Exogenous interferences with Jaffe creatinine assays: addition of sodium dodecyl sulfate to reagent eliminates bilirubin and total protein interference with Jaffe methods.

BACKGROUND: The study evaluated the impact of interferences on the analytical specificity of three commercial and commonly used creatinine methods (two Jaffe and one enzymatic). METHODS: Manufacturer creatinine methods plus modified methods were tested with the following interferences: spiking serum with bilirubin, albumin, glucose, hemoglobin and lipid, and patient sera with maximum concentrations of bilirubin, 1,090 micromol/l and protein, 117.8 g/l. RESULTS: Hemoglobin, 7.5 g/l and lipaemic with triglyceride concentration of 6.27 mmol/l, did not interfere with all assays. Glucose >33.3 mmol/l increased creatinine recovery for Dimension method. Samples spiked with bilirubin imparted a negative bias for Dimension and Architect methods but imparted a positive bias for Vitros assay. However, using patient sera, negative bias with bilirubin was found for all methods, from which Architect method gave the highest effect (R(2)=0.861), followed by Vitros (R(2)=0.239) and Dimension (R(2)=0.163). Protein provided the positive bias for all creatinine measurements that increased with increasing concentration (R(2) ranging from 0.104 to 0.182, P<0.0001). Addition of sodium dodecyl sulfate (SDS) in alkaline-picrate reagent reduced the effect of bilirubin and protein for kinetic Jaffe method. Although adding potassium ferricyanide was well effective for eliminating negative interference of bilirubin, it was prone to interference from protein. CONCLUSIONS: Endogenous interferences continue to plague creatinine accuracy measurement in both Jaffe and enzymatic methods, and consequentially the estimated glomerular filtration rate. The addition of SDS to the alkaline-pirate reagent was shown to be effective in reducing bilirubin and protein interferences.

How to use linear regression and correlation in quantitative method comparison studies.

Linear regression methods try to determine the best linear relationship between data points while correlation coefficients assess the association (as opposed to agreement) between the two methods. Linear regression and correlation play an important part in the interpretation of quantitative method comparison studies. Their major strength is that they are widely known and as a result both are employed in the vast majority of method comparison studies. While previously performed by hand, the availability of statistical packages means that regression analysis is usually performed by software packages including MS Excel, with or without the software programe Analyze-it as well as by other software packages. Such techniques need to be employed in a way that compares the agreement between the two methods examined and more importantly, because we are dealing with individual patients, whether the degree of agreement is clinically acceptable. Despite their use for many years, there is a lot of ignorance about the validity as well as the pros and cons of linear regression and correlation techniques. This review article describes the types of linear regression and regression (parametric and non-parametric methods) and the necessary general and specific requirements. The selection of the type of regression depends on where one has been trained, the tradition of the laboratory and the availability of adequate software.

von Willebrand factor binding to platelet GpIb initiates signals for platelet activation.

The hypothesis that von Willebrand factor (vWF) binding to platelet membrane glycoprotein Ib (GpIb) initiates intracellular pathways of platelet activation was studied. We measured the biochemical responses of intact human platelets treated with ristocetin plus vWF multimers purified from human cryoprecipitate. vWF plus ristocetin causes the breakdown of phosphatidylinositol 4,5-bisphosphate, the production of phosphatidic acid (PA), the activation of protein kinase C (PKC), increase of ionized cytoplasmic calcium ([Ca2+]i), and the synthesis of thromboxane A2. PA production, PKC activation, and the rise of [Ca2+]i stimulated by the ristocetin-induced binding of vWF multimers to platelets are inhibited by an anti-GpIb monoclonal antibody, but are unaffected by anti-GpIIb-IIIa monoclonal antibodies. Indomethacin also inhibits these responses without impairing platelet aggregation induced by vWF plus ristocetin. These results indicate that vWF binding to platelets initiates specific intraplatelet signaling pathways. The mechanism by which this occurs involves an arachidonic acid metabolite-dependent activation of phospholipase C after vWF binding to platelet membrane GpIb. This signal then causes PKC activation and increases of [Ca2+]i, which promote platelet secretion and potentiate aggregation.

Protease-activated receptor-induced Akt activation--regulation and possible function.

BACKGROUND: Thrombin induces the activation of the platelet serine/threonine kinase Akt. Akt activation is dependent on its phosphorylation at Thr308 and Ser473. The mechanism by which thrombin induces Akt phosphorylation is controversial, as is the role of Akt in platelet function. OBJECTIVES: To investigate how protease-activated receptors (PARs) stimulate Akt and the role that Akt plays in human platelet function. METHODS: Platelets were stimulated through PAR1 or PAR4. Specific inhibitors were used to evaluate, by Western blotting, signaling pathways regulating Akt phosphorylation, and the role of activated Akt was evaluated by aggregometry and flow cytometry. RESULTS: Phospholipase C (PLC) controls Akt phosphorylation elicited by PARs. Stimulation of PAR1 or PAR4 resulted in rapid Akt phosphorylation, independently of secreted ADP and phosphatidylinositol-3-kinase (PI3K) activation. Akt phosphorylation approximately 60 s after PAR1 stimulation became entirely dependent on the purinergic receptor P2Y(12) and the activation of PI3K. In contrast, PAR4 partially sustained Akt phosphorylation independently of P2Y(12) and PI3K for up to 300 s. Pharmacologic inhibition of Akt reduced P-selectin expression and fibrinogen binding in platelets stimulated through PAR1, and delayed platelet aggregation in response to submaximal PAR1 or PAR4 stimulation, although aggregation at 300 s was unaffected. CONCLUSIONS: Platelet PAR stimulation causes rapid Akt phosphorylation downstream of PLC, whereas with continuous stimulation, ADP and PI3K are required for maintaining Akt phosphorylation. Activated Akt regulates platelet function by modulating secretion and alpha(IIb)beta(3) activation.

Extended pharmacologic thromboprophylaxis in oncologic liver surgery is safe and effective.

Essentials The risk for venous thromboembolism after liver surgery remains high in the modern era. We evaluated the safety/efficacy of extended anticoagulation in liver surgery. This protocol reports zero venous thromboembolism events in 124 liver surgery patients. Extended anticoagulation after oncologic liver surgery is safe and effective. SUMMARY: Background The incidence of venous thromboembolism (VTE) after liver surgery remains high. Objective To evaluate the safety and efficacy of extended pharmacologic thromboprophylaxis after liver surgery for the prevention of VTE. Patient/Methods From August 2013 to April 2015, 124 patients who underwent liver resection for malignancy were placed on an extended pharmacologic thromboprophylaxis protocol. Intraoperative VTE prophylaxis included thromboembolic deterrent hoses and sequential compression devices. Once hemostasis had been ensured following hepatectomy, daily anticoagulant VTE prophylaxis was initiated for the duration of hospitalization. After hospital discharge, the large majority of patients (114, 91.9%) continued to receive anticoagulant thromboprophylaxis (enoxaparin) to complete a total course of 14 days after minor/minimally invasive hepatectomy or 28 days after major hepatectomy or a history of VTE. Results The cohort included 39 (31.2%) major hepatectomies and 38 (31.5%) minor/minimally invasive approaches. The intraoperative, postoperative and overall transfusion rates were 5.6%, 8.1%, and 10.5%, respectively. Pharmacologic thromboprophylaxis was started on postoperative day (POD) 0 for 40 (32.3%) patients and on POD 1 for 84 (67.7%) patients. During 90 days of follow-up, no postoperative symptomatic deep vein thrombosis or pulmonary embolic events were diagnosed. Standard-protocol computed tomography scans of the chest, abdomen and pelvis that were obtained for 112 (90.3%) study patients showed no pulmonary emboli, or other thoracic, splanchnic or ileofemoral vein thromboses. Two (1.6%) patients had minor bleeding events that resolved after discontinuation of enoxaparin, requiring neither blood transfusion nor reoperation. The severe complication rate was 5.6%, with no 90-day mortalities. Conclusions These preliminary data suggest that extended pharmacologic thromboprophylaxis for liver surgery patients is safe and effective.

Control of platelet protein kinase C activation by cyclic AMP.

Experiments were performed to elucidate the role of adenosine 3': 5'-cyclic monophosphate (cAMP) in the control of platelet protein kinase C (PKC) activation. Platelet aggregation and secretion in response to 4 beta-phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl-2-acetylglycerol (OAG) were inhibited by dibutyryl cAMP in a dose-dependent manner. Inhibition of these functional activities paralleled a decrease in the PMA-induced phosphorylation of the Mr 47,000 substrate (p47) of PKC by pre-incubation of platelets with dibutyryl cAMP. These changes were also observed when platelet cAMP was increased by prostacyclin (PGI2), forskolin, or theophylline. The ADP scavenger creatine phosphate/creatine phosphokinase (CP/CPK) and the cyclooxygenase inhibitor indomethacin also diminished the aggregation and p47 phosphorylation responses to PMA or OAG. Pre-incubation of platelets with dibutyryl cAMP significantly potentiated the inhibition of aggregation and p47 phosphorylation effected by CP/CPK and indomethacin. These results are consistent with the model that PMA- or OAG-induced activation of platelets is amplified by secreted ADP and that the response to secreted ADP is inhibited by cAMP. Furthermore, the findings that increased intracellular cAMP inhibits PMA- or OAG-induced p47 phosphorylation in excess of that due solely to CP/CPK, and that cAMP significantly potentiates the effects of ADP removal and inhibition of cyclooxygenase in blocking p47 phosphorylation suggest that cAMP also exerts non-ADP-mediated inhibitory effects on PKC in intact platelets.

Regulation of the phosphoinositide cycle by Na+/H+ exchange and intracellular pH in human platelets.

We have found that thrombin-induced activation of protein kinase C (PKC) in platelets, measured by phosphorylation of the 47 kDa protein, is synergistically enhanced by the amiloride analogue ethylisopropylamiloride (EIA), a specific inhibitor of Na+/H+ exchange. This EIA effect was further synergistically enhanced by lowering intracellular pH (pHi) with either nigericin or sodium propionate, and reversed by raising pHi with monensin or ammonium chloride. The synergistic enhancement of thrombin-activated PKC by EIA plus nigericin was not observed when PKC was directly activated by phorbol esters. EIA and EIA plus nigericin caused a 3- to 6-fold increase in thrombin-induced diacylglycerol (DAG), but not phosphatidic acid (PA), production. EIA and nigericin also caused a marked increase in thrombin-induced breakdown and inhibition of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2). In summary, we have presented evidence that inhibition of Na+/H+ exchange causes primarily a H(+)-mediated interruption of the phosphoinositide cycle in activated platelets, including the accumulation of DAG associated with the enhancement of PKC activation, the inhibition of conversion of DAG to PA, and increased PIP2 breakdown. These data suggest a model in which Na+/H+ and pHi play an important regulatory role in permitting the phosphoinositide cycle to proceed in thrombin-activated platelets.

Monoclonal antibody AG-1 initiates platelet activation by a pathway dependent on glycoprotein IIb-IIIa and extracellular calcium.

The biochemical responses of intact human platelets to the monoclonal antibody (mAb) AG-1 were investigated. AG-1 is a murine IgG mAb that recognizes a series of platelet membrane glycoproteins (Gp) from M(r) 21,000 to 29,000, one of which is the M(r) 24,000 (p24) receptor for anti-CD9 mAbs. AG-1 causes platelet aggregation and secretion. Platelets binding AG-1 demonstrate a dose- and time-dependent breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), production of diacylglycerol, and generation of phosphatidic acid (PA). These events are associated with the activation of protein kinase C (PKC), an increase in intracellular calcium, and fibrinogen binding. Platelet PA generation and PKC activation in response to AG-1 are inhibited by mAbs to platelet GpIIb-IIIa or by extracellular EGTA, but not by a mAb to platelet GpIb or by inhibiting platelet Na+/H+ exchange with 5-(N-ethyl-N-isopropyl)amiloride. Platelet cytoplasmic free calcium ([Ca2+]i) is elevated in response to AG-1, and this elevation is inhibited by mAbs to GpIIb-IIIa, an RGDS peptide or by chelating extracellular calcium. These results suggest that AG-1 binding to a unique platelet-surface glycoprotein initiates platelet responses through the activation of PIP2-specific phospholipase C, and that this occurs through a signal pathway that is dependent on GpIIb-IIIa and extracellular calcium.

von Willebrand factor binding to platelet glycoprotein Ib-IX-V stimulates the assembly of an alpha-actinin-based signaling complex.

BACKGROUND: Pathological shear stress induces platelet aggregation that is dependent on von Willebrand factor (VWF) binding to glycoprotein (Gp)Ib-IX-V and phosphatidylinositol 3-kinase activation. We tested the hypothesis that pathological shear stress stimulates phosphatidylinositol 3,4,5-trisphosphate (PIP3) synthesis by directing the assembly of a molecular signaling complex that includes class IA phosphatidylinositol 3-kinase (PI 3-KIA). METHODS: Platelets were subjected to 120 dynes cm-2 shear stress in a cone-plate viscometer. Resting and sheared platelets were lyzed, immunoprecipitations of PI 3-KIA performed, or lipids extracted for PIP3 measurements. alpha-Actinin was incubated with phosphatidylinositol 4,5-bisphosphate (PIP2), immunoprecipitated, and used as a substrate for in vitro PI 3-KIA activity. RESULTS: Pathological shear stress induces biphasic PIP3 production. In resting platelets, PI 3-KIA associates with alpha-actinin and PIP2. After exposure to shear stress, alpha-actinin and PIP2 rapidly disassociate from PI 3-KIA. PI 3-KIA then gradually re-associates with PIP2 and alpha-actinin, and this complex becomes linked to GpIb alpha through the cytoskeleton. PIP3 production and the observed changes in the association between alpha-actinin, PIP2, and PI 3-KIA are inhibited when VWF binding to GpIb alpha is blocked. In a cell-free system, alpha-actinin binds PIP2 and when the alpha-actinin-PIP2 complex is added to platelet PI 3-KIA, PIP3 production is stimulated. CONCLUSIONS: These results suggest that pathological shear-induced VWF binding to GpIb-IX-V stimulates PIP3 production through the assembly of an alpha-actinin-based complex that colocalizes PI 3-KIA with substrate PIP2.

Regulation of interleukin-1-beta-stimulated inducible nitric oxide synthase expression in cultured vascular smooth muscle cells by hemostatic proteins.

Experiments were performed to examine the mechanism by which specific hemostatic proteins regulate the release of nitric oxide (NO) from interleukin-1 beta (IL-1beta) stimulated cultured rate aortic smooth muscle cells. Treatment of smooth muscle cells with IL-Beta stimulated inducible nitric oxide synthase (iNOS) mRNA expression, which preceded the release of NO (as measured by the accumulation of nitrite in the culture media). The cytokine-stimulated production of nitrite was blocked by the protein synthesis inhibitor cycloheximide, the transcriptional inhibitor actinomycin D, and the competitive inhibitor of NOS nitro-L-arginine. However, only actinomycin D inhibited IL-1beta-stimulated iNOS mRNA expression, Treatment of smooth muscle cells with IL-1beta in the presence of platelet derived growth factor or thrombin resulted in the inhibition of cytokine-stimulated expression of iNOS mRNA and NO release. The inhibitory effect of thrombin was reversed by hirudin and was mimicked by a 14 amino acid thrombin receptor activating peptide. In contract, the concomitant exposure of smooth muscle cells to IL-1beta-and plasmin resulted resulted in the potentiation of both IL-1beta-stimulated iNOS expression and NO generation. Finally, treatment of smooth muscle cells with IL-1beta in the presence of the hemostatic proteins did not affect the half-life of iNOS mRNA. These results demonstrate that specific protein components of the hemostatic system regulate IL- 1beta-stimulated iNOS mRNA expression in vascular smooth muscle cells. The capacity of hemostatic proteins to modulate the induction of vascular iNOS activity may play an important role in governing the release of NO and regulating thrombogenesis in vivo.

Lack of interference with creatinine assays by four cephalosporin-like antibiotics.

Some cephalosporin-like antibiotics have been shown to interfere with creatinine assays performed by the commonly used Jaffe methods. The authors show that four new drugs of this type--moxalactam, cefoperazone, cefotaxime, and ceftazidime--do not interfere with this test. A table is presented summarizing all literature data regarding interference with creatinine assays of cephalosporin-like drugs.

Standardization of lipoprotein reporting.

We wanted to ascertain whether the current format of lipid laboratory reports seemed adequate to promote identification and treatment of patients with dyslipidemia. In a random survey of lipid laboratory reports from 25 laboratories, we found great inconsistencies among reporting formats and contents. Fewer than half the laboratories correctly reported the ranges for cholesterol, only 4 correctly reported ranges for high-density lipoprotein cholesterol, only 2 correctly reported ranges for triglycerides, and none presented low-density lipoprotein cholesterol ranges in terms of risk factors for coronary heart disease. Reports typically were disjointed and difficult to read. The current practice of reporting results for lipid panels is confusing and does not follow the National Cholesterol Education Program (NCEP) guidelines. We recommend that reporting of results be standardized, and a "model" standardized report is presented herein, based on consensus from a team of experts. The standardized report uses current recommendations for ranges, follows the flowcharts of the NCEP guidelines, and takes the patient's clinical condition (the number of risk factors and the presence of coronary heart disease) into consideration. Standardizing lipid reports should decrease confusion and perhaps increase application of the guidelines and patient compliance with treatment.

Rapid rise of serum acid phosphatase after irradiation of metastatic carcinoma of prostate.

An eighty-two-year-old man with metastatic prostatic adenocarcinoma was treated with radiation therapy to the lumbar region of the spinal column. A rapid rise in his acid phosphatase activities developed, increasing thirty-eight-fold in two days. He died on the second day post-therapy of hemorrhagic complications. The rapid increase in acid phosphatase activity was due to release from injured or dying prostatic adenocarcinoma cells.

Bone to total alkaline phosphatase ratios improve sensitivity and specificity of bone alkaline phosphatase immunoassays.

OBJECTIVES: Evaluate the ability of two bone alkaline phosphatase (ALPB) immunoassays (Ostase, Hybritech Inc and Alkphase-B, Metra Biosystems) to clinically differentiate between osseous and non-osseous ALP sources. DESIGN AND METHODS: Specimens from patients with either liver or bone disease (Paget's disease or metastatic cancer) were analyzed by both methods. RESULTS: There was a good correlation between these two assays. Values for ALPB, whether determined as a concentration by the Ostase assay or as an activity by the Alkphase-B assay, were similar for subjects with liver disease or bone disease. However, total ALP (ALPT) activity was higher in liver disease compared to bone. When ALPB was expressed in relation to ALPT, ratios were significantly greater in subjects with bone disease than in those with liver disease. ALPB/ALPT ratios improved the specificity of the Ostase assay from 52% to 86% and the Alkphase-B assay from 58% to 74%. CONCLUSIONS: These two ALPB assays have good analytical performance and their clinical utility can be enhanced by expressing ALPB values in relation to ALPT activity.

M(r) 6,400 aurin tricarboxylic acid directly activates platelets.

ATA is a novel anticoagulant polymeric anionic aromatic compound that inhibits von Willebrand factor binding to platelet glycoprotein Ib and thereby prevents ristocetin- and shear stress-induced platelet aggregation. To investigate its mechanism of action, ATA fractions of homogeneous M(r) have been prepared by size exclusion chromatography. ATA fractions of M(r) > or = 2,500 are most effective at inhibiting vWF-mediated platelet aggregation, and ATA of M(r) = 2,500 also inhibits thrombin-induced platelet activation. Paradoxical results were observed in studies of ATA with M(r) = 6,400. This fraction of ATA stimulates aggregation of washed platelets or platelet-rich-plasma. The dose/response of aggregation shows a bell-shaped curve with maximal aggregation at approximately 2 micrograms/ml. Platelet aggregation is associated with phosphoinositide turnover and protein kinase C- and calcium-dependent protein phosphorylation. Platelet signalling responses to ATA are inhibited by platelet pretreatment with PGI2 or dibutyryl-cyclic AMP, but are unaffected by inhibiting platelet cyclooxygenase with aspirin. These results suggest that M(r) 6,400 ATA directly activates platelet phospholipase C to initiate platelet aggregation. This effect, unique to M(r) 6,400 ATA, could potentially mitigate ATA's beneficial anti-thrombotic effect on vWF-mediated platelet responses, and should be considered when analyzing results of experiments that utilize unfractionated ATA.

Glycoprotein Ib/IX/V binding to the membrane skeleton maintains shear-induced platelet aggregation.

The extracellular domain of glycoprotein (Gp) Ibalpha serves as the von Willebrand factor (vWf) receptor that triggers shear stress-dependent platelet aggregation. Its intracellular domain associates with actin-binding protein-280 (filamin 1a) that binds directly to filamentous actin, thereby linking the membrane skeleton to GpIbalpha. We examined the functional significance of GpIbalpha interactions with actin during platelet aggregation in response to 120 dyn/cm(2) shear stress. Lysates of resting and sheared platelets were centrifuged at approximately 13,000xg for 15 min, and GpIbalpha was immunoprecipitated from the lysate supernatant. GpIbalpha and coimmunoprecipitated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with antibodies specific for GpIbalpha and actin. We observed a significant increase in the amounts of actin coimmunoprecipitating with GpIbalpha as platelets aggregated in response to shear stress. Actin/GpIbalpha interactions reached a maximum after 90 s of shear stress. Monoclonal antibody (mAb) blockade of vWf binding to GpIbalpha inhibited shear stress-induced platelet aggregation and actin associating with GpIbalpha. Pretreatment of platelets with cytochalasin D resulted in the inhibition of actin binding to GpIbalpha in sheared platelets and in an increase in the rate and magnitude of platelet disaggregation. These data indicate that shear stress causes changes in the association between GpIbalpha and the actin-based membrane skeleton. The increased interaction between GpIbalpha and the actin-based membrane skeleton results from shear-induced vWf binding to GpIbalpha and is mechanoprotective in that it maintains shear-induced aggregation of activated platelets.

Linear regression estimation of minimal detectable concentration. Thyrotropin as an example.

BACKGROUND: Minimal detectable concentration is an important analytic feature of certain clinical immunoassays. We believe that accuracy is an important component of the minimal detectable concentration; for a given observed concentration to be meaningful, it should reflect a consistent linear relationship with the amount of analyte actually present. METHODS: To evaluate the minimal detectable concentration, we developed a linearity regression protocol based on accuracy and also accounting for between-run variability. Using serial twofold dilutions of serum samples, we regressed the log of concentration (x) and of dilution (y) with linear, second-, and third-order polynomials. Initially, we evaluated two elements to find the linear region of the dataset, establishing the statistical significance of the beta coefficients with a t test and the reduction of the sum of square of the residuals between the linear regression and the higher-order regressions by means of an F test. As needed, we successively eliminated the lowest point until the linear regression was the best fit. Once we found the best fit, we added the most recently removed point back and calculated the difference between the value predicted by the first-order regression and the observed value. If the difference was not analytically significant, then we considered the point to be part of the linear set; otherwise, it was not included. In either case, the lowest included point was considered to be the minimal detectable concentration. RESULTS: We applied the technique in evaluating two automated systems for serum thyrotropin. One system appeared linear and accurate down to 0.02 mU/L, or better, approximately 77% of the time, and to 0.01 mU/L 68% of the time. The second system was linear infrequently and appeared to be useful down to 0.02 mU/L, or better, only about 20% of the time. CONCLUSIONS: This accuracy-based approach to determining the minimal detectable concentration is an attractive alternative to current empiric approaches, which are based only on interassay variability.

Trabecular carcinoma of the skin: further clinicopathologic and morphologic study.

We studied the clinical aspects of 30 cases of trabecular carcinoma of the skin. Twenty-three patients were followed up for more than one year. Trabecular carcinoma of the skin is often misdiagnosed as a metastatic malignant tumor. The average age at the time of diagnosis was 68 years; most tumors occurred during the seventh and eighth decades of life. Most initial lesions were located in the head, neck, and upper extremities. Lymph node metastases developed in 13 patients, three of whom eventually died of the disease. Local recurrence developed in ten patients, four of whom died of metastatic trabecular carcinoma. The overall mortality was five of the 30 patients. Three of the patients in whom generalized metastases developed also suffered from some other severe systemic disorder. Nearly half of the 23 patients are free of disease. Sweat gland differentiation was observed in two cases, which indicates that the cell of origin is a multipotential unit capable of both neuroendocrine and sudoriferous differentiation.

Evaluation of the extent of nonlinearity in reportable range studies.

OBJECTIVES: To extend the polynomial method for evaluating linearity in 2 ways. First, we developed a screen to ascertain whether the data were precise enough to permit a reliable evaluation of linearity and therefore eliminate findings of linearity due to low statistical power. Second, we assessed whether the degree of nonlinearity detected by the polynomial method was clinically relevant using a statistically rigorous method. METHODS: Because we assessed linearity relative to a clinically determined level of importance instead of the default value of zero, we used sampling theory based on the noncentral chi(2) distribution. Using statistical power calculations, we incorporated a screen for imprecision that guarantees that the probability of correctly identifying nonlinear methods is at least 80%. RESULTS: With the described methods, we achieved a sensitivity of at least 80% and a specificity of at least 95%. When the data were too imprecise to achieve a sensitivity of 80%, no determination of linearity was made. This procedure mimics the practice in manual inspection of flagging data that appear imprecise by visual inspection and halting the evaluation. CONCLUSIONS: Formal statistical tests for precision and amount of nonlinearity are advantageous because they allow us to quantify and limit classification errors. By formalizing these various aspects of linearity assessment, we maintain some of the complex features of manual methods while making the linearity assessment feasible to apply to a high volume of assessments and removing the between-analyst variability.

Biological variation of glucose and insulin includes a deterministic chaotic component.

Serial data of glucose and insulin values of individual patients vary over short periods of time; this phenomenon has been called biological variation. The classic homeostatic control model assumes that the physiological mechanisms maintaining the concentrations of glucose and insulin are linear. The only deviations over a short period of time one should observe are in relation to a glucose load or major hormonal disturbance. Otherwise, the values of these analytes should be constant and any variations seen are due to random disturbances. We investigated previously published serial data (three for glucose and one for insulin) with nonlinear analytical methods, such as embedding space, correlation dimension, Lyapunov exponents, singular value decomposition and phase portraits, as well as linear methods, such as power spectra and autocorrelation functions. The power spectra failed to show dominant frequencies, but the autocorrelation functions showed significant correlation, consistent with a deterministic process. The correlation dimension was finite, around 4.0, the first Lyapunov exponent was positive, indicative of a deterministic chaotic process. Furthermore, the phase portraits showed directional flow. Therefore, the short-term biological variation observed for analytes arises from nonlinear, deterministic chaotic behavior instead of random variation.