U.-H. Stenman

In vitro stability of free prostate-specific antigen (PSA) and prostate-specific antigen (PSA) complexed to α1-antichymotrypsin in blood samples

OBJECTIVES To study the in vitro stability of free and complexed forms of prostate specific antigen (PSA) in blood samples in order to establish guidelines for specimen handling, in particular for the clinical utility of the analysis of percentage free PSA. METHODS Blood samples were collected and processed to generate serum, heparin plasma, and EDTA plasma. Three different two-site immunoassays were used to measure the concentrations of total PSA (PSA-T), free form of PSA (PSA-F), and PSA-alpha 1-antichymotrypsin complex (PSA-ACT) in order to determine the effect of repeated freezing and thawing, delayed separation of serum from blood cells, and stability during storage at 4 degrees C and 30 degrees C. RESULTS Five cycles of freezing and thawing introduced no statistically significant changes in the measured concentrations of PSA-T, PSA-F, or PSA-ACT. The effect of storing blood samples at room temperature for 1-6 h before separation of serum revealed a statistically significant decrease only for PSA-F after 5.5 h of storage (mean decrease 3.5%). PSA-T and PSA-ACT showed good stability in both serum and plasma samples, whereas PSA-F, after 1 week of storage at 4 degrees C, decreased on average by 28.8%, 7.8%, and 5.6%, respectively, in serum, heparin plasma, and EDTA plasma. The decreases of PSA-F at 4 degrees C were statistically significant (P < 0.05) relative to the controls (samples stored at -20 degrees C) after storage for 23 h in serum, 86 h in heparin plasma, and 71 h in EDTA plasma. When the same samples were stored at 30 degrees C for 24 h, only the mean decrease of PSA-F (4.8%) in serum was statistically significant. CONCLUSIONS PSA-F in blood samples is less stable than PSA-ACT. It is not advisable to store samples on the clot, especially if time and temperature cannot be controlled. Serum samples should be stored frozen if not analyzed during the same day. After thawing, samples can be stored up to 23 h at 4 degrees C prior to analysis. The use of plasma samples improves the stability of free PSA.

The ISOBM TD-7 Workshop on hCG and Related Molecules

The ISOBM TD-7 hCG Workshop was established to characterize the molecular epitope structure and specificities of a panel of diagnostically relevant monoclonal antibodies (MAbs) directed against human chorionic gonadotropin (hCG) and its derivatives, and to consider how this information could be used to improve comparability of immunoassay results for these analytes. In this multicenter study, 27 MAbs have been characterized in detail as to their main and fine specificities by direct binding-, competitive- and sandwich-RIA, -ELISA, BIAcore® and Western blotting. Antigens used in the study included the upcoming first WHO reference reagents for immunoassay, i.e. nick-free hCG (hCG), nicked hCG (hCGn), hCG α-subunit (hCGα), hCG β-subunit (hCGβ), nicked hCG β-subunit (hCGβn), hCG β-core fragment (hCGβcf), synthetic peptides of hCGβ C-terminal peptide (hCGβCTP), and homologous hormones, luteinizing hormone (LH) and subunits (LHβ) from various species. Correct classification of blinded internal controls demonstrated the reliability of the MAb referencing approach. Three-dimensional molecular epitope assignment was possible in many instances by comparing immunoreactivity of the ISOBM MAbs (n = 27) to a large panel of MAbs (n = 18) previously well characterized in the Innsbruck (P.B.) and Paris (J.M.B.) laboratories. All three major antibody specificities (α, n = 1; β, n = 21; αβ, n = 5) were represented in the TD-7 MAb panel. HCGβ MAbs could further be subdivided into (i) those recognizing hCGβ only (epitopes: β6, n = 1; β7, n = 2; β14, n = 1) and (ii) those recognizing hCGβ + hCG (β1, β2, β4, β5, n = 10; β8 and β9, n = 9). Members of the latter group were specific either for hCG + hCGβ + hCGβcf (β1, n = 3) or hCG + hCGβ + hCGβCTP (β8, n = 6; β9, n = 1) or in addition to hCG + hCGβ + hCGβcf recognized hLH/hLHβ to a minor (β2, n = 3; β4, n = 3) or similar degree (β5, n = 1). Epitopes were (i) located on the first and third loops protruding from the cystine knot of hCGβ (β2–β6, aa hCGβ20–25 and 68–77), (ii) presumably centered around the knot itself (β1), or (iii) on hCGβCTP (epitope β8 = hCGβ141–144, β9 = hCGβ113–116). The ISOBM panel of MAbs represents all major epitope specificities suitable for the design of specific sandwich immunoassays. High analyte variability in serum and urine during the course of pregnancy and tumor development favors certain epitope combinations. For routine diagnostic purposes, assays recognizing a broad spectrum of hCG/hCGβ variants such as hCG + hCGn + hCGβ + hCGβn + hCGβcf + –CTPhCG + –CTPhCGβ may be useful. Low cross-reactivity against related glycoprotein hormones (e.g. hLH) and their derivatives is mandatory. These criteria are best met by combinations of MAbs directed against epitopes located around the cystine knot (β1) and against those encompassing the top of loops 1 and 3 on hCGβ (β2, β4). The first WHO reference reagents for immunoassay of hCG and hCG-related molecules being prepared by the IFCC should facilitate characterization of what assays for ‘hCG’ are measuring. The next step towards improving between-laboratory comparability of measurements of hCG/hCG derivatives in pregnancy and oncology is provided by results of this TD-7 Workshop.

Summary Report of the TD-3 Workshop: Characterization of 83 Antibodies against Prostate-Specific Antigen

Twelve research groups participated in the ISOBM TD-3 Workshop in which the reactivity and specificity of 83 antibodies against prostate-specific antigen (PSA) were investigated. Using a variety of techniques including cross-inhibition assays, Western blotting, BIAcore, immunoradiometric assays and immunohistochemistry, the antibodies were categorized into six major groups which formed the basis for mapping onto two- and three-dimensional (2-D and 3-D) models of PSA. The overall findings of the TD-3 Workshop are summarized in this report. In agreement with all participating groups, three main antigenic domains were identified: free PSA-specific epitopes located in or close to amino acids 86–91; discontinuous epitopes specific for PSA without human kallikrein (hK2) cross-reactivity located at or close to amino acids 158–163; and continuous or linear epitopes shared between PSA and hK2 located close to amino acids 3–11. In addition, several minor and partly overlapping domains were also identified. Clearly, the characterization of antibodies from this workshop and the location of their epitopes on the 3-D model of PSA illustrate the importance of selecting appropriate antibody pairs for use in immunoassays. It is hoped that these findings and the epitope nomenclature described in this TD-3 Workshop are used as a standard for future evaluation of anti-PSA antibodies.

Low-dose oral methotrexate with expectant management of ectopic pregnancy

Objective To investigate recovery times and need for laparoscopy in women with ectopic pregnancy who were treated for 5 days 2.5 mg/day of oral methotrexate or placebo. Methods Sixty women with ectopic pregnancy among patients of an outpatient clinic specializing in early pregnancy disorders were selected for medical treatment in a double-blind, placebo-controlled study. The diagnosis was made by transvaginal sonography and serum hCG determinations, either at admission or after repeated examinations. Women were recruited for the study if they had mild symptoms: the hCG increase was less than 50% within 2 days, the diameter of the ectopic pregnancy was less than 40 mm, there were no signs of intra-abdominal bleeding by transvaginal sonography, and there were no secondary reasons for laparoscopy. Either 2.5 mg of methotrexate or placebo was given orally for 5 days. Serum hCG was determined after 2 days, and hCG, red blood cell count, white blood cell count, platelet count, and serum glutamic-oxaloacetic transaminase were measured; transvaginal sonography was performed after 5 and 12 days. Expectant management was continued individually with check-ups at 1–3-week intervals. Laparoscopy was performed if the patient developed abdominal pain or intra-abdominal hemorrhage, as seen by transvaginal sonography. Statistical analysis was by paired or unpaired t test, Mann-Whitney U test, regression analysis, and repeated measures analysis of variance. Results Seventy-seven percent of the patients recovered without the need for laparoscopy in both groups, and there were no significant differences in recovery times or the need for laparoscopy between groups. Conclusion Oral methotrexate, 2.5 mg for 5 days, does not appear to be more effective than placebo in the treatment of ectopic pregnancy in women eligible for expectant management.

Tumor-associated trypsin inhibitor in normal and malignant renal tissue and in serum of renal-cell carcinoma patients.

Tumor‐associated trypsin inhibitor (TATI) is a 6‐kDa peptide, which is identical to the pancreatic‐secretory‐trypsin inhibitor (PSTI). TATI is produced by several tumors and cancer cell lines, and is used as a serum marker for mucinous ovarian cancer. Elevated serum levels of TATI have also been observed in renal‐cell carcinoma (RCC). However, it is unclear whether the increase of serum TATI in this disease is caused by production of TATI by the tumor tissue, by the acute‐phase reaction frequently associated with cancer, or by impaired renal function. We examined the expression of TATI in malignant and histologically normal renal tissue by immunohistochemistry, in situ hybridization and reverse‐transcriptase‐polymerase‐chain reaction (RT‐PCR). Furthermore, we measured pre‐operative serum TATI levels in 21 patients with RCC. Immunohistochemically, TATI was detected in 13 of 20 histologically normal renal‐tissue samples, but not in 32 tissue samples from RCC. By RT‐PCR, TATI mRNA was detected in all of 10 histologically normal kidneys and in 6 of 11 RCCs, while in situ hybridization analysis gave negative results. Pre‐operative serum TATI was elevated in 57% of RCC patients. We also studied expression of TATI mRNA and protein in 7 renal‐cancer cell lines, by RT‐PCR and immunofluorometric assay respectively: 6 cancer cell lines were positive for TATI mRNA, while 4 of them also produced TATI protein at low levels. These results indicate that TATI is synthesized by the histologically normal renal tissue and by some renal cancers, and suggest that the elevation of serum TATI associated with renal‐cell carcinoma may be caused by the release of TATI produced by the tumor. Int. J. Cancer 83:486–490, 1999.

Reactivity of 77 antibodies to prostate-specific antigen with isoenzymes and complexes of prostate-specific antigen.

Seventy-seven antibodies submitted to the ISOBM TD-3 PSA Workshop (TD-3.1 and TD-3.2) were characterized by measuring their reactivity with isoenzymes of free prostate-specific antigen (PSA), PSA complexed to α1-antichymotrypsin (PSA-ACT) and α1-proteinase inhibitor (PSA-API). Antibodies were classified into 15 distinct groups according to their reaction profiles with the various isoenzymes. Some antibodies recognizing both free and complexed PSA were inaccurate in measuring total PSA. Eight of the 9 free PSA-specific antibodies cross-reacted more with PSA-API than with PSA-ACT, while 1 antibody reacted less with PSA-API than PSA-ACT. From the panel of antibodies 39 reacted with both free and complexed PSA and were classified as total PSA antibodies.

Preoperative Serum hCGβ as a Prognostic Marker in Primary Fallopian Tube Carcinoma

Objectives: It was the aim of this study to evaluate the prognostic value of the pretreatment serum concentrations of the β-subunit of human chorionic gonadotropin (hCGβ), CA 125 and tumour-associated trypsin inhibitor (TATI) in primary fallopian tube carcinoma (PFTC). Methods: The pretreatment serum concentrations of hCGβ, CA 125 and TATI were analyzed in serum samples from 60 women with a mean age of 61 years, treated for PFTC between 1985 and 2000. Of the 91 patients treated during this period, 31 were excluded because no serum sample was available. The patients were followed-up for recurrence and survival until February 14, 2003. The prognostic value of the serum markers were compared with those of stage, grade and histological type. Results: The median survival time was 27 months and the overall 5-year survival rate 33%. Stage and size of the residual tumour (<1 vs. ≧1 cm) predicted both overall and disease-free survival (p < 0.050). Histology (serous vs. others) (p = 0.023) also influenced overall survival. Overall 5-year survival was 38% when serum hCGβ was below 3.5 pmol/l, while it was 18% when the level was higher (p = 0.052). The corresponding disease-free 5-year survival was 38 and 20%, respectively (p = 0.014). Patients with CA 125 values above 1,017 kU/l had an overall 5-year survival of 39% as compared with 14% for those with lower values (p = 0.009), while the disease-free survival was 37 and 23%, respectively (p = 0.096). Serum TATI was not a prognostic marker. Serum concentrations of hCGβ and CA 125 correlated significantly with stage (p = 0.049 and p = 0.050, respectively). In multivariate Cox proportional hazards regression analysis, only hCGβ, stage and histology emerged as independent prognostic factors. Conclusions: Clearly elevated serum concentrations of hCGβ and CA 125 predict survival in fallopian tube carcinoma, but in multivariate analyses, only hCGβ is a prognostic factor independent of stage and histology.

Quality assessment for prostate-specific antigen (PSA) in relation to ERSPC: report of the PSA Committee

To assess the application of a quality control scheme for total prostate‐specific antigen (PSA) as used for participants of the European Randomized Study for Screening of Prostate Cancer (ERSPC) during 1996–2002.

Reactivity of Anti-PSA Monoclonal Antibodies with Recombinant Human Kallikrein-2

Seventy-nine monoclonal antibodies submitted to the ISOBM TD-3 PSA Workshop were tested for their reactivity with recombinant human kallikrein-2 (rhK2). A sandwich immunofluorometric assay using polyclonal anti-prostate-specific antigen (PSA) antiserum-coated plates was used to capture rhK2 and subsequently the test antibody. The response of each test antibody was compared with 3 reference antibodies (H50, H117 and 5E4) known to react with hK2. Nine antibodies from the workshop panel failed to react with purified PSA and rhK2 in this assay and were subsequently excluded. From the remaining panel of antibodies, 11/70 showed strong reactivity with rhK2, 9/70 showed weak reactivity with rhK2, while 50/70 antibodies did not react with rhK2 in this assay format. All antibodies binding to rhK2 recognized both free and complexed PSA.