USAGE OF HISTOLOGICAL METHODS IN DETERMINING THE PRESCRIPTION OF SPLEEN INJURIES IN FORENSIC MEDICAL PRACTICE
Olena P. Babkina1, Dmytro O. Matyukhin2, Svitlana I. Danylchenko3, Edouard A. Glazkov2, Oleksiy O. Galmyz4
¹OO Bohomolets National Medical University, Kyiv, Ukraine
²Luhansk State Medical University, Rubizhne town, Luhansk region, Ukraine
3Petro Mohyla Black Sea National University, Mykolaiv, Ukraine
4Lugansk Regional Clinical Oncologic Dispensary, Kremennaia, Ukraine
Introduction: The article presents data from literary sources and a statistical analysis of one’s own research on the nature, mechanism and prescription of spleen injury in the case of mechanical trauma and the absence of alcohol intoxication.
The aim: To study the dynamics of changes in the histological parameters of the spleen injured tissues in case of mechanical trauma depending on the prescription of injury.
Materials and methods: The material of the study was the spleen tissue of 56 males and females aged from 20-60 who died at known and unknown time in the presence and absence of alcohol in the blood. We used histological, histochemical methods, and carried out a statistical analysis of the results.
Results: The obtained results showed that during the mechanical injury of spleen there often developed a capsule and a parenchyma with hematoma in the area of injury. Our records showed that during the first 6 hours after injury, there appeared a hematoma in the center of the injury. Hemolysis of the erythrocyte particles was observed in the center of the hematoma. There were isolated leukocytes and fibrin tissues closer to the edge of the hematoma.
Conclusions: The obtained results indicate that there are several histological changes in the damaged spleen tissues area which directly depend on the time which passed from the moment of injury.
KEY WORDS: forensic medical examination, trauma, prescription, spleen, histological parameters
Wiad Lek 2019, 72, 9 cz II, 1752-1756
The study of the mechanism of development and the prescription of injury to the abdominal cavity, in particular the spleen, is thoroughly researched by specialists in various fields of medicine, as these issues are extremely relevant, both in terms of diagnosis and treatment of patients, and in the preventive aspect of injury . The prescription of injuries determination in the deceased, the life expectancy of victims after injury, the mechanism and morphological characteristics of injuries of the abdominal cavity were not deliberately studied and systematized, despite the fact that these cases are encountered very often in forensic medical practice. In recent years, many domestic and foreign authors , studying the possibility of solving this problem, have begun paying serious attention to the study of various biological objects with the following laboratory methods: histological, histochemical, biochemical, immunological, etc. The comprehensive methods for assessing the prescription of injury, based on the consideration of the case circumstances, in combination with the results of the forensic examination of the corpse and laboratory data are prospective. From this point of view, histological research methods turned to be very informative, as they were most often used in forensic medical practice while determining the prescription of injuries. The dynamics of organ and tissue changes after injury, in particular of the skin, and less attention was paid to the study of internal organs . Nowadays the spleen injury occurs quite often and among the injuries of the abdominal cavity occupies one of the leading places. According to the literature the frequency of the spleen injuries is from 15 to 33% .
Microscopic changes in the damaged zone of spleen from the view point of the prescription of their formation have not been studied in details. Moreover, the features of the spleen damaged in cases of combined injury with other organs  have not been described. One of the methods for determining the prescription of spleen injury is a histological method.
The purpose of this work was to study the dynamics of changes in the histological parameters of the spleen injured tissues in case of mechanical trauma depending on the prescription of injury.
MATERIALS AND METHODS
The material of the study was the spleen tissue of 56 males and females aged from 20 to 60 who died in the presence and absence of alcohol in the blood and were subjected to an autopsy in the anatomical department of the Forensic Medical Examination Bureau. The collection of tissues of traumatized organs was carried out in the morgue at air temperature from +16 to 25 C, relative humidity 40-60%. In the course of research, we used histological and histochemical methods to detect the dynamics of regeneration processes of histological changes in the spleen tissues, and carried out a statistical analysis of the results.
The work was carried out in accordance with the requirements of the “Instruction on conducting forensic medical examination” (Order of the Ministry of Health of Ukraine No. 6 dated January 17, 1995), in accordance with the requirements and norms, the standard provisions on ethics of the Ministry of Health of Ukraine No. 690 dated September 23, 2009, “The procedure for the removal of biological objects from the dead, whose bodies are subject to forensic examination and pathological anatomical investigation, for scientific purposes” (2018).
RESULTS AND DISCUSSION
In literary sources there is a large amount of data , indicating the presence of specific features of the spleen damage depending on the type of traumatic action, the place of external force use, the anatomical structure of the organ, topography, the state of the surrounding organs, resulting in damage or the capsule rupture and tissue breakage in a linear, stellate or zigzag form, focal hematoma under the spleen capsule and in the cellular gates, tissue ruptures in the gate area and on the back of the spleen, breakages in the spleen connection, its partial or complete disruption in the gate with its shifting into the peritoneal.
The main mechanisms for the formation of spleen injuries are stroke, compression, shaking of the body, or their combination. Up to now, there was no single morphological classification of spleen injuries, nor a single approach to the expert assessment of spleen injuries, including the nature of injuries, their shape, orientation, localization, volume, and morphological manifestations of trauma. Some authors singled out subcapsular hematomas, capsule ruptures, parenchymal ruptures, central damage, and two-stage ruptures. Thus, M. A. Sapozhnikova  distinguishes among the traumatic spleen injuries surface ruptures or cracks of the capsule, crossover hematomas with the preservation of the capsule integrity, crossover hematomas and breaks in the capsule integrity, tears and smashing of the organ, damage to its vascular leg. The author points out that there are specific features of the spleen injury depending on the type of traumatic effect: the formation of capsule breaks in traffic injuries and falling from high altitude; subcapsular ruptures and hematomas as a result of the organ concussion with a significant blood flow to the pulp; crossover hematomas splitting the spleen and tearing off its legs which occurs in case of direct kicks in the abdomen.
The classification of morphological manifestations of blunt trauma of the spleen was developed according to other data. The classification includes the nature of lesions, their shape, and orientation of the organ, localization, volume and morphological manifestations of the spleen trauma: hematomas, breaks, destruction of the organ, full or partial tearing. It is necessary to pay attention to the classification of the spleen injuries with traumatic blunt objects developed by Yu. I. Sosedko  which includes description of signs and constituent elements of objects. The author classifies the nature of the damage as the classification signs, which includes: constituent elements in the form of hematomas, breaks, fractures, subcapsular lesions, complete separation of the organ or its partial destruction, damage of connection; orientation of damages in the direction. The spleen injury with a traumatic blunt object with a limited surface depends on the place of external force application, direction of blow and energy of traumatic action. However, in the above classification, authors indicated trauma from blunt objects, despite the fact that a combined injury and injuries from sharp objects and from firearms. Yu. I. Sosedko  considers setting the spleen injury prescription with the help of microscopy to be very questionable. In the author’s opinion, it is especially difficult to diagnose the early terms of injury, where the main diagnostic feature is the cellular response of the spleen tissue, which is due to its anatomical structure, contains a significant number of cellular elements. An objective indicator of the spleen subcapsular hematoma prescription is a leukocyte reaction, which can be reliably determined in the damaged area 2-3 hours after injury. Granulocytes gradually formed a torus demarcationis, which was visible under a microscope 12 hours after injury. A torus demarcationis completed its formation in one day. Granulocytes decay in the spleen damage area began on the 2nd-3rd day. 4-5 days after injury there occured a massive disintegration of granulocytes.
Attention should be also paid to the peculiarities of the formation and morphology of the subcapsular spleen ruptures, in particular the hematomas under the organ capsule or the depths of its parenchyma with a rupture or no apparent rupture of parenchyma, which begin to appear at the time of splitting the spleen capsule at the site of the hematoma and gradually accumulate in the distant periods after the trauma. This process often leads to diagnostic errors in medical practice. The spleen capsule is not damaged. Cases when hematoma under the capsule accumulates and at different time after the trauma leads to the break of capsules and bleeding into the abdominal cavity, are called two-stage or two-phase. Two-stage, or “delayed”, spleen ruptures according to the literature develop from 3 to 30 days and make up 10 to 30% of all injuries. Statistical data show that 50% of ruptures occur during the first week, not earlier than on the 2nd day after injury, 25% occur during the 2nd week, and 10% can occur in a month. The mechanism of the subcapsular spleen ruptures formation, the time interval of their formation and rupture, is partially reflected in the works of a number of authors . The morphological features of the subcapsular damage depend on the nature and extent of damage to the spleen parenchyma in the first stage of the injury and the term of the second stage of the injury, namely the capsule rupture in the place of the capsule hematoma accumulation. Taking into account the peculiarities of spleen injuries morphology, their volume, depth of distribution, duration of trauma, the author identified several types of subcapsular rupture of this organ.
There also arises a question of spleen injury prescription, which, despite the large number of conducted studies, was not finally resolved. Rusakova T. I.  established the dynamics of the inflammatory and reparative process occuring in the area of spleen damage and revealed its basic laws. The author found out that in spleen injury with a fatal end at the place of accident, they histologically determined only hematomas with unchanged erythrocytes in areas of tissue destruction. 2 hours after the injury, they often revealed rupture of the capsule and parenchyma with bleeding in the form of hematoma in areas of pulp destruction. Ruptures were represented by clots of blood, consisting mainly of unchanged red blood cells, white blood cells, among which a moderate amount of unchanged granulocytes was determined. In some fields of view, under the microscope, there could be seen a few clusters of tender, loose grainy masses of fibrin. Approximately 7-8 hours after injury there was a small amount of granulocytes in a state of disintegration among unchanged granulocytes. Small clusters or structures like torus demarcationis were formed near the edges of the hematoma granulocytes
Sapozhnikova M. A.  defined the spleen injuries prescription taking into account blood changes in hematoma and the surrounding tissue reaction. It is not possible to use a ferous pigment, which is formed in a pulp, knowing that in the normal spleen there is a destruction of the formed blood elements that are deposited, with hemosiderin grains discharge. As a result of the studies, the author described a histological picture of the changes observed in the spleen and parenchyma hematomas area at different periods of trauma occurence. It was noted that erythrocytes with clearly visible boundaries accumulated initially under the capsule or in the pulp of the spleen. Perifocal edema and fulminate sinusoids of the pulp develop around the prevailing hematoma. On the 2nd-3rd day, there occured plasma and fibrin separation from the formed blood elements where the signs of hemolysis beginning in the center of hematoma were observed. Impregnation the spleen pulp with blood did not damage its structure. The red blood cells destruction occured at the same time as in subcapsular hematoma. The first signs of hematoma organization began to appear before the 5th day after the injury, when the fibroblasts from the capillaries walls were proliferating.
The obtained results  showed that mechanical ruptures of the spleen often caused ruptures in capsules and parenchyma as well as hematomas in the injured area. According to our data, during the first 6 hours after injury, and a histological examination there appeared a hematoma in the center of the injury. It had the form of a cluster of orange erythrocytes with clear contours. Hemolysis of the erythrocyte particles was observed in the center of the hematoma. There were isolated leukocytes and fibrin tissues closer to the edge of the hematoma. It should be noted that there appeared a perifocal edema along the edge of the hematoma. Leukotases and perifocal clumps of leukocytes were observed in the vessels. In the vicinity of the vessels, in sinuses, and near damaged areas we revealed small clumps of leukocytes not connected with vessels (Fig. 1).
6-12 hours after injury, most of the erythrocytes in the center of the injury had fuzzy contours with their widespread hemolysis. Unaffected erythrocytes were sometimes found only along the edge of the hematoma. The number of granulocytes in the center of hematoma increased and their destruction began. Granulocytes continued to accumulate on the periphery of the hematoma; most of them were in decay. Fibers appeared in the form of clusters forming a torus demarcationis, which clearly separated the areas of the damaged parenchyma from intact. There were focal clusters of granulocytes in the perifocal zone in the sinuses.
During 12-24 hours after injury we observed hemolyzed red blood cells. The granulocytes were completely destroyed; the formed torus demarcationis was represented by destroyed granulocytes; macrophages were with intracellular contents of hemosiderin grains; fibrin strands were on the border of the hematoma with intact tissues; thrombi were found in the vessels. There was an anemia of red pulp (Fig. 2). 2-3 days later, there began a resorption of erythrocytes destruction products and the formation of siderophages. The gistio-fibroblast cells proliferation and platelets organization in vessels (fibroblasts appearance and blood vessels in the thrombus) started on the edge of the hematoma, on the border with intact tissue. The formation of a torus demarcationis continued in the form of fuzzy strains of the fibrin mass which grew in the histo-fibroblastic cells and formed soft calcium fibers, most of which were hemosiderophages. Perifocal proliferative and leukocyte responses decreasd and were practically not expressed. Extracellular hemosiderin was occasionally present in light yellow color (Fig. 3).
If the prescription of injury is 4-6 days, there is a prevalence of connective tissue elements (lymphocytes, histiocytes, plasma cells); there is a massive disintegration of granulocytes and signs of blood clots organization in the spleen vessels (Fig. 4). The hematoma was represented by erythrocytes, which were completely hemolyzed; there was a large amount of leukocyte nuclear detritus with the disturbed integrit; the fibrin fibers were densified and clearly distinguish hematoma from damaged tissues. Multiple strains of histio-fibroblast cellular elements (fibroblasts, fibrocytes) grew into hematoma from parenchyma. They can be arranged both in order and chaotically. Delicate collagen fibers began to form a capsule. It should be noted that during this period the number of siderophages is sharply increased, and when colored with medications, small seeds of hemosiderin of light brown color are observed (Fig. 5).
The capsule formation lasted up to 1-2 months from the moment of injury. It should be noted that in cases of two-stage spleen ruptures, we observed the formation of a subcapsular hematoma, which grew in its volume and resulted in the destruction of parenchyma. As a result of the aforementioned, the morphological picture could differ from the above, which should be taken into account when establishing the prescription of injury.
Thus, mechanism and prescription of spleen injury in the case of mechanical trauma and the absence of alcohol intoxication, microscopic changes in the damaged zone of spleen from the view point of the prescription of their formation have of combined not been studied in details authors.
Thus, the obtained results indicate that there are several histological changes in the damaged spleen tissues area which directly depend on the time which passed from the moment of injury. It is appropriate to use the histological method to determine the prescription of injury both in cases of isolated spleen injury and in cases of combined injury of the abdominal organs as it enables establishing the prescription of injury more precisely.
Prospects for further research: Further research of spleen injuries, in particular the diagnosis of its mechanism, nature and time, is necessary for the development and application of such injuries prevention.
1. Dubrov SO. Osnovnye printsipy intensivnoy terapii patsientov s politravmoy na rannem etape. Puti optimizatsii [The basic principles of intensive therapy of patients with polytrauma at an early stage. Ways of optimization]. Science Rise. 2015; 5(4): 117-23. (Russian)
2. Boyko VV, Lelytsya AV, Mylovydova HE, Sotskyi AM. Pislyaoperatsiyni vnutrishnocherevni krovotechi u postrazhdalykh z abdominalnoyu travmoyu [Postoperative intra-abdominal bleeding in patients with abdominal injury]. Kharkiv Surgical School. 2014; 1: 67-70. (Ukrainian)
3. Brown MA, Casola G, Sirlin CB, Patel NY, Hoyt DB. Blunt abdominal trauma: Screening US in 2,693 patients. Radiology. 2001; 218(2): 352-8.
4. Politravma. Rukovodstvo dlya vrachey [Polytrauma. A guide for doctors]. In 2 volumes. Vol. 2. Ed by Boyko VV. 2nd ed., Revised. and additional. H: Factor; 2011. 688 p. (Ukrainian)
5. Babkina OP. Doslidzhennya travmy orhaniv cherevnoi porozhnyny laboratornymy metodamy [Duplication of injuries to organs of the empty light by laboratory methods]. LAP LAMBERT Academic Publishing is a trademark of International Book Market Service Ltd. Member of OmniScriptum Publishing Group. 17 Meldrum Street, Beau Bassin 71504, Mauritius. 148 p. (Ukrainian)
6. Ershova NV. Sudebno-meditsinskaya diagnostika podkapsulnykh povrezhdeniy selezenki pri travme tupymi predmetami [Forensic diagnosis of subcapsular spleen injuries in blunt-force trauma]: Abstr. PhDr. (Med.). M; 2005. 27 p. (Russian)
7. Sapozhnikova MA. Morfologiya zakrytoy travmy grudi i zhivota [Morphology of the closed injury of the chest and abdomen]. M: Medicine; 1988. 160 p. (Russian)
8. Sosedko YuI. Sudebno-meditsinskaya diagnostika davnosti povrezhdeniy pri tyazheloy tupoy travme [Forensic diagnostics of damage in severe blunt trauma]. Forensic Medical Examination. 1984; 2: 15-8. (Russian)
9. Samchuk VV. Sudebno–meditsinskaya diagnostika v sluchayakh travmy organov pishchevaritelnogo trakta tupymi predmetami [Forensic diagnosis in cases of injury to the digestive tract with blunt objects]: Abstr. PhDr. (Med.). M; 2001. 23 p. (Russian)
10. Rusakova TI. Sudebno-meditsinskoe opredelenie davnosti povrezhdeniy selezenki, prichinennoy tupymi predmetami [Forensic determination of the duration of damage to the spleen caused by blunt objects]: Abstr. PhDr. (Med.). M; 2007. 28 p. (Russian)
11. Babkina OP, Herasymenko OI, Kazimirko NK, Shevchenko VV, Shevchenko LA. Vyznachennya davnosti vynyknennya travmy orhaniv cherevnoi porozhnyny ta zaocherevynnoho prostoru u travmovanykh ta zahyblykh vnaslidok spoluchenoi travmy [Establishing the limitation period for damage to the abdominal cavity and retroperitoneal space in injured and killed persons due to a combined injury]. Lugansk: SPD Reznikov VS; 2013. 152 p. (Ukrainian)
The study was carried out within the framework of the department research: “Pathogenetic substantiation of correction of pathophysiological disturbances in the human body under the influence of endogenous and exogenous factors”, state registration number is 0118U004006
According to the order of the Authorship.
Olena P. Babkina – 0000-0001-8120-6526
Dmytro O. Matyukhin – 0000-0002-4429-4720
Svitlana I. Danylchenko – 0000-0001-5312-0231
Edouard A. Glazkov – 0000-0002-7087-8455
Oleksiy O. Galmyz – 0000-0001-7066-8100
Conflict of interest:
The Authors declare no conflict of interest.
Olena P. Babkina
Department of Forensic Medicine and Medical Law
OO Bohomolets National Medical University
Kyiv, 01601, Ukraine
tel: +38 093 065 42 39
Fig. 1. Hematoma in the form of red blood cells accumulation, perifocal edema, small clumps of the leukocytes in the spleen in a woman, 24 years old, who died from a mechanical trauma. The prescription of injury is 6 hours. Colored with hematoxylin-eosin. ZB.: x100
Fig. 3. Single hemosiderin granules in the spleen in a man, 60 years old, who died from a mechanical trauma. The prescription of injury is 30 hours. Colored with hematoxylin-eosin. ZB.: x 100
Fig. 2. Clear torus demarcationis, a large number of granulocytes in the stage of decay in the spleen of 40 year old man who died from a mechanical trauma. The prescription of the injury is 20 hours. Colored with hematoxylin-eosin. ZB.: x200
Fig. 4. Prevalence of connective tissue elements, the spleen granulocytes disintegration in a man, 54 years old, who died from mechanical injury. The prescription of the injury is 5 days. Colored with hematoxylin-eosin. ZB.: x 200
Fig. 5. Sideroblasts in the spleen tissues of a woman, 20 years old, who died from a mechanical trauma. The prescription of injury is 7 days. Colored with hematoxylin-eosin. ZB.: x200