Immunoassay for trypsinogen-4.

Trypsin has been identified as a pancreatic protease comprising three isoenzymes, trypsin-1, -2, and -3. However, the gene for trypsinogen-3, PRSS3, also gives rise to additional variants, trypsinogen-4A and B, which differ from trypsinogen-3 only with respect to the leader-peptide part, and when activated are identical to trypsin-3. The unique overlapping leader peptides of trypsinogen-4A and B allowed us to develop a specific sandwich-type immunofluorometric assay that detects both these isoforms, but not trypsinogen-3 or activated trypsinogen-4. We measured the concentrations of trypsinogen-4 in various cell line lysates and bile of primary sclerosing cholangitis patients. Lysates of cell lines MDA-MB-231 and PC-3, and astrocytes contained trypsinogen-4, while the conditioned media from these cells did not, suggesting that trypsinogen-4, lacking a classical signal sequence, is not secreted from the cells. Interestingly, 5.7% of the 212 bile samples analyzed contained measurable (>2.4 µg/l) trypsinogen-4. In conclusion, we have established a specific assay for trypsinogen-4 and demonstrated that trypsinogen-4 can be found in biological samples. However, the clinical utility of the assay remains to be established.

UriSed 3 PRO automated microscope in screening bacteriuria at region-wide laboratory organization.

BACKGROUND AND AIMS: We assessed the possibility to rule out negative urine cultures by counting with UriSed 3 PRO (77 Elektronika, Hungary) at Helsinki and Uusimaa Hospital District. MATERIALS AND METHODS: Bacteria counting of the UriSed 3 PRO automated microscope was verified with reference phase contrast microscopy against growth in culture. After acceptance into routine, results of bacteria and leukocyte counting from 56 426 specimens with eight UriSed 3 PRO instruments were compared against results from parallel samples cultured on chromogenic agar. Laboratory data including preanalytical details were accessed through the regional database of the Helsinki and Uusimaa Hospital District. RESULTS: A combined sensitivity of 87-92% and a negative predictive value of 90-96% with a specificity of 54-50% was reached, depending on criteria. Preanalytical data (incubation time in bladder) combined with the way of urine collection would improve these figures if reliable. CONCLUSIONS: Complex patient populations, regional logistics and data interfases, and economics related to increased costs of additional particle counts against costs of screening cultures of all samples, did not support adaptation of a screening process of urine cultures. This conclusion was made locally, and may not be valid elsewhere.

Verification of UriSed 3 PRO automated urine microscope in regional laboratory environment.

BACKGROUND AND AIMS: Ten UriSed 3 PRO automated microscopes (77 Elektronika, Hungary) were verified for nine HUSLAB laboratories with 160 000 annual urine samples. MATERIALS AND METHODS: Particle counting of the primary UriSed 3 PRO instrument (77 Elektronika, Hungary) was verified against reference visual microscopy with 463 urine specimens, and against urine culture on chromogenic agar plates with parallel 396 specimens. Nine secondary instruments were compared pairwise with the primary instrument. RESULTS: Relative imprecisions compared to Poisson distribution, R(CV), were estimated to be 1.0 for white blood cell (WBC) and 1.5 for red blood cell (RBC) counts, respectively. Spearman's correlations against visual microscopy were rS = 0.94 for WBC, rS = 0.87 for RBC, and rS = 0.82 for squamous epithelial cell (SEC) counts. Agreement with visual microscopy (Cohen's weighted kappa) was 0.94 for WBC, 0.89 for RBC, 0.88 for SEC, 0.59 for combined casts, and 0.49 for non-squamous epithelial cells (NEC). Bacteria were detected with a sensitivity of 90% and specificity of 39 against culture at 107 CFB/L (104 CFU/mL). Created flagging limits allowed automated reporting for 70-75% of patient results. CONCLUSIONS: UriSed 3 PRO instruments were adopted into routine use after acceptance of the verification.

Dramatic increase in serum trypsinogens, SPINK1 and hCGß in aortic surgery patients after hypothermic circulatory arrest.

The concentrations of several diagnostic markers have been found to increase dramatically in critically ill patients with a severe disturbance of normal physiological homeostasis, without indication of the diseases they are normally associated with. To prevent false diagnoses and inappropriate treatments of critically ill patients, it is important that the markers aiding the selection of second-line treatments are evaluated in such patients and not only in the healthy population and patients with diseases the markers are associated with. The levels of trypsinogen isoenzymes, the trypsin inhibitor serine peptidase inhibitor Kazal type 1 (SPINK1), hCG and hCGß, which are used as pancreatitis and cancer markers, were analyzed by immunoassays from serum samples of 17 adult patients who have undergone surgery of the ascending aorta during hypothermic circulatory arrest (HCA) with optional selective cerebral perfusion. Highly elevated levels of trypsinogen-1, -2 and -3, SPINK1 and hCGß were observed in patients after HCA. This was accompanied by increased concentrations of S100ß and NSE. In conclusion, this study highlights the importance of critically evaluating the markers used for aiding selection of second line of treatments in critically ill patients.

Tumor expression of human chorionic gonadotropin beta mRNA and prognosis of prostate cancer treated by radical prostatectomy.

The beta subunit of human chorionic gonadotropin (hCGß) is encoded by six genes (CGB) classified as type I and type II. CGB mRNA is produced in large amounts by trophoblastic tissues and in small amounts by several cancerous tissues including prostate cancer and by a few benign tissues, including the prostate. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) was used to study the expression levels of all CGB mRNAs together (total CGB mRNA) and the two types of CGB mRNA separately in non-cancerous (n = 74) and cancerous prostatic tissue obtained by radical prostatectomy (n = 193). RNA was isolated from formalin-fixed paraffin-embedded (FFPE) samples and mRNA levels of CGB were correlated with disease-specific survival. Total CGB mRNA concentrations were significantly lower (p < .0001) in cancerous than non-cancerous prostatic tissue. Separate analysis of type I CGB and type II CGB mRNA showed that both type I CGB (p < .0001) and type II CGB mRNA (p = .007) are lower in cancerous tissue than in non-cancerous tissue. Low type II CGB mRNA level in cancerous tissue was associated with shorter cancer-specific survival (p = .001) of prostate cancer patients treated by radical prostatectomy.

Expression of human chorionic gonadotropin in testicular germ cell tumors.

BACKGROUND: We have shown that most patients with seminomas have elevated serum concentrations of the free ß subunit of human chorionic gonadotropin (hCGß) and that in nonseminomatous testicular cancer, most of the hCG in the serum is hyperglycosylated (hCG-h). However, the tissue expression of hCG-h or hCGß in germ cell tumors (GCTs) has not been reported. Our objective was to study the expression and diagnostic value of hCG-h and hCGß in testicular GCTs. METHODS: We studied the immunohistochemical expression of hCG, hCG-h, hCGß, and the free α subunit of hCG (hCGα) in GCTs from 154 patients. We compared the tissue expression with serum concentrations and evaluated the correlation between staining intensity, established prognostic variables, and outcome. RESULTS: The expression varied between tumor types. All forms of hCG, including hCG-h, were detected in embryonal carcinomas (22%) and mixed GCTs (48%). Polyclonal hCG and monoclonal hCGß antibodies detected immunoreactivity in some seminomas (7%). No form of hCG was found in spermatocytic seminomas, pure teratomas, or a yolk sac tumor. The serum concentrations correlated with the corresponding tumor expression. The staining intensities of hCG, hCGß, hCG-h, and hCGα correlated with disease stage but not significantly with relapse, disease-related mortality, or progression-free survival. CONCLUSION: Trophoblastic tissue expresses hCG, hCG-h, and free subunits together whereas seminoma tissue occasionally expresses hCGß. This difference might aid in differential diagnosis of some difficult-to-classify cases.

Hyperglycosylated human chorionic gonadotropin in serum of testicular cancer patients.

BACKGROUND: Hyperglycosylated human chorionic gonadotropin (hCG-h) contains larger and more complex carbohydrate chains than regular human chorionic gonadotropin (hCG). hCG-h is thought to be the major form of hCG produced by testicular cancers and it has been suggested to play a key role in tumor invasion, but studies on hCG-h in testicular cancer are limited. We studied whether serum hCG is hyperglycosylated, and whether measurement of hCG-h in serum offers clinical value in the management of testicular cancer. METHODS: We determined the serum concentrations of hCG-h, hCG, and the free ß subunit of hCG (hCGß) by time-resolved immunofluorometric assays in 176 serum samples (preoperative n = 67, relapse n = 20, follow-up n = 89) obtained from 84 testicular cancer patients. We analyzed the association between preoperative serum concentrations of hCG, hCG-h, and hCGß with known prognostic factors and progression-free survival time. RESULTS: A major proportion of hCG was hyperglycosylated preoperatively, at relapse, and shortly after treatment. The serum concentrations of hCG-h and hCG correlated strongly with each other and had similar diagnostic value. The preoperative serum concentration of hCG-h correlated with prognostic factors and outcome in the same way as hCG. Increased preoperative hCGß concentration predicted shorter progression-free survival. CONCLUSIONS: Most of the hCG expressed by testicular cancers is hyperglycosylated and therefore it is important that hCG assays used for management of testicular cancer recognize hCG-h.

Loss of human chorionic gonadotropin in urine during storage at -20°C.

BACKGROUND: Quantitative determination of human chorionic gonadotropin (hCG) in urine is used in population studies of pregnancy disorders and in doping control. For these purposes samples are often stored at -20°C, which in our experience may cause loss of hCG. METHODS: We redetermined hCG in 17 pregnancy urine samples stored at +4, -20 and -80°C for 3-10 months and in 74 cancer urine samples stored at -20°C for 5-14 years. We further studied the effect of added urea, pH and four preservatives on hCG immunoreactivity during storage at +4, -20 and -80°C. RESULTS: At -20°C, twenty to 100% of hCG immunoreactivity was lost in 15 of 17 pregnancy urine samples and in 43 of 74 cancer urine samples. At -20°C, added urea (0.2-1.0 mol/L) caused a rapid decrease in hCG immunoreactivity, which glycerol (5-10%) prevented. CONCLUSION: hCG immunoreactivity is lost in many urine samples during storage at -20°C. Urea most likely plays a role in this process, but other mechanisms contribute to the loss. Urine should not be stored at -20°C before analysis of hCG.

Free beta-subunit of human chorionic gonadotropin in serum is a diagnostically sensitive marker of seminomatous testicular cancer.

BACKGROUND: We studied whether measurement of the free beta subunit of human chorionic gonadotropin (hCGbeta) in serum offers additional diagnostic information compared to determination of intact hCG alone in testicular cancer. METHODS: We determined hCG and hCGbeta with ultrasensitive assays in 94 serum samples obtained preoperatively, 22 samples obtained during relapse, and 3687 samples obtained during routine follow-up of 351 patients with testicular tumors. RESULTS: In preoperative samples, isolated increases of hCGbeta were seen in 40% of the samples from seminoma patients (n = 42) and in 8% of those from patients with nonseminomatous testicular cancer (NSGCT) (n = 51). Both markers were increased in 12% of the seminoma and 71% of the NSGCT patients and were within reference intervals in 43% of the seminoma and 20% of the NSGCT patients. Specific determination of hCGbeta increased the frequency of marker-positive seminomas from 17% to 57% and of marker-positive relapses from 32% to 59% (n = 22). Theoretically, about 40% of marker-positive seminomas and relapses would have been missed with an assay measuring hCG and hCGbeta together. Preoperative hCG and hCGbeta concentrations correlated with stage, tumor histology, and disease-related mortality. Additionally, hCGbeta correlated with tumor size. CONCLUSIONS: hCGbeta is a diagnostically sensitive marker for testicular cancer. In patients with seminomatous testicular cancer, hCGbeta is superior to hCG, and in some NSGCT patients it provides additional information.