Kinetic immunoturbidimetric measurement of thyroxine binding globulin

We describe the extension of kinetic immunoturbidimetry to measurement of low-concentration proteins in serum, specifically thyroxine binding globulin. Method performance is superior to that of currently available direct assays for this protein by radioimmunoassay or the Laurell "rocket" techniques. The assay is rapid (1-min measurement period), economical, and results compare well with those by established techniques. The technique has wide applicability to instruments with kinetic measuring ability and could provide a useful support to other non-isotopic thyroid-function tests. The assay further emphasizes the sensitivity and usefulness of kinetic immunoturbidimetry for measuring low concentrations of proteins.     

Kinetic immunoturbidimetry: the measurement of pregnancy specific beta 1 glycoprotein.

The extension of kinetic immunoturbidimetry to the measurement of low concentration proteins has been described using the protein pregnancy-specific beta 1 glycoprotein. The technique has a precision superior to that of all other methods currently available. The assay is rapid, cheap and compares well with other published methods. The assay further emphasises the usefulness of the technique of kinetic immunoturbidimetry.     

Fingerstick glycosylated hemoglobin, plasma protein, and albumin

We have developed techniques that permit the affinity-chromatographic determination of glycosylated hemoglobin, plasma protein, and albumin on fingerstick samples of whole blood. The fingerstick glycohemoglobin technique takes advantage of the high sensitivity of measurement of hemoglobin by absorbance at 414 nm. The glycosylated plasma protein is assayed by a highly sensitive method based on binding of Coomassie blue. An enzyme-linked immunosorbent assay is used to measure albumin in the bound and nonbound fractions of an aminophenylboronic acid chromatographic separation. The fingerstick method for assay of glycosylated plasma albumin gives results that are approximately 40% higher than comparable values obtained on the same patient with a 1-ml plasma sample determined with the bromcresol green technique. There is good correlation of fingerstick glycoalbumins with fingerstick glycohemoglobins and glycosylated plasma protein values. These procedures should be useful for children and for large-scale ambulatory screening for diabetes mellitus.     

Quantitative relationships of the fourth complement component in human cerebrospinal fluid.

A technique for the measurement of cerebrospinal fluid C4 concentration in unconcentrated specimens has been developed with the methods of electroimmunodiffusion and immunofixation. The method has proved to be reproducible and requires only microliter volumes of undiluted cerebrospinal fluid (CSF). The mean value for CSF C4 concentrations in 16 neurologically normal individuals was 325 +/- 32 mug/100 ml. A positive correlation between CSF C4 concentration and the concentration of CSF albumin and total protein was observed. The positive correlation between the concentrations of CSF C4 and albumin was, however, more clearly defined than the relationship of CSF C4 to total CSF protein.     

血清前白蛋白对糖尿病患者营养状况的评价

目的：探讨血清前白蛋白对糖尿病患者营养状况评价的临床价值。方法：随机选择我院2006年3月～11月收治的178例糖尿病患者（糖尿病组）及健康人50例（对照组）,均抽取静脉血,采用免疫比浊法进行血清前白蛋白（PA）及白蛋白（ALB）测定。结果：糖尿病患者PA与ALB水平均明显降低,与正常对照组比较差异显著（P〈0．01）,178例糖尿病患者PA降低率（47．2％）明显高于ALB降低率（29．8％）。结论：血清前白蛋白灵敏性优于白蛋白,血清前白蛋白的测定简便快速,是早期评价糖尿病患者营养状况的良好指标。     

Three turbidimetric methods for determining total protein compared

We used human serum protein fractions to evaluate the sensitivity and bias of three turbidimetric methods for determining concentrations of proteins. Each fraction (Cohn Fractions II, III, IV, and V) was assigned a protein concentration value that was determined by the biuret method, which we calibrated with purified monomer of human serum albumin. All three turbidimetric methods (those involving sulfosalicylic acid/sodium sulfate, trichloroacetic acid, and alkaline benzethonium chloride) gave acceptable results for Fraction V with crystallized human serum albumin as the reference material, but there was bias by each of the three methods for the three globulin fractions. The method involving alkaline benzethonium chloride with measurement at 450 nm had the best sensitivity within the range of linearity and the most consistent bias among the three globulin fractions. These results define the dilemma for valid calibration of these methods for total serum protein in cerebrospinal fluid and urine.     

A comparative evaluation of the use of immunoturbidimetry in clinical practice]

A variant of immunoturbidimetry available for any clinical diagnostic laboratory has been developed. The data of measurements of immunoglobulins, albumin, alpha 2-macroglobulin and some other proteins of pregnancy in the blood serum and other biologic fluids, carried out by immunoturbidimetry, coincided with the findings of radial immunodiffusion and rocket immunoelectrophoresis, but immunoturbidimetry is more rapid and simple.     

Possibility of determining the urinary content of albumin by capillary electrophoresis

An assay has been developed to determine urinary albumin by capillary electrophoresis. The detection limit for albumin is 6.0 mg/l; its correlation coefficient is 5.3%; the calibration dependence is linear in the range of albumin concentrations of up to 150 mg/l. The findings are in good agreement with the results of immunoturbidimetry (r = 0.996). In addition of albumin, the proposed procedure can detect the presence of other proteins in urine.     

The variable influence of an alpha component in pregnancy plasma on four different assay systems for the measurement of pregnancy-specific beta1 glycoprotein

Four methods (radioimmunoassay, electroimmunoassay, laser nephelometry and kinetic immunoturbidimetry) have been evaluated for the measurement of proteins with pregnancy-specific beta1 glycoprotein immunoreactivity. The influence of two maternal plasma proteins with different electrophoretic mobilities (alpha2 and beta1) but with similar antigenic determinants has been assessed in each assay system. The radioimmunoassay method, which utilises a homogeneous preparation of pregnancy-specific beta1 glycoprotein for the preparation of iodinated tracer, and the kinetic immunoturbidimetric assay which measures antigen-antibody complex formation over the first minute of the reaction, were both considerably more specific for the beta1 component of pregnancy specific beta1 glycoprotein immunoreactivity than the laser nephelometric or electroimmunoassay methods.     

Microplate measurement of urinary albumin and creatinine

We describe microplate methods for measurement of human urinary albumin (HUA) by competitive enzyme-linked immunosorbant assay (ELISA) and creatinine with a modified commercial enzymatic kit. Incorporation of substrate mixing into the competitive ELISA changes the dynamic absorbance-concentration response, greatly simplifying calculations and improving sensitivity and accuracy. Measurement of creatinine in urine and plasma samples with a commercially available enzymatic kit modified for analysis by use of an inexpensive microplate reader produced values comparable in precision and accuracy to those obtained by an automated kinetic Jaffé method.     

Apparent loss of urinary albumin during long-term frozen storage: HPLC vs immunonephelometry

BACKGROUND: Urinary albumin detection by immunonephelometry is decreased by approximately 30% in samples that have been frozen at -20 degrees C. An HPLC method for assessment of urinary albumin that detects immunoreactive and immunochemically nonreactive albumin has been introduced as an alternative to immunonephelometry. We investigated whether this technique is affected by sample temperature, particularly freezing. METHODS: Urine samples (n = 295) were collected from the general population (Prevention of Renal and Vascular End-Stage Disease Study). Samples were assessed by both immunonephelometry and HPLC when fresh and after storage at -20 degrees C for 4, 8, and 12 months and at -80 degrees C for 12 months. RESULTS: With immunonephelometry, storage for 4, 8, and 12 months at -20 degrees C resulted in mean (SD) urine albumin changes of -21% (29%), -28% (29%), and -34% (31) (P <0.001 for trend). Storage at -80 degrees C resulted in a 5% (19%) change after 12 months of storage (not significant). With HPLC, storage for 4, 8, and 12 months at -20 degrees C resulted in urine albumin changes of -33% (28%), -43% (24%), and -55% (21%; P <0.001 vs immunonephelometry). Storage at -80 degrees C resulted in a -29% (29%) change (P <0.001 vs immunonephelometry). CONCLUSION: Loss of albumin after freezing urine depends not only on freezing temperature but also on detection method. Detection of albumin by immunonephelometry appears to be significantly less influenced by freezing than detection by HPLC. Storage at -80 degrees C appears to prevent loss when using immunonephelometry, whereas HPLC still shows considerable loss even when urine is frozen at -80 degrees C. We propose that for reliable measurement of urine albumin, fresh samples should be used.     

Screening school children for albuminuria, proteinuria and occult blood with dipsticks.

Beginning in 1974, the Japanese Ministry of Health Welfare directed the screening of schoolchildren for proteinuria. We studied their procedure and methods in 6197 school children and also evaluated a new urine dipstick that measures albumin concentrations down to about 10 mg/l and creatinine down to about 300 mg/l. We used specimens from adult in- and outpatients to test the accuracy of the dipsticks. Based on the quantitative results, we set as cutoffs < 150 mg/l for protein and < 30 mg/l for albumin as the concentrations representing "low risk." The quantitative values were assumed to be correct, and the dipstick results were judged accordingly, i.e., a dipstick protein of > or = "150" mg/l or an albumin of I "30" mg/l indicated increased risk of developing or having a genitourinary disorder. The sensitivity/specificity of the protein dipstick was 95.1%/95.5%, and the same for the albumin dipstick was 83.8%/93.8%. The cut-off for the albumin dipsticks probably should be set somewhat lower to reduce the number of false negatives and increase the sensitivity of the dipstick. When we compared the quantitative albumin to the protein dipsticks with the above cut-offs, we found the sensitivity/specificity to be 79.3%/94.4%, i.e., much like the albumin dipstick results. The many reports on the association of albuminuria and risk of renal disease recommend that screening should be done for albumin rather than protein. Based on the data from the school children, we estimate that a dipstick albumin of "30" mg/l is borderline increased risk, and that a protein dipstick of "150" mg/l is the same. If we call the dipstick "10" mg/l albumin, "30" mg/l albumin and the "150" mg/l protein results "low risk," then we estimate the prevalence of albuminuria in the school children to be about 2.1% and proteinuria to be about 4.3%. Children with these values should have a quantitative test for albumin and protein. We also tested a dipstick for creatinine and found increasing values with increasing age in both genders; the older boys had significantly higher creatinine values than the older girls and younger boys. For the albumin/creatinine ratio, we found 6028 children with a ratio of < 30 mg/g indicating low risk and 159 children with a ratio of > or = 30 mg/g indicating increased risk. The ratio may be more useful owing to the likely reduction of the number of false negatives and false positives.     

Variations in prostate‐specific antigen free/total ratio in acute stress

Serum prostate‐specific antigen complexed to alpha2‐macroglobulin is occult and is not detected by conventional immunoassays. Conditions affecting alpha2‐macroglobulin levels may alter the specificity of prostate‐specific antigen free/total ratio in predicting prostate cancer. A group of patients (n = 24) undergoing surgical stress due to a coronary artery bypass grafting was followed pre‐ and postoperatively up to 6 days. Total and free prostate‐specific antigen, alpha2‐macroglobulin, and C‐reactive protein were measured by electrochemiluminescence, immunonephelometry, and immunoturbidimetry, respectively. Total prostate‐specific antigen and C‐reactive protein increased significantly postsurgery and remained elevated. Free/total ratio correlated negatively with C‐reactive protein only (p = 0.000) using xtgee panel data analysis, after correction for plasma volume changes using albumin. Increased C‐reactive protein may reflect falsely decreased free/total ratio. Therefore, prostate‐specific antigen free/total ratio would be more reliable if interpreted in combination with information about CRP. However, it is recommended to defer the measurement of free/total ratio if CRP is highly elevated.     

Relationship between plasma Cystatin C and creatinine in chronic renal diseases and Tx-transplant patients

OBJECTIVES: Glomerular filtration rate (GFR) is the best overall index of renal function in health and disease. Recently, Cystatin C (Cyst C), a low molecular weight protein freely filtered through the glomerulus, and almost completely reabsorbed and catabolized by tubular cells, has been proposed as a new and very sensitive serum marker of change in GFR. This study investigated the relationship between Cyst C and creatinine (CR) in renal disease patients. METHODS: Serum Cyst C was determined by particle-enhanced immunoturbidimetry using the Cyst C PET-kit. The results could be obtained within 1 h. Cuvettes were washed before the Cyst C assay as recommended. Serum CR, BUN and albumin were determined by auto-analyzer. RESULTS: A strong correlation was observed between Cyst C and CR (P = 0.001, r = 0.764 and P = 0.0001, r = 0.888, respectively) in prehemodialysis (pre-HD) and kidney transplantation (Tx-kidney), whereas there was a weak correlation in continuous ambulatory peritoneal dialysis (CAPD) (P = 0.05, r = 0.535). CONCLUSION: These findings suggest that serum Cyst C may be considered as a sensitive predictive parameter for reduced GFR. It is of value for the laboratory diagnosis of chronic renal failure and should be preferentially used for CR clearance.     

Comparison of prognostic nutrition indices in preoperative detection of risk patients. A prospective trial

Malnutrition must be considered as a factor of risk in surgery and therefore it has to be taken into account in surgical planning. Many authors aggregated several measurements into an index or another mathematical model by stepwise regression or discriminant analysis. Hitherto none of these approaches has been subjected to a critical analysis designed to determine whether the information gained differentiates patients with increased operative risk from those without, to a degree that is clinically relevant. In a prospective study the predictive values of nutritional assessment techniques of various authors were examined in 246 surgical patients undergoing a major surgical procedure. The specificity, sensitivity, and validity of each assessment technique were determined. The statistical analysis showed that none of the assessment techniques separated patients who were at high risk from those who were at low risk in a statistically significant predictive power. Serum albumin level was a quite accurate prognostic indicator of postoperative morbidity and mortality. The mean complication rate in this study was 26.8%. Concerning the specificity, sensitivity, and validity the single measurement of the serum albumin had a predictive value as high as all other determined assessment techniques in this study. We contend that combining measurements into a statistically derived index is time-consuming and expensive and does not produce an assessment technique with sufficient predictive power to identity high risk patients in a clinically relevant fashion.     

Monitoring urinary orosomucoid in acute inflammation: observations on urinary excretion of orosomucoid, albumin, alpha1-microglobulin, and IgG

BACKGROUND: Inflammation-associated proteinuria in acute, nonrenal disease is a common but poorly understood phenomenon. We performed an observational study of the urinary excretion of orosomucoid (alpha(1)-acid glycoprotein), albumin, alpha(1)-microglobulin (protein HC), and IgG to obtain quantitative and temporal data on these 4 proteins. METHODS: Urine samples were collected at daily intervals for up to 23 days from 6 patients with surgery-induced inflammation and at hourly intervals for a 24-h period from 7 sepsis patients. Urinary protein concentrations were assessed by immunoturbidimetry. RESULTS: During surgery-induced inflammation, the increase and decrease in orosomucoid excretion mirrored changes in plasma C-reactive protein. Values for all 4 urinary proteins were increased in sepsis patients. The observed maximum increases in urinary protein excretion relative to the upper reference values were 280-fold for orosomucoid, 98-fold for alpha(1)-microglobulin, 33-fold for albumin, and 26-fold for IgG. CONCLUSIONS: Orosomucoid, usually present in plasma and urine in much lower concentrations than albumin, is increased in urine to concentrations equal to or higher than albumin in proteinuria associated with acute inflammation. The pathophysiologic mechanisms responsible for this markedly increased excretion are unknown. Monitoring of urinary excretion of orosomucoid and other specific proteins, expressed as protein/creatinine ratios, may provide a window for clinically relevant real-time observation of changes in acute inflammatory processes. Orosomucoid in urine may be a more informative marker than albumin for inflammation.     

Activated protein C (APC)-resistance: automated detection of the point mutation at position 1691 in the factor V gene.

Proteolytic cleavage of factor Va, caused by activated protein C, is an important mechanism in limiting clot formation in normal haemostasis. A single point mutation in the factor V gene has been demonstrated to cause resistance of factor Va to proteolytic cleavage by activated protein C. With an 8-fold increased risk of thrombosis and a 2 to 13% prevalence in the Caucasian population for the heterozygous state of this mutation, knowledge of the patient's genetic disposition is of great importance in conditions such as pregnancy, surgery, use of oral contraceptives and immobilization. Therefore we have developed an automated test for the detection of the factor V mutation. This PCR based test makes use of the disappearance of an Mnl 1 restriction site if the mutation is present. The assay has been developed for the widely used ES-systems of Boehringer Mannheim. The test discriminates between the heterozygous and the homozygous state. Because of its low costs and easy handling the assay can be used as a screening test and can be performed in routine clinical laboratories.     

高压液相色谱检测糖尿病患者尿白蛋白的应用

目的探讨高效液相色谱法（HPLC）测定糖尿病（DM）患者尿白蛋白的应用。方法收集85例DM患者及40名健康体检者（正常对照组）尿液,分别用HPLC和免疫比浊法检测尿白蛋白,并将检测结果使用回归和Bland—Ahman进行一致性分析。结果HPLC线性方程为Y=2．9482X＋38．601,r2=0．9944;标准品和样本的保留时间（RT）均为2．42min;尿白蛋白浓度为40．1和152．6mg／L的样本其批内和批间变异系数（CV）分别为4．1％、4．8％和5．6％、6．5％;最低检测限为2mg／L。DM组尿白蛋白HPLC结果为2t．0（3．4～678．9）mg／L,高于免疫比浊法[8．2（2．0—442．2）mg／L]（P〈0．01）,但偏高的倍数不一致（1．3～6．1倍）;但正常对照组2种方法测定结果差异无统计学意义（P〉0．05）。将2种方法的测定结果用回归分析和Bland—Ahman进行一致性分析,显示2种方法的一致性较差。85例DM患者中用HPLC和免疫比浊法检测达到微量蛋白尿诊断标准[尿白蛋白排泄率（AER）〉30mg／24h]的阳性例数分别为48例（56．5％）和27例（31．8％）。结论HPLC能检测尿中的包括免疫性和非免疫性的总的尿白蛋白,与免疫比浊法相比更能早期检出尿白蛋白。     

Pleural adenosine deaminase in the separation of transudative and exudative pleural effusions

OBJECTIVE: The purpose of this study was to evaluate the usefulness of a new parameter, pleural adenosine deaminase (PADA), for separating transudative pleural effusion from exudative pleural effusion, and to compare the results with other tests (albumin gradient and protein gradient). METHODS: From November 2001 to January 2003, 359 consecutive patients with pleural effusion who underwent a diagnostic thoracentesis were included in the study. Effusions were individually classified as transudates or exudates after the careful evaluation of all clinical data and biochemical parameters of pleural fluid and serum of patients on the basis of Light's criteria. The means and standard deviations of PADA, pleural/serum ADA (P/S ADA) ratio, albumin gradient and protein gradient were evaluated for transudative and exudative effusions. The best cut-off values for each test were identified by using the receiver operating characteristic (ROC) curve. The optimum cut-off level was determined by selecting points of test values that provided the greatest sum of sensitivity and specificity. RESULTS: There were 113 transudates and 246 exudates. For each test, differences in mean value between the transudate group and the exudate group were statistically significant (t test, P<0.001). The optimum cut-off levels for PADA and P/S ADA were 15.3 U/L and 0.66 U/L, respectively. ROC analysis confirmed previous recommendations for albumin gradient (12 g/L) and protein gradient (31 g/L). For detecting exudates, the PADA test yielded a sensitivity and specificity of 85.8% and 82.3%, respectively. Sensitivity and specificity of the albumin gradient were found to be 88.5% and 79.3%, and of the protein gradient 85% and 83.2%, respectively. The areas under the curve (AUC) data and accuracy demonstrated similar discriminative properties in the examined tests. CONCLUSIONS: The measurement of PADA is suggested as a reliable test in the separation of pleural exudates from transudates with accuracy similar to that of the albumin gradient and protein gradient.