Fertility Diagnostics
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1 Fertility Diagnostics Fertility hormones measured on PATHFAST For internal use only Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 1
2 Content: page 1. Fertility hormones - general aspects 1.1 Reproductive system of females Marker system in blood Assays on the PATHFAST analyzer 5 2. Fertility hormones - details 2.1 Follicle stimulation hormone (FSH) Luteinizing hormone (LH) Estradiol (E2) Progesteron Testosterone Prolactin (PRL) hcg Fertility hormones - measurement on PATHFAST 3.1 Immunological test principle Details of test performance Survey of abbreviations 39 Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 2
3 1.1 Fertility hormones Reproductive system of females Hormonal regulation - Follicular and luteal phase Follicle-stimulating hormone (FSH) stimulates the growth and recruitment of immature follicles in the ovary Luteinizing hormone (LH) triggers ovulation Estradiol (E2) reflects the activity of the ovaries Progesterone stimulates the development of the fetus Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 3
4 1.2 Fertility hormones Marker system in blood Measurement of hormone concentration Performance of hormone assays clinical laboratory Basis of measuring system heterogeneous immunoassay Measuring principle Ag-Ab reaction with a bound/free separation step Ag-Ab complex is bound to the tube wall or to magnetic particles The specific antibody is marked with an enzyme The enzyme triggers a biochemical reaction Measurement photometrical or with Chemiluminescent technology see PATHFAST Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 4
5 1.3 Fertility hormones Assays on the PATHFAST analyzer Portfolio (origin) FSH (Pituitary gland) LH (Pituitary gland) Estradiol (Ovaries and corpus luteum) Progesterone (Follicle and corpus luteum) Testosterone (Testes) Prolactin (Pituitary gland) hcg preg (Developing embryo and placenta) Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 5
6 2.1 Follicle stimulating hormone (FSH) Follicular phase of monthly cycle (28 days) Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 6
7 2.1.1 Follicle stimulating hormone (FSH) - release Brain Pituitary gland FSH is produced and secreted by the gonadotropes in the pituitary gland and interacts with the FSH receptor Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 7
8 2.1.2 Follicle stimulating hormone (FSH) - effect Follicles Ovary Effect in females: FSH regulates the development, growth and recruitment of immature follicles in the ovary Seminiferous tubules Testis Corpus luteum Epididymis Effect in males: FSH together with Testosterone cause the Sertoli cells in the Seminiferous tubules to stimulate sperm production The sperm become mature in the Epididymis In both - females and males - FSH stimulates the maturation of germ cells Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 8
9 2.1.3 Follicle stimulating hormone (FSH) - assay Specification of the FSH assay on PATHFAST : Assay range: miu/ml Measuring time: 26 minutes Standardization: traceable to WHO 2nd IPR 94/632 Specificity: LH, TSH, HCG, Prolactin and hpl: not detectable Imprecision / total CV in %: QC-L = 3.7%, QC-M = 4.0%, QC-H = 5.1% Correlation* vs. Elecsys: y = 0.940x+ 2.21, r = 0.985, n = 88 Correlation* vs. VIDAS: y = 0.930x , r = 0.965, n = 88 * Diagram of method comparison page 10 Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 9
10 2.1.4 Follicle stimulating hormone (FSH) - method comparison PATHFAST vs. Elecsys PATHFAST vs. VIDAS PATHFAST miu/ml y = 0.940x r = n = 88 PATHFAST miu/ml y = 0.930x r = n = ECLusys miu/ml VIDAS miu/ml Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 10
11 2.2 Luteinizing hormone (LH) Luteal phase of monthly cycle - LH maximum at time of ovulation Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 11
12 2.2.1 Luteinizing hormone (LH) - release Hypothalamus Pituitary gland Ovaries Testes Biochemical background: The release of LH from the gonadotropic cells of the pituitary gland is controlled by pulses of the Gonadotropin-releasing Hormone (GnRH) from the hypothalamus. LH is transported via the blood stream to the ovaries (to stimulate the growth and maturation of follicles) and to the testes (to stimulate the production of testosterone) Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 12
13 2.2.2 Luteinizing hormone (LH) - effect egg Effect in females An acute rise of LH (the LH surge) triggers ovulation at about day 14 in the monthly cycle and induces formation of the corpus luteum Detection of the LH rise (with immunological tests) is useful for persons with the intension of conceiving Effect in males LH stimulates the Leyding cells of the testes to release the male sex hormone Testosterone Section through seminiferous tubule of the testes Leyding or Interstitial cells In both females and males LH is essential for reproduction Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 13
14 2.2.3 Luteinizing hormone (LH) - assay Specification of the LH assay on PATHFAST : Assay range: miu/ml Measuring time: 26 minutes Standardization: traceable to WHO 2nd IS 80/552 Specificity: FSH, HCG, HGH and hpl: n.d., TSH: 0.12% Imprecision / total CV in % : QC-L = 4.1%, QC-M = 4.8%, QC-H = 5.6% Correlation* vs. Immulite 2000: y = 1.00x , r = 0.985, n = 112 Correlation* vs. Elecsys: y = 0.932x , r = 0.990, n = 112 * Diagram of method comparison page 15 Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 14
15 2.2.4 Luteinizing hormone (LH) - method comparison PATHFAST vs. Immulite 2000 PATHFAST vs. Elecsys LH LH PATHFAST miu/ ml y = 1.00x r = n = 112 PATHFAST miu/ ml y = 0.932x 1.64 r = n = Immulit e2000 miu/ ml ECLusys miu/ ml Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 15
16 2.3 Estradiol (E2) Follicular and luteal phase of monthly cycle (28 days) Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 16
17 2.3.1 Estradiol (E2) - release E2 derives from Cholesterol Estradiol is the most potent naturally occurring estrogen, produced in the gonads (ovaries and testes) but also in the brain and in arterial walls Steroid hormone Estradiol Testosterone in men and women can be converted to Estradiol Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 17
18 2.3.2 Estradiol (E2) - effects egg whole in follicle filled with liquid mature follicle stimulates E2 production Sustentacular or Sertoli cells Section through the seminiferous tubule of the testis Effect in females: Estradiol (E2) reflects primarily the activity of the ovaries E2 acts as a growth hormone for tissue of the reproductive organs E2 concentration during fertility therapy is an important basis for the monitoring of IVF treatment E2 is the most important indicator to define timeframe of IVF Effect in males: The effect of estradiol upon male reproduction is complex Estradiol is produced in the Sertoli (Sustentacular) cells of the testes There is evidence that estradiol prevents apoptosis (cell death) of male germ cells In both females and males FSH stimulates the maturation of germ cells Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 18
19 2.3.3 Estradiol (E2) - assay Specification of the E2 assay on PATHFAST : Assay range: pg/ml Measuring time: 26 minutes Standardization: traceable to CRM 576, 577,578, LC-MS/MS Specificity: Progesterone, Testosterone and Estradiol < 1% Imprecision / total CV in %: QC-L = 9.4%, QC-M = 9.5%, QC-H = 9.1% Correlation* vs. Immulite 2000: y = 1.031x , r = 0.982, n = 53 * Diagram of method comparison page 20 Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 19
20 2.3.3 Estradiol (E2) - method comparison PATHFAST vs. Immulite 2000 (preliminary data) 2,500 2,000 y = 1.031x r = n = 53 PF (pg/ml) 1,500 1, Immuiyze (pg/ml) Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 20
21 2.4 Progesterone Luteal phase of monthly cycle (28 days) Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 21
22 2.4.1 Progesterone - release and regulation Progesterone Progesterone is a steroid hormone It is mainly formed of in the follicle wall and in cells of the corpus luteum A rise in the progesterone level is observed one day prior to ovulation Steroid hormone Progesterone Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 22
23 2.4.2 Progesterone - effect Effect in females Increased synthesis of progesterone occurs during luteal phase It prepares the uterus for implantation of the embryo It prevents further maturation of follicles in case of fertilization If pregnancy does not occur Progesterone level decreases In case of pregnancy, progesterone is produced in the placenta Process of follicle growth, ovulation and formation of corpus luteum: Corpus luteum Egg (Primary oocyte) Egg (Secondary oocyte) Corpus luteum triggers release of Progesterone Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 23
24 2.4.3 Progesterone - assay Specification of the Progesterone assay on PATHFAST : Assay range: ng/ml Measuring time: 26 minutes Standardization: traceable to USP Specificity: Corticosterone, Testosterone, Estradiol and others < 1% Imprecision / total CV in %: QC-L = 24.2%, QC-M = 6.6%, QC-H = 3.4% Correlation* vs. Elecsys: y = 0.997x , r = 0.983, n = 28 * Diagram of method comparison page 25 Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 24
25 2.4.4 Progesterone - method comparison PATHFAST vs. Elecsys (preliminary data) 30 y = 0.997x r = n = 28 T PATHFAS ECLusys 2010 Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 25
26 2.5.1 Testosterone - release Testosterone - like other steroid hormones (e.g. E2) - derives from Cholesterol The largest amount of testosterone is produced by the testes in men in the Interstitial or Leyding cells Section through the seminiferous tubule of the testes Interstitial or Leyding cells Testosterone is also synthesized in women, however in far smaller quantities Testosterone in men and women can be converted to Estradiol Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 26
27 2.5.2 Testosterone - effects Anabolic effect Growth of muscle mass and strength Impact on bone density and strength Stimulation of growth and bone maturation Activation of androgen receptors Virilizing effect Maturation of sex organs (prenatal) Biological development of sex differences (postnatal) e.g. deepening of voice, growth of the bird Development of sperm Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 27
28 2.5.3 Testosterone - assay Specification of the Testosterone assay on PATHFAST : Assay range: ng/ml Measuring time: 26 minutes Reference range: x.xx - x.xx (2.5th th percentile) Standardization: traceable to USP Specificity: Corticosterone, Cortisol, Cortisone and others <1% (see insert) Imprecision / total CV: <1 ng/ml = <20%, 3-7 ng/ml <15.0%, 10 ng/ml <10% Correlation vs. Immulite 2000: on trial a = 1+0.1, r > 0.95, n = xx Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 28
29 2.6.1 Prolactin (PRL) - release Prolactin is a peptide hormone (199 amino acids) Synthesized & secreted in the pituitary gland by so-called lactotrope cells Secretion is regulated in the hypothalamus Release is stimulated by PRH (Prolactin Releasing Hormone), mainly during the night Release is stimulated by breastfeeding (breast is filled with milk lactogenesis) Release is inhibited by PRIH (Prolactin Release Inhibiting Hormone) = Dopamine, mainly during the day Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 29
30 2.6.2 Prolactin (PRL) - effects Females - Stimulation of the mammary glands to produce milk (lactation) - Contribution to surfactant synthesis of the fetal lungs (end of pregnancy) - Contribution to immune tolerance of the fetus by the maternal organism Males and females Unusually high amounts of PRL are indicator for impotence and loss of libido High concentration of Prolactin decreases normal levels of sex hormones (Estrogen in women and Testosterone in men) Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 30
31 2.6.3 Prolactin (PRL) - assay Specification of the Prolactin assay on PATHFAST : Assay range: ng/ml Measuring time: 26 minutes Standardization: traceable to WHO 3rd IS 84/500 Specificity: LH, TSH, FSH, HCG, hpl: n.d., HGH: 0.02% Imprecision / total CV in%: QC-L = 3.3%, QC-M = 4.1%, QC-H = 4.4% Correlation* vs. Architect: y = 0.091x , r = 0.985, n = 98 Correlation* vs. Elecsys: y = 1.08x , r = 0.989, n = 100 * Diagram of method comparison page 32 Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 31
32 2.6.3 Prolactin (PRL) - method comparison PATHFAST vs. Architect PATHFAST vs. Elecsys PATHFAST y = 0.991x r = n = PATHFAST PRL ng/ ml y = 1.08x r = n = Architect ECLusys PRL ng/ml Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 32
33 2.7.1 Human Chorionic Gonadotropin (hcg) - release hcg is a glycoprotein hormone (244 amino acids) It is produced in pregnancy - by the developing embryo after conception - and later on by the placenta hcg is also produced by some kinds of tumor Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 33
34 2.7.2 Human Chorionic Gonadotropin (hcg) - role and effect hcg = marker for early detection of pregnancy Indication of presence of an implanted embryo Maintenance of the corpus luteum during the beginning of pregnancy, causing it to produce/secrete Progesterone hcg = tumor marker hcg is used in the tumor diagnosis Embryo - 16 cell stage hcg has a high similarity with LH It can be used clinically for fertility treatment to trigger ovulation in the ovaries produce Testosterone in the testes Structure of a glycoprotein hormone alpha -polypeptide Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 34
35 2.7.3 Human Chorionic Gonadotropin (hcg) preg - assay Specification of the hcg preg assay on PATHFAST : Assay range: miu/ml Measuring time: 26 minutes Standardization: traceable to WHO 4th IS 75/589 Specificity: FSH, Prolactin: n.d., LH: 0.76%, TSH: 0.11% Imprecision / total CV in%: QC-L = 2.4%, QC-M = 2.7%, QC-H = 2.7% Correlation* vs. PATHFAST HCG: y = 1.028x , r = 0.992, n = 106 Correlation* vs. VIDAS: y = 0,994x , r = 0.984, n = 106 * Diagram of method comparison page 36 Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 35
36 2.7.4 Human Chorionic Gonadotropin (hcg) preg - method comparison PATHFAST HCG preg vs. PATHFAST HCG PATHFAST HCG preg. vs. VIDAS PATHFAST HCG preg(miu/ml) PATHFAST HCG preg (miu/ml) PATHFAST HCG (miu/ml) minividas (miu/ml) Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 36 36
37 3.1 Principle of immunological reaction on PATHFAST : Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 37
38 3.2 Measurement of fertility markers (hormones) on PATHFAST : Details of assay & test performance: Enzyme-immunoassay Ag-Ab-Ag (sandwich principle) Use of monoclonal antibodies Chemiluminescent technology with magnetic particles Sample material: 100 µl whole blood, hep. plasma, serum 6 samples and/or tests simultaneously per run Turn Around Time per assay 26 min Traceability to reference standard or method is given Kit size 60 tests per kit 2 calibrators in kit low and high Calibration frequency 4 weeks Controls available from Biorad Reagent stability 12 months Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 38
39 4. Survey of abbreviations: FSH Follicle Stimulating Hormone E2 Estradiol hcg human Chorionic Gonadotropin LH Luteinizing Hormone LTH Luteotropic Hormone PRL Prolactin PRH Prolactin Releasing Hormone PRIH Prolactin Release Inhibiting Hormone PoC Point of Care Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 39
40 Thanks for your attention Diagnostics PATHFAST Chemiluminescence-immuno-analyzer 40
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