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In order to increase the pathway of the light inside a gel (or autoradiogram) during scanning, it is placed on top of a mirror and the densitometry is performed in a vertical reflection mode. As the light passes the gel a second time after being reflected by the mirror, the absorbance is nearly doubled. This increase in absorbance results in higher sensitivity and resolution.
About 15% of monoclonal components or paraproteins are associated with malignancy when detected by simple electrophoresis on cellulose acetate membranes or agarose gel followed by protein staining. More sensitive methods for detecting monoclonal components result in a lower frequency of association with recognisable underlying disease. The sensitivity is dependent on the system used. Isoelectric focusing is 10 to 40 times more sensitive than simple electrophoresis at detecting monoclonal components. Electrophoretically separated bands may be quantitated densitometrically and at the present time this is the most satisfactory method for measuring monoclonal component concentration. Immunochemical methods for quantitating monoclonal components are limited by failure to react in a similar manner to polyclonal standards and giving problems of antigen excess resulting both in falsely elevated and low results. Electrophoretic methods must always be used in conjunction with these. The normal polyclonal ratio of immunoglobulin kappa:lambda light chain is disturbed by the presence of a monoclonal component. In 260 patients with myeloma we found that serum electrophoresis alone detected 90% while a disturbance in the kappa:lambda ratio detected 98%. However, 50% of monoclonal components less than 3 g/L were missed. Log kappa:lambda ratio correlates well with the densitometric scan of monoclonal components, both in vitro and in patients being monitored for treatment response in myeloma. This approach may complement or even replace densitometry for this purpose in the future.
A protein determination procedure which involves the densitometry of silver stained polyacrylamide gels is described. It involves calibration with bovine serum albumin and molecular weight markers on the same gel with the protein to be quantitated. The procedure is simple, rapid, reproducible and accurate and is more sensitive than other procedures for protein determination. The procedure is particularly useful in quantitating proteins purified in small amounts since the determination can be performed on the same gel used to check the purification. It avoids interference by detergents and other substances usually present in solutions of purified proteins. The procedure has been applied to the quantitation of a recently identified protein, aquaporin (CHIP28), assumed to be a major water channel in the red blood cell membrane. A quantitative analysis of a purified fraction of this protein shows that the 28 kDa component represents approximately two thirds of the protein content of the sample, with the remainder comprising a glycosylated, high molecular mass component. The procedure may be useful for quantitating proteins revealed on silver stained gels and could be included as a standard part of any protocol for protein purification.
Osteoporosis of the spine and femoral neck is a major problem in aging populations, but detection prior to a fracture remains a challenge. Forearm bone mineral content has been advocated as a useful screening test in this situation. We have examined the correlation between distal forearm bone mineral content, by single photon absorptiometry, and lumbar vertebral and femoral neck bone mineral density, by dual photon absorptiometry. Eighty women aged 20 to 76 years were studied and significant correlations (p less than .001) were found between the measurements on the forearm and at the two axial sites (r = 0.66 and 0.69, respectively). However, forearm bone mineral content was an unreliable predictor of axial bone mineral density. For prediction of lumbar spine osteopenia with a sensitivity of 88%, the false positive rate was 91%. Conversely, to achieve a specificity of 82%, the false negative rate was 65%. Similarly, for prediction of femoral neck osteopenia at a sensitivity of 92%, the false positive rate was 87%, and at a specificity of 90%, the false negative rate was 33%. These data demonstrate that forearm bone densitometry cannot be used as a screening procedure for osteopenia of the lumbar spine or femoral neck.
The purpose of this investigation was to determine the ability of three bone densitometry techniques to discriminate subjects with mild vertebral deformities from those with definite compression fractures. We determined bone mineral density (BMD) in 68 postmenopausal women by quantitative computed tomography (QCT) and dual-photon absorptiometry (DPA) of the spine, as well as single-photon absorptiometry (SPA) of the radius. Forty four individuals were classified as having mild deformities of the spine and 24 were considered to have definite vertebral compressions. Several statistical approaches were used to compare these subgroups and to estimate the relative risk of vertebral fracture. Included among these were percent decrements and zeta-scores, ROC curves, odds ratio estimations, and logistic regression analysis. Individuals with definite vertebral fractures had lower bone mineral density at all sites, but measurement of radial compact bone by SPA failed to reach significance. Using ROC analysis to distinguish mild deformities from true compressions, we found that measurement of spinal trabecular bone by QCT to be the most sensitive discriminator; although measurement of spinal integral bone by DPA also gave satisfactory discrimination, whereas assessment of radial compact bone did not adequately differentiate patients with mild deformities from those with definite compressions. Likewise, we found determination of spinal trabecular bone to be the most robust predictor of relative risk of definite fracture using either odds ratios or logistic regression analysis. Measurement of BMD in the peripheral cortical skeleton offered no predictive power for true vertebral fracture. We concluded that direct assessment of the spine, particularly of the trabecular portion, offered the strongest discrimination and relative risk prediction for definite osteoporotic fractures compared with milder forms of this condition.
Major technical progress in the development of computer-based image analysis has made possible the entry of autoradiography and immunohistochemistry into a new era where quantification by densitometry has become easily accessible. Autoradiography could become quantitative and displayed adequate reproducibility with the help of emulsion-coated films and the use of scales of standards of known radioactivity exposed and analyzed in parallel to the tissue sections. Immunohistochemistry after revelation by a color-based enzymatic technique can also become quantitative, providing that standardization of the crucial steps of the procedure and calibration through a parallel treatment of a scale of antigen standards can be ensured. Such an approach is described here in the rat with reference to tyrosine hydroxylase (TH), the main synthesizing enzyme for catecholamines, and with dopamine (DA) itself, a catecholaminergic neurotransmitter. The different parts of the procedure, which can influence the results, such as the fixation of the animals by perfusion and the evaluation of the fluctuations via the calibration curve, are discussed in detail. Biological validation of the proposed procedure is described by reference to experiments already well documented biochemically, such as the induction effect of reserpine on TH in the rat locus coeruleus and the depleting effect of alpha-methyltyrosine (AMPT), a well-known blocker of TH activity, on rat striatal DA content. Finally the importance of restricting the measurements to the (pseudo)linear portion of the calibration curve is illustrated by the autoradiographic identification of the differential intrastriatal repartition of the dopaminergic D1 and D2 receptor sites, particularly the dual patch-matrix compartments.
The quantitation of stained Western blots by reflectance and transmission scanning was explored using a blot of monoclonal immunoglobulin G probed with anti-mouse antibody. The so-called "scanned absorbance" output from a document scanner was found to be directly proportional to the fraction of light absorbed rather than obeying the logarithmic relationship expected for true spectrophotometric absorbance. This explains observations in the literature of a strongly curved relation between loading and "absorbance." Laser transmission densitometry of a blot immersed in a clarifying solvent mixture showed that peak area was linearly related to loading over a wide range. In reflectance mode the document scanner also gave equally linear quantitation of dry blots, providing that a logarithmic correction curve was applied during scanning. It was found advantageous to interpose a red acetate filter sheet between the blot and the scanning table to aid detection of weakly stained bands. The document scanner gave less satisfactory results when used in transmission mode on a clarified blot because weak bands were poorly quantitated.
Bone scintigraphy and densitometry (iodine-125 photon absorptiometry) were performed in eight patients with symptomatic haemodialysis bone disease. The bone scintiscan showed either hot spots or hyperactivity as a feature of metabolic bone disease. The bone density (BMC/W) was reduced, but could not be distinguished from the degree of demineralisation found in asymptomatic patients on long-term haemodialysis. Therefore, bone density measurements require critical interpretation. Complementary bone scintigraphy should be used in symptomatic haemodialysis bone disease for assessing the extent of the disease.
Urinary pyridinoline (pyr) and deoxypyridinoline (dpyr) are new markers for bone resorption, and serum osteocalcin reportedly indicates osteoblastic activity. Recently, a new ultrasound bone densitometer instrument has been developed that measures ultrasonic properties of the os calcis, namely, the speed of sound (SOS), broadband ultrasound attenuation (BUA), and stiffness index. The effects of menopause on biochemical markers and ultrasound densitometry were investigated in 40 healthy females, 36-39 years, with regular menstruation, and in 117 healthy perimenopausal females, 47-57 years, who were divided into a premenopausal group and a postmenopausal group. Significantly elevated values of pyr, dpyr, and serum osteocalcin were found for the postmenopausal group as a whole compared with the premenopausal group. We examined postmenopausal groups 48-57 years of age stratified into 2-year intervals (within 2 years of the menopause, 2-4 years postmenopause and 4-6 years postmenopause). Elevated values of urinary pyr, dpyr, and serum osteocalcin were evident even in the first 2 years postmenopause compared with the premenopausal group, and these higher values were exhibited until 6 years after menopause. We found a significant decrease in SOS, BUA, and stiffness index of the postmenopausal group as a whole, compared with those of the premenopausal group. SOS, BUA, and stiffness index of the group within 2 years of menopause significantly decreased compared with those of the premenopausal group. The Z-scores of the increase in biochemical markers and the decrease in stiffness index in the postmenopausal group were approximately 0.7-1.3 compared with the premenopausal group.(ABSTRACT TRUNCATED AT 250 WORDS)
The present study examines the use of photographic densitometry combined with atomic absorption spectrophotometry for the quantitation of enzyme activities (SDH and ATPase) in fresh frozen sections of rat tibialis anterior muscles. The technique eliminates some difficulties which are inherent in other methods. The reliability of the technique was found to be in the 98% range; the results were precise for all samples studied. The use of SDH to separate muscle fibers into "types" was found to be totally inaccurate since a full spectrum of activities was observed. ATPase activities could separate easily into two groups, but a continuum of ATPase activities was observed in the fast-twitch fibers. The simultaneous use of both enzymes was capable of separating the FG, FOG and SO fibers; however, variation within a single type was considerable and a great deal of information was lost when using any classification system. The continuum of SDH activities indicates the motor units are arranged as a spectrum of fatigue-resistant contractile units. The range of ATPase activities observed is comparable to ranges of motor unit contraction times emphasizing the importance of this enzyme in the regulation of contraction speed.
The relative performance was determined of two different photographic films (Kodak Panatomic-X and Kodak Technical Pan) for densitometry readings from retinal photographs of patients who had or were suspected of having glaucoma. The raw data from the two films were significantly different. The high contrast film was particularly sensitive to external variables; however, when normalised in terms of the standardised deviation, the data from the two films were comparable (P < 0.005). We also measured the pattern of nerve fibre layer loss using digital image analysis of red-free photographs of normal, ocular-hypertensive, glaucoma-suspect and glaucomatous eyes. Several mathematical techniques were used to characterise the data from each eye and then to compare these data to the mean photographic density of the normal eyes. Results showed that it was possible to separate normal eyes from glaucomatous eyes. The highest sensitivity achieved was 100% for right and 88% for left eyes, and the highest specificity was 100% for both eyes.
The present report describes a new approach in soft laser scanning densitometry performed on opaque electrophoregrams. The method employs a light reflectance system instead of absorption, which is commonly used. The data of this preliminary study were obtained by directly scanning a photograph of an electrophoregram exhibiting viral proteins separated by SDS-PAGE. Comparison of the two methods clearly demonstrate that laser reflectance offers finer resolution than laser absorbance when opaque materials are scanned.
OBJECTIVE: To evaluate the agreement between cardiac output (CO) measurements obtained by a new dye dilution technique using pulse dye densitometry (PDD) and thermodilution (TD) and the direct Fick method (F). DESIGN AND SETTING: Prospective clinical study in a university hospital, cardiac surgery intensive care unit. PATIENTS: Fifty-eight cardiac surgery patients after admission to the intensive care unit (six were excluded due to a low pulse signal quality using the PDD method). MEASUREMENTS AND RESULTS: Mean CO was 5.3+/-1.8 l/min for PDD, 5.7+/-1.68 l/min for TD, and 6.16+/-1.66 l/min for F. There was a good correlation between PDD and TD ( r(2)=0.93) and between PDD and F ( r(2)=0.77). Bias and precision between PDD and TD were -0.39+/-0.5 l/min and -0.69+/-0.85 l/min between PDD and F. In general, PDD determined lower CO values than TD and F. Especially in patients with CO below 5 l/min PDD underestimated CO in comparison to TD and F (bias and precision: -0.51+/-0.40 l/min and -0.83+/-1.0 l/min). CONCLUSION: Comparison between PDD and TD showed good agreement for the normal to high CO range. However, agreement was poor in patients with low CO. In the latter patient group PDD showed relevant underestimation of CO compared to TD and F. Due to these limitations PDD cannot entirely replace the pulmonary artery catheter for CO determination.
OBJECTIVE: To determine the accuracy and repeatability of pulse dye densitometry (PDD) in measuring blood volume (BV) by comparing it with the conventional method using 51Cr-labeled red blood cells (RI method) and by assessing sequential measurements. DESIGN: Prospective clinical study. SETTING: University hospital. PATIENTS AND PARTICIPANTS: Eleven adult ICU patients who received cardiac surgery (1st ICU day). INTERVENTIONS: None. MEASUREMENTS AND RESULTS: After injecting indocyanine green (10 or 20 mg) into the right atrium, its arterial concentration was continuously monitored at the nose and finger by PDD, and BV was calculated by back extrapolating the logarithmic dye concentration on the dye elimination curve between 2.5 and 5.5 min after mean transit time to each mean transit time with the least squares method. These measurements were repeated in eight patients and performed only once in the other three, and the BV was measured concurrently by the RI method one time. The Bland-Altman method was used for evaluating differences between methods and within methods. The (percentage) biases and standard deviations between the PDD and RI methods and between the successive measurements by PDD at the finger and nose were 0.26 +/- 0.491 (8.8 +/- 15.3%) and 0.004 +/- 0.251 (0.06 +/- 5.9%) with the probe on a nostril, and 0.16 +/- 0.561 (2.5 +/- 14.4%) and 0.19 +/- 0.311 (4.7 +/- 7.3%) using the finger probe. The bias between methods was less than 10%, and the repeatability of PDD was better. CONCLUSIONS: As PDD can measure BV with good repeatability and with a small bias compared to the RI method, serial changes in BV can be evaluated at the bedside of critically ill patients noninvasively and repeatedly.
PURPOSE: Effective hepatic blood flow (EHBF) is thought to reflect splanchnic perfusion and the metabolic state of the liver. This study was conducted to examine the relationship between cardiac output (CO) and EHBF using pulse dye-densitometry (PDD) in nonseptic and septic patients, and to assess the prognostic value of this relationship. METHODS: The subjects were 33 critically ill patients, 16 of whom met the criteria for sepsis. Indocyanine green (ICG) was given via a central venous catheter to each patient. CO (l/min) and EHBF (l/min) were assessed with PDD. RESULTS: CO and EHBF were significantly correlated in the nonseptic patients ( r = 0.92, P < 0.001), but not in the septic patients ( r = 0.38, P = 0.15). The ratio of EHBF to CO (EHBF/CO) in the septic patients was significantly lower than that in the nonseptic patients (0.08 +/- 0.04 vs 0.22 +/- 0.05; P < 0.001). Moreover, in the septic patients, the EHBF/CO ratios of nonsurvivors were significantly lower than those of survivors (0.06 +/- 0.04 vs 010 +/- 0.02; P < 0.01). CONCLUSIONS: In nonseptic patients, the EHBF decreased in relation to the CO. However, the EHBF/CO ratio of septic patients was lower than that of nonseptic patients, suggesting that inadequate splanchnic perfusion or metabolic change occurs in septic patients. Furthermore, the lower EHBF/CO ratio was related with a fatal outcome in septic patients. PDD could be a clinically useful method of assessing splanchnic conditions in critically ill patients.
OBJECTIVE: Pulse-dye densitometry (PDD) could be a suitable, low-invasive alternative to thermodilution using a pulmonary artery catheter (PAC) for monitoring cardiac output. The aim of our study was to assess the reproducibility and validity of PDD compared to PAC-thermodilution. METHODS: In 43 post-cardiac surgery patients, the mean of triplicate readings of cardiac output was assessed using both methods. In a subgroup of 26 patients, a second set of measurements was obtained on average 2 h later. RESULTS: Reproducibility of consecutive measurements was slightly better for PAC-thermodilution than for PDD (median coefficient of variation of the triplicate measurements: 3.5% versus 5.4%, P < 0.01). Both methods correlated well (r = 0.84, p < 0.001). Using Bland and Altman analyses with PAC-thermodilution as the reference method, PDD showed a bias of -0.68 +/- 0.82 L/min, mainly due to differences in higher ranges of cardiac output (>6.5 L/min). Measured changes in cardiac output were 81% concordant (i.e. <1 L/min different) between both methods. CONCLUSION: PDD correlates well with PAC-thermodilution and thus deserves consideration as a low-invasive alternative for measurement and follow-up of cardiac output.
We evaluated foveal cone photopigment kinetics by retinal densitometry in 14 patients (12 men and two women, ranging in age from 30 to 54 years) with central serous choroidopathy. At the initial examinations when 12 of the 14 patients showed active leaks, the measured two-way density of pigment was low and the time-course of regeneration was slow. At later examinations, we found low two-way densities with normal regeneration times in five patients without active leakage. Near normal two-way densities occurred only in three patients who had complete clinical recoveries.
Protein content of a wide variety of sample types may be determined by adsorption of microliter samples onto nitrocellulose, followed by amido black staining and laser densitometry. The method is quite rapid, sensitive, and selective, and dispenses with precipitation and transfer steps.