Measurement of temporal changes in contusion color by colorimeter
Purpose: To explore the pattern of skin color change of contusion and to find suitable indicators to infer the time of contusion formation and initial degree. Methods Using negative pressure suction, areas of skin contusion of different degrees were simulated bilaterally on the medial forearms of 41 volunteers. Measurements were made using a colorimeter every 0.5 d for the first 3 d after the injury and every 1 d after 3 d until the color difference in the contused area was indistinguishable to the naked eye. Curve fitting was performed for the six color difference indicators ΔL*, Δa*, Δb*, ΔE*ab, ΔC*, and Δh. For the five characteristic time indicators, ta peak, tb negative, tb peak, tC peak, and ttotal, the Mann-Whitney U test was used to compare gender differences, and the paired-sample mean t test was used to analyze differences in the degree of contusion. Results: The regression equation for the Δh indicator had the best fit (R2=0.6395). ta peak, tb negative, tb peak, tC peak, and ttotal 5 indicators were not related to gender (P>0.05),3 indicators of ta peak, tb peak, and ttotal, and the rate of change of the color (ΔE*ab slope of the fitted curve) were correlated with the degree of contusion (P<0.05), but the 2 indicators of tb negative and tC peak were not influenced by the degree of contusion (P<0.05). Indicators were not affected by the degree of contusion (P>0.05). tb negative appeared at 1.72±0.90 d, i.e., Δb* was positive after 19h. Conclusion Δh and Δb* values can be used to infer the time of contusion formation and initial extent, and the positive and negative Δb* values can determine whether the contusion was formed within 19h.
What is contusion?
Contusion (bruise) is an injury mainly characterized by intra- and subcutaneous hemorrhage and is most common in various types of violence. Especially in incidents of domestic violence, sexual assault, and child and elder abuse, it is important to make inferences about the time of contusion formation. Research on the formation and evolution mechanism of contusion helps us to analyze the site and time of violence, determine whether the injury was formed simultaneously, or analyze the time of death and post-injury survival time to reconstruct and restore the case. Forensic doctors have always relied on visual observation and empirical analysis to determine the formation time of contusion. However, this model is limited by the experience and ability level of the forensic workers; there are large errors, and different individuals have different subjective feelings about the same color. Therefore, it is unreliable to infer the time of contusion through vision, and we need to quantify the color and digitize the color change through instruments.
A colorimeter is an instrument designed to measure color deviation by simulating the process of light entering the human eye. It can measure the color signal under the specified light source conditions, and the software calculates the color values of different color spaces. At present, there are very few domestic studies using colorimeters in the field of forensic science. Still, using colorimeters and spectrophotometers abroad studied the color change of skin contusion and quantitatively explored other color changes in forensic science, such as necrotic spots and the color of cadaver blood. However, there are fewer such studies in general, lacking data on Asian yellow adults, and there is a gap in this field, especially in domestic studies.
1. Materials and Methods
Main instruments: CS-410 portable spectral colorimeter (Hangzhou CHNSpec Technology Co., Ltd.); No. 5 (outer diameter 3.7cm, inner diameter 2.5cm) spiral vacuum extractor (Shuangjin Health Care Instrument Factory); Canon EOS M50 camera; Philips MASTER TL-D90 Graphica36W/965 lamps.
41 volunteers (18 males and 23 females) were recruited, aged 19-42 years old, with a mean age of 23.27±4.06 years. Volunteers’ inclusion criteria:
- (1) No coagulation system abnormality, including not taking any anticoagulants, antiplatelets, anti-inflammatory drugs, and steroids within 2 months;
- (2) No major trauma or surgery within 6 months, and no donated blood or blood products;
- (3) No obvious pigmentation or scars on the inner side of the forearms bilaterally;
- (4) No important organ disorders, such as heart, liver, kidney, etc., or any other diseases affecting recovery from injury.
All volunteers were familiar with the contents of the experiment and the possible consequences and signed an informed consent form.
1.2.1 Contusion model
On one side of the subject’s inner forearm near the elbow fossa, a spiral vacuum copper was used to apply negative pressure for 7 minutes. In contrast, on the other side, the same number of rotations was applied for 15 min. After removing the negative pressure, a circular contusion area with a diameter of about 3 cm was formed.
Sit still for 1h after the completion of suction, and take the first measurement after the dilated and bruised capillaries have fully recovered. Set the instrument parameters SCE mode (excluding specular reflected light to measure color), 10° field of view angle, light source condition D65, and automatically take the average value after 5 consecutive measurements. Take 5 positions with similar skin color within 2cm around the contusion (measuring aperture 8mm) for measurement, and the obtained value was set as the control value. The measurement of the contusion site was repeated 5 more times, and the obtained value was the experimental value. To minimize sampling error, the experiments were measured by the same operator. The color of some contusion areas was not uniform; the color of one side of the circular area was darker, the other side was lighter, or the local area color was darker, and the area with the heavier color was taken as the measurement point. Measurements were taken every 0.5 d in the first 3 d after injury and every 1 d after 3 d until the color difference in the contused area was difficult to distinguish with the naked eye. Immediately after each measurement, the contusion area was photographed under the conditions that the light source was 85cm high, the camera height was 45cm, and the camera was at an angle of 15° to the target with the camera parameters fixed.
1.2.3 Color indicators
CIE Lab color space (also written as L*a*b*) was published by the International Commission on illumination (CIE) in 1976. It is most commonly used in scientific research because it contains the most colors and is unaffected by light and equipment. The L* value indicates the light and dark channels. The value range is 0-100 (0 is the darkest, 100 is the brightest); a* value indicates the red-green channel, the value range -128-127 (-128 is dark green, 0 is gray, 127 is bright pink); b* value indicates the yellow-blue channel, the value range -128-127, (-128 is dark blue, 0 is gray, 127 is yellow). l* value in the LCh color space is the same as that L* value in the LCh color space is the same as L* in the CIE Lab color space, C* represents the saturation value (0-100), and h represents the hue angle value (0-360). LCh value can be completely converted from CIE Lab value, but its expression is more closely related to the practical applications in life. Therefore, the two color space values of CIE Lab and LCh are selected for study, and the software is set to export the corresponding color values.
1.2.4 Color difference formula
Use the software Color QC2 (version: V18.104.22.168) that comes with the instrument to calculate the color difference values, and the formulas involved are as follows.
Note: (1=experimental value; 2=control value)
1.2.5 Data Analysis
The raw data were exported from Color QC2 software, and Excel software was used for preliminary statistics of the appearance time of each characteristic value. Graph Pad Prism software was used to make graphs, analyze the trend of different indicators, and fit the regression curve of all samples and each sample to get the regression equation. SPSS 26.0 software was used to describe the emergence of eigenindicators and slopes statistically and the time of the emergence of eigenindicators was expressed as x+s. Unilateral data variances were compared for gender differences using the Mann-Whitney U test (Mann-Whitney U test), and two-sided comparisons were made using the paired samples mean t-test (paired-test). The test level α = 0.05.
Among the 41 cases, 7 missed the measurement on the 6th day, and 1 missed the measurement on the 5th and 6th days, and these missing data were recorded as blank data. Because there were cases where the actual effect of negative pressure suction was not related to the duration of negative pressure suction, the side with the larger ΔE*ab measured for the first time was recorded as the “heavy side”, and the other side was recorded as the “light side”. The reasons for this will be analyzed in the discussion section.
2.1 Overall trends
The overall trend of the six indicators is shown in Table 1. The initial value of ΔC* can be positive or negative depending on the degree of contusion, and the trend of Δh slows down when the value is different, so it decreases non-linearly. For the “heavy side” data, Graph Pad Prism software was used to make scatter plots and regression curves, and the results are shown in Figure 1.
Table.1 Trends and ranges of the 6 parameter indicators
|Parameter indicators||Positive and negative values||Changing trends||Extreme range|
|Minimum value||Maximum value|
|AL*||Negative||Linear increase||-21.93 ~ -3.74||—|
|Aa*||Positive value||Linear reduction||—||2.03 ~ 13.08|
|Ab*||From negative to positive||Increase first and then decrease||-10.02 ~ -0.08||0.77 ~ 6.64|
|AE*ab||Positive value||Linear reduction||—||4.73 ~ 25.35|
|AC*||From negative to positive||Increase first and then decrease||-6.07 ~ 1.74||1.10 ~ 7.67|
|Ah||Negative||Nonlinear reduction||—||-35.98 ~ -2.78|
Note: “-” means that the value tends to be 0.
Figure.1 Scatterplot and regression curve of 6 color indicators of “heavy side”
Note: Changes in the values of the 6 indicators over 7d.
The same volunteer was photographed in fixed conditions on both sides of the contusion area, and the time points of 0, 1, 3, 5, and 7d were selected for comparison, and the results are shown in Fig.2.
Figure.2 Color changes in both sides of the contusion region within 7d
2.2 Characteristic time points
There are time points where the Δb* value changes from positive to negative, the time of the last negative value is noted as tb negative, the time of the highest peak of the Δb* value is noted as tb peak, and the time of the highest peak of the ΔC* value is noted as tC peak. In addition, by analyzing the curves in each case, it was found that Δa* had a smaller peak based on the linear variation, and the time of its appearance was noted as the ta peak. Because of the sampling error in the location of the selected point during measurement, the relative error of the data was larger when the contusion was lighter in color, and it wasn’t easy to distinguish the color difference with the naked eye, the time of the first ΔE*ab value <3 was designated as the time of disappearance of the contusion, ttotal. The mean and standard deviation of each time were counted by SPSS software, and the results are shown in Table 2.
Table.2 Mean appearance time of characteristic time indicators (d)
|ta peak||tb negative||tb peak||tc peak||ttotal|
|Overall on both sides||1.96±0.80||1.72±0.90||4.00±1.44||3.73±1.35||7.47±3.22|
2.3 Fitting the regression curve
According to the time of disappearance of individual contusion, the data within 7d were analyzed for each index, and the “heavy side” and “light side” data were processed separately. Because the initial value of the “light-side” part of the sample was too low, the relative error of the data was large, so the “heavy-side” data of the 41 samples were fitted. The regression equations of the 6 indexes are shown in Table 3, with the best fit being Δh (R2=0.6395), the worst fit being Δh (R2=0.6395), and the best fit being Δh (R2=0.6395), and the worst fit being Δh (R2=0.6395). The best fit was ΔC* (R2=0.3451), and the worst was ΔC* (R2=0.3451). Because of the large difference in the simulation effect of contusion during negative pressure suction, the initial values differed greatly, and the effect caused by the initial value and sampling error could be reduced if all the data at the same time point were averaged. After taking the mean value, the color values with time change law are obvious, and the regression equation has excellent goodness of fit; the best fit is Δh(R2=0.9980), and the worst fit is ΔC*(R2=0.9089).
ΔE*ab represents the total color difference, and the slope of the regression equation indicates the speed of color change, so the slope of the regression equation for the ΔE*ab indicator can indicate the degree of speed of color change. All samples with ΔE*ab maxima <5 and one case of subcutaneous hemorrhage that affected the stability of the data were excluded, and the slopes of the regression equations of ΔE*ab for all remaining individuals (40 cases on the “heavy side” and 35 cases on the “light side”) were counted to analyze the differences in color change between the two sides of the contusion. Differences in contusion color change.
Table.3 Regression equations for the overall and mean values of the six indices for the “heavy side
|Color difference index||Overall||Mean value|
|Regression equation||R2||Regression equation||R2|
Note: x denotes time, and y denotes color difference values.
2.4 Gender differences
Because the variance of the 5 indicators of ta peak, tb negative, tb peak, tC peak, and ttotal were not homogeneous between men and women of the 41 cases of the heavier side, comparisons were made using the Mann-Whitney U-test. P-values were all >0.05, i.e., there was no significant gender difference in the appearance of these characteristics at the time.
2.5 Differences in degree
Excluding the samples with ΔE*ab maximum <5 on the “light side” and one case of subcutaneous hemorrhage on the “heavy side”, the paired t-test was used to compare the ta peak, tb minus, tb peak, tC peak, and ttotal on both sides of the remaining 35 samples. ta peak, tb minus, tb peak, tC peak, ttotal, tb peak, ttotal 3 indicators P value <0.05, the time of appearance of the three indicators and the degree of contusion significantly correlated. tb negative, tC peak, P value >0.05, the correlation between these two indicators and the degree of contusion is insignificant. ΔE * ab slope P value <0.05, that is, the degree of contusion and the speed of color change are significantly correlated, and the greater the degree of contusion, the faster the speed of change.
3.1 Significance of each index
After erythrocytes escape from blood vessels to subcutaneous or intradermal, the skin at the contusion site will change color due to the inflammatory reaction and the metabolic process of hemoglobin. The color of the early contusion site depends mainly on the number of escaped erythrocytes and the depth of the erythrocyte site from the skin. Hemoglobin near the skin’s surface will appear red, but the skin will appear blue when the escaped blood goes deeper into the tissue. This phenomenon is caused by a combination of Rayleigh scattering, the light absorption coefficient of the skin, and our visual system. After a contusion occurs, although neutrophils are the first inflammatory cells to arrive, they do not degrade hemoglobin. Mononuclear phagocytes phagocytose erythrocytes contain the enzyme heme oxygenase that breaks down hemoglobin into biliverdin and releases carbon monoxide and iron. Biliverdin is a green pigment rapidly converted to bilirubin by the enzyme biliverdin reductase. Free iron combines with ferritin to form ferric hemoflavin, and bilirubin can also accumulate locally to form yellow bilirubin crystals.
The ΔL* value showed a negative value after injury, that is, the color of the contusion site deepened by the naked eye, and the color gradually became lighter during the recovery process. The Δa* value reflects the red-green index, that is, the hemoglobin is degraded while the green biliverdin is generated, so the Δa* value gradually decreases with time. The appearance of ta peak may be related to the rate of biliverdin production and the rate of bilirubin conversion, but the specific mechanism is unclear. The Δb* value represents the yellow-blue axis. When the contusion occurs, the hemoglobin in the deeper part appears blue. Therefore, the Δb* value is negative after contusion, and the Δb* value becomes larger as these hemoglobins are degraded. Subsequently, due to the formation of hemosiderin and bilirubin, the contusion site gradually showed yellow visible to the naked eye. Negative tb does not mean the time when hemosiderin and bilirubin begin to be produced. It should be understood as the time when the yellow value can cover the blue value of deep hemoglobin, that is, the amount of hemosiderin and bilirubin accumulated to a certain extent. The tb peak appears because the decomposition of iron-containing blood yellow and bilirubin exceeds its formation rate. ΔE*ab represents the total color difference, reflecting the color of the contusion area from the initial abnormality to the final and the surrounding normal skin color is basically the same. ΔC* and Δh can be transformed from CIE Lab color space, so the meaning represented by their values is more complex and difficult to be explained by a single material change.
3.2 Inference of the time and degree of contusion
This experiment verifies that gender does not affect the color elimination speed or the appearance of characteristic time points of contusion. In contrast, the 3 indicators of ta peak, tb peak, and ttotal and the color difference elimination speed are related to the degree of contusion, and the 2 indicators of tb negative and tC peak are not affected by the degree of contusion. Among them, the process of Δb* value from negative to positive appeared in 1.72±0.90d, so Δb* turned positive at least after 19h. tC peak appeared in 3.73±1.35d, but it requires several measurements to determine, which is difficult to apply in practice. The time at which the Δb* value turns negative to positive is recommended as the primary indicator of the newness of the contusion.
The goodness of fit of each color difference index of the “heavy side” is the best with Δh (R2=0.6395). Still, in the application process, many software does not support the LCh color mode and need to be converted to get, and h represents the hue angle of beginners is more difficult to understand, so it is only recommended to use it when the conditions allow. Δb* regression curve of R2 = 0.6203 also has a high degree of fit, and Δb* represents a clear change in the specific substance, so Δb* is more suitable for the precise inference of the damage time.
The generally low goodness of fit of the “heavy side” fitting curves was mainly due to the different initial damage degrees, and fitting all the data at the same time after averaging could eliminate the differences in damage degrees between individuals and reflect the rate of change of each color difference index. The goodness of fit of all six color difference indicators for the mean values was >0.9, indicating a significant correlation between the rate of change of contusion color and time.
Because both the initial degree of contusion and the time of contusion affect contusion color, the precise time of contusion can be inferred only from photographs taken a short time after the presence of a contusion, or the initial degree of contusion if the time of contusion is determined.
3.3 Evaluation of Contusion Models
Common contusion models include negative pressure suction, paintball gun strikes, artificial compression, and subcutaneous injection. Although the degree of negative pressure attraction is controllable and the time of action is controllable, it was found during the experiment that the effect of contusion is still different for different individuals. This may be due to the richness of blood vessels in the medial forearm and the differences in distribution between individuals, so the final effect is different. In addition, the sebum thickness of the medial forearm was greater in the volunteers with smaller initial contusions, which was not favorable for negative pressure suction, consistent with the fact that contusions are less likely to occur in real-life scenarios with greater sebum thickness. Therefore, the experimental model needs to be improved by avoiding superficial veins as much as possible and controlling the appropriate pressure for suction.
In summary, the color change of contusions is not related to gender but to the initial degree of contusion. The color data can be quantified by measuring the color index of the contusion using a colorimeter to determine the degree of newness of the contusion, i.e., the Δb* value turned positive at least 19h after the occurrence of the contusion. Both Δh and Δb* values can be used when inferring the time of contusion formation and initial degree.
In this study, we only examined the color change of contusions by gender and degree of contusion and did not consider age, sebum thickness, and contusion site. We need to study further the effects of these factors on contusion, as well as continue to expand the sample size and search for new color indicators to make the method more accurate.
Author: Yao Zewei