How much insulin-like growth factor I (IGF-I) circulates? Impact of standardization on IGF-I assay accuracy

There is a significant systematic difference between the normal range obtained from ethylenediamine tetraacetate plasma samples using the Genentech total insulin-like growth factor I (IGF-I) RIA and normal ranges for other total IGF-I RIAs. To determine whether the quality of the assay standard was the cause of this systematic difference, we analyzed commercially available preparations of recombinant human IGF-I (rhIGF-I) typical of those used as IGF-I immunoassay standards along with our own well characterized rhIGF-I assay standard. For the commercial standards, high performance liquid chromatography-derived purities were low, and some vendor-assigned protein concentrations were inconsistent with values from quantitative amino acid analysis. The Genentech rhIGF-I assay standard was highly pure and quantitatively correct. However, the poor quality of some commercial rhIGF-I preparations was not the primary reason for the systematic discrepancy between the Genentech total IGF-I RIA normal range and most other normal ranges. Most assays for total IGF-I are calibrated against the WHO International Reference Reagent (IRR) for IGF-I Immunoassays (87/518). The Genentech total IGF-I RIA is not calibrated against WHO IRR 87/518. The protein content assigned to WHO IRR 87/518 was a consensus value from a multicenter collaborative study. Physicochemical analyses showed that WHO IRR 87/518 is Met(-1)-IGF-I of low purity (44%), and that the assigned protein content is higher than the value determined by quantitative amino acid analysis. Thus, assays that are calibrated against WHO IRR 87/518 will report total IGF-I concentrations in excess of actual values. We believe that calibration against WHO IRR 87/518 is the cause of the systematic discrepancy between the Genentech IGF-I assay normal range and most other normal ranges, and that much of the plasma IGF-I concentration data in the literature are of questionable accuracy.     

Dissolution of malachite in aqueous ethylenediaminetetraacetate solution

The kinetics of malachite dissolution in aqueous ethylenediaminetetraacetate (EDTA) solution has been investigated in the temperature range of 298 to 318 K. The dissolution rate of malachite determined under the present set of experimental conditions was found to be independent of agitation speed. The dissolution rate increased with increasing EDTA concentration, but leveled off at higher concentrations. At constant EDTA concentration, an increase in dissolution rate was detected at higher temperatures. A dissolution mechanism involving Langmuir-type EDTA adsorption was proposed, in which the dissolution rate of malachite is controlled by the removal of Cu(II)-EDTA complex from the malachite lattice. The proposed mechanism can explain the dependency of the dissolution rate on EDTA concentration. The activation energies determined at pHs 5, 7.5, and 10 were found to be 51.4, 50.2, and 57.5 kJ mol⁻¹, respectively. The calculated enthalpy changes of EDTA adsorption equilibrium were −43.2, −35.2, and −45.0 kJ mol⁻¹ for pHs 5, 7.5, and 10, respectively. These values are in agreement with the proposed mechanism. Formerly Graduate Student, Department of Metallurgy, Kyoto University, is Formerly Professor, is Professor Emeritus, Department of Metallurgy, Kyoto University.     

C-reactive protein concentration in dogs with inflammatory leukograms

Serum C-reactive protein (CRP) concentration was measured, using an automated immunoturbidimetric assay, in 44 clinically normal dogs and 67 dogs with band neutrophil count > or = 10(9) cells/L, and values were found to be significantly (P < or = 0.05) different. Correlation of serum CRP concentration and band neutrophil count in the 67 dogs with > or = 10(9) band neutrophils/L resulted in a statistically significant (P < or = 0.05), but low correlation coefficient of 0.34. Serum CRP concentration and CBC values were determined for 6 clinically normal dogs undergoing anesthesia (controls) and 6 clinically normal dogs undergoing anesthesia and ovariohysterectomy. Significant alterations in CBC results and serum CRP concentration, compared with baseline values, were lacking in dogs of the control group. Serum CRP concentration was significantly (P < or = 0.05) increased above baseline values in dogs undergoing surgery and was significantly (P < or = 0.05) increased, compared with values in control dogs by 12 hours after surgery. In dogs undergoing surgery, serum CRP concentration was also significantly (P < or = 0.05) different from values in control dogs at 28 and 36 hours, but not at the 76- and 124-hour sample collection times. Alterations in CBC values compatible with possible or convincing inflammation were detected in 83% of the dogs undergoing surgery at the 8- and 12-hour postsurgery sample collection times, 100% of dogs at 16, 22, 28, and 36 hours after surgery, 83% of dogs at 52 and 76 hours after surgery, 67% of dogs at 100 hours after surgery, and 0% of dogs at 124 hours after surgery.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine structure of extracellular matrix and basal laminae in two types of abnormal collagen production: L-proline analog-treated otocyst cultures and disproportionate micromelia (Dmm/Dmm) mutants

The diagnostic implications of different procedures of DNA extraction were examined for the detection of HCMV DNA from sera and plasma of immunosuppressed patients. The detection limit of HCMV plasmid DNA from cell free seronegative plasma and serum by limiting dilution nested PCR decreased in the following sequence: phenol/chloroform > NaI-single tube method > proteinase K digestion equal to amplification of native sera and plasma. Nested PCR from native sera and plasma performed well and surpassed the proteinase K method in sensitivity for detection of serum DNAemia. Semi-quantitative determination of HCMV DNA titer present in native sera of immunosuppressed patients did not seem to be correlated to HCMV disease. When compared to the persistence of leukoDNAemia, the viral DNA titer in native plasma could only be observed in the acute phase of HCMV infection, an important phenomenon for diagnostic purposes. Correlation of serum DNAemia to virus culture revealed low positive and high negative predictive values. Predictive values of nested PCR from native sera for HCMV infection were not lower than those following organic DNA extraction. Despite its low correlation to viremia and virus isolation from any site, nested PCR from organic DNA extracts of serum or plasma is the most sensitive diagnostic tool of an ongoing HCMV infection. Additionally, semi-quantitative end point dilution nested PCR from native serum or plasma promises to be a rapid and easy tool for the monitoring of antiviral therapy.     

Tumor-host interaction in non-random metastatic pattern distribution

In a prospective study, white and red blood cell counts, hematocrit, erythrocyte sedimentation rate (ESR), albumin, alpha-1 acid glycoprotein, C-reactive protein (CRP), and transthyretin (TTR) values were determined by serial measurements during 23 days in 80 patients with an open fracture of the lower limb. Postoperative reference profiles were defined in 74 patients without septic complications. In the six remaining patients, serum CRP and TTR concentrations were found efficient for the early diagnosis of postoperative infections: a CRP/TTR mass concentration ratio higher than 0.6 from the 8th day after surgery was sensitive (100%) and specific (93%). Variations of CRP and TTR concentrations often preceded the clinical diagnosis in patients with early infection. ESR was found unreliable with regard to postoperative infection because of its high dependence with respect to red blood cell count.     

False increase in C-reactive protein attributable to heterophilic antibodies in two renal transplant patients treated with rabbit antilymphocyte globulin

Increased serum C-reactive protein (sCRP) is a sensitive marker of renal graft rejection. We describe the cases of two children with uncomplicated renal transplantation who had false-positive sCRP values on analyzers using rabbit anti-CRP but values within the reference range with anti-CRP from other animal species. Cross-reaction with heterophilic antibodies was suggested by clinical and biological signs of serum sickness and daily treatment with rabbit antilymphocyte globulin (ALG). The interference depended on the serum concentration of the cross-reactant and was removed by subtotal IgG adsorption to Protein A or Protein G or by immunoadsorption using rabbit ALG or total IgG in non-immune rabbit serum. Anti-rabbit IgG and IgM antibodies were detected in both patients. These are the first reported cases of cross-reaction with heterophilic antibodies in a turbidimetric CRP assay.     

Comparison of the quasifree charge-exchange reaction for 12C and 54Fe

Serotonin (5-hydroxytryptamine, 5-HT) in blood is stored in platelets and has vascular and platelet stimulating effects when released into plasma. Accurate measurements of 5-HT in plasma are complicated by inadvertent platelet activation causing sampling artifacts and by analytical problems when determining trace levels. We developed an assay for plasma 5-HT based on solid-phase extraction (Sep-Pak C18), aqueous acetylation, pentafluoropropionylation, and negative ion chemical ionization gas chromatography-mass spectrometry. The method was able to recover 5-HT from plasma by > 90% and to quantitate with a precision of 7.5% at a level of 0.5 nmol/l. It was used to define blood sampling and sample handling procedures giving low and consistent values for 5-HT. A good blood sampling technique, adequate platelet stabilization in the test tube, and rapid high speed centrifugation of the blood resulted in low plasma levels of both 5-HT and beta-thromboglobulin (a platelet release product). Using these procedures plasma 5-HT levels in healthy volunteers were found to be 0.77 +/- 0.38 (mean +/- S.D.; range 0.27-1.49) nmol/l (n = 18), which is 4-100-fold lower than previously reported values.     

Estimates of the virological benefit of antiretroviral therapy are both assay- and analysis-dependent

OBJECTIVE: To assess the potential discrepancies in reported changes in plasma viral load (PVL) depending on how values below the detection limit of the assay are handled in the data analysis phase of a randomized controlled clinical trial. DESIGN: Data from a recently completed clinical trial comparing combinations of zidovudine, didanosine and nevirapine were analysed. In this trial, PVL was measured using an assay with a lower quantification limit of 400 HIV-1 RNA copies/ml initially. All PVL values less than 500 copies/ml were retested with a more sensitive assay with a lower quantification limit of 20 copies/ml. METHODS: Several summary measures for assessing change in PVL were calculated using three different methods to adjust for PVL values less than the quantification limit of the assay. The differences between these measures were evaluated. RESULTS: We found that the magnitude of the discrepancy between summary measures used to report changes in PVL depended on the proportion of subjects with PVL less than the quantification limit of the assay, how those observations were handled in the data analysis, and the relative difference between the quantification limits of the conventional and more sensitive assay. CONCLUSION: The lack of consensus in reporting of PVL data in the literature makes the interpretation of published trial results difficult. In the absence of agreement on the most appropriate summary measure of PVL data, we recommend that all summaries include information on the quantification limit of the assay used, the proportion of observations at or below the quantification limit and how these observations were handled in the data analysis.     

Assessment and modification of alcoholics drinking behavior in controlled laboratory settings: a cautionary note

A group of 10 prepubertal boys was studied during prolonged exercise (60 min) on bicycle ergometer and on treadmill at two levels of work load (appr. 40% and 60% VO2 max). The hematocrit, serum proteins, Cl- and K+ were followed, and from the blood hematocrit changes the plasma volume changes were calculated. At the exercises of lower intensity of both types a slight hemodilution was found (appr. +5% increase in plasma volume), at higher intensity practically no changes could be demonstrated. These findings are supported by the values of serum protein concentration, where no increase was found, and by the fact that at the lower work loads a rather decreasing trend was found for this blood constituent. These findings are at variance with those in adults under similar conditions. The authors suggest that different changes of plasma volume during exercise in boys than in adults could be related to the disparate lactate production and fate in these age groups.     

A novel isoform of the neurofibromatosis type-1 mRNA and a switch of isoforms during murine cell differentiation and proliferation

There are different recommendations for the handling of blood samples for analyses of the kallikrein-kinin or complement system, respectively. C1 inhibitor (C1-INH) takes a crucial part in both systems. In order to establish recommendations for blood specimen collection and transport for making the diagnosis of hereditary angioedema (HAE), the effect of time, temperature and different additives on C1-INH function and antigen was determined. We used blood samples from normals and patients suffering from HAE type I. Plasma containing EDTA, heparin, sodium citrate or polybrene-EDTA, and serum were assayed after incubations at 4 degrees C or 37 degrees C for 6 or 24 h. In addition, pooled serum was incubated for up to 5 days at room temperature. A modest decrease in C1-INH function was observed as an effect of storage-time in samples from normals (p = 0.039) and a substantial decrease was seen for the HAE patients (p = 0.0002). No significant effect of temperature (4 degrees C or 37 degrees C) was found. Clotting did not reduce C1-INH activity. Plasma containing heparin or polybrene interfered with the functional assay, yielding falsely high and low values, respectively. C1-INH functional assay performed within 24 h in serum, EDTA-treated or citrated plasma discriminated well between HAE patients and normals. This was also the case for serum kept at room temperature for up to 5 days, although a modest fall in C1-INH function was seen in the incubation period. For practical purposes we recommend serum as the sample of choice, preferably received within 48 h.     

Development of monkey C-reactive protein (CRP) assay methods

Monkey-specific C-reactive protein (CRP) assay methods (enzyme-linked immunosorbent assay (ELISA) and turbidimetric immunoassay (TIA)) were developed. The anti-monkey CRP serum was prepared by immunization of rabbits with the immune complex formed between the acute-phase serum from turpentine oil-inoculated monkeys and goat anti-human CRP serum. The specificity of the rabbit anti-monkey CRP serum was confirmed by immunoelectrophoresis and Western blotting. The purity of monkey CRP prepared by chromatography procedures was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The serum CRP levels in nine normal monkeys, as measured by sandwich ELISA were ranged from 0.26 to 1.42 microg/ml (mean 0.71+/-0.37). The CRP levels in five acute-phase sera of turpentine oil-inoculated monkeys were 248-451 microg/ml (mean 371.2+/-73.8). This monkey-specific CRP assay method was found more sensitive than the human-specific CRP assay method in detecting monkey CRP by TIA.     

Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis

We have developed a real-time quantitative PCR assay to measure the concentration of fetal DNA in maternal plasma and serum. Our results show that fetal DNA is present in high concentrations in maternal plasma, reaching a mean of 25.4 genome equivalents/ml (range 3.3-69. 4) in early pregnancy and 292.2 genome equivalents/ml (range 76. 9-769) in late pregnancy. These concentrations correspond to 3.4% (range 0.39%-11.9%) and 6.2% (range 2.33%-11.4%) of the total plasma DNA in early and late pregnancy, respectively. Sequential follow-up study of women who conceived by in vitro fertilization shows that fetal DNA can be detected in maternal serum as early as the 7th wk of gestation and that it then increases in concentration as pregnancy progresses. These data suggest that fetal DNA can be readily detected in maternal plasma and serum and may be a valuable source of material for noninvasive prenatal diagnosis.     

Decreased erythrocyte Na+,K(+)-ATPase activity associated with cellular potassium loss in extremely low birth weight infants with nonoliguric hyperkalemia

To determine whether a shift of potassium ions from the intracellular space to the extracellular space accounts, in part, for the hyperkalemia seen in extremely low birth weight infants, we examined potassium concentration in serum and erythrocytes from extremely low birth weight infants with hyperkalemia (n = 12) or with normokalemia (n = 27). In addition, to determine whether the shift of potassium was associated with low sodium-potassium-adenosinetriphosphatase (Na+,K(+)-ATPase) activity, we studied the activity of ATPase in the last 16 infants enrolled in the study. Fluid intake and output were measured during the first 3 days of life. Infants were considered to have hyperkalemia if the serum potassium concentration was 6.8 mmol/L or greater. Blood was obtained daily for intracellular sodium and potassium levels by means of lysis of erythrocytes. The remaining erythrocyte membranes were frozen and analyzed for Na+,K(+)-ATPase activity. There were significantly lower intracellular potassium/serum potassium ratios in the infants with hyperkalemia for each day of the 3-day study (p < 0.001). In the hyperkalemic group, there was lower Na+,K(+)-ATPase activity than in the infants with normokalemia (p = 0.006). Low Na+,K(+)-ATPase activity was associated with lower intracellular potassium/serum potassium ratios (p = 0.006), higher serum potassium values (p = 0.02), and lower intracellular potassium concentration (p = 0.009). The urinary data demonstrated that there was no difference in glomerulotubular balance between the two groups. We conclude that nonoliguric hyperkalemia in extremely low birth weight infants may be due, in part, to a shift of potassium from the intracellular space to the extracellular space associated with a decrease in Na+,K(+)-ATPase activity.     

Inflammation enhances cardiovascular risk and mortality in hemodialysis patients

BACKGROUND: Atherosclerosis, a major problem in patients on chronic hemodialysis, has been characterized as an inflammatory disease. C-reactive protein (CRP), the prototypical acute phase protein in humans, is a predictor of cardiovascular mortality in the general population. We hypothesize that several of the classic, as well as nontraditional, cardiovascular risk factors may respond to acute phase reactions. An activated acute phase response may influence or predict cardiovascular risk. METHODS: In 280 stable hemodialysis patients, serum lipids, apolipoproteins (apo) A-I and B, lipoprotein(a) [Lp(a)], fibrinogen, and serum albumin (Salb) were determined in relation to CRP and serum amyloid A (SAA), two sensitive markers of an acute phase response. Mortality was monitored prospectively over a two year period. RESULTS: Serum CRP and SAA were found to be elevated (more than 8 and more than 10 mg/liter, respectively) in 46% and 47% of the patients in the absence of clinically apparent infection. Patients with elevated CRP or SAA had significantly higher serum levels of Lp(a), higher plasma fibrinogen, and lower serum levels of high-density lipoprotein cholesterol, apo A-I, and Salb than patients with normal CRP or SAA. The rise in Lp(a) concentration was restricted to patients exhibiting high molecular weight apo(a) isoforms. During follow-up, 72 patients (25.7%) had died, mostly due to cardiovascular events (58%). Overall mortality and cardiovascular mortality were significantly higher in patients with elevated CRP (31% vs. 16%, P < 0.0001, and 23% vs. 5%, P < 0.0001, respectively) or SAA (29% vs. 19%, P = 0.004, and 20 vs. 10%, P = 0.008, respectively) and were also higher in patients with Salb of lower than 40 g/liter (44% vs. 14%, P < 0.0001, and 34% vs. 6%, P < 0.0001, respectively). Univariate Cox regression analysis demonstrated that age, diabetes, pre-existing cardiovascular disease, body mass index, CRP, SAA, Salb, fibrinogen, apo A-I, and Lp(a) were significantly associated with the risk of all-cause and cardiovascular mortality. During multivariate regression analysis, SAA, fibrinogen, apo A-I, and Lp(a) lost their predictive values, but age and CRP remained powerful independent predictors of both overall death and cardiovascular death. CONCLUSION: These results suggest that a considerable number of hemodialysis patients exhibit an activated acute phase response, which is closely related to high levels of atherogenic vascular risk factors and cardiovascular death. The mechanisms of activated acute phase reaction in patients on chronic hemodialysis remain to be identified. A successful treatment of the inflammatory condition may improve long-term survival in these patients.     

Optimal seat suspension design based on minimum "simulated subjective response"

The perfusion of serum, citrated whole blood and citrated plasma, through a simple tube system resulted in a significant loss of large von Willebrand factor (vWf) multimers, without decrease in antigen levels. Maximum loss of large multimers was observed at a shear rate of 15,000 s-1 for 15 min. Heparin, aprotinin, soybean trypsin inhibitor, phenylmethylsulphonylfluoride, N-ethylmaleimide, leupeptin or calpain inhibitor peptide could not prevent the loss of large vWf multimers in citrated plasma. The addition of EDTA calcium salt partially prevented it, and it was totally prevented by EDTA without calcium. Perfusion of purified vWf did not induce the loss of large multimers, but this did happen after the addition of either whole serum or a plasma fraction. The activity of this plasma fraction disappeared at pH < 6.8. Besides, we have found that the binding to subendothelium of purified vWf diluted in dialyzed serum was lower at pH 7.2 than at pH 6.0. Chromatographic studies demonstrated that the loss of large vWf multimers, induced by high shear rates, involves a plasma substance(s) of molecular weight larger than 200 kD; calpain and granulocyte or cysteine proteases do not seem to be this plasma substance(s).     

Carbohydrate-deficient transferrin is associated with insulin sensitivity in hypertensive men

An elevated concentration of carbohydrate-deficient transferrin in serum (CDT) has been reported to indicate excessive ethanol consumption. However, in hypertensive men, we found low values for diagnostic sensitivity and specificity. Furthermore, in the individuals with high CDT values, the concentrations of serum triglycerides and blood glucose were low rather than high, indicating that factors related to insulin/glucose metabolism may be operative. The current study addresses this issue by examining 48 patients with treated hypertension and at least 1 of following: hypercholesterolemia, history of smoking, and diabetes mellitus. We determined serum CDT, fasting plasma insulin, and glucose disposal rate during hyperinsulinemic euglycemic clamp. Seven patients had elevated CDT concentrations. This group of patients had higher glucose disposal rates than the others (mean difference, 19 mumol/min.kg lean body mass; 95% confidence interval, 5-33 mumol/min.kg lean body mass; P = 0.0096), but did not differ in body mass index or alcohol intake. Serum CDT correlated positively with glucose disposal rate (r = 0.55; P = 0.0004) and negatively with fasting plasma insulin (r = -0.43; P = 0.0039). These relationships remained after exclusion of 8 patients with diabetes mellitus and adjustment for potentially confounding factors. We conclude that the serum CDT concentrations in our patients were associated with insulin sensitivity.     

The porphyromonas gingivalis prtP/kgp homologue exists as two open reading frames in strain 381

Plasma atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) levels increase in patients with heart failure with the progression of clinical symptoms and with the deterioration of hemodynamics; consequently, assay methods for these peptides may be useful in the follow-up of cardiac patients. Non-competitive immunoradiometric assay (IRMA) methods for ANP or BNP do not generally require preliminary extraction and/or purification of the plasma sample, and so may be more suitable than competitive immunoradiometric assay (RIA) methods for the routine assay of plasma peptide concentrations. We evaluated the analytical characteristics and clinical usefulness of two IRMAs for plasma ANP and BNP, to verify whether these methods may be considered suitable for the follow-up of patients with heart failure. Both methods are based on the solid-phase sandwich IRMA system, which uses two monoclonal antibodies prepared against two sterically remote epitopes of peptide molecule; the first antibody was coated on the beads solid-phase and the second was radiolabeled with 125I. Blood samples were collected from a brachial vein in ice-chilled disposable polypropylene tubes containing aprotinin and EDTA after the patient had rested for at least 20 min in the recumbent position. Plasma samples were immediately separated by centrifugation and stored at -20 C until assay. The IRMA methods showed a better sensitivity and a wider working range sensitivity (about 2 ng/l) than those of RIA methods. Moreover, the normal range found with these methods (ANP = 16.1 +/- 8.6 ng/l, 5.2 +/- 2.8 pmol/l, BNP = 8.6 +/- 8.2 ng/l, 2.5 +/- 2.4 pmol/l) was similar to that generally reported using the most accurate methods, such as the other IRMAs or RIAs, using a preliminary extraction and purification of plasma samples with chromatographic procedures. Our results obtained in patients with different degrees of heart failure indicate that plasma ANP and BNP increase with the progression of clinical symptoms (NYHA class) (ANOVA p < 0.0001). Indeed, circulating levels of ANP (R = -0.701, no. = 86) and BNP (R = -0.745, no. = 55) were significantly (p < 0.0001) and negatively correlated with the left ventricular ejection fraction values. Furthermore, a close curvilinear regression (R = 0.960, no. = 215) was found between ANP and BNP values, because plasma BNP progressively increases more than plasma ANP in patients with different stages of heart failure. In conclusion, IRMA methods are preferable for the measurement of plasma ANP and BNP for experimental studies and routine assay because they are more practicable, sensitive and accurate than RIA procedures. Finally, BNP assay appears to be better than ANP for discriminating between normal subjects and patients with different degrees of heart failure.     

Are current methods of measurement of erythropoietin (EPO) in human plasma or serum adequate for the diagnosis of polycythaemia vera and the assessment of EPO deficiency?

Current methodology for the immunoassay of erythropoietin (EPO) in human plasma or serum is reviewed, with an emphasis on measurement of EPO concentrations in the low and normal ranges, analytical interference and blood sampling requirements. In only 2 out of 8 research or in-house immunoassays reported since 1987 was there evidence that patients with polycythaemia vera (PV) could be identified, PV being an EPO-independent form of polycythaemia in which EPO concentrations are low in untreated cases. The same was true for only 1 out of 13 currently available kit methods. Remarkable differences in sample stability have been observed with different methods. Measurement of EPO in serum is recommended in most published articles. However, only EDTA plasma seems to be acceptable for the one generally available method with proven high diagnostic sensitivity for PV. It is concluded that most EPO assay methods have not been shown to be adequate for the measurement of the low EPO concentrations, and thus have poor diagnostic sensitivity for PV. It is inferred that they might not be appropriate to assess states of EPO deficiency. Only when a sufficiently sensitive diagnostic method becomes generally available will it be possible to define the various causes of low EPO concentrations. As in other fields of polypeptide hormone measurement, further developments in the field of EPO assay may be expected to be important in diagnostic medicine.     

An EDTA-associated anti-B agglutinin: the role of ionized calcium

BACKGROUND: It is believed that EDTA-dependent panagglutination is associated with free carboxylic acids that support reactions of rare autoagglutinins. CASE REPORT: An ABO typing discrepancy occurred in an 88-year-old patient. The specificity of his autoagglutinin was demonstrated by panel cell study and absorption tests using normal donors' red cells or immunoadsorbents coated with A, B, or O substances. Inhibition assays were performed to determine whether the autoagglutinin was inhibited by ionized calcium or carboxylic acids. The autoagglutinin had anti-B specificity when tested in the presence of EDTA. It was neutralized by group B secretor saliva and adsorbed by crystalline silica coated with simple B substances with or without EDTA, although it was absorbed by group B red cells only in the presence of EDTA. The agglutinating activity was stronger at 25 degrees C (titer 64) than at 37 degrees C (titer 16) and was destroyed by treatment of the serum with dithiothreitol, which suggests that the autoagglutinin is IgM. This activity also appeared in the patient's serum after dialysis and in an eluate obtained after adsorption with simple B substances, and it was inhibited by the addition of CaCl2 at 0.5 mM or higher concentrations. This suggests that the agglutination is not dependent on EDTA but, rather, on the concentration of ionized calcium. The autoagglutinin failed to react with group B red cells treated with glutaraldehyde for 10 minutes. CONCLUSION: An anti-B autoagglutinin was shown to have caused an ABO typing discrepancy in the presence of EDTA. These results suggest that autoagglutination requires an environment with low levels of ionized calcium, but not the presence of carboxyl groups.     

Relative activation of milk lipoprotein lipase by serum of cows fed varying amounts of fat

A routine laboratory assay to evaluate relative concentrations of lipoprotein lipase activator (apo C-II) in cow serum was developed. The assay was linear for at least 120 min after an initial, unexplained, lag ime of 13 to 15 min. Half-maximal activation was in the range of 1 to 2% serum in the assay. Inhibition of activation was indicated at high amounts (10%) of serum. Activation from plasma was half that from serum, presumably caused by an increase in substrate Km in the presence of heparin. Use of glyceryl tri[9,10-3H] oleate yielded excessively high blanks; [2-3H] glyceryl triolein is suggested for routine assay. Relative amounts of activator were not different between dry and lactating cows fed "conventional" diets. Activator concentration increased linearly with increasing dietary fat and was related to concentration of total lipid in plasma. The assay may provide a useful adjunct in studies on lipoprotein metabolism.