Multicenter analytical performance evaluation of a fully automated anti-Müllerian hormone assay and reference interval determination.

OBJECTIVES: Anti-Müllenria hormone (AMH) is an established biomarker for assessing ovarian reserve and predicting response to controlled ovarian stimulation. Its routine clinical use is hampered by the variability and low-throughput of available enzyme-linked immunosorbent assays (ELISA). The presented study examined if a fully automated AMH electrochemiluminescence assay (ECLIA; Elecsys® AMH assay, Roche Diagnostics) was suitable for measuring AMH levels in healthy women and in those diagnosed with polycystic ovary syndrome (PCOS). DESIGN AND METHODS: Five European laboratories evaluated the Elecsys® AMH assay independently under routine conditions over eight months. Within-run imprecision, repeatability, intermediate precision, linearity and functional sensitivity were assessed. The Elecsys® AMH assay was compared to a manual ELISA microtiter plate format test (AMH Gen II ELISA, modified version; Beckman Coulter Inc.) using 1729 routine serum samples. AMH reference intervals were determined in 887 healthy women with regular menstrual cycle aged 20­50 years, and 149 women diagnosed with PCOS. RESULTS: The fully automated Elecsys® AMH assay showed excellent precision, linearity, and functional sensitivity. The coefficient of variation was 1.8% for repeatability and 4.4% for intermediate precision. Values measured with the Elecsys® AMH assay were highly correlated with the manual ELISA method (modified version) but 24­28% lower. Reference intervals showed the expected AMH decline with age in healthy women and increased AMH levels in women with PCOS. CONCLUSIONS: The Elecsys® AMH assay demonstrated good precision under routine conditions, and is suitable for determining AMH levels in serum and lithium-heparin plasma.

Low-dose mistletoe lectin-I reduces melanoma growth and spread in a scid mouse xenograft model.

This study investigates the effects of mistletoe lectin-I (ML-I) on melanoma growth and spread in vivo. The human melanoma cell line MV3 was xenografted into severe combined immunodeficient mice and vehicle solution or purified ML-I was administered at 30, 150 and 500 ng per kg body weight (20 mice per group) daily. After 19 days, mice were killed, primary tumours (PTs) and lungs were dissected out, and tumour weights, number of lung metastases (LMs), number of tumour-infiltrating dendritic cells (DCs), and apoptosis rates in the melanoma cells and in the DCs were assessed. A 35% reduction of PT weight (P=0.03) and a 55% decrease in number of LMs (P=0.016) were evident for low-dose ML-I (30 ng kg(-1)) treatment but not for higher doses. Mistletoe lectin-I increased apoptosis rates in the melanoma cells of PTs at all doses, while no induction of apoptosis was noted in the LMs. Low-dose ML-I significantly increased the number of DCs infiltrating the PTs (P<0.0001) and protected DCs against apoptosis, while higher doses induced apoptosis in the DCs (P<0.01). Our results demonstrate that low-dose ML-I reduced melanoma growth and number of metastases in vivo, primarily due to immunomodulatory effects.

[Typical bacteria in an intensive care burn unit in severely burned patients and their importance with regard to mortality: retrospective study 1995 - 2004]. / Typische Bakterien auf einer Schwerbrandverletztenintensivstation und ihre Bedeutung für die Mortalität - Retrospektive Studie 1995 - 2004.

The purpose of this study was to identify risk profiles for wound infection of severely burned patients in a retrospective analysis of patients of an intensive care burn unit during 1995 - 2004. The goal of this study was to identify risk factors on wound infection in severely burned patients. Possible influences on mortality were to be discussed. Inclusion criteria of the study population was a minimum age of 18 years and a body surface area burned of at least 40 % during the time period 1995 - 2004. 912 patients were screened and 96 patients were enrolled. Logistic regression was performed to investigate factors influencing wound infection and mortality in the study population. The initially detectable bacteria in the burn wounds were Staphylococcus aureus (21.1 %), Staphylococcus epidermidis (16.2 %) and Enterococcus faecalis (16.2 %). Of all swabs taken the most frequent initial discovered bacteria were Staphylococcus aureus (18.2 %), Staphylococcus epidermidis (12.7 %), Enterococcus faecalis (12.7 %) and Escherichia coli (13.3 %). The majority of positive swabs were the burn wound followed by nose and tracheal secretion. The risk of a wound infection was more likely in the period 2000 - 2004 in comparison to 1995 - 1999 with an Odds Ratio of 0.17 (95 % KI [0.05 - 0.63], p = 0.008). Wound infection was promoted by longer hospitalization on the burn intensive care unit with an Odds Ratio of 2.62 (95 % KI [1.34 - 5.11], p = 0.005) and by bacterial detection in the unburned parts of the body with an Odds Ratio of 5.36 (95 % KI [1.30 - 22.24], p = 0.02). Death was significantly promoted by age (over 50 years) with an Odds Ratio of 11.62 (95 % KI [2.76 - 48.92], p = 0.0008), wound infection with an Odds Ratio of 0.12 (95 % KI [0.03 - 0.52], p = 0.004) and inhalation injury with an Odds Ratio of 5.95 (95 % KI [1.72 - 20.55], p = 0.005). During the study period a rise of wound infections could be notified. Promoting factors were longer hospitalization on the burn intensive care unit and bacterial detection in the unburned parts of the body. Regarding mortality, higher age, wound infection and inhalation injury were prognostic factors.

Clinically proven markers of metastasis predict metastatic spread of human melanoma cells engrafted in scid mice.

Metastasis formation is a complex process and as such can only be modelled in vivo. As markers indicating metastatic spread in syngenic mouse models differ significantly from those in man, this study aimed to develop a human melanoma xenograft mouse model that reflects the clinical situation. Six human melanoma cell lines (LOX, MV3, FEMX-1, G361, MeWo and UISO-Mel6) were xenografted into severe combined immunodeficient mice and tumour growth, metastatic behaviour and number of lung metastases were assessed. Tumours and metastases were analysed for HPA binding and expression of CEACAM-1 and L1, all markers indicative of metastasis in clinical studies. Development of primary tumour nodules ranged from 3 weeks (MV3) to 3 months (MeWo). Whereas G361 and FEMX-1 rarely formed lung metastases, MeWo, MV3 and LOX were moderately and UISO-Mel6 was highly metastatic. Similar to clinical studies, HPA, CEACAM1 and L1 indicated metastatic spread in the xenograft melanoma model, but were not all simultaneously expressed in all cell lines. Considering the strongest expression of one marker combined with an absent or low expression of the other two markers, we conclude that LOX is the cell line of choice for analyses of the functional role of HPA-binding glycoconjugates, UISO-Mel6 is ideally suited to study CEACAM1 function in melanoma spread and L1 function can best be modelled using MeWo.

PAS-positive loops and networks as a prognostic indicator in cutaneous malignant melanoma.

Recently, microvascular channels, as detected by PAS histochemistry, were positively correlated with poor prognosis in uveal malignant melanoma. Since uveal melanomas are not penetrated by lymphatic vessels, while cutaneous melanomas are, the question arises as to whether these loops and networks are also of prognostic relevance in cutaneous melanoma. Histochemically and immunohistochemically detected loops and networks in 100 cases of cutaneous malignant melanoma were correlated with the occurrence of metastasis in a 10-year follow-up study. To detect these patterns, the significance of various methods (PAS reaction with/without nuclear counterstain, anti-laminin immunohistochemistry) was investigated. The presence of loops and networks was a highly significant prognostic marker (p<0.0001) for metastasis in cutaneous malignant melanoma. The presence of these patterns proved to have higher prognostic relevance for metastasis than Breslow's tumour thickness, especially for stage IB and stage IIA tumours (intermediate thickness/risk). PAS reaction without nuclear counterstain proved to be the best method to detect these patterns. Compared with the conventional staging of Breslow's tumour thickness, and especially so for stage IB and IIA melanomas, the determination of PAS-positive loops and networks in cutaneous malignant melanoma provides additional prognostic information.

Binding of mistletoe lectins to cutaneous malignant melanoma: implications for prognosis and therapy.

BACKGROUND: Glycoconjugates, as detected by lectin histochemistry, have been implicated in metastasis formation in many neoplasias. However, no data concerning the three mistletoe lectins (MLs) and the spread of malignant melanoma have been published. MATERIALS AND METHODS: The binding status of ML-I, -II and -III was histochemically assessed in 100 malignant melanomas and correlated with metastasis in a 10 year follow-up period. Furthermore, the staining intensity of the three MLs, scored from negative (-) to very intense (+ + +), was evaluated. RESULTS: Kaplan-Meier analsis revealed that very intense binding (+ + +) of ML-I was positively-correlated with metastasis (p=0.044). CONCLUSION: Since ML-I is specific for galactose, high density galactose expression in malignant melanoma is a predictor of poor prognosis.

Lectin binding to cutaneous malignant melanoma: HPA is associated with metastasis formation.

Changes in protein glycosylation of tumour cells, as detected by lectin histochemistry, have been associated with metastasis formation in several human malignancies. This study analysed the association between lectin binding and metastasis in cutaneous malignant melanoma. In a 10-year retrospective study, sections of 100 primary cutaneous malignant melanomas were histochemically stained for the following 5 lectins: HPA, SNA-I, MAA, WGA and PHA-L, differing in their carbohydrate specificity. Since differences in the results of HPA binding depending on methodology have been reported, an indirect and a biotinylated method were employed for HPA. Kaplan-Meier analysis of time to first metastasis revealed a positive correlation between HPA binding and metastasis for both methods, with the biotinylated HPA method (P< 0.0001) being superior to the 'indirect' method (P = 0.0006). Cox regression analysis demonstrated that even after adjustment for stage, HPA positivity is an independent predictor for metastasis. The results of the present study indicate that N -acetyl-galactosamine/-glucosamine residues, recognized by HPA, are linked to metastasis in malignant melanoma. In contrast, beta1-6 branched oligosaccharides or sialic acid residues, both of which were correlated with metastasis in other malignancies, are of no functional importance for metastasis formation in malignant melanoma. Thus, HPA proved to be a useful and independent prognostic marker for the metastatic phenotype of melanoma.

Age-related changes in T- and B-lymphocyte subpopulations in the peripheral blood.

Quantitation of T and B lymphocytes in infants and children is an important test in the diagnosis of a suspected immuno-deficiency. Previous studies indicated that the absolute and relative numbers of lymphocyte subpopulations vary with age, but these data in the pediatric age group are incomplete and often contradictory. We reviewed the literature and investigated the relationship between age and lymphocyte subpopulations in healthy infants and children using common methods and recent methodologic improvements. We found that absolute numbers of T and B cells followed the same trend as the total lymphocyte count, which was elevated at birth, increased in the first six months, and then gradually decreased to adult levels at approximately 13 years of age. Compared with adult values, the percentage of B cells also was higher at birth and continued to increase for six months, followed by a gradual decrease to adult levels by late childhood or early adolescence. The percentage of T cells gradually increased to adult levels by the same age range.

Age-related changes in mitogen-induced lymphocyte function from birth to old age.

This study examines the relationship between age and mitogen-induced lymphocyte blastogenesis. Lymphocytes were stimulated with PHA; Concanavalin A (Con A), and PWM in 156 normal, healthy subjects ranging in age from birth to 75 years. The findings indicate a gradual but significant decrease in PHA- and Con A-induced blastogenesis with increasing age. The decrease in Con A and PHA induced in vitro lymphocyte function begins in early childhood and young adulthood, respectively, and continues throughout the age range studied. In addition, there appears to be a decreased range of lymphocyte functional capacity among the 50-75-year-old subjects. The clinical and laboratory implications of these observations are discussed.

Perivascular myofibroblasts and microvascular occlusion in hypertrophic scars and keloids.

Microvessels in normal skin, granulation tissue, hypertrophic scar, keloid, and mature scar from human subjects were studied by transmission electron microscopy. Comparative observations suggested that most microvessels in hypertrophic scar and keloid are occluded or partially occluded, apparently owing to an excess of endothelial cells. Endothelial cell contraction was also supported by the observations, and perivascular satellite cells (pericytes), some of which were identified as myofibroblasts, were observed in hypertrophic scars and keloids. Among findings from statistical analyses were that 1) the patency of microvessels in hypertrophic scar and granulation tissue is similar, as is that of microvessels in keloid and mature scar, but the patency of all these microvessels is significantly less than that of microvessels in normal skin, and 2) endothelial cell density is greater in nonpatent vessels than in patent vessels. The observed extent of microvascular occlusion supports a previously published theory that hypoxia is involved in the generation of hypertrophic scar.

Correlations between amino acid concentrations in brains of seizure-susceptible and seizure-resistant rats.

We have examined the relationship between free amino acid concentrations in the brains of genetically seizure-susceptible and seizure-resistant rats. The concentrations of free amino acids in the two strains do not differ significantly in the inferior colliculus or the cortex. However, animal-to-animal variations in the concentrations of numerous amino acid pairs are highly correlated. Glutamic acid decarboxylase activities did not vary between the two strains. We conclude that the strong correlations reported between glutamate and taurine levels in several species are not unique to this amino acid pair. Furthermore, unlike the situation with some experimentally-induced epilepsies, genetic epilepsy is not associated with major disturbances in free amino acid concentrations. The high correlations between amino acid pairs in some cases may reflect variations in cellular and subcellular compartment sizes that are shared by several amino acids.

The effect of lymphocyte recovery on lymphocyte typing results.

This study examines the relationship between percent recovery of lymphocytes and T- and B-cell typing results. Lymphocytes were recovered from heparinized whole blood by density gradient centrifugation. T- and B-cells were enumerated by spontaneous sheep rosetting and direct fluorescent antibody staining for surface immunoglobulin, respectively. The findings indicate that at low recovery levels ( less than 80%) there is a significant increase in variability of lymphocyte typing results and a significant increase in IgG-bearing B-lymphocytes. Possible explanations for these findings are discussed. These results suggest that lymphocyte typing results should be interpreted in relation to recovery level and that recovery levels of greater than or equal to 80% are desirable.

Prevalence of mastoid infection in prehistoric Arizona Indians.

In view of th reported high prevalence of otitis media and mastoiditis in the present-day Indian inhabitants or Arizona, when an opportunity arose to examine the skulls of prehistoric Indians in the collection of the Arizona State Museum, University of Arizona, Tucson, Arizona, it was thought that determining the prevalence of mastoiditis in them might be helpful in the treatment and prevention of mastoid infections in the present-day population. Our findings are compared with those of two other studies of mastoiditis in prehistoric Indians in another area of the United States.

Thoracic kyphosis: range in normal subjects.

Thoracic kyphosis was measured on chest radiographs of 316 "normal" subjects by means of a modification of the Cobb technique for measuring scoliosis. Patients were accepted as "normal" if they had no thoracic or spinal complaints or radiographic abnormalities in the chest including the thoracic spine. A total of 159 males and 157 female subjects 2-77 years old was studied. The relation among age, gender, and kyphosis were determined using least squares fits of first-order linear mathematical models. These results were also used to determine the expected ranges of kyphosis for a "normal" patient of a given age and gender. The degree of kyphosis increased with age and the rate of increase was higher in females than in males. Clinical explanations for this differential increase are discussed.

Immunochemical determination of human immunoglobulins: use of kinetic turbidimetry and a 36-place centrifugal analyzer.

We describe the measurement of human immunoglobulins IgG, IgA, and IgM in diluted sera, with diluted commercial monospecific antisera, with use of a 36-place centrifugal analyzer (RotoChem IIa; American Instrument Co., Silver Spring, MD 20920). The assay involves a two-point kinetic turbidimetric technique. The changes in absorbance at 340 nm between 10 and 255 s are taken for standard curve construction with use of a computer-generated cubic least-squares fit. Patients' samples are quickly calculated from the stored curve. Multiple time-interval readings are taken to observe the reaction kinetics. The lowest detectable concentrations are: IgG, 5 mg/L; IgA, 20 mg/L; and IgM, 20 mg/L. Correlation with radial immunodiffusion was excellent. Precision, accuracy, linearity, and sensitivity were very acceptable. Antigen or antibody excess can be easily detected. The accuracy of the proposed method when measuring idiotypic monoclonal proteins is greater than radial immunodiffusion. Up to 30 patients' samples can be analyzed at one time, and calculation of test results by use of the computer program is efficient and rapid.

Evaluation of a new multichannel analyzer, "Astra-8".

We evaluated a new multiple-channel chemistry analyzer, the Beckman "Astra-8," which is controlled by a microprocessor with 52K bytes of memory. The instrument we tested performed the following tests: Na, K, CO2, chloride, urea nitrogen, glucose, and creatinine. We compared the Astra-8 to a continuous-flow (SMA 6/60) and a discrete (Du Pont aca, urea nitrogen only) analyzer. The Astra-8 demonstrated excellent precision, linearity, accuracy, analytical recovery, lack of interference, ease of operation, and satisfactory comparison to values obtained by the comparison methods.