Renal immunohistochemical investigation for the differentiation of the cause of multiple trauma fatalities

Article Outline

• Abstract
• 1. Introduction
• 2. Materials and methods
  • 2.1. Cases
  • 2.2. Immunohistochemical staining
  • 2.3. Immunohistochemical scoring
  • 2.4. Statistical analysis
• 3. Results
• 4. Discussion
• 5. Conclusion
• 6. Conflict of interest
• References
• Copyright

Abstract 

In fatalities with multiple traumatic injuries, it is important to determine the severity of trauma, the main damaged organ, and the antemortem pathophysiological condition. We examined 63 cases within 48h of the postmortem interval, which included assaults, slips and falls and falls from heights, traffic accidents, and sharp instrumental injuries. Immunohistochemically, each kidney was stained against hemoglobin (Hb), myoglobin (Mb), superoxide dismutase (SOD), 8-hydroxy-2′-deoxyguanosine (8-OHdG), 150kDa oxygen regulated protein (ORP150), pulmonary surfactant A (SP-A), and liver-type fatty acid binding protein (L-FABP). Bleeding or circulatory failure induced ORP150, 8-OHdG, and L-FABP in the kidney. Statistical analysis of the immunoreactivity revealed that in battered and/or abused cases, Hb could be considered a specific marker. Hb and Mb were observed in the cases with general severe trauma, such as slips and falls and falls from heights. In traffic accidents, ORP150 could reflect general circulatory failure with bleeding. SP-A was observed in the cases with severe thoracic injuries, such as lung injuries and multiple thoracic fractures. L-FABP appeared in cases with renal circulatory failure as well as renal injury. These findings suggest that immunohistochemical observation of the kidneys could be a useful tool in determining several key factors, such as the severity of injury, the specific damaged organ, and the pathological condition after injury.

Keywords: L-FABP, SP-A, Multi-trauma fatality, Kidney, Immunohistochemistry

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1. Introduction 

When investigating the cause of death in cases with multiple traumatic injuries, we consider the traumatic shock as a summation of multiple injuries. It is especially necessary to clarify the difference between the conditions of hemorrhagic shock, due to hemorrhaging from many wounds, and traumatic shock. Moreover, domestic abuse cases have increased in recent years. Thus, the clarification of the causal relationship between the injuries and cause of death in cases of abuse of adult women and the elderly is essential.

With the above factors in mind, it is important to diagnose the specific cause of death and mechanisms of trauma. It is also necessary to obtain useful information about the severity of the trauma, the main damaged organ or site, and the pathological condition after receiving the wound. Various investigations [1], [2], [3], [4], especially immunohistochemical studies [5], [6], [7], [8], [9], [10], about multiple traumas have been reported. Previously we reported that if Hb and Mb were observed in battered child cases, that this immunohistochemically reflected the additive trauma of repeated abuse [6]. We also reported that 8-OHdG and SOD, as a reaction to peroxidative damage, were induced in the kidneys by Mb in the case of rhabdomyolysis [11].

In the present study, we aimed to clarify the cause of multiple injuries and the post-traumatic pathophysiological condition or course. To this end, we observed the kidneys immunohistochemically with ORP150, L-FABP, SP-A and other biomarkers, in forensic autopsy cases.

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2. Materials and methods 

2.1. Cases 

Sixty-three forensic autopsy cases were examined, all within 48h of the postmortem interval. To demonstrate the difference between the conditions of hemorrhagic shock and traumatic shock, a control group (group A), consisting of 20 cases of death by bleeding from sharp instrumental injuries, was created. The multiple blunt injury cases were divided into three groups as follows: Group B–battered and/or abused, eight cases; Group C–slips and falls or falls from heights, 11 cases; and Group D–traffic accidents, 24 cases. We observed cases involving falls separately from traffic accident cases in order to determine if they could be distinguished immunoreactively, even though both involve high energy wounds generated from multiple injuries. A summary of the examined cases is shown in Table 1. We estimated the severity of the trauma using the abbreviated injury scale (AIS) [12].

Table 1. Summary of the examined cases.

Group		A		B		C		D		Total
n	(clinical corse)	20	(8)	8	(2)	11	(10)	24	(4)	63	(24)
Sex	(M/F)	11/9		2/6		10/1		17/7		40/23
Age	(Min–max)	50.2±14.2	(27–71)	53.3±19.0	(26–79)	48.9±15.9	(27–69)	63.3±19.6	(2–84)	55.4±18.1	(2–84)
PMI	(Min–max)	19.5±11.5	(7–48)	23.1±13.4	(6–50)	32.5±13.6	(14–48)	18.0±7.5	(7–36)	21.6±11.8	(6–50)
PTSI	(Min–max)	4.4±9.8	(1–36)	7.7±12.0	(1–36)	2.7±2.5	(1–6)	9.3±34.1	(1–168)	6.5±22.0	(1–168)
ISS	(Min–max)	31.8±18.0	(10–75)	28.3±10.8	(11–42)	40.0±21.6	(10–75)	53.8±19.5	(16–75)	41.2±21.0	(10–75)

The cases transported to the hospital are shown in ().

PMI: postmortem interval.

PTSI: post-traumatic survival interval.

ISS: injury severity score.

There were no significant differences in age, postmortem interval (PMI), and post-traumatic survival interval (PTSI) between the groups. However, the injury severity scores (ISS) in group D were significantly higher than group A and B (p<0.05).

This study was executed in accordance with the privacy policy of the Japanese Society of Legal Medicine [13].

2.2. Immunohistochemical staining 

The kidneys were fixed in phosphate-buffered formalin, embedded in paraffin and sectioned at 5μm. Hematoxylin-eosin (HE) stain and Azan stain were used for conventional staining. Immunostaining was performed with antibodies against hemoglobin (Hb, 1:800, Dako, Japan), myoglobin (Mb, 1:800, Dako, Japan), 8-hydroxy-2′-deoxyguanosine (8-OH-dG, 1:200, JICA, Japan), superoxide dismutase Cu/Zn enzyme (SOD, pre-diluted, Lab Vision, USA), 150kDa oxygen regulated protein (ORP150, 1:500, IBC, Japan), pulmonary surfactant type A (SP-A, 1:200, Dako, Japan), and liver-type fatty acid binding protein (L-FABP, 1:50, abcam, Japan). The immunostaining was carried out using the ENVISION kit/HRP (DAB) (Dako, Japan), according to the manufacturer’s instructions. The immunostaining was visualized by incubation for 3–5min with the DAB solution. For positive control tissue slices, muscle was used for Mb, liver was used for 8-OHdG and SOD, lung was used for SP-A, and kidney was used for L-FABP. Staining specificity was checked using negative control slides omitting the primary antibody. Additionally, tissue specimens other than positive control tissues were used in a negative control study. The coverslip was mounted on a glass slide, examined and photographed. Controls for the specificity of the immunohistochemistry involved omission of the primary antibody.

2.3. Immunohistochemical scoring 

The immunoreactivity was screened semi-quantitatively by observing the pattern and the number of positive cells in all fields of a whole section. Immunostaining patterns were divided into two grades as follows: (1) the diffuse staining type, where the positive cells were spread out across the section (D-type, 2 points); and (2) the focal staining type, where the positive cells were clustered (F-type, 1 point). They were then evaluated according to the number of immunopositive cells, and classified into two grades as follows: (1) a large number of immunopositive cells (L-type, 2 points); and (2) a small number of immunopositive cells (S-type, 1 point). No immunoreactivity was assessed as negative, and given zero points. A final value was assigned by multiplying the points from the immunostaining pattern with the points from the number of immunopositive cells, and this value was adopted as the immunoreactive score [14].

The pathological findings of the kidneys were observed separately by two pathologists, and were then screened.

2.4. Statistical analysis 

The Mann–Whitney U test was utilized to compare the immunoreactive scores against the decedent’s sex. A p-value of less than 0.01 was considered statistically significant. The correlation coefficients were analyzed by Spearman’s rank coefficient of correlation between the immunoreactive score and each of the following: the injury severity score, the decedent’s age, the postmortem interval, and the post-traumatic survival interval. The immunoreactivity scores of each group were analyzed with the Mann–Whitney U test.

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3. Results 

The immunoreactivities against each antigen are summarized in Table 2. In addition to the data of Table 2, in the 21 SP-A positive cases, lung injuries were present in 12 of them (57.1%). Seventeen cases had an AIS score of four or more (81.1%). In the eight liver injury cases, L-FABP was positive in 75% of them (six cases). However, L-FABP was observed in all six of the kidney injury cases (100%).

Table 2. Immunoreactivities and their score of biomarkers in each group.

Score: immunoreactive score.

The localization of each Hb, Mb, ORP150, and L-FABP was observed in the cytoplasm of tubular cells, and the lumen of the tubules and the glomerulus. 8-OHdG was observed in the nucleus of both tubular and glomerulus cells. The localization of SOD was observed in the cytoplasm and nucleus of tubular cells, and the lumen of the tubule. The localization of each SP-A, was observed only in the lumen of tubulus.

The immunoreactive patterns for the antigens Hb (Fig. 1a), SOD (Fig. 1d), and SP-A (Fig. 1f) displayed the F-type pattern about 60% or more of the time. For both ORP150 (Fig. 1e) and L-FABP (Fig. 1g), the D-type pattern was observed about 60% or more of the time. In Mb (Fig. 1b) and 8-OHdG (Fig. 1c), there was no great difference between the frequency of D-type and F-type patterns. Regarding the number of immunopositive cells, most of the antigens were of the S-type, except for Mb and 8-OHdG, in which the S-type and L-type frequencies were almost the same.

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• Fig. 1. 

  Immunoreactivity of each marker. Bar=50μm, arrows: SP-A. Antigen/group/age/sex/ pattern/number. (a) Hb/group B/42 yo/female/L/F, (b) Mb/group C/68 yo/male/L/D, (c) 8-OHdG/group D/75 yo/male/L/D, (d) SOD/group A/29 yo/female/S/F, (e) ORP150/group B/72 yo/female/L/D, (f) SP-A/group C/68 yo/female/L/F, (g) L-FABP/group C/68 yo/male/L/D.

No correlation was found between the immunoreactivity scores for these antibodies and the cadaver’s age, injury severity score, the postmortem interval, or post-traumatic survival interval. There was also no significant difference between the immunoreactivity scores for these antibodies and the cadaver’s sex (data not shown).

The Hb score in group A was significantly low (p<0.01), and the Mb score in group C was significantly high (p<0.01). The immunoreactivity scores of the other antigens showed no significant differences.

In group A (bleeding), Hb and Mb were hardly stained. 8-OHdG and ORP150 and L-FABP were positive in high frequencies, over 70% each. SP-A was positive in seven cases, and four of them had lung trauma.

In group B (battered and/or abused), Hb was positive in 100% of cases, but Mb only in 25%. 8-OHdG and ORP150 were positive in at least 75% of the cases. SP-A was positive in only two cases.

In group C (slips and falls or falls from heights), Hb, Mb, 8-OHdG, ORP150 and L-FABP were positive at a high rate, over 70%. In the four cases where SP-A was positive, two cases had lung injuries and the other two cases were relatively long survivors.

In group D (traffic accidents), Hb was positive in 62.5% of the cases, but Mb was negative in all the samples. SP-A positive was positive in only 33.3% of the cases. Lung injuries were observed in four of the eight cases. In contrast, 8-OHdG, ORP150 and L-FABP were positive in over 70% of the cases in this group.

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4. Discussion 

We had Hb as the marker of bleeding and hemolysis, and Mb as the marker of muscle damage [6], [8], [9]. 8-OHdG and SOD are known to be markers of oxidative damage [5], [11], [15], [16], [17], [18], [19]. Extreme hypoxia induces the enhanced synthesis of oxygen regulated proteins (ORPs), thus ORP150 was used as the marker of circulatory failure [20], [21], [22]. SP-A [23], [24], [25] and L-FABP [26], [27], [28] was observed as the markers of lung and abdominal injuries, respectively. L-FABP was also considered an important cellular antioxidant during oxidative stress, such as circulatory failure [29].

Hb-immunoreactive score was significantly low, and Mb was also hardly stained in group A (bleeding). These results may reflect that the inflow of Hb and Mb from the lesion to the bloodstream did not occur from the sharp instrument injury. In a previous study [6], Hb was detected in the kidneys of battered children who had sustained severe or moderate external injuries, whereas Mb was not found. Our results from group B (battered and/or abused) were concordant with this report. Therefore, it was considered that Hb in the kidney reflects a large amount of subcutaneous damage.

Mb-immunoreactive score in group C (slips and falls or falls from heights) showed significantly higher than any other group. Because the ISS was also high in this group (Table 1), it was thought that the high positive rate of Hb and Mb reflected general severe subcutaneous and intramuscular bleeding and damage [6], [8], [9].

Both Hb and Mb were highly observed in group C; however, only Hb was positive in group B. The ISS of group C was much higher than in group B, so it was considered that intra-muscle and deep wounds induce both Hb and Mb in the kidney, but only Hb appears when the injuries were limited to the subcutaneous layer.

The high positive rates of ORP150, L-FABP and 8-OHdG were observed in all groups. Those immunoreactivities reflected circulatory failure accompanying hypovolemia, as in the bleeding cases [19], [20], [21], [28].

8-OHdG, ORP150, and L-FABP were observed with high ratios in group D (traffic accidents). This group had the highest ISS of all the groups; however, there were no Mb-positive cases. It was considered that in traffic injuries the effect of circulatory failure was more dominant than the traumatic damage.

SP-A was observed as the marker for lung injuries. In our results, it is thought that the appearance of SP-A in the kidney reflects not only the presence of a lung injury, but also a severe thoracic wound [23], [24], [25].

L-FABP had been discovered in the liver (hence the designation ‘L’-FABP), but later it was shown to exist mainly in the kidney. Therefore, the relationship between L-FABP and injuries of the liver and the kidney was examined. It was concluded that renal injuries also contribute to the manifestation of L-FABP [28]. The appearance of L-FABP in the kidney may be due to renal circulatory failure as well as renal injuries.

In multiple trauma fatalities, the immunohistochemical observation of the kidneys may prove to be a useful tool in determining the influence of circulatory failure and organ damage on the pathological condition that contributed to the death.

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5. Conclusion 

In this report we examined the immunohistochemical study of multiple trauma fatalities. The following results were obtained:

The immunoreactivities of each antigen might be a useful tool in investigating the cause of multiple trauma and the post-traumatic physiological condition or course.

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6. Conflict of interest 

The authors have declared no conflict of interest.

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