Reference intervals for eight enzymes in blood of adult females and males measured in accordance with the International Federation of Clinical Chemistry reference system at 37 degrees C: part of the Nordic Reference Interval Project.

As part of the Nordic Reference Interval Project we present reference intervals for alanine transaminase (ALT), aspartate transaminase (AST), creatine kinase (CK), lactate dehydrogenase (LD), alkaline phosphatase (ALP), gamma-glutamyltransferase (GT), amylase (AMY) and pancreatic type of AMY in blood of adult males and females. A total of 3036 reference persons, all of whom considered themselves to be in good health, were recruited by 102 Nordic clinical biochemical laboratories. Exclusions were undertaken on the basis of predefined biochemical and clinical criteria. Enzyme activities in serum and plasma were measured in the different laboratories using various commercially available routine measurement systems at 37 degrees C. Only results obtained with the International Federation of Clinical Chemistry (IFCC) compatible measuring systems were selected for estimation of the enzyme reference intervals. The final number of results on each enzyme varied from 459 (LD) to 2300 (ALT). The 2.5 and 97.5 percentile reference limits were calculated by a non-parametric method in accordance with the IFCC recommendations, using the Refval 4.0 data program. Statistical partitioning testing was undertaken to decide whether the reference intervals ought to be partitioned according to gender and/or age. For most of the enzymes, but not for all, the upper reference limits were found to be higher than those that have been in general use until now.

Spermatogenesis after unilateral orchiectomy for testicular cancer in patients following surveillance policy.

PURPOSE: Only limited information is available on the natural course of spermatogenesis in patients with testicular cancer who underwent unilateral orchiectomy and surveillance. We analyze long-term exocrine function of the remaining testicle in patients following surveillance policy. MATERIALS AND METHODS: Sperm counts and serum follicle-stimulating hormone (FSH) levels were available in 60 nonrelapsing cases approximately 3 weeks (baseline), 1 year and 2 years or greater after orchiectomy. Contralateral testicular cancer subsequently developed in 2 men. RESULTS: At baseline 36 patients were normospermic (10 or greater x 106/ml.), 7 were azoospermic and 17 were oligospermic. After 1 year 45 patients were normospermic. Mean sperm concentrations increased significantly from 26 to 39 x 106/ml. during year 1 after orchiectomy. Elevated serum FSH at baseline was associated with incomplete recovery of spermatogenesis, although sperm counts improved in 3 of 7 patients. Furthermore, in the 2 initially oligospermic patients with subsequent contralateral testicle cancer transient normospermia was observed after 1 year. After orchiectomy fatherhood was recorded in 28 men and was assisted by fertilization using fresh semen in 2. CONCLUSIONS: In nonrelapsing testicular cancer cases on surveillance, initially reduced spermatogenesis recovers during year 1 after orchiectomy especially if baseline serum FSH is normal. Transient recovery also occurs in patients in whom contralateral testicular cancer subsequently develops. In high risk patients and in initially oligospermic patients with plans for future fatherhood, the period of improved spermatogenesis may be used for multiple semen cryopreservations enabling subsequent assisted fertilization.

Prediction of posttreatment spermatogenesis in patients with testicular cancer by flow cytometric sperm chromatin structure assay.

The hypothesis to be tested was that abnormal sperm chromatin structure is related to disturbed spermatogenesis in patients with testicular cancer. After orchiectomy but before further treatment ("pretreatment"), semen samples from 39 patients with testicular cancer were analyzed for sperm concentration by light microscopy and by the sperm chromatin structure assay (SCSA). In 28 patients assessment of sperm concentration was repeated 12-26 months after orchiectomy ("posttreatment"). The pretreatment SCSA results for the patients were compared to those from 18 healthy semen donors and assessed for correlation with the patients' posttreatment sperm concentration. Twenty-three patients displayed an abnormal chromatin structure in their pretreatment sample. For the nine evaluable patients on the surveillance program, the pretreatment SCSA results were not correlated with the posttreatment concentration. The results from 19 evaluable patients undergoing cytotoxic treatment (radiotherapy, 13; chemotherapy, 6) indicate that posttreatment recovery of spermatogenesis (recovery in 4 of 5 patients) is observed more often in patients with a normal pretreatment chromatin structure than in those with abnormal SCSA values before treatment (recovery in 2 of 14 patients; P = 0.02). The results of SCSA display sperm characteristics beyond those of light microscopically assessed sperm concentration. Pretreatment SCSA results might help clinicians to identify those testicular cancer patients with a high risk of long-lasting posttreatment disturbance of spermatogenesis.

In vitro stability of the reticulocyte count.

A collaborative study was undertaken to investigate the effect of storage conditions on the measured reticulocyte count in venous blood samples. It was designed to cover variation in the three main determinants, namely time, temperature and anticoagulant. The aim was to determine the time for which samples could be stored for subsequent analysis for clinical purposes.

Indicators of haematopoietic recovery after bone marrow transplantation: the role of reticulocyte measurements.

The aim of this project was to study haematological recovery in patients following different types of bone marrow transplantation (BMT). Forty-three patients were analysed. The duration of haematopoietic suppression following conditioning regimens was much shorter in 12 peripheral blood stem cell transplantation patients than in 19 allogeneic BMT patients and in 12 autologous BMT patients. Among 12 different variables, the parameters with the highest specificity or predictive value for monitoring recovery in these patients were the absolute neutrophil count (ANC) at the classical cut-off point of 0.5 x 10(9)/l, an absolute reticulocyte count (RET) above 20 x 10(9)/l and a high fluorescent reticulocyte fraction (HFR) above 5%,. It is suggested that these parameters be used as the reference measurements for monitoring haematological recovery in similar studies. Among these variables, the HFR fraction was the earliest and most sensitive index of engraftment in 79.1% of patients, HFR recovery requiring a median time of 13 days after infusion, in comparison with a median period of 19 and 18 days, respectively, for RET and ANC (P<0.0001).

Evaluation of monocyte counting with two automated instruments by the use of CD 14-specific immunomagnetic Dynabeads.

Accurate determination of monocytes can be a problem with automated haematological analysers as well as with visual microscopy. We have compared monocyte counts from our two routine automated instruments, Technicon H*1 and Sysmex NE-1500. In the NE-1500 the monocyte counts appear to increase with increasing concentrations of neutrophils, compared with the H*1 results. The accuracy of the monocyte counts was assessed using Dynabeads M-450 CD14. These uniform magnetizable polystyrene beads coated with monoclonal antibody specific for CD14 membrane antigen isolated monocytes from the blood samples. The influence of this depletion on the monocyte counts and histograms from the two instruments and by visual microscopy were compared. On average 90% of the monocytes counted by visual microscopy were removed from six blood samples. In the same samples the beads removed an average of 75% of cells identified as monocytes by the H*1 but only 49% of those identified as monocytes by the NE-1500. These Dynabeads can be a useful tool in evaluating monocyte identification in different haematological situations and for manufacturers developing technology for automated leucocyte differential counting.

Clinical and biochemical long-term toxicity after postoperative cisplatin-based chemotherapy in patients with low-stage testicular cancer.

In this case-control study the long-term chemotherapy-related morbidity was assessed in testicular cancer patients (> or = 5 years after treatment). The case group consisted of 47 patients with nonseminomatous testicular cancer who had undergone retroperitoneal surgery and 3-4 cycles of conventional-dose cisplatin-based chemotherapy (accumulated cisplatin dose: 300-400 mg/m2), also containing vinblastine and bleomycin. The results of the clinical and biochemical investigations and the patients' responses to a questionnaire were compared to similar observations from a control group comprising 47 patients treated with surgery only. In the case group the serum magnesium values and the levels of FVIIvWF were significantly lower (but within the normal range) than in the control group. Serum cholesterol values were distributed similarly in both groups. About 40% of the patients from the chemotherapy group still suffered from Raynaud-like phenomena and/or sensoric neurotoxicity as compared to 10% of the patients from the control group. Chemotherapy resulted in a significant elevation of serum LH within the normal range, probably associated with a slight subclinical Leydig cell dysfunction. Serum FSH levels were similar in the case and control groups, indicating an absence of long-term disturbance of spermatogenesis after chemotherapy. No increased cardiovascular morbidity was observed as a result of chemotherapy. Raynaud-like phenomena and sensoric neurotoxicity represent the principal clinical long-term side effects after 3-4 cycles of cisplatin-based chemotherapy containing vinblastine and bleomycin. Serum LH is slightly elevated after chemotherapy, whereas long-term spermatogenesis seems to be unaffected.

Experiences of the Janus Serum Bank in Norway.

The ongoing JANUS project was started in 1973. The serum bank comprises 424,938 serum samples consolidated from 293,692 donors. The specimens are stored at -25 degrees C. From 1 to 13 consecutive samples are available from each donor. Up to October 1993 about 14,000 of the donors had developed some form of cancer. Frozen serum samples collected from a few months to 19 years prior to clinical recognition of their disease are available for research purposes. The principle aim of the JANUS project is to search in the premorbid sera for chemical, biochemical, immunological or other changes that might be indicative of cancer development at early stages. Gas chromatography-mass spectrometry and two-dimensional protein electrophoresis have been used to evaluate the stability of the frozen sera. Some recent findings are: CA-125 may be elevated months prior to the diagnosis of ovarian cancer; serum thyroglobulin may be a preclinical tumor marker in subgroups of thyroid cancer; low levels of selenium in serum reflects increased risk of thyroid cancer; raised antibodies in serum against Epstein-Barr virus is a risk factor for development of Hodgkins disease; prostate-specific antigen may be elevated years prior to clinical diagnosis of prostate cancer; and linoleic acid in serum phospholipids is inversely related to breast cancer risk. The serum bank is, in principle, suitable for environmental studies, e.g., human exposure assessment. The steering committee of the JANUS project is open to suggestions for collaborative research on this topic.

[Tumor marker analyses--valuable tools if used correctly]. / Tumormarkøranalyser--nyttig verktøy ved riktig bruk.

The tumour marker assays in routine use today are not cancer-specific. Most markers are normal products of epithelial tissues, usually excreted from the intact organ. The infiltrating malignant growth allows release directly into lymph and blood, where the product is measured as a "tumour marker". Many tumour markers are glycoproteins depending on the liver function for their catabolism. These mechanisms explain the slight to moderate increases in tumour marker values found in various non-malignant diseases. Therefore, in general, such assays are of no use for screening healthy populations, and a normal value does not exclude cancer. Their main use is in the primary staging of patients known to have cancer, to evaluate the completeness of primary surgery, to follow-up patients for early detection of relapses, and to monitor the effect of cytotoxic or radiation therapy of advanced disease. In all these applications, good clinical judgement is the necessary basis for using these assays.

Pre-treatment DNA ploidy of sperm cells as a predictive parameter of post-treatment spermatogenesis in patients with testicular cancer.

OBJECTIVE: To evaluate DNA flow cytometry (DNA FCM) as a means of predicting post-treatment spermatogenesis in patients with testicular cancer. SUBJECTS AND METHODS: After unilateral orchidectomy and before further treatment (pre-treatment), light microscopic sperm cell analysis and FCM were performed in 96 patients in whom post-treatment sperm cell counts were available > or = 2 years after therapy. Twenty-nine patients had not received any cytotoxic treatment after orchidectomy, whereas 64 had received either abdominal radiotherapy [37] or 3-4 cycles of cisplatin-based chemotherapy [27]. Three patients had both chemotherapy and radiotherapy. A control group consisted of 26 healthy age-matched men. RESULTS: In the pre-treatment situation 43% of the patients had low sperm counts (< 10 x 10(6)/ml). A median of 38 months after treatment the median sperm cell density had increased significantly, independent of treatment, but was still less than that of the control group. All control specimens, but only 66% of the patients' ejaculates, contained cells of which more than 90% were classified as being condensed DNA haploid sperm cells. The median percentages of cells classified as non-condensed DNA haploid, as DNA diploid or as being above DNA diploid cells were increased in the patients' samples as compared with those of the controls, both before and after treatment. CONCLUSION: Spermatogenesis is quantitatively and qualitatively impaired in patients with newly diagnosed testicular cancer. Long-term recovery is relatively independent of routine treatment but is related to the pre-treatment sperm cell density. For the whole series of patients DNA FCM parameters did not yield independent parameters which predicted recovery of spermatogenesis. In patients in whom no sperm cells were found by light microscopy of the seminal fluid the demonstration of > or = 30% condensed DNA haploid cells was correlated with post-treatment recovery of spermatogenesis.

Host markers and prognosis in recurrent rectal carcinomas treated with radiotherapy.

The value of blood tests as prognostic factors in patients with recurrent rectal carcinomas treated with radiotherapy was studied in one retrospective (n = 114, 1976-1984) and one prospective (n = 100, 1985-1989) group of patients. The retrospective group was used for validation of the results from the prospective group. In univariate survival analyses, 19 of totally 38 variables significantly correlated to the survival. Of 13 significant blood parameters, lactate dehydrogenase (LD), erythrocyte sedimentation rate (ESR), alpha 1-, alpha 2-globulin, fibrinogen, carcinoembryonic antigen (CEA), C-reactive protein (CRP), haptoglobin, granulocytosis and thrombocytosis were the most important ones (p < or = 0.01). In the multivariate analyses (Cox regression) of the prospective group, LD, alpha 1-globulin, diagnosed liver metastases and CEA were found to be significant predictors of survival. A prognostic index was derived from the prospective group including ESR, LD and relapse-free interval. This clearly separated the patients in the retrospective group into one low- and one high-risk group.

The JANUS serum bank.

The on-going JANUS project was initiated by the Norwegian Cancer Society in 1973. The serum bank comprises close to 0.5 million serum samples collected from 170,000 donors. From 2-16 consecutive samples are available from each donor. The sera are stored at -25 degrees C. At regular intervals the JANUS-collection is matched against the files of the Norwegian Cancer Registry. From 1973 to 1991 almost 5000 of the donors have developed some form of cancer. Frozen serum samples collected from a few months to 18 years prior to clinical recognition of their disease are consequently available for research purposes. The aim of the JANUS-project is to search in these premorbid sera for chemical, biochemical, immunological or other changes that might be indicative of cancer development at early stages. Gas chromatography-mass spectrometry and two-dimensional protein electrophoresis have been used to evaluate the stability of the frozen sera. Some recent findings are: CA125 is elevated several months prior to diagnosis of ovarian cancer; serum thyroglobulin may be a preclinical tumour marker in subgroups of thyroid cancer; low level of selenium in serum reflects increased risk of thyroid cancer; and raised antibodies in serum against Epstein-Barr virus is a risk factor for development of Hodgkin's disease. On-going research includes trace elements and cancer, and studies on lipid-profiles, diet and cancer. The serum bank may in principle be used for other purposes, e.g. in environmental studies. Analysis of sequential sera may determine chemical substances in the sera that might reflect differences in exposure to environmental pollutants in the period 1973-1991.

[Reticulocytes--new possibilities with automated counting]. / Retikulocytter--automatisert telling gir nye muligheter.

A fully automated reticulocyte counter, Sysmex R-1000 has been evaluated at three Norwegian hospitals. The instrument measures fluorescence-labelled RNA in the reticulocytes by flow cytometry. It also generates information on reticulocyte maturity. Instrument precision was superior to that achieved with the routine visual counting method. Samples preserved at room temperature remain stable for one day, whereas samples preserved at 5 degrees C remain stable for four days. An increasing number of reticulocytes is often the first sign of marrow regeneration after bone marrow transplantation or treatment of leukaemia with cytostatica. In patients with chronic renal failure who are receiving erythropoietin, the increase in reticulocytes is seen within days, and precedes the increase in haemoglobin by 2-3 weeks.

Dry reagent technology. Kodak Ektachem 700 XR in clinical enzymology.

The comparability of results for enzyme activity concentrations estimated with the methods recommended by the Scandinavian Committee on Enzymes(SCE) or the subcommittee on Enzymes of the European Committee for Clinical Laboratory Standards(ECCLS), and with the Kodak Ektachem methods, are discussed. As the Ektachem system in principle uses the IFCC methods at 37 degrees C as a reference, the results compare reasonably well for the alanine amino-transferase(ALAT), aspartate aminotransferase(ASAT), creatine kinase(CK) and gamma-glutamyltransferase(GT). The Ektachem reference method for lactate dehydrogenase(LD) is in principle the SCE method. The reasons for lower values with the SCE method are discussed. The interference on the Ektachem alkaline phosphatase (ALP) method from high concentrations of methotrexate(MTX) in serum, is shown. Results from our investigations on the interference by heparin in enzyme determinations on Ektachem are given. The problems with CK determinations in cancer patients regarding myocardial diseases are well known. When CK-BB is present in serum, the CK-B concentration is apparently lower using the Ektachem CK method than with use of the CK-MB slide, probably relating to the fact that chelator is omitted from the CK slide, while EGTA is supplied in the CK-MB slide. The user's dependence on the manufacturer for the choice of methods and the quality of the reagents, makes it important for laboratories and the Kodak Company to collaborate with the intention to continuously improve the Ektachem methodologies. The future possibilities of this technology seem to be nearly unlimited.

Follow-up of breast cancer patients stage I-II: a baseline strategy.

In 430 stage I-II breast cancer patients the cost-benefit of investigations during follow-up have been studied. Median follow-up time was 8 years and 128 patients had relapsed, 91 with metastatic disease. High costs of routine chest X-ray, limited skeletal X-ray and bone scan examinations were associated with low incidence of diagnosed relapses not suspected otherwise. In the eight blood analyses examined, increases of more than 10 mm/h in erythrocyte sedimentation rate (ESR), 20 U/l in gamma-glutamyltransferase (GT) or 60 U/l in alkaline phosphatase (ALP) resulted in a combined sensitivity of 55% and specificity of 91% for relapses with distant metastases. Elevation of at least two blood tests gave a combined sensitivity of 31% and a specificity of 98%. The importance of using individual reference values in screening for recurrences is emphasised. Symptomatic relapse or relapse detected at interval visits were not independent prognostic factors. The blood tests ALP, ESR and GT were strong predictors of survival measured from relapse which increase their legitimacy in follow-up. A more frequent follow-up for patients with 4+ involved nodes is proposed: three visits annually the first 5 years vs. two visits annually for the others. We conclude that history, clinical examination, ALP, ESR and GT are sufficient as a baseline screening for relapse in breast cancer patients.

Continuous intravenous interleukin-2 infusion and subcutaneous interferon-alpha in metastatic renal cell carcinoma.

In a phase II trial patients with metastatic renal cell carcinoma (MRCC) received two induction cycles each consisting of 24-h intravenous infusions of interleukin-2 (IL-2) 18 x 10(6) U/m2/day and interferon (IFN) 3 x 10(6) U/m2/day given subcutaneously on days 1-5 and 8-12 of a 2-week cycle. Between cycles 1 and 2 there was a 3-week treatment-free interval. Maintenance therapy consisted of four monthly cycles of IL-2 and IFN. Due to considerable toxicity the trial was prematurely closed after inclusion of 16 of 23 scheduled patients. Three partial responses were observed. Nine events of severe or life-threatening side-effects occurred and 8 patients were transferred to the intensive care unit. The combination of continuous intravenous high-dose infusions of IL-2 and subcutaneously given IFN is moderately effective, but too toxic for routine treatment of MRCC.

Safety and tolerability of iodixanol in healthy volunteers with reference to two monomeric X-ray contrast media.

The low osmolar, non-ionic X-ray contrast media have shown a lower frequency of adverse events than the older ionic ones. In this study changes in routine clinical-chemical parameters in blood and urine, vital signs and adverse events were recorded in six groups of 10 healthy male volunteers receiving either iodixanol, a new non-ionic, dimeric X-ray contrast medium for general vascular use, or one of the two non-ionic, monomeric contrast media iopentol and iopamidol. Minor decreases were observed in the values for haemoglobin, haematocrit and erythrocytes 5 min and 3 days after injection of iodixanol. A minor increase was seen in platelets and total protein after 3 days. A transient increase in serum osmolality was seen 5 min after the injections of iopentol and iopamidol. This was not seen in any iodixanol group. The level of thyrotropin showed an increase in all groups at 3 days. It was back to normal within 21 days. No changes of clinical importance were seen regarding blood pressure, heart rate or ECG in any volunteer. No severe adverse events were reported. All events were of short duration, and of mild or moderate intensity. The results, however, may indicate a lower frequency of adverse events/discomfort after the administration of the dimeric iodixanol than the 2 monomeric contrast media iopentol and iopamidol.

Glomerular filtration rate, 131I-hippuran clearance and estimated creatinine clearance in cancer patients.

Glomerular filtration rate (GFR), 131I-Hippuran clearance and estimated creatinine clearance were investigated in 34 patients with cancer. For Hippuran clearance and GFR, analysed with the X-ray contrast (iohexol) and fluorescence technique, the least square linear regression coefficient was 5.01 +/- 0.41 (r = 0.91). This value concurs with the five to one ratio between GFR and renal plasma flow known from normal physiology and supports that Hippuran clearance is a valid measure of renal function. When the individual values of Hippuran clearance were divided by 5.01, the mean difference between the methods was 0.4 ml min-1 1.73 m-2 with standard deviation 13.4 ml min-1 1.73 m-2. The lower and upper limits of agreement were -26.7 and 25.9 ml min-1 1.73 m-2, respectively. Comparing creatinine clearance estimated from the serum creatinine level with GFR, the limits of agreement were -29.4 and 21.6 ml min-1 1.73 m-2. These agreement limits are in the same range as those which can be calculated from the data from other studies.