Turbidimetric detection of clot in plasma is a major assay principle adopted by many coagulometersused in clinical laboratories. We derived local maxima of 1st and 2nd derivatives of turbidimetriccurves of activated partial thromboplastin time as introduced in other studies as peak 1 and peak 2respectively.
Peak 1 and peak 2 values were derived from APTT data stored in the operating computer. TheAPTT turbidime try data were converted into csv file format and analyzed using a spreadsheet program.Each series of first or second derivative values were scanned for local maxima, which were then chosenas peak 1 and 2 values, respectively.
The coefficient of variations of peak 1 and 2 of within-run repeats were 3.8% and 8.3% for normalAPTT plasma and 4.8% and 14.8% for prolonged APTT plasma. Peak 1 inversely correlated with APTTbut the relation was not strong (r: -0.5324; 95% confidence interval: -0.7143 to -0.2832) whereas peak 2showed stronger inverse correlation (r: -0.8257; 95% CI: -0.9009 to -0.7024). The reference intervals werederived from APTT data from 115 apparently healthy individuals. There was no significant difference indistribution of peak 1 or 2 values between both sexes and the reference intervals were determined to 150.0 to 290.7 TU/sec for peak 1 and 488.4 to 1,026.3 TU/sec^{2} for peak 2.
Our study is a starting point that will aid in the study of hemostatic meaning of the new coagulometerparame ters peak 1 and 2 and on the evaluation of their clinical usefulness.
Turbidimetry is a major assay principle of measurement of clotting time and is used in many coagulometers in current use in clinical laboratories. Briefly, as fibrinogen is converted to fibrin, it acquires a structure favorable to lateral aggregation, and a branching fibrin clot can be formed, increasing the viscosity of the plasma to that of turbid gel. The relation between microscopic web-like fibrin clot structures and increase in turbidity was demonstrated in detail by Chernysh et al. [
Every APTT test was conducted using an automated coagulometer ACL TOP 700 (Instrumentation Laboratory, USA). During an APTT test, the coagulometer measures the turbidity of a plasma-reagent mixture more than 1,000 times. Thus, for each APTT test, there is a series of normalized turbidity values plus first and second derivative values obtained through numerical methods along with time points of measurements. The first and second derivative values are scanned, and the maximum values that are in line with adjacent time point values are chosen to be named as peak 1 and peak 2. A typical turbidimetric curve with overlying first and second derivative curves is shown in
Two pooled plasma samples were prepared from patient referred to our clinical laboratory for APTT. One was pooled from samples with normal APTT results, and the other from those with APTT longer than 60 seconds. Simple short-term repeatability tests were performed by repeating APTT 20 times in a single run for each pooled sample. APTT data from 45 patients with results distributed evenly over the measurable range were selected, and peak 1 and peak 2 values were extracted for correlation analysis. Reference values were collected by derivation of peaks 1 and 2 from stored APTT data from 115 apparent healthy adults.
All relevant statistical analyses including calculation of statistics (mean, standard deviation, coefficient of variation), correlation analyses with scatter plot representations, analyses of parameter distribution, comparison of means and variances, and determination of reference intervals were conducted using MedCalc ver. 15.10.1 (MedCalc Software, Belgium).
Within run repeatability of peak 1 and peak 2 was examined from two preparations of pooled plasma, one with normal and the other with moderately prolonged APTT. As summarized in
Reference intervals of Peak 1 and 2 values were derived from 115 apparently healthy individuals including 60 females. Because the origin of the data was APTT included in health check-up tests, we could not include the data from pediatric patients. Patient age ranged from 26 to 79 years, and no parameter included in the analysis showed significant correlation with age. Both peaks 1 and 2 as well as APTT were normally distributed. However, peaks 1 and 2 were closer to normal distribution than APTT, which showed high degree of peakedness (
Fibrin clot formation is an essential component of hemostasis and can be assessed and characterized in a number of ways. As the weblike micro-structure of a fibrin clot forms in plasma, it progressively increases the turbidity such that the light transmittance through the plasma decreases accordingly. This can be exploited to detect fibrin clot formation (
The present findings are different from our previous observation where much lower or similar peak 1 and 2 CVs compared to APTT CVs were obtained from coagulation factor VIII calibrators. The calibrators included in that study almost lacked factor VIII (factor VIII activities 0.0 to 2.0%) such that APTTs were much longer than those observed in the present study. Possibility of improved precision of peak 1 and 2 in plasmas with more prolonged APTT needs to be examined in detail. Peak 1 value inversely correlated with APTT, but the relation was not strong. Peak 2 showed a stronger relation to APTT. It is understandable that plasma with a more active coagulation system more quickly reaches a certain point of most precipitous clot formation. However, from our results, it is evident that high fibrin formation rate does not necessarily mean normal clotting time. For protection from bleeding, a fully organized fibrin structure is necessary. Our results indicate that, although clotting starts somewhat later (prolonged clotting time) than the upper reference limit, the hemostatic amount of fibrin clot can still be achieved if the subsequent clot formation rate is sufficiently high. It would be valuable to prove this hypothesis for predicting and managing bleeding risk. No case of normal APTT with low peak 1 was found in our study but deserves to be monitored continuously in clinical samples because this might represent a novel indicator of bleeding risk.
Peak 2 was different from peak 1 with regard to overlap between reference and prolonged APTT groups. The possible explanation is that the persistence of a certain level of acceleration and thus the driving force of clot formation, even if its highest peak is not that high, can still produce a significantly high clotting rate peak (peak 1). This also needs to be further analyzed with mathematical consultation. Finally, we derived reference intervals for peak 1 and peak 2 from apparently healthy individuals. Although the reference intervals were determined in adults, and an age effect needs to be examined more thoroughly, Our study is a starting point that will aid in the study of hemostatic meaning of the new coagulometer parameters peak 1 and 2 and on the evaluation of their clinical usefulness.
Turbidimetry curve of APTT and its first and second derivatives. From the turbidimetry curve of APTT, the first and second derivatives can be derived using a numeric method. From those derived curves, the local maxima of peak 1 and peak 2 can be obtained.
Correlations between APTT and peak 1 and 2. APTT and peak 1 showed a weak inverse correlation that was not strengthened by log transformation of peak 1 values (A). Peak 2 showed a more evident inverse correlation with APTT that was strengthened through log transformation and took a linear form (B). Peak 1 correlated well with peak 2 in an almost linear fashion (C).
Comparison of peak 1 and 2 values between reference and prolonged APTT groups. Peak 1 values between the two groups overlapped significantly because many peak 1 values from plasma with prolonged APTT were in the reference interval (A). Peak 2 was different from peak 1 in that the values of the two groups were clearly separate, with only a few overlap (B).
Within-run coefficients of variation of APTT, peaks 1 and 2 measured from two pools of plasma with normal and prolonged APTT
APTT (sec) |
Peak 1 (TU/sec) |
Peak 2 (TU/sec^{2}) |
||||
---|---|---|---|---|---|---|
Pool 1 | Pool 2 | Pool 1 | Pool 2 | Pool 1 | Pool 2 | |
Mean | 32.8 | 76.5 | 189.3 | 114.8 | 642.7 | 243.1 |
Within-run standard deviation | 0.52 | 0.68 | 7.2 | 5.5 | 53.2 | 35.9 |
Within-run coefficient of variation (%) | 1.59 | 0.88 | 3.8 | 4.8 | 8.3 | 14.8 |
Reference intervals of peak 1 and 2 derived with parametric and nonparametric methods
Peak 1 (TU/sec) | Peak 2 (TU/sec^{2}) | |
---|---|---|
Range | 138.1-317.8 | 372.5-1,109.7 |
Mean | 220.4 | 757.4 |
Standard deviation | 35.9 | 137.2 |
Median | 218.5 | 747.4 |
Reference interval | ||
Parametric method | 150.0-290.7 | 488.5-1,026.3 |
Non-parametric method | 152.4-306.9 | 512.6-1,038.6 |