperprolactinemic patients (P < 0.05). Conclusions The prevalence of macroprolactin in Chinese patients with hyperprolactinemia was described for the first time. Monomeric prolactin concentration, along with a reference interval screening with PEG precipitation, provides a diagnostic approach for hyperprolactinemia with improved accuracy.

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Establishment of Reference Intervals of Monomeric Prolactin To Identify Macroprolactinemia in Chinese Patients with Increased Total Prolactin

10.21203/rs.3.rs-590536/v1 ◽

2021 ◽

Author(s):

Yao Hu ◽

Jiajin Ni ◽

Buyue Zhang ◽

Wei Cheng ◽

Huating Zhang ◽

...

Keyword(s):

Gel Filtration ◽

Reference Interval ◽

Reference Intervals ◽

Chinese Patients ◽

Clinical Feature ◽

Specific Reference ◽

Healthy Female ◽

Roche Diagnostics ◽

Improved Accuracy ◽

First Time

Abstract Background Macroprolactin is responsible for pseudohyperprolactinemia and is a common pitfall of the prolactin immunoassay. We aimed to determine the frequency of macroprolactinemia in Chinese hyperprolactinemic patients using monomeric prolactin discriminated by precipitation with polyethylene glycol (PEG). Methods Post-PEG monomeric prolactin gender-specific reference intervals were established for the Elecsys immunoassay method (Roche Diagnostics) using sera from healthy female (n = 120) and male (n = 120) donors. The reference intervals were validated using 20 macroprolactinemic (as assessed by gel filtration chromatography (GFC)) sera samples, and presence of monomeric prolactin was discriminated by GFC. Patients with high total prolactin were then screened by PEG precipitation to analyze macroprolactin. The demographic and biochemical details of patients with true hyperprolactinemia and macroprolactinemia were compared. Results Reference intervals for monomeric prolactin in females and males were 3.4–18.5 and 2.7–13.1 ng/mL, respectively. Among 1140 hyperprolactinemic patients, macroprolactinemia was identified in 261 (22.9%) patients while the other 879 (77.1%) patients were diagnosed with true hyperprolactinemia. Menstrual disturbances were the most common clinical feature in both groups. Galactorrhea, amenorrhea, and visual disturbances occurred more frequently in true hyperprolactinemic patients (P < 0.05). Conclusions The prevalence of macroprolactin in Chinese patients with hyperprolactinemia was described for the first time. Monomeric prolactin concentration, along with a reference interval screening with PEG precipitation, provides a diagnostic approach for hyperprolactinemia with improved accuracy.

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The Origin of Reference Intervals

Archives of Pathology & Laboratory Medicine ◽

10.5858/2007-131-348-toori ◽

2007 ◽

Vol 131 (3) ◽

pp. 348-357 ◽

Cited By ~ 2

Author(s):

Richard C. Friedberg ◽

Rhona Souers ◽

Elizabeth A. Wagar ◽

Ana K. Stankovic ◽

Paul N. Valenstein

Keyword(s):

Pediatric Patients ◽

Statistical Tests ◽

Reference Intervals ◽

Thyroid Stimulating Hormone ◽

Slight Variation ◽

Healthy Individuals ◽

Site Testing ◽

Clinical Laboratories ◽

External Sources ◽

Calcium Magnesium

Abstract Context.—Standards have been developed for establishing reference intervals, but little is known about how intervals are determined in practice, interlaboratory variation in intervals, or errors that occur while setting reference intervals. Objectives.—To determine (1) methods used by clinical laboratories to establish reference intervals for 7 common analytes, (2) variation in intervals, and (3) factors that contribute to establishment of “outlier” intervals. Design.—One hundred sixty-three clinical laboratories provided information about their reference intervals for potassium, calcium, magnesium, thyroid-stimulating hormone, hemoglobin, platelet count, and activated partial thromboplastin time. Results.—Approximately half the laboratories reported conducting an internal study of healthy individuals to validate reference intervals for adults. Most laboratories relied on external sources to establish reference intervals for pediatric patients. There was slight variation in intervals used by the central 80% of study laboratories, but some laboratories outside the central 80% had surprisingly low and high limits for their reference intervals. In some cases the intervals used by 2 laboratories had no overlap. For example, one laboratory considered a hemoglobin of 13.8 g/dL in a woman to be “low” while another considered the same value to be “high.” Three percent of reference intervals contained a limit that qualified as an “outlier” using standard statistical tests; we could not identify any practice associated with adoption of outlier intervals. Conclusions.—Many laboratories adopt reference intervals from manufacturers without on-site testing of healthy individuals. Reference intervals used by facilities that forgo on-site testing are not statistically different from intervals validated with on-site studies.

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Variability in the detection of macro TSH in different immunoassay systems

Acta Endocrinologica ◽

10.1530/eje-15-0883 ◽

2016 ◽

Vol 174 (1) ◽

pp. 9-15 ◽

Cited By ~ 15

Author(s):

Naoki Hattori ◽

Takashi Ishihara ◽

Akira Shimatsu

Keyword(s):

Hormone Replacement Therapy ◽

Polyethylene Glycol ◽

Subclinical Hypothyroidism ◽

Hormone Replacement ◽

Elevated Serum ◽

Gel Filtration ◽

Cross Reactivity ◽

Serum Samples ◽

Gel Filtration Chromatography ◽

Serum Tsh

DesignMacro TSH is a large molecular-sized TSH that is mostly a complex of TSH and IgG. Patients with macro TSH have elevated serum TSH and normal free thyroxine levels, mimicking subclinical hypothyroidism. The aim of this study was to clarify the degree of cross-reactivity of macro TSH to different commercial immunoassay systems.MethodsScreening for macro TSH was done using a polyethylene glycol (PEG) method and confirmed with gel filtration chromatography in serum samples from 1901 patients with subclinical hypothyroidism. Interference due to human anti-mouse antibodies (HAMA) was examined using HAMA blockers. TSH was measured with an enzyme immunoassay for the analysis of macro TSH. Serum TSH values in patients with macro TSH were also determined with the widely used commercial immunoassay platforms Elecsys, Centaur and Architect, and the detectability of macro TSH was compared among them.ResultsGel filtration chromatography was performed with 174 serum samples with PEG-precipitable TSH ratios >75%. Twenty serum samples were found to contain large molecular-sized TSH, five of which were due to interference by HAMA. The prevalence of macro TSH was eventually 0.79% (15/1901). Commercial immunoassay systems variably recognized macro TSH. The Architect TSH immunoassay platform was the least reactive to macro TSH, but still recognized it in 60% of macro TSH-containing serum samples.ConclusionsThere were no commercial TSH immunoassay platforms that did not cross-react with macro TSH. Screening for macro TSH should be performed before hormone replacement therapy is initiated for subclinical hypothyroidism.

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