A Comparative Analysis of the Efficacy of CD117 and Special Stains as Indicators of Mechanical Asphyxial Deaths

Abstract

Background:

Determining the cause of death in suspected asphyxia is often difficult when external findings are absent. Mast cells (MCs) may provide objective histological evidence of hypoxia.

Objective:

To compare the efficacy of immunohistochemistry (IHC) using CD117 with Toluidine Blue (TB) and Giemsa (GM) stains in detecting mast cells in asphyxial and non-asphyxial lung tissues.

Materials and Methods:

Lung samples from 164 medicolegal autopsies at AIIMS Jodhpur were studied, 57 asphyxial and 107 non-asphyxial deaths. Peribronchial and perialveolar regions were examined using TB, GM, and IHC (CD117). Mean mast cell counts were compared using Mann–Whitney U and Kruskal-Wallis tests.

Results:

Mast cell counts were significantly higher in asphyxial cases. Peribronchial counts: IHC 2.27 ± 1.02, TB 2.29 ± 1.19, GM 0.84 ± 0.59. Perialveolar counts: IHC 0.74 ± 0.12, TB 0.10 ± 0.19, GM 0.01 ± 0.51. IHC was more sensitive than GM (P < 0.001), while TB showed comparable efficacy to IHC.

Conclusion:

CD117-based IHC is the most sensitive method for identifying mast cells in asphyxial lungs, though TB remains a valuable low-cost option. Mast cell analysis can serve as an adjunct marker in diagnosing mechanical asphyxia.

INTRODUCTION

Asphyxia-related deaths are regularly encountered by forensic pathologists, and many times, ascertaining the cause of death is not a substantial challenge.[1,2] In the prevailing forensic pathology services, the collaborative teamwork of the forensic pathologist and law enforcement agencies often forms the core of investigative procedures. The importance of the crime scene analysis cannot be lesser emphasized. While certain macroscopic signs, such as strangulation marks, cyanosis, petechial hemorrhage, and lung edema, can provide valuable insights, there can be situations in which, due to the paucity of these gross findings, it becomes difficult to conclude the cause of death.[3] In such ambiguous situations, a forensic pathologist may require additional ancillary investigations, such as histopathological examination and postmortem thanatobiochemical techniques. However, these subsidiary investigations may prove to be of adjunct importance in concluding a diagnosis of asphyxia.

Recent studies have underscored the significant role of histopathological investigation, especially the examination of mast cells (MCs), in identifying the cause of death in cases of suspected mechanical asphyxia.[4] MCs are highly abundant producers of enzymes that break down proteins, including specific proteases such as tryptase, carboxypeptidase A, and chymase. In hypoxia, MCs play a crucial role in restructuring pulmonary arteries.[,6,7,8] Their involvement in asphyxia-induced pathophysiological processes has garnered attention from forensic pathologists seeking additional objective markers to support their conclusions.

MCs are present in human lung tissues, the pulmonary epithelium, and the bronchial lumen. These cells contain secretory granules in which specific mediators are stored and released in response to acute asphyxia. Acute asphyxia has been shown to trigger increased MC activity and degranulation in many studies. This response may be attributed to the hypoxic conditions and mechanical stress experienced by the tissues during the event.[,10,11,12] Thus, MC analysis may help explain asphyxial fatalities. Histopathologists use specific stains, such as toluidine blue (TB), GM, hematoxylin–eosin, and Oil Red O (also known as Sudan III-red-B-stain), to demonstrate MCs in both antemortem and postmortem specimens. MCs are typically stained with TB and GM. TB stains tissue purple to red, and the backdrop appears blue, whereas the GM stain shows MCs in purple.[13]

Immunohistochemistry (IHC) utilizes precise antibody–antigen interactions to locate and identify antigens in cells and tissues. These antigens are found and analyzed using this method. IHC markers can identify and characterize MCs in postmortem tissue samples. CD117 (c-kit) is one of the common IHC markers employed for analyzing the distribution and characterization of MCs in clinical and forensic practice.[14] IHC staining targeting CD117 allows for the specific identification and quantification of MCs, providing objective data that can assist forensic pathologists in determining the cause of death in suspicious asphyxial cases.[,16,17,18,19,20,21,22]

The present study has employed IHC using the CD117 marker and special stains, such as TB and GM stains, which may assist in determining the cause of death in asphyxial deaths with ambiguous findings, depending on the investigation circumstances. This study will explore the role of MCs in hypoxia pathophysiology and examine the application of histological methods in forensic investigations.

A statistical comparative analysis is also conducted in the present study to highlight the efficacy of the techniques mentioned above, considering the accuracy and cost-effectiveness of these techniques in resource-limited settings.

MATERIALS AND METHODS

The research was conducted in the Department of Forensic Medicine and the Department of Pathology and Laboratory Medicine at All India Institute of Medical Sciences (AIIMS), Jodhpur (India). Approval was sought from the institutional ethics committee vide approval no. (AIIMS/IEC/2017/370). The research included a total of 164 instances involving medicolegal autopsies. Lung tissue specimens were collected from deceased human individuals at the AIIMS mortuary facility in Jodhpur. Informed consent was obtained from the next of kin.

The cases were divided into two groups. Asphyxia cases made up the first group (total n = 57: hanging (n = 50), drowning (n = 2), postural asphyxia (n = 1), strangulation (n = 2), smothering (n = 2). 107 fatalities, which were represented as control cases [total n = 107: road crashes (n = 37); railway traffic accidents (n = 18); sudden death (n = 30); head injury (n = 5); fall from height (n = 3); poisoning (n = 7); electrocution (n = 4); burns (n = 2); transfusion lungs injury (n = 1)]. Cases with pulmonary pathologies, such as diffuse alveolar damage, bronchiolitis, pulmonary hemorrhage, and pulmonary edema, were excluded from the study. We examined gross lung edema and petechial hemorrhages in the case and control groups. Cases with these findings were included, and controls with the same findings were excluded from the study. The cases with a history of atopy/hypersensitivity were also excluded from the study. Only cases in which the time between death and sample collection was <48 h were used to decrease the impact of autolytic changes as a confounding factor.

Histopathological examination using special stains

The right and left lung tissue from each case was received in 10% neutral buffered formalin (NBF). Representative tissue slices were prepared in an automated Leica tissue processor. Leica’s automated slide stainer treated 4–5 µ tissue slices with hematoxylin and eosin (H and E) stain. To highlight MCs, representative slices were manually stained with TB. TB stain was prepared using a TB stock solution (pH: 2–2.5) and sodium chloride (1%). The staining procedure involved deparaffinizing and hydrating sections in distilled water, staining them for 2–3 min, washing them in distilled water, dehydrating them quickly with 95% ethanol and two changes of 100% ethanol, and clearing them in xylene. The staining was then covered with mounting media.

GM stain was prepared using a stock solution of glycerol, methanol, and 3.89 g of GM powder. The stock solution was used after 2 weeks. The GM working solution was prepared by mixing a stock solution and distilled water in a ratio of 1:9. The staining procedure involved deparaffinizing and hydrating sections with water, staining them for 8–10 min, washing them with tap water, and then drying and mounting the stained slide. The microscope was used to examine the presence and number of MCs in five fields, each with a diameter of 0.65 mm and a field area of 0.332 mm², and was recorded at ×400. The mean MC count per field was calculated.

Immunohistochemical analysis

Lung tissue samples from each lobe’s peribronchial and perialveolar regions in each case were fixed in a 10% NBF solution and cut into 5-μm slices from each paraffin-embedded block. Nikon Eclipse (Model Eclipse Ci-L) microscopes analyzed lungs from all asphyxial and nonasphyxial fatalities. Representative H and E-stained sections from all patients were chosen for IHC. IHC slides were poly-L-lysine-coated. Polyclonal rabbit antihuman CD117, c-kit (Dako, code A4502) antibody was diluted 1:500 and utilized in an automated IHC stainer, Leica Bond-Max, with diaminobenzidine substrate chromogen solution. The pathologist blindly examined IHC slides to determine the cause of death. All instances had MC CD117 IHC-positive sections. MCs expressed CD117 both cytoplasmically and on their membranes. MCs were examined in the perialveolar and peribronchial regions. The mean MC counts in five random microscopic fields, each with a diameter of 0.65 mm and a field area of 0.332 mm², were recorded at ×400.

Statistical analysis

Data were recorded in Microsoft Office Excel Sheet 2019 (Microsoft Corporation, Redmond, WA, USA) and were analyzed and processed using IBM SPSS Statistics for Windows, Version 26.0 (IBM Corp., Armonk, NY, USA). software.

RESULTS

Table 1 shows that the mean MC count in the lungs’ peribronchial region of asphyxial fatalities using IHC (2.270) was significantly higher than in nonasphyxial deaths (0.310), with asphyxial deaths exhibiting a significantly higher variability in MC counts (standard deviation [SD]: 1.027) compared to nonasphyxial deaths (SD: 0.372). TB staining revealed that the mean MC count in asphyxial deaths (2.288) was significantly higher than in nonasphyxial deaths (0.241), with asphyxial deaths showing higher variability in MC counts (SD: 1.193) compared to nonasphyxial deaths (SD: 0.318). GM-stained asphyxial fatalities had a mean MC count of 0.839, compared to 0.099 in nonasphyxial deaths. Asphyxial deaths exhibited significantly greater variability in MC counts (SD: 0.595) than nonasphyxial deaths (SD: 0.218). IHC, TB, and GM stain MCs in the perialveolar area of asphyxial and nonasphyxial fatalities [Table 1]. Asphyxial fatalities using IHC had a higher perialveolar MC count (0.740) than nonasphyxial deaths (0.022), with asphyxial deaths had a higher MC count variability (SD: 0.123) than nonasphyxial deaths (SD: 0.063). TB staining of asphyxial deaths had a higher mean MC count in the perialveolar area (0.105) than nonasphyxial deaths (0.024), with asphyxial deaths had higher MC count variability (SD: 0.196) than nonasphyxial deaths (SD: 0.065). GM stain MCs demonstrated that asphyxial fatalities had a higher perialveolar MC count (0.014) than nonasphyxial deaths (0), and asphyxial fatalities had a higher MC count variability (SD: 0.515) than nonasphyxial deaths (SD: 0).

Table 1.

Descriptive statistics of immunohistochemistry, toluidine blue, and Giemsa stain mast cells in the peribronchial and perialveolar area of asphyxia and nonasphyxial deaths

Region	Staining method	Group	n	Range	Meanein
PB	IHC mast cells	Asphyxial	57	0.2hyxi	2.270x1.027
	IHC mast cells	Nonasphyxial	107	0.2asph	0.310phyxia
PB	TB mast cells	Asphyxial	57	0.0hyxi	2.288xialel
	TB mast cells	Nonasphyxial	107	0.0asph	0.241phyxia
PB	GM mast cells	Asphyxial	57	0.0hyxi	0.839xialel
	GM mast cells	Nonasphyxial	107	0.0asph	0.099phyxia
Perialveolar	IHC mast cells	Asphyxial	57	0.0hyxi	0.740xialls
	IHC mast cells	Nonasphyxial	107	0.0asph	0.022phyxia
Perialveolar	TB mast cells	Asphyxial	57	0.0hyxi	0.105xialel
	TB mast cells	Nonasphyxial	107	0.0asph	0.024phyxia
Perialveolar	GM mast cells	Asphyxial	57	0.0hyxi	0.014xialel
	GM mast cells	Nonasphyxial	107	0.0asph	0.000phyxia

n=Number of samples from each lung, SD=Standard deviation, TB=Toluidine blue staining, IHC=Immunohistochemistry, GM=Giemsa staining, PB=Peribronchial tissue

Mann–Whitney U-test results, as shown in Table 2, reveal the difference in MC visualization between three staining techniques: IHC, TB, and GM in cases of asphyxial death. There was no significant difference in the staining of IHC and TB within the peribronchial area, as indicated by a Z-score of − 0.051 and P = 0.959. The mean ranks were 57.34 for IHC and 57.66 for TB, reflecting that the two techniques perform equally well in visualizing MCs in this region. However, there was a very significant difference observed between IHC and GM staining, with a Z-score of − 7.066 and P < 0. The IHC showed a significantly higher mean rank of 79.32 compared to GM’s 35.68, indicating that IHC is more efficient than GM in identifying MCs in the peribronchial area. In the perialveolar area, no difference was found between IHC and TB staining, with a Z-score of 0.661 and P = −0.438. The mean ranks were 56.39 for IHC and 58.61 for TB, proving equal efficacy in MC visualization in this area.

Table 2.

The Mann hial tis U-test was employed to determine the differences in mast cell visualization between immunohistochemistry (CD117) and toluidine blue stain, as well as between immunohistochemistry (CD117) and Giemsa stain in mast cells located in the peribronchial and perialveolar regions of asphyxial fatalities

Region (mast cells)	Staining method	n	Mean rank	Z-score	P
PB (mast cells)	IHC mast cells	57	57.34	−0.051	<0.959
PB (mast cells)	TB mast cells	57	57.66
Perialveolar (Mast cells)	IHC mast cells	57	56.39	−0.438	<0.661
Perialveolar (mast cells)	TB mast cells	57	58.61
PB (Mast cells)	IHC mast cells	57	79.32	−7.066	<0.000
PB (mast cells)	GM mast cells	57	35.68
Perialveolar (mast cells)	IHC mast cells	57	64.57	−3.335	<0.001
Perialveolar (mast cells)	GM mast cells	57	50.43

TB=Toluidine blue staining, IHC=Immunohistochemistry, PB=Peribronchial tissue, GM=Giemsa staining

Nevertheless, a significant difference was found between GM staining and IHC, with a Z-score of − 3.335 and P < 0.001. IHC yielded a higher mean rank of 64.57 compared to GM’s 50.43, suggesting that IHC is more effective than GM in visualizing perialveolar MCs. The Kruskal–Wallis test compares ordinal dependent variables across three or more groups in asphyxial and nonasphyxial parabronchial and perialveolar samples, demonstrating that GM stain differs significantly from TB and IHC. At the same time, TB and IHC show strong agreement, reinforcing that TB and IHC are more consistent and effective methods for identifying MCs in forensic lung tissue analysis [Table 3].

Table 3.

Immunohistochemistry, toluidine blue, and Giemsa stain mast cells P-substantial value in the peribronchial and perialveolar area of asphyxia and nonasphyxial deaths were calculated using the Kruskal–ruskal were

Stain/area	Asphyxial samples (n) (PB)	P (PB)	Nonasphyxial samples (n) (PB)	P (PB)	Asphyxial Samples (n) (Perialveolar)	P (perialveolar)	Nonasphyxial samples (n) (perialveolar)	P (perialveolar)
GM-TB	57	0.000	107	0.000	57	0.001	107	0.001
GM-IHC	57	0.000	107	0.000	57	0.003	107	0.002
TB-IHC	57	0.899	107	0.152	57	0.678	107	0.799

n=Number of samples from each lung, TB=Toluidine blue staining, IHC=Immunohistochemistry, GM=Giemsa staining, PB=Peribronchial tissue

Figure 1 shows the box and whisker plots for IHC, TB, and GM stain MCs in asphyxial patients for the peribronchial and perialveolar regions. Box and whisker plots for IHC, TB, and GM stain MCs in nonasphyxial patients are shown in Figure 2 for the peribronchial and perialveolar regions. The immunohistochemical technique using CD117 was used to selectively identify and visualize the peribronchial and perialveolar MCs, as shown by the red arrows [Figure 3]. The peribronchial and perialveolar MCs were highlighted with the TB stain [Figure 4] and the GM stain [Figure 5], as shown by the red arrow. The magnification for Figures 1-5 is ×400.

Figure 1.

Box and whisker plots for immunohistochemistry, Giemsa, and toluidine blue stain mast cells in asphyxial cases in the peribronchial and perialveolar region

Figure 2.

Box and whisker plots for immunohistochemistry, Giemsa, and toluidine blue stain mast cells in nonasphyxial cases in the peribronchial and perialveolar region

Figure 3.

Immunohistochemistry for CD117 highlights (a) peribronchial mast cells (red arrows), which should not be confounded with the coarse, dense anthracotic pigment seen in the image above the arrows, and (b) perialveolar mast cells (red arrows). These should not be confused with the alveolar macrophages, which have darker, coarse cytoplasmic pigment, ×400

Figure 4.

(a) Peribronchial mast cells and (b) Perialveolar mast cells are highlighted with the special stain toluidine blue (the red arrow); ×400

Figure 5.

(a) Peribronchial mast cells and (b) Perialveolar mast cells are highlighted with the special stain Giemsa stain (the red arrow, ×400)

DISCUSSION

In real-life forensic pathology practice, the conclusions regarding the cause and manner of death in cases of asphyxia have a potential direct effect on civil and criminal proceedings. In recent times, the research has focused on utilizing various techniques in cases of asphyxia, where the findings are nonspecific. Histochemical techniques and laboratory analysis play a crucial role in medicolegal investigations, serving as invaluable tools that are frequently essential in reaching a conclusive diagnosis, particularly in cases where there is no apparent macroscopic evidence.[,24,25] In this context, various research endeavors in clinical asphyxia have focused on understanding the precise role and functional implications of MCs in tissue remodeling processes.[26,27] Upon examination of the lungs of mice subjected to chronic hypoxia, Williams et al. discerned the existence of a heterogeneous assembly of MCs.[7,28]

A reduction in the level of inspired oxygen characterizes mechanical asphyxia.[29,30] Gutjahr and Madea found conventional and atypical hanging as a brief period of agony: following complete airway obstruction by an extrusion device, oxygen reserves are depleted at rest within 5 min. Prolonged asphyxia, observed in instances such as choking and strangling, was presumed in cases of partial airway obstruction or insufficiently inhibited respiratory movements, allowing for the inhalation of minimal oxygen quantities, hence extending survival to 12–15 min.[31] Studies have shown that MCs mediate the microvascular inflammatory changes in response to the systemic hypoxia produced due to low levels of inspired oxygen.[32] In the context of systemic hypoxia, there is an interaction between leukocytes and endothelial adhesion molecules. This interaction leads to leukocyte rolling and adherence within systemic venules, ultimately resulting in the migration of leukocytes into the surrounding tissue. In addition, during systemic hypoxia, an increase in vascular permeability to plasma proteins is observed.[4,33,34] This phenomenon is associated with the inflammatory response occurring at the microvascular level, mediated by MCs.[,36,37]

A range of methodologies has been utilized thus far for the identification and detection of MCs, encompassing both conventional techniques, such as special staining methods, such as TB and GM, as well as more advanced methodologies, including flow cytometry, IHC, enzyme-linked immunosorbent assay, reverse transcriptase polymerase chain reaction (RT-PCR), and spectroscopy.[13,39,40,41]

Steiner et al. provided compelling evidence supporting the role of MC activation in forming a chemotactic gradient during systemic hypoxia and its subsequent impact on microvascular inflammation. Their conclusion was drawn based on their observation that systemic hypoxia led to the degranulation of MCs, as evidenced by the increased uptake of ruthenium red by these cells. This indicated that MCs were undergoing degranulation, releasing their stored substances.[42,43] Reisinger et al. studied asphyxiation in badly deteriorated remains. The researchers examined tissues from 107 asphyxia-related fatalities using Oil Red O stain. Oil Red O stain histology showed tiny droplets of fatty degeneration.[44] Morphometric analysis examined pulmonary polynuclear large cells as a histological marker of asphyxiation.[29]

Previous investigations have undertaken numerous studies to identify MCs using GM and toluidine stains in different medical scenarios, including tumors, yielding notable findings.[13,39,46,47] Our study employed both of these staining approaches to detect MCs in mechanical asphyxia, an understudied condition. Our descriptive data show that asphyxia and nonasphyxial mortality differ in MC numbers in the perialveolar and peribronchial regions. MCs were examined using toluidine staining in the peribronchial area and found to be considerably higher in asphyxial deaths than in nonasphyxial deaths. MCs stained with GM stain showed no statistically substantial change in the average MC count in the same research sample.

Immunohistochemical methodologies have widespread applications across diverse research contexts, enabling the detection and investigation of cellular or tissue antigens, ranging from proteins to distinct populations of cells.[,48,49] In the context of mechanical asphyxia, several markers have been studied to date utilizing these techniques, such as hypoxia-inducible factor-1 (HIF-1), P-selectin, vascular endothelial growth factor, calbindin-D28K, GFAP, and vimentin.[11,50] Grellner et al. examined alveolar macrophages and pulmonary giant cells in lethal asphyxia. Six monoclonal antibodies were used for IHC on lung tissues from various lobes. The monoclonal antibodies including PG-M1 (for macrophage detection) and 25 F9 (for late-stage inflammation) showed positive immunohistochemical results. LN-4, 27 E10, AMH 152, and MIB 1 did not provide statistically substantial findings.[10] In the context of MCs, the HIF-1 is a pivotal regulator of the cellular response to hypoxic conditions.[51,52] It has been observed that HIF-1 expression is upregulated in human MCs in response to hypoxia.[,54,55,56,57,58]

In their original study, Cecchi et al. found HIF-1α, a subunit of HIF-1 (oxygen-sensitive, regulated by cellular oxygen levels), in the pulmonary arteries of suffocation victims. In their later analysis, they found HIF-1α expression in the pulmonary vasculature of suffocation victims. In several situations, CD117 + MCs colocalized with arteries that expressed HIF-1α. Hypoxia-induced HIF-1α expression in mast and endothelial cells. These findings provide a potential explanation for the observed quantitative increase in MCs and the notable elevation of HIF-1α expression in arteries of varying diameters, as previously reported.[3] In a similar study, Bogomolov et al. employed conventional histological and immunohistochemical techniques to examine the expression of the CD117 antigen. Their investigation revealed an upregulation of CD117 in lung tissue obtained from cases of mechanical strangulation asphyxia. However, they did not compare their findings with those using special stains and MCs in the peribronchial and perivascular regions of the lung.[,60,61]

In our study, the immunohistochemical analysis of MCs in the perialveolar and peribronchial region demonstrated a substantially higher mean MC count in cases of asphyxial fatalities than in nonasphyxial deaths. The present study’s findings on comparative analysis revealed that MCs stained with IHC and TB exhibited a substantially higher mean count in cases of asphyxial deaths than in nonasphyxial cases. On the other hand, MCs stained with GM showed a slightly higher mean count in asphyxial deaths, although the difference was not statistically significant. These results suggest MCs may play a role in the pathophysiological processes associated with asphyxial deaths. The differential response of MCs to different staining methods (TB vs. GM) indicates potential variations in their activation and degranulation patterns in asphyxiation-related conditions. Further investigation is warranted to elucidate the mechanisms underlying MC involvement in asphyxial deaths and determine the diagnostic and prognostic value of MC counts and activation patterns in forensic pathology.

CONCLUSION

In our research, we conducted a comparative analysis between the staining techniques, namely GM and TB. Our findings suggest that TB staining is superior to GM for detecting MCs in fatalities related to asphyxia. Upon further comparison, IHC techniques utilizing the CD117 marker yielded more specific information regarding the involvement of MCs in asphyxia-related alterations in lung tissue specimens compared to the aforementioned histological staining techniques, which is consistent with the findings of previous studies. Nevertheless, it is essential to contemplate the expensive cost of immunohistochemical techniques. Consequently, in resource-limited settings, special tissue staining methods can be utilized effectively for the characterization of MCs in asphyxia-related fatalities.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

The study was financially supported by the Intramural Project, All India Institute of Medical Sciences, Jodhpur, India.