Virtual staining of colon cancer tissue by label-free Raman micro-spectroscopy.

The great capability of the label-free classification of tissue via vibrational spectroscopy, like Raman or infrared imaging, is shown in numerous publications (review: Diem et al., J. Biophotonics, 2013, 6, 855-886). Herein, we present a new approach, virtual staining, that improves the Raman spectral histopathology (SHP) images of colorectal cancer tissue by combining the integrated Raman intensity image in the C-H stretching region (2800-3050 cm-1) with the pseudo-colour Raman image. This allows the display of fine structures such as the filamentous composition of muscle tissue. The morphology of the virtually stained images is in agreement with the gold standard in medical diagnosis, the haematoxylin-eosin staining. The virtual staining image also represents the whole biochemical fingerprint, and several tissue components including carcinoma were identified automatically with high sensitivity and specificity. For fast tissue classifications, a similar approach was applied on coherent anti-Stokes Raman scattering (CARS) spectral data that is faster and therefore potentially more suitable for clinical applications.

Raman fiber-optical method for colon cancer detection: Cross-validation and outlier identification approach.

Endoscopy plays a major role in early recognition of cancer which is not externally accessible and therewith in increasing the survival rate. Raman spectroscopic fiber-optical approaches can help to decrease the impact on the patient, increase objectivity in tissue characterization, reduce expenses and provide a significant time advantage in endoscopy. In gastroenterology an early recognition of malign and precursor lesions is relevant. Instantaneous and precise differentiation between adenomas as precursor lesions for cancer and hyperplastic polyps on the one hand and between high and low-risk alterations on the other hand is important. Raman fiber-optical measurements of colon biopsy samples taken during colonoscopy were carried out during a clinical study, and samples of adenocarcinoma (22), tubular adenomas (141), hyperplastic polyps (79) and normal tissue (101) from 151 patients were analyzed. This allows us to focus on the bioinformatic analysis and to set stage for Raman endoscopic measurements. Since spectral differences between normal and cancerous biopsy samples are small, special care has to be taken in data analysis. Using a leave-one-patient-out cross-validation scheme, three different outlier identification methods were investigated to decrease the influence of systematic errors, like a residual risk in misplacement of the sample and spectral dilution of marker bands (esp. cancerous tissue) and therewith optimize the experimental design. Furthermore other validations methods like leave-one-sample-out and leave-one-spectrum-out cross-validation schemes were compared with leave-one-patient-out cross-validation. High-risk lesions were differentiated from low-risk lesions with a sensitivity of 79%, specificity of 74% and an accuracy of 77%, cancer and normal tissue with a sensitivity of 79%, specificity of 83% and an accuracy of 81%. Additionally applied outlier identification enabled us to improve the recognition of neoplastic biopsy samples.

Artifactual elevation of serum creatinine level due to fasting.

Abnormal serum creatinine (1.6 mg/dL) and creatinine clearance (33 mL/min) levels found in a 50-year-old woman during fasting were corrected with refeeding. Five healthy subjects who fasted for 96 hours demonstrated an increase in their mean serum creatinine level from 1.0 +/- 0.08 to 1.7 +/- 0.11 mg/dL as determined by Jaffé's method. This increase was probably an artifact caused by the rise in the serum acetoacetate level during fasting. The serum creatinine level determined by an enzymatic method and serum urea nitrogen level did not change substantially during the fast. We conclude that fasting may cause an artifactual increase in the serum creatinine level determined by Jaffé's method, the method used by most clinical laboratories.

Long-term, ambulatory, subcutaneous insulin infusion versus multiple daily injections in brittle diabetic patients.

We compared the blood glucose control of four intact and eight kidney recipient, metabolically unstable, ketosis-prone, insulin-dependent diabetic patients under two different regimens: (a) intensive conventional treatment with two to four insulin injections daily (48 patient-months) and (B) subcutaneous, portable insulin delivery system (IDS) (54 patient-months). Both regimens included frequent home blood glucose and 24-h urine glucose determinations and daily telephone follow-up to maximize compliance with treatment. Analyzed as a group the fasting blood glucose for intact patients (A: 172 +/- 13 mg/dl; B: 141 +/- 12, P less than 0.02) and the nonfasting blood glucose for kidney recipient patients (A: 165 +/- 10; B: 138 +/- 5, P less than 0.01) were significantly lower during treatment with the IDS than with multiple injections. Six out of 12 patients (2/4 intact and 4/8 kidney recipient patients) showed significant and consistent improvement of blood glucose concentrations. Four showed marginal and inconsistent improvement. Two patients (one intact and one kidney recipient) improved on the IDS but maintained the improvement when changed back to conventional treatment. The 24-h urine glucose, maximal glucose excursions, number of blood glucoses less than or equal to 40 mg/dl, and glycosylated hemoglobin decreased significantly in some patients on the pump. We conclude that subcutaneous, portable insulin delivery devices can significantly improve the metabolic control of some ambulatory, unstable diabetic patients during long-term treatment beyond that obtained with intensive, multiple-injection, conventional treatment. Normalization of the metabolic control, however, is not obtained. These infusion systems still pose several problems during ambulatory use, which could have serious consequences in patients less compliant and/or followed less closely than ours.

Enzyme inactivation in serum before determination of total bile acids.

Endogenous NADH-generating enzymes must be inactivated before total serum bile acids can be measured accurately by the direct enzymic method. To do this, we pretreat the sera with NaOH, in a final concentration of 0.1 mol/L. Consequently, lactate dehydrogenase activity at least as high as 30 000 U/L is destroyed, obviating blank determinations. Values for bile acid in serum, so obtained, agree with values obtained after pretreatment with heat, an alkali-methanol solution, or sodium pyruvate, but our pretreatment has the advantages of ease, speed, economy, and negligible blank values.

Improved method for identifying alpha 1-antitrypsin Pi M subtypes by isoelectric focusing in agarose.

We describe an improved method for the classification of alpha 1-antitrypsin variants by isoelectric focusing in agarose. Identification of the three Pi M subtypes can now be made by using a narrow-range carrier ampholyte (pH 4.2-4.9) and pretreating serum with dithioerythritol-iodoacetic acid to enhance band resolution. Phenotype results for two groups of Pi M homo- and heterozygotes are compared to illustrate the improved accuracy of the new method.

Determination of inorganic phosphorus using a centrifugal analyzer.

A method involving the molybdenum blue reaction for measurement of serum phosphorus using a centrifugal analyzer is described. The need to measure a separate serum blank is eliminated by measuring the rate of color development compared to that of a known standard. The procedure which uses o-phenylenediamine as a reductant is precise and compares well with a continuous flow automated procedure.

A simple and precise method of determining true sodium, potassium, and chloride concentrations in hyperlipemia.

Sodium, potassium, and chloride levels are artifactually depressed in hyperlipemic sera. Accurate electrolyte levels are needed for management of patients with hyperlipemia, but present methods for correcting the values (serum water and/or osmolality determinations) either are technically cumbersome or fail to provide accurate data to correct the falsely low levels. Alternatively, to determine true sodium, potassium, and chloride concentrations in hyperlipemic sera, only the required electrolyte values and the triglycerides are measured. The percentage by which the measured electrolyte levels in the hyperlipemic sample must be increased to approximate the true values is given by the following equation: per cent increase = 2.1 X triglycerides (Gm./dl.) - 0.6.

Direct assays of lactate, pyruvate, beta-hydroxybutyrate, and acetoacetate with a centrifugal analyzer.

Methods are described for direct assays of lactate, pyruvate, beta-hydroxybutyrate, and acetoacetate in plasma with the GEMSAEC centrifugal analyzer. The methods for lactate, beta-hydroxybutyrate, and acetoacetate are kinetic and ratiometric, eliminating the need for specimen-blank assays. The pyruvate method is an end-point assay, because endogenous lactate dehydrogenase interferes in a kinetic pyruvate assay. The methods are precise and accurate and 1-min of analysis time is adequate for each assay. Rapid assessment and monitoring of metabolic acidosis is possible with these methods, as is illustrated by examples.

Improved simultaneous gas-chromatographic analysis for homovanillic acid and vanillylmandelic acid in urine.

We describe an improved gas-chromatographic method for the simultaneous quantitation of the catecholamine metabolites, homovanillic acid (3-methoxy-4-hydroxyphenylacetic acid) and vanillylmandelic acid (3-methoxy-4-hydroxymandelic acid). Our improvements in the method of Muskiet et al. (Clin. Chem. 23: 863, 1977) include a shorter program time and a longer silylation interval. Recovery and precision data obtained by this improved technique are similar to those of Muskiet et al. Vanillylmandelic acid results (y) were compared with those by the method of Pisano et al. (Clin. Chim. Acta 7: 285, 1962). The relation is expressed by the equation y = 0.52 + 1.05x (Sy . x = 2.33 mg/24 h and r = 0.997). Results for homovanillic acid (y) were compared with those by the method of Knight and Haymond (Clin. Chem. 23: 2007, 1977); the equation was y = 0.84 + 0.90x (Sy . x = 2.04 and r = 0.97). Retention times are also reported for several phenolic acids and other related compounds found in urine.

Nephelometric assay of apolipoprotein A-I with a centrifugal analyzer.

We developed an automated immunonephelometric assay for quantification of human apolipoprotein A-I (apo A-I) with a fluorescence light-scattering microcentrifugal analyzer. The presence of polyethylene glycol and Tween 20 in the reaction mixture ensures maximum exposure of the antigenic sites of the apoprotein so that immune complex formation occurs more rapidly (reaction is complete within 2 min) and to a greater extent. Lipemia and hemolysis do not interfere with the measurement of apo A-I. The method requires only 10 microL of specimen and is fast and easy to perform. Results vary linearly with apo A-I concentrations to 2.5 g/L. Assay precision (CV) was 3.1% for a specimen with an apo A-I concentration of 1.45 g/L, and the lower limit of detection was 0.15 g/L. Values for a candidate Reference Material agree well with those reported in an international survey (Clin Chem 1985;30:223-8).

A comparison of the Murphy-Pattee and the Seligson methods for serum thyroxine.

We compared two methods for serum thyroxine measurement by competitive protein binding - the Murphy and Pattee and the Seligson and Seligson methods. We found the Seligson and Seligson method, which requires less sample volume and analysis time, to be the method of choice on the basis of sensitivity, extraction efficiency, precision, and accuracy. Standard plots of time/10,000 counts versus thyroxine concentration are linear to 20 ug/dl, extraction efficiency is 99.6 per cent, within-day S.D. plus or minus 0.28 ug/dl, and 98.3 per cent of added thyroxine is recovered in the Seligson and Seligson method. The Seligson and Seligson method is superior to the Murphy and Pattee method with respect to all these parameters. The methods were also compared with correlation and normal value studies.

Effect of protein on the determination of total bile acids in serum.

We measured total serum bile acids on a fluorescence-light-scattering micro centrifugal analyzer by the direct enzymatic method with 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) and with resazurin as a fluorogenic electron acceptor. We found that serum protein has an inhibitory effect on the measurement of bile acids, but this effect was eliminated by adding bovine serum albumin to the reaction mixture in a final protein concentration (12.2 g/L) that was high compared with that contributed by a normal serum specimen. The assay is a sensitive method that reaches equilibrium in 5 min. The method is microscale (5 microL of sample, 150 microL of working reagent), is easy to perform, and is accurate (analytical recovery = 104.1%) and precise (CV = 11.1 and 5.7% on specimens with bile acid concentrations of 7.6 and 35.4 mumol/L, respectively). Normal values are 1-12 and less than 9 mumol/L on nonfasting and fasting individuals, respectively. Pure 3 alpha-hydroxysteroid dehydrogenase must be used: we found several enzyme preparations that gave falsely high values for bile acid.

Contribution of monocyte-macrophage system to serum alpha 1-antitrypsin.

The major serum antiprotease is alpha 1-antitrypsin (A1AT). Deficiency of A1AT can result in infantile cirrhosis and premature emphysema, both of which have a high degree of morbidity and significant mortality. Although synthesized primarily by the liver, A1AT has been histochemically localized in monocytes and macrophages in vitro and has been shown to be produced in tissue culture of monocyte-macrophage origin. This study was planned to quantitatively and qualitatively assess the in vivo monocyte-macrophage system contribution to serum A1AT. We used bone marrow transplantation (BMT) as an experimental method because there is commanding evidence that after engraftment, the monocyte-macrophage system of the recipient is replaced by that of donor origin. Protease inhibitor (Pi) typing was done on 150 potential BMT recipients and on their potential donors before transplantation. From these initial recipients, 92 eventually underwent transplantation, and 11 recipient-donor pairs, in which each donor's Pi type contained a band not in the recipient's Pi type, were chosen for the study. Six recipients survived beyond 100 days after BMT, and in these cases the donor contained either an S or an M2 band in his or her Pi type not present in the recipient. Using a silver stain method on diluted serum of known M1M2 and MS types, we were able to detect a 2% dilution of the S band and a 25% dilution of the M2 band. When the same method was applied to gels used in typing recipient Pi after BMT, we were unable to detect any contribution to serum A1AT by the donor monocyte-macrophage system.

Two-point determination of plasma ammonia with the centrifugal analyzer.

A simple, convenient, and rapid method for determining ammonia in plasma by the glutamate dehydrogenase reaction is described for the centrifugal analyzer. The measuring principle is fixed-time, with NADH as the coenzyme. ADP is added to stabilize glutamate dehydrogenase and prevent interference from endogenous plasma ADP. The reaction is linear to 400 mumol of ammonia per liter. The plasma sample volume is 100 microliter and the whole procedure takes only 25 min, including the 15-min preincubation. The normal range for venous plasma was 44 +/- 13.5 (SD) mumol of ammonia per liter.

Immunonephelometry of apolipoprotein B with a centrifugal analyzer.

We developed an automated immunonephelometric assay for the measurement of apolipoprotein B (apo B) with a light-scattering microcentrifugal analyzer. Pretreating specimens with a dilute solution of Tween 20 or triglyceride lipase decreased the nephelometric response of apo B. Polyethylene glycol is included in the reaction mixture, and the reaction is complete within 4 min. The method is precise (CV = 6.5%, mean = 0.68 g/L) and the standard curve is linear to an apo B concentration of 2.8 g/L. Lipemia does not interfere with the method if grossly lipemic specimens are centrifuged to remove chylomicrons.

Response of serine antiproteases to growth hormone therapy in growth hormone deficient children.

Growth hormone regulates the hepatic mRNA levels of alpha 1-antitrypsin and two contrapsin-like mRNAs in the rat. To determine whether growth hormone regulates similar serine protease inhibitors in humans, we measured serum alpha 1-antitrypsin, alpha 1-antichymotrypsin, and antithrombin III by radioimmunodiffusion in 16 growth hormone deficient children before and after growth therapy. Of the 19 determinations made, 17/19 showed an increase in alpha 1-antitrypsin after administration of growth hormone, 198.6 +/- 39.1 mg/dl before growth hormone and 239.4 +/- 44 mg/dl after growth hormone (p = 0.005). Specificity of the response for alpha 1-antitrypsin was indicated by the fact that neither alpha 1-antichymotrypsin or antithrombin III values changed after growth hormone (p = 0.6 and 0.5, respectively). These data are compatible with the hypothesis that growth hormone regulates serine protease inhibitors in humans and suggests that investigation of other members of the serpin gene family might prove fruitful in defining additional growth hormone target genes.