Effects of Flunitrazepam Drug on the Decomposition Process of Pigs Liver in PMI Determination: A Histopathology Perspective

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Muge" }, { "@type": "Person", "name": "Peter W. Kinyanjui" }, { "@type": "Person", "name": "James M. Mbaria" }, { "@type": "Person", "name": "John K. Kiai" }, { "@type": "Person", "name": "Nichodemus M. Kamuti" }, { "@type": "Person", "name": "Peter K. Gathumbi" } ], "publisher": { "@type": "Organization", "name": "F1000Research", "logo": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 480, "width": 60 } }, "image": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 1200, "width": 150 }, "description": " Background Decomposition can be retarded by several processes, including physical and chemical barriers and climatic factors. The presence of flunitrazepam (Rohypnol®) in the bodies may interfere with the decomposition process by inhibiting enzymatic activity, particularly in the liver making it difficult for experts investigating a forensic case to determine the post mortem interval. This study investigated the effect of flunitrazepam in alcohol (40%) on the decomposition of pig liver to provide a guiding standard for postmortem interval (PMI) determination in cases involving flunitrazepam ingestion. Methods Two pigs (Sus Scrofa L.), aged six months and weighing 24.6 kg and 25.2 kg, were selected to ensure physiological uniformity. Using two pigs balanced logistical constraints while providing sufficient data for preliminary analysis. One pig served as a control, while the experimental pig was administered 2 mg of flunitrazepam dissolved in 250 ml of vodka to mimic a common practice of spiking in a bar setting. The pigs were euthanized using an electric stunning method, ensuring a humane procedure. Liver samples measuring 2 cm x 2 cm were randomly excised daily from the caudal lobe and preserved in 10% neutral buffered formalin. Sampling was monitored every 24 hours over 16 days. Results These findings underscore flunitrazepam’s significant impact on decomposition, aiding forensic PMI determination. Histopathology was conducted on all liver samples to determine the structural and cellular changes consistent with autolysis daily for 16 days. The study found, flunitrazepam slowed down the decomposition process of pigs’ liver in PMI determination in the period between day 1 and day 6. However, the effect of flunitrazepam drugs on the decomposition process of pigs’ liver was absent after day 6. Conclusions Forensic investigators should account for flunitrazepam’s impact on postmortem interval (PMI) when determining time of death in forensic cases. 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F1000Research 2025, 14 :712 ( https://doi.org/10.12688/f1000research.163436.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article Effects of Flunitrazepam Drug on the Decomposition Process of Pigs Liver in PMI Determination: A Histopathology Perspective [version 1; peer review: 1 approved] Daniel Gachuiri Njau https://orcid.org/0000-0001-7287-7256 1 , Edward K. Muge 1 , Peter W. Kinyanjui 1 , [...] James M. Mbaria 2 , John K. Kiai 3 , Nichodemus M. Kamuti 4 , Peter K. Gathumbi 4 Daniel Gachuiri Njau https://orcid.org/0000-0001-7287-7256 1 , Edward K. Muge 1 , [...] Peter W. Kinyanjui 1 , James M. Mbaria 2 , John K. Kiai 3 , Nichodemus M. Kamuti 4 , Peter K. Gathumbi 4 PUBLISHED 21 Jul 2025 Author details Author details 1 Department of Biochemistry, University of Nairobi, Nairobi, 00100-30197, Kenya 2 Department of Public Health, Pharmacology & Toxicology, University of Nairobi, Nairobi, 00100-30197, Kenya 3 Department of Veterinary Anatomy and Physiology, University of Nairobi, Nairobi, 00100-30197, Kenya 4 Department of Veterinary Pathology, Microbiology and Parasitology, University of Nairobi, Nairobi, 00100-30197, Kenya Daniel Gachuiri Njau Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Resources, Software, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Edward K. Muge Roles: Project Administration, Resources, Supervision, Validation, Writing – Review & Editing Peter W. Kinyanjui Roles: Project Administration, Resources, Supervision, Validation, Writing – Review & Editing James M. Mbaria Roles: Project Administration, Resources, Supervision, Validation, Writing – Review & Editing John K. Kiai Roles: Investigation, Resources, Validation, Writing – Review & Editing Nichodemus M. Kamuti Roles: Investigation, Resources, Validation, Writing – Review & Editing Peter K. Gathumbi Roles: Investigation, Resources, Supervision, Validation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS Abstract Background Decomposition can be retarded by several processes, including physical and chemical barriers and climatic factors. The presence of flunitrazepam (Rohypnol®) in the bodies may interfere with the decomposition process by inhibiting enzymatic activity, particularly in the liver making it difficult for experts investigating a forensic case to determine the post mortem interval. This study investigated the effect of flunitrazepam in alcohol (40%) on the decomposition of pig liver to provide a guiding standard for postmortem interval (PMI) determination in cases involving flunitrazepam ingestion. Methods Two pigs ( Sus Scrofa L. ), aged six months and weighing 24.6 kg and 25.2 kg, were selected to ensure physiological uniformity. Using two pigs balanced logistical constraints while providing sufficient data for preliminary analysis. One pig served as a control, while the experimental pig was administered 2 mg of flunitrazepam dissolved in 250 ml of vodka to mimic a common practice of spiking in a bar setting. The pigs were euthanized using an electric stunning method, ensuring a humane procedure. Liver samples measuring 2 cm x 2 cm were randomly excised daily from the caudal lobe and preserved in 10% neutral buffered formalin. Sampling was monitored every 24 hours over 16 days. Results These findings underscore flunitrazepam’s significant impact on decomposition, aiding forensic PMI determination. Histopathology was conducted on all liver samples to determine the structural and cellular changes consistent with autolysis daily for 16 days. The study found, flunitrazepam slowed down the decomposition process of pigs’ liver in PMI determination in the period between day 1 and day 6. However, the effect of flunitrazepam drugs on the decomposition process of pigs’ liver was absent after day 6. Conclusions Forensic investigators should account for flunitrazepam’s impact on postmortem interval (PMI) when determining time of death in forensic cases. READ ALL READ LESS Keywords Histopathology, Flunitrazepam, Decomposition, Carcasses, Postmortem Interval, Pigs Corresponding Author(s) Daniel Gachuiri Njau ( [email protected] ) Close Corresponding author: Daniel Gachuiri Njau Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2025 Njau DG et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Njau DG, Muge EK, Kinyanjui PW et al. Effects of Flunitrazepam Drug on the Decomposition Process of Pigs Liver in PMI Determination: A Histopathology Perspective [version 1; peer review: 1 approved] . F1000Research 2025, 14 :712 ( https://doi.org/10.12688/f1000research.163436.1 ) First published: 21 Jul 2025, 14 :712 ( https://doi.org/10.12688/f1000research.163436.1 ) Latest published: 21 Jul 2025, 14 :712 ( https://doi.org/10.12688/f1000research.163436.1 ) Introduction Understanding the processes of autolysis (postmortem self-destruction) in biologic systems is very important in forensic science. 1 It represents the rotting of tissue without any vital reactions. It is the decay of an organism or different compartments of the organism by cellular enzymes or non-bacterial decomposition of tissue through intercellular enzyme activity and named auto-digestion by its own enzymes. 2 The assessment of the time since death is one of the most challenging tasks in forensic pathology. 3 The alterations in the tissue composition offer opportunities that can be utilized in determining the time of death, but the accurate estimation of the postmortem interval (PMI) has remained a challenge to forensic pathologists. 4 One of the organs used in the estimation of PMI is the liver. The liver is a vital organ, which is the largest internal organ in the body and is situated in the right upper abdomen; it measures about 1.5 kg. It has four lobes, which include the right lobe, left lobe, quadrate lobe, and caudate lobe. 5 Features of its body organization and its body location in the abdomen reduce the chances of infestation with maggots. 6 The liver is regarded as an organ with a critical metabolic function and anatomical closeness to the gastrointestinal tract, which makes it have the highest density of bacterial taxa in all internal organs after death. 7 It is also very unstable towards enzymatic activity being digested by pancreatic secretions and body fluids. The estimation of PMI is an important factor in forensic science, though it is affected by several chemicals. Barbiturates and benzodiazepines act through modifications of autolytic enzyme activity to slow down decomposition. In contrast, substances such as cocaine raise metabolic and thermal activity before death, leading to rapid decomposition that hampers PMI assessment. 8 Different substances may affect tissue decomposition in various ways; for instance, heroin influences bacterial development and fluid distribution, whereas preservatives, such as formaldehyde, hinder normal decomposition. 6 , 9 Knowledge of these chemical effects is imperative when conducting PMI in forensic investigations. The postmortem interval can be roughly estimated when considering decomposition begins soon after death, but the process slows with time. Different factors can slow the process of decomposition, such as chemicals, physical obstruction, and environmental factors. 10 The presence of flunitrazepam (Rohypnol®) in the body can also affect the process of decomposition in another way and pose a challenge to forensic examination. This research focuses on estimating the post-mortem interval (PMI) of a case involving flunitrazepam in pig liver samples. Methods Research design This study was part of a larger research project and it adopted case control as the experimental research design. Two (24.6kg and 25.2kg) domestic pigs ( Sus scrofa Linnaeus ) were acquried from veterinary swine center at Kabete, Kanyariri, Kiambu County, Kenya. One of the pigs was used as a control and the other was experimental. The pigs used in this study were issued by the University of Nairobi Faculty of Veterinary Medicine veterinary farm, where animals are specifically bred and maintained for research and training purposes. As such, no private ownership was involved, and institutional authorization was granted for their use in accordance with ethical and welfare regulations. Ethical approval for animal use was provided by Biosafety, Animal Use and Ethics Committee of the Department of Veterinary Anatomy and Physiology, Faculty of Veterinary Medicine, University of Nairobi. REF: FVM BAUEC/2019/203. Study site The study was carried out in the upper Kabete region of Kiambu County, situated within the veterinary farm of the University of Nairobi (Kenya). The study site was a soft wood tree forest characterized by a black soil and low tree cover. The site was located at an altitude of 1900m a.s.l, representing a typical montane region. Experimental animals Two pigs (Sus Scrofa L.) weighing 24.6 and 25.2kg each were used as human models to determine effect of flunitrazepam on the decomposition of liver in an open area of thickest forest at the upper Kabete campus farm, University of Nairobi, Kenya in the month of July 2021 (16 Days). One pig was used as a control and the other one as an experimental pig. Both pigs were allowed a three-day acclimatization period to adapt to the environmental conditions before the study commenced. On the evening of June 30, 2021, the experimental pig was administered 2 mg of flunitrazepam dissolved in 250 ml of vodka (40% ethanol composition). On the morning of 1st July, 2021, at 5:00 a.m., the pigs were euthanized using an electric stunning method which involved applying an electric current through the brain. This is a commercially allowed method in slaughterhouses in Kenya by the Directorate of Veterinary Services in protecting the welfare of animals to ensure a humane and rapid procedure. The abdomen was then opened to expose the liver for sampling the then placed on an open ground and secured against preditors with metal cages as shown in Figure 1 . Figure 1. Dissected Control and Experimental Carcasses in their Cages: (a) represents the control pig while (b) represents experimental pig. Data collection Small pieces of liver tissue samples were obtained from both control and experimental animal daily and preserved in 15 ml of 10% formalin (Sup: Loba chemie, Cat No: 00146) per sample from day 1 to day 16 as shown in Figures 2 and 3 respectively. The control and experminetal slides were labelled control histopathology day (CHD) and experimental histopathology day (EHD) (From day 1 to day 16) respectively. These tissue samples were then processed to assess histopathological changes and the impact of flunitrazepam on the decomposition process. The changes occurring at both tissue and cellular levels were observed using an electron microscope to determine the degree of autolysis/decomposition over time, and images were captured along with descriptions. Figure 2. Fixed liver tissues in 10% neutral buffered formalin from Day 1 to Day 16 in the control specimen. Figure 3. Fixed liver tissues in 10% neutral buffered formalin from Day 1 to Day 16 in the experimental specimen. Tissue processing & histopathological analysis Following fixation, the liver tissues were routinely processed for sectioning according to protocol by Gunasegaran. 11 The samples were placed in a glass slide in 10% neutral buffered formalin (NBF), (pH = 7.0) and allowed to fix flat. These samples were then placed in 2.0 ml snap cap eppendorf tubes in 10% NBF and fixed at 4 °C for between 16–24 hr. Following fixation, samples were rinsed in running tap water overnight then dehydrated in 250 ml ascending concentrations of ethanols (50%, 70%, 80%, 90%, 95% and 100%) (sup: Scharlau, Cat No: ET00052500). 12 The samples were then cleared in two changes of methyl benzoate, infiltrated and embedded in molten paraffin wax then left to solidify overnight. Blocking was done by attaching the wax embedded samples onto wooden blocks. Tissue sections 5 to 7 μm thick were then cut from the embedded blocks 13322 a leitz Weitzar ® rotary microtome. 13 The sections were then mounted on glass slides, deparaffinised using 250 ml of xylene (Sup: Loba Chemie PVT LTD, Cat No: 00370), rehydrated in a 250 ml through decreasing concentrations of ethanol (100%, 90%, 70% and 50%) and finally in distilled water. 14 The rehydrated sections were then subjected to staining protocol; Hematoxylin & Eosin (H&E) and Masson’s trichrome staining (Sup: Loba Chemie PVT LTD). The stained sections were then examined and photographed using a Leica ® DM 500 light microscope. Results Histopathology Day One The main structural appearance of the liver in day one are illustrated in Figure 4 , liver tissue of the control specimen showed an intact cuboidal epithelium of bile ducts on the basement membrane within the portal tract in the first day. Furthermore, the endothelium of hepatic artery and portal vein were intact, and the portal connective tissue was well-organized with evidence of portal congestion. 15 In the lobules, the lobular integrity was preserved, and hepatocytes appeared intact with a visible nucleus. The sinusoids were distended with red blood cells (RBCs). Additionally, a few hepatocytes displayed early stages of autolysis, characterized by the loss of details, such as cytoplasmic boundaries. Figure 4. Histopathology; liver of control pig in day 1: Photomicrograph of pig liver showing (a) congested portal veins (blue arrow) (X40 magnification; H&E staining). (b) congested portal veins (blue arrow) (X100 magnification; H&E staining). (c) congested portal veins (blue arrow) and intact portal vein endothelium (yellow arrow) (X400 magnification; H&E staining). (d) Scattered hepatocytes showing early degree of autolysis (X400 Magnification; H&E staining). Histopathological features of the liver in experimental pig are illustrated in Figure 5 . Similar to the control, the experimental liver tissue exhibited an intact cuboidal epithelium of bile ducts on the basement membrane within the portal tract. The hepatic artery endothelium and portal vein endothelium were also intact, and evidence of portal congestion was observed, with the portal connective tissue being well-organized. 16 The lobular integrity was preserved, with intact hepatocytes displaying a visible nucleus and distended sinusoids filled with red blood cells. Scattered hepatocytes in the lobules showed early stages of autolysis, characterized by a loss of details, such as cytoplasmic boundaries. However, the experimental liver also exhibited large foci of lost hepatocellular details, indicating more advanced stages of autolysis. Figure 5. Experimental Histopathology Day 1: Photomicrograph of pig liver showing (a) normal portal veins (blue arrow) (X40 magnification; H&E staining). (b) normal portal veins (blue arrow) and intact cuboidal epithelium of the bile ducts (yellow arrow) (X100 magnification; H&E staining). (c) normal portal veins with intact endothelium (blue arrow) (X400 magnification; H&E staining). Day Two In Day two, as shown in Figure 6 , the portal tract lesions in the control specimen replicated those of Day one. However, the lobules showed loss of cell boundaries, leakage of cytoplasm and free nuclei trapped in eosinophilic coagulum of leaked cytoplasm. Figure 6. Control Histopathology Day 2: Photomicrograph of pig liver showing (a) congested portal veins (blue arrow) and intact lobular pattern (yellow arrow) (X40 magnification; H&E staining). (b) congested portal veins (blue arrow) and intact interlobular connective tissue (yellow arrow) (X100 magnification; H&E staining). (c) congested portal veins (blue arrow) and intact portal vein endothelium (yellow arrow) (X400 magnification; H&E staining). In the experimental specimen, as illustrated in Figure 7 , the liver tissue within the portal tract exhibited intact blood vessels, bile ducts, and connective tissue. However, pools of red blood cells were observed outside the blood vessels, indicating hemorrhage. Furthermore, the lobular pattern was mainatained and the lobules displayed a loss of hepatic columns with individualization of hepatocytes and lost integrity of sinusoids and borderless pools of red blood cells, indicating further hemorrhage. The hepatocytes showed individualization with a loss of cell-cell boundaries, with the majority exhibiting a clear cell structure and boundaries. However, nuclear pyknosis was frequently observed, along with occasional instances of nuclear abnormalities. Figure 7. Experimental Histopathology Day 2: Photomicrograph of pig liver showing (a) congested portal vein (blue arrow), intact lobular pattern with intact interlobular connective tissue (yellow arrow) (X100 magnification; H&E staining). (b) congested portal vein (blue arrow), intact lobular pattern with intact interlobular connective tissue (yellow arrow) (X100 magnification; H&E staining). (c) congested portal vein (blue arrow) with intact endothelium (X400 magnification; H&E staining). Day Three On day three, as shown in Figure 8 , the control specimen portal tract lesions remained similar to those observed on day one, with intact connective tissue, arterial and venous structures, and congested blood vessels. The bile duct epithelium remained intact, maintaining its orderly cuboidal structure on the basement membrane, although slight cellular swelling was noted. Furthermore, the lobular pattern remained intact, but the hepatic columns were absent, and there was a loss of cell-to-cell boundaries with a predominance of ghost hepatocytes. The sinusoids showed distension with red blood cells, and nuclear pyknosis was observed in scattered hepatocytes, indicating progressive autolytic changes. 17 The results also revealed prominent nucleus changes, such as pyknosis, and individualized hepatocytes, with some clumped into multinucleated hepatocytes. The loss of sinusoidal arrangements was also observed, along with the occurrence of brown pigments (bile pigments), RBC swelling, and hemolysis. Figure 8. Control Histopathology Day Three: Photomicrograph of pig liver showing (a) congested portal vein (blue arrow), intact lobular pattern with intact interlobular connective tissue (yellow arrow) (X100 magnification; H&E staining). (b) congested portal vein (blue arrow), intact lobular pattern with intact interlobular connective tissue (yellow arrow) (X100 magnification; H&E staining). (c) congested portal vein (blue arrow) with intact endothelium (X400 magnification; H&E staining). (d) hemorrhage (blue arrow) (X400 magnification). The Histopatology of liver in experimental pig on day three are illustrated in Figure 9 . Mainly, the portal tract was intact, with distended veins filled with blood, intact connective tissue, and bile ducts. The lobular pattern was preserved, but the hepatic columns were lost, leaving ghost-like remnants of dead cells. Furthermore, a pink coagulum of leaked cytoplasm was observed in the lobules, along with prominent distension of sinusoids with red blood cells. Scattered free bile pigment was also observed, and the majority of the red blood cells appeared swollen and mostly hemolyzed. Figure 9. Experimental Histopathology Day 3: Photomicrograph of pig liver showing (a) congested portal veins (blue arrow) and intact lobular pattern and liver sinusoids distended with red blood cells (yellow arrow) (X40 magnifications; H&E staining). (b) congested portal veins (blue arrow) and intact lobular pattern and liver sinusoids distended with red blood cells (yellow arrow) (X100 magnifications; H&E staining). (c) congested portal veins (blue arrow) (X400 magnifications; H&E staining). (d) congested liver sinusoids (yellow arrow) (X400 magnifications; H&E staining). Day Four On day four, as shown in Figure 10 , the portal tract in control specimen exhibited a lack of bile ducts, and the blood vessels were shells that lacked a clear endothelial lining. Additionally, the arterial blood vessel wall had loose spongy tissue of non-viable cells, and there was a loss of connective tissue, leaving empty spaces in the wall. In the lobules, the hepatocyte columns were lost, and there were loosely sequestrated clumps of ghost hepatocytes, which were elongated in shape. Furthermore, there was no evidence of red blood cells and hepatic sinusoids were indistinct. The structural changes were extensive and widespread throughout the lobules. Figure 10. (a) Histopathology of liver in control pig in day 4: Photomicrograph of pig liver showing, (a) shell of blood vessels with no clear endothelial lining (blue arrow) and loss of lobular pattern with loss of hepatic columns and interlobular connective tissue (X40 magnifications; H&E staining). (b) shell of blood vessels with no clear endothelial lining and loss of lobular pattern with loss of hepatic columns and interlobular connective tissue (X100 magnifications; H&E staining). (c) shell of blood vessels with no clear endothelial lining with loose spongy mass of non-viable cells (blue arrow) (X40 magnifications; H&E staining). (d) loosely sequestrated clumps of ghost hepatocytes (elongated) (X400 Magnification; H&E staining). Histological features of liver in experimental specimen on day four are illustrated in Figure 11 . The portal tract displayed shells of blood vessels with non-viable cells and a lack of clear endothelium Furthermore, no evidence of red blood cells was observed within the lumen, and the bile ducts were not evident. The connective tissue framework around the lobules was reduced, with areas of absence, causing adjacent lobules to collapse into each other. In the lobules, the hepatic column was lost, and there were no viable hepatocytes or sinusoids. Amorphous to elongated clumps of ghost hepatocytes were observed, along with pink-orange pools of clumps of hemolyzed red blood cells. Figure 11. Experimental Histopathology of liver in day 4: (a) Photomicrograph of pig liver showing shells of blood vessels with non-viable cells and no clear endothelium with no RBC in the lumen and loss of hepatic lobular pattern with adjacent lobules collapsing into each other (yellow arrow) (X40 magnifications; H&E staining). (b) Shells of blood vessels with non-viable cells and no clear endothelium with no RBC in the lumen (blue arrow) (X40 magnifications; H&E staining). (c) Shells of blood vessels with non-viable cells and no clear endothelium with no RBC in the lumen (blue arrow) (X100 magnifications; H&E staining). (d) Photomicrograph of pig liver showing amorphous to elongated clumps of ghost hepatocytes (X400 magnifications; H&E staining). Day Five In the control specimen, as shown in Figure 12 , the liver tissue’s portal tract exhibited shells of blood vessels, but these structures lacked viable cells and showed no evidence of bile ducts or connective tissue. The bile duct epithelium was absent, and the remnants of the blood vessels were surrounded by loosely organized debris. Within the lobules, there was a complete absence of interlobular connective tissue, resulting in a loss of the characteristic lobular pattern. Sinusoids and red blood cells were no longer visible, reflecting advanced autolytic changes. The hepatocytes were represented by enlarged clumps of ghost cells, with no discernible parenchyma or cellular details. These findings highlight a significant progression in tissue degradation compared to earlier days, characterized by the near-total breakdown of structural integrity and cellular components. Figure 12. Control Histopathology Day 5: Photomicrograph of pig liver showing (a) shells of blood vessels with no viable cells (blue arrow) and loss of lobular pattern (yellow arrow) leaving empty spaces (X40 Magnification; H&E Staining). (b) shells of blood vessels with no viable cells (blue arrow) and loss of lobular pattern (yellow arrow) leaving empty spaces (X100 Magnification; H&E Staining). (c) shells of blood vessels with no viable cells (blue arrow) (X400 Magnification; H&E Staining). (d) enlarged clumps of ghost hepatocytes with no parenchyma between them in the entire liver tissue (blue arrow) (X400 Magnification; H&E staining). In the experimental liver tissue portal tract, the blood vessel “shells” were non-viable, and there were no bile ducts or viable connective tissues ( Figure 13 ). In the lobules, minimal interlobular non-viable connective tissues were observed, with surrounding lobular patterns being non-viable, and no evidence of sinusoids or intact hepatocyte columns. 18 The results indicated the presence of non-viable, amorphous clumps of ghost hepatocytes, with shadows of nucleus and cytoplasm. Furthermore, no visible parenchyma was observed between hepatocytes, and there was no evidence of red blood cells, with isolated pink-orange pigments indicating hemolyzed red blood cells. Figure 13. Histopathology of experimental liver at day 5: (a) shells of blood vessels with no viable cells (blue arrow) and loss of lobular pattern (yellow arrow) leaving empty spaces (X40 Magnification; H&E Staining). (b) shells of blood vessels with no viable cells (blue arrow) (X100 Magnification; H&E Staining). (c) shells of blood vessels with no viable cells (blue arrow) (X400 Magnification; H&E Staining). Day Six On day six, as shown in Figure 14 , the control tissue’s results remained consistent with those observed on day five (CHD5), with the addition of blue/basophilic staining clumps observed in the blood vessels, which were consistent with bacterial colonies. Figure 14. Control Histopathology Day 6: Photomicrograph of pig liver showing (a) shells of blood vessels with no viable cells (blue arrow) and loss of lobular pattern (yellow arrow) leaving empty spaces (X100 Magnification; H&E Staining). (b) shells of blood vessels with no viable cells with basophilic bacterial colonies in the lumen (blue arrow) (X40 Magnification; H&E Staining). (c) clumps of ghost hepatocytes (X400 Magnification; H&E Staining). (d) hells of blood vessels with no viable cells (blue arrow) (X400 Magnification; H&E Staining). The results, as shown in Figure 15 , indicated that in the portal tract of the experimental liver tissue, the blood vessels were without a viable endothelium, the bile duct was lost, and the portal connective tissue was lost. In addition, the interlobular connective tissue was lost, there was no lobular pattern, no sinusoids and no RBCs in blood vessels and the amorphous hepatocytes were separated by empty spaces (no parenchyma). Figure 15. Experimental Histopathology Day 6: Photomicrograph of pig liver showing (a) shells of blood vessels with no viable (blue arrow) (X40 Magnification; H&E Staining). (b) shells of blood vessels with no viable (blue arrow) (X100 Magnification; H&E Staining). (c) shells of blood vessels with no viable (blue arrow) (X400 Magnification; H&E Staining). (d) clumps of ghost hepatocytes (blue arrow) (X400 Magnification; H&E Staining). Day Seven The results, as shown in Figure 16 , show that in the portal tract of the control tissue on day seven, there were shell of blood vessels, many artifacts, no RBCs in the vessels, no bile ducts and no connective tissue. The results also show that there was no lobular pattern, no interlobular connective tissue, there were dead individualized hepatocytes, there were large empty spaces separate hepatocytes (no parenchyma) and the shape of ghost hepatocytes was polymorphic. Figure 16. Control Histopathology Day 7: (a) Photomicrograph of pig liver showing shells of blood vessels with no viable (blue arrow) and loss of lobular (yellow arrow) (X100 Magnification; H&E Staining). (b) Photomicrograph of pig liver showing shells of blood vessels with no viable (blue arrow) (X100 Magnification; H&E Staining). (c) Photomicrograph of pig liver showing shells of blood vessels with no viable (blue arrow) (X400 Magnification; H&E Staining). (d) Photomicrograph of pig liver showing clumps of ghost hepatocytes (blue arrow) (X400 Magnification; H&E Staining). The findings revealed that in the experimental specimen liver tissue portal tract, the blood vessel wall remained intact, with clumped red blood cells and dark blue clumps in the lumen consistent with bacterial colonies ( Figure 17 ). 19 No evidence of bile ducts was observed, with debris present in the lumen. Concerning the lobules, the interlobular demarcation of lobules by fibrous tissue was present, with non-viable hepatocytes, individualized ghosts of hepatocytes, and a lack of hepatic columns. Figure 17. Experimental Histopathology Day 7: Photomicrograph of pig liver showing (a) shells of blood vessels with no viable (blue arrow) (X40 Magnification; H&E Staining). (b) shells of blood vessels with no viable (blue arrow) (X100 Magnification; H&E Staining). (c) shells of blood vessels with no viable (blue arrow) (X400 Magnification; H&E Staining). (d) clumps of ghost hepatocytes (blue arrow) (X400 Magnification; H&E Staining). Day Eight On day eight, the examination of the control liver tissue portal tract revealed the presence of non-viable shells of blood vessels, with no bile ducts observed ( Figure 18 ). Moreover, the interlobular connective tissue was lost, and the hepatocytes had undergone necrosis, resulting in individualized ghost cells with no observable hepatic columns. Figure 18. Control Histopathology Day 8: Photomicrograph of pig liver showing (a) shells of blood vessels with no viable cells (blue arrow) and loss of lobular pattern (yellow arrow) (X40 Magnification; H&E Staining). (b) shells of blood vessels with no viable cells (blue arrow) (X100 Magnification; H&E Staining). (c) shells of blood vessels with no viable cells (blue arrow) (X400 Magnification; H&E Staining). (d) clumps of polymorphic ghost hepatocytes (blue arrow) (X400 Magnification; H&E Staining). As illustrated in Figure 19 , the examination of the experimental specimen liver tissue portal tract showed the presence of non-viable shells of blood vessels, with no evidence of bile ducts observed. Additionally, there was a loss of interlobular connective tissue, resulting in hepatocytes undergoing necrosis and the formation of individualized ghost cells with no observable hepatic columns. Figure 19. Experimental Histopathology Day 8: Photomicrograph of pig liver showing (a) shells of blood vessels with no viable cells (blue arrow) and loss of lobular pattern (yellow arrow) (X40 Magnification; H&E Staining). (b) shells of blood vessels with no viable cells (blue arrow) (X100 Magnification; H&E Staining). (c) clumps of polymorphic ghost hepatocytes (blue arrow) (X400 Magnification; H&E Staining). (d) shells of blood vessels with no viable cells (blue arrow) (X400 Magnification; H&E Staining). Day Nine In the portal tract of the control’s liver tissue on day nine, there were shell of blood vessels, there were no bile ducts, there was loss of interlobular connective tissue, the hepatocytes necrotized and there were individualized ghost cells (no hepatic columns) ( Figure 20 ). In addition, there was collapse of wall of arterial blood vessels diminishing the luminal volume sometimes to total absence. Figure 20. Control Histopathology Day 9: Photomicrograph of pig liver showing (a) shells of blood vessels with no viable cells (blue arrow) and loss of lobular pattern (yellow arrow) (X40 Magnification; H&E Staining). (b) shells of blood vessels with no viable cells (blue arrow) (X100 Magnification; H&E Staining). (c) shells of blood vessels with no viable cells (blue arrow) (X400 Magnification; H&E Staining). (d) clumps of polymorphic ghost hepatocytes (blue arrow) (X400 Magnification; H&E Staining). As demonstrated in Figure 21 , the outcomes indicated that in the experimental tissue, there were clumps of amorphous eosinophilic masses of non-viable hepatocytes. Furthermore, the contours of blood vessels with clear lumens were observed, and the interlobular connective tissue was disintegrating, resulting in shells of empty spaces in between. Additionally, there were individualized ghosts of scattered hepatocytes separated by conspicuous empty spaces, with the density of ghost hepatocytes being less than in EHD8. The dissolution at EHD9 was more noticeable. Figure 21. Experimental Histopathology Day 9: Photomicrograph of pig liver showing (a) shells of blood vessels with no viable cells (blue arrow) and loss of lobular pattern (yellow arrow) (X40 Magnification; H&E Staining). (b) shells of blood vessels with no viable cells (blue arrow) and loss of lobular pattern (yellow arrow) (X100 Magnification; H&E Staining). (c) shells of blood vessels with no viable cells (blue arrow) (X100 Magnification; H&E Staining). (d) clumps of polymorphic ghost hepatocytes (blue arrow) (X400 Magnification; H&E Staining). Day Ten On day 10, as indicated on Figure 22 , observations showed that blood vessels on the control’s liver tissue had contracted. The connective tissue among the interlobules had dissolved but not bile ducts, therefore, providing a ghost hepatocytes appearance. Figure 22. Control Histopathology Day 10: Photomicrograph of pig liver showing (a) collapsed blood vessels (blue arrow) and loss of lobular pattern (yellow arrow) (X40 magnification; H&E staining). (b) collapsed blood vessels (blue arrow) and loss of lobular pattern (yellow arrow) (X100 magnification; H&E staining). (c) clumps of polymorphic ghost hepatocytes (blue arrow) (X400 Magnification; H&E Staining). The results revealed that in the experimental liver tissue on day 10 ( Figure 23 ), there was a complete loss of hepatocytes, and the ghost hepatocytes were more densely packed with no discernible individualization. Furthermore, the interlobular connective tissue had disintegrated, leaving empty shells. 20 Additionally, the results showed that dark blue clumps were present in the lumen of the ghost blood vessels, which indicates the presence of bacteria as determined by staining. Figure 23. Experimental Histopathology Day 10: Photomicrograph of pig liver showing (a) loss of lobular pattern (blue arrow) (X40 Magnification; H&E Staining). (b) loss of lobular pattern (yellow arrow) and shells of portal veins with basophilic bacterial colonies (blue arrow) (X40 Magnification; H&E Staining). (c) shells of portal veins with basophilic bacterial colonies (blue arrow) (X100 Magnification; H&E Staining). (d) shells of portal veins with basophilic bacterial colonies (blue arrow) (X400 Magnification; H&E Staining). (e) clumps of polymorphic ghost hepatocytes (blue arrow) (X400 Magnification; H&E Staining). The study findings revealed that on day 11, the liver tissue of the control exhibited clumped eosinophilic masses, loss of interlobular connective tissue, clumps of necrotic cells, and absence of blood vessels and bile ducts. 21 Additionally, the results indicated the presence of amorphous clumps of dead tissue with varying degrees of eosinophilic staining, dispersed within noticeable empty spaces. The findings were that on day 11, no evidence of connective tissue, vessels, and bile ducts was seen. 22 The findings also revealed that there were groups of dead tissue (eosinophilic) and big/profuse clusters of blue staining masses in ghost of vessels. On day 12, the results revealed that the liver tissue of the control specimen displayed shells of blood vessel walls, debris, and empty spaces. Furthermore, there were multifocal basophilic staining masses distributed in the shells of blood vessels. 23 The interlobular connective tissue had completely dissolved, resulting in a loss of tissue eosinophilic staining. On day 12, the experimental specimen exhibited significant findings. The liver tissue showed eosinophilic staining with dissolved interlobular connective tissue and extensive tissue loss. Moreover, the remnants of blood vessel walls were observed, albeit devoid of any blood cells. The bacterial colonies were identified through basophilic staining. Additionally, all hepatocytes were determined to be dead, leaving behind only ghost remnants. On day 13, the liver tissue of the control specimen exhibited loss of tissue eosinophilia, coagulum of dead tissue, absence of interlobular pattern and connective tissue, and lack of blood vessels/traces. The results also showed a significant occurrence of multifocally distributed basophilic masses (bacterial colonies) not clearly marked by vessels boundaries. Furthermore, there were conspicuous empty spaces in the tissue. Similarly, experimental tissue within the specimen exhibited loss of tissue eosinophilia, necrotic tissue coagulum, absence of interlobular pattern, absence of connective tissue, and absence of vessels/traces. Additionally, there were many empty spaces, but unlike with the control specimen, there were no multifocally distributed basophilic masses (colonies of bacteria) with indistinct vessel boundaries. On Day 14, control specimen liver tissue exhibited characteristics as on Day 13, including tissue eosinophilia loss, absence of interlobular pattern, absence of connective tissue, and absence of vessels/traces. Still, there were a number of empty spaces and few stained basophilic masses (multifocally distributed; bacterial colonies). Furthermore, tissue debris was separated by empty spaces and there was no visible eosinophilia. The liver tissue in the experiment specimen on day 14 was characterized by scattered eosinophilia of dead tissue, tissue debris separated by empty spaces, no basophilic masses and no connective tissue or blood vessels. On day 14, the liver tissue in the experimental specimen exhibited scattered eosinophilia of dead tissue with tissue debris separated by conspicuous empty spaces. Notably, there were no basophilic masses, connective tissue, or blood vessels present. On day 15, the liver tissue in the control specimen demonstrated the presence of silhouettes of blood vessels with basophilic staining masses (bacterial colonies), scanty eosinophilia, and tissue debris separated by conspicuous empty spaces. Furthermore, there was a loss of interlobular connective tissue, resulting in the absence of the characteristic lobular pattern. On Day 16, the liver tissue in the control specimen was similar to that of Day 12, characterized by multiple empty spaces separated by debris, multifocally scattered basophilic masses (bacterial colonies), and a lack of eosinophilia. On day 16, the liver tissue in the experimental specimen exhibited clumped masses of dead tissue with conspicuous empty spaces due to autolysis and complete loss of tissue architecture. The tissue also showed coagulum of eosinophilic debris with numerous empty spaces, while the clumped basophilic masses were absent, indicating the absence of bacterial colonies. Changes in the integrity of the liver tissue From the results, as shown in Figure 24 , EHD1, EHD2, EHD3, EHD4 and CHD1, CHD2 show normal liver tissue with normal architecture. The hepatocytes arranged in plates, polygonal cells with round, centrally located nuclei and abundant eosinophilic cytoplasm with fine basophilic granules; are pleomorphic and multinucleated. 24 Kupffer cells are located in the sinusoids and are mononuclear; they are phagocytic cells (macrophages) that respond to cell injury. 25 The cells are in bean shaped nucleus with abundant cytoplasm and star shaped cytoplasmic extensions. Endothelial cells lining sinusoids as well as blood vessels with indistinct cytoplasm and small elongated nuclei were observed. Figure 24. Percentage Integrity of the Liver Tissue: Control Histopathology Day (CHD) (Blue line) represents decline in the architecture of the liver tissue from control. Experimental Histopathology Day (EHD) (Red line) represents decline in the architecture of the liver tissue from experimental. CHD3 demonstrates the ratio of liver tissue organization is preserved at 70%, and hepatocytes along with Kupffer cells and endothelial cells are also identified. CHD4 and EHD5 indicate that the architecture of the liver tissue remains at 60% and hepatocytes, Kupffer cells, and endothelial cells are visible. CHD5 and EHD5 indicate that the architecture of liver tissue is retained at 50% while hepatocytes, Kupffer cells, and endothelial cells are visible. CHD6 and EHD6 indicate that 45% of liver tissue architecture is maintained and hepatocytes, Kupffer cells, and endothelial cells are presented. CHD7 and EHD7 retains the liver tissue organization at 35%, and hepatocytes, Kupffer cells, and endothelial cells are present. The hepatocyte arrangements of the liver are preserved at 30% while the cellularity of the tissue is distorted at 80%. CHD9 and EHD9 depict that the porosity of the liver tissue architecture is at 25% and the cellularity is altered at 85%. CHD10 and EHD10 exhibited a fully disrupted structure of the tissue along with ghost cells. The total tissue, as shown in CHD11, CHD12, CHD13, CHD14, CHD15, CHD16, EHD11, EHD12, EHD13, EHD14, EHD14, EHD15 and EHD16, is unrecognizable. Discussion This study sought to establish the contribution of the flunitrazepam drug in relation to the decomposition process of the pig liver so as to identify the PMI. The findings showed that the flunitrazepam drug reduced the rate of decomposition or putrefaction of pigs’ liver tissues during the PMI ranging between one and five days. This is in accordance with the study conducted by Early and Goff; 26 it was evident that the mechanical and chemical barriers reduce the rate of decomposition of the liver tissue. The findings showed that the liver tissue under the experimental structure showed normal architecture with hepatocytes in plates and polygonal-shaped cells with a round central nucleus and abundant eosinophilic cytoplasm containing fine basophilic granules. These findings align with the description of normal liver histology, 27 though it is important to note differences in cellular morphology under experimental conditions. Kupffer cells, critical components of the sinusoidal system, were present and demonstrated their typical bean-shaped nuclei and abundant cytoplasm with star-shaped cytoplasmic extensions, as noted in previous studies. 28 However, the liver tissue architecture in the experimental specimen remained normal only on the first and second day of observation, thereby depicting the chronological health erosion capability of flunitrazepam. 18 Histological comparison between control and experimental tissue specimens revealed that the hippocampus of the control specimen was well preserved up to day three with approximately 70% of the actual liver tissue architecture preserved together with most cells, including hepatocytes, Kupffer, and endothelial cells, intact. 29 In the experimental specimen, the architecture was already facing vulnerability, particularly reflecting in the diminished cellular structure. Starting from day four, gradual transformations started occurring in both specimens. On day six, the hepatic structure in the two specimens was significantly damaged to the extent of 50% with shattered hepatocytes, Kupffer cells, and endothelial cells. After seven days, the control specimen had only 35% of its architectural integrity preserved, while the experimental had further deterioration in terms of cell structure. On the eighth day, the architecture of both specimens was minimally observed at 30% while there was 80% cellular distortion. There was a decrease in the liver tissue architecture to about 25% on Day 9, and the liver tissue samples demonstrated severe cellular distortion. 30 On day ten, the tissue was cellularized by ghost cells, which signified that the process of tissue breakdown was almost complete. In subsequent days (11 to 16), the architecture became entirely unrecognizable. 31 These results support the usefulness of the comparison and contrast, in terms of the temporal structure of histological changes between the control and the experimental specimen. Conclusion The study observed that the presence of flunitrazepam affected the process of decomposition in the pig’s liver during determination of PMI. The features of the liver tissue, such as hepatocytes, Kupffer cells, and sinusoidal endothelial cells, were well preserved on day one and day two of the experiment. However, between days three and five, there was a gradual decline in the two test specimens, but the interior configuration of the experimental specimen was slightly superior to that of the control specimen. On day five, both specimens exhibited 50% preservation of cellular structures. From day six onwards, a marked decline in tissue architecture was evident, with the experimental specimen maintaining 35% integrity by day seven compared to 30% in the control specimen. By day nine, cellular architecture was severely distorted at 85% in both specimens. On day ten, complete architectural loss was observed in both specimens, characterized by ghost cells. By days 11 to 16, the liver tissue in both specimens was unrecognizable. These findings demostrate that flunitrazepam delay the early stages of liver decomposition, but its effects diminish as decomposition progresses. The drug’s impact on liver histology should be considered in forensic investigations, especially in cases involving rhohypnol ingestion. Further studies using approved human specimens across varied geographical and environmental conditions are recommended to validate these findings. Ethical considerations Ethical approval for animal use was provided by Biosafety, Animal Use and Ethics Committee of the Department of Veterinary Anatomy and Physiology, Faculty of Veterinary Medicine, University of Nairobi. REF: FVM BAUEC/2019/203 on 12/03/2019. Data and software availability Mendeley Data: Histological Dataset on the Effects of Flunitrazepam on Liver Decomposition for Postmortem Interval Estimation. https://doi.org/10.17632/sssgjcss42.1 . 32 This project contains the following underlying data: Data file 1. Histopathology Data.doc Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Reporting guidelines Mendeley Data: ARRIVE 2.0 checklist for ‘Effects of flunitrazepam Drug on the Decomposition Process of Pigs Liver in PMI Determination: A Histopathology Perspective’. https://doi.org/10.17632/32cvm9rc5y.1 . 33 Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Acknowledgement The author would like to thank Mrs. Phyllis Mwai, Mr. Caleb Musango and Francis Okumu for their technical support and Veterinary Farm, Department of Biochemistry, Department of Veterinary Anatomy and Physiology and Department of Veterinary Pathology, Microbiology and Parasitology University of Nairobi for providing the study site and all the required instruments, equipment and reagents that were required for this research. The author also would wish to acknowledge the assistance of Miss. Khadija Osman in reviewing the manuscript write up. 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Comments on this article Comments (0) Version 1 VERSION 1 PUBLISHED 21 Jul 2025 ADD YOUR COMMENT Comment Author details Author details 1 Department of Biochemistry, University of Nairobi, Nairobi, 00100-30197, Kenya 2 Department of Public Health, Pharmacology & Toxicology, University of Nairobi, Nairobi, 00100-30197, Kenya 3 Department of Veterinary Anatomy and Physiology, University of Nairobi, Nairobi, 00100-30197, Kenya 4 Department of Veterinary Pathology, Microbiology and Parasitology, University of Nairobi, Nairobi, 00100-30197, Kenya Daniel Gachuiri Njau Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Resources, Software, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Edward K. Muge Roles: Project Administration, Resources, Supervision, Validation, Writing – Review & Editing Peter W. Kinyanjui Roles: Project Administration, Resources, Supervision, Validation, Writing – Review & Editing James M. Mbaria Roles: Project Administration, Resources, Supervision, Validation, Writing – Review & Editing John K. Kiai Roles: Investigation, Resources, Validation, Writing – Review & Editing Nichodemus M. Kamuti Roles: Investigation, Resources, Validation, Writing – Review & Editing Peter K. Gathumbi Roles: Investigation, Resources, Supervision, Validation, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information The author(s) declared that no grants were involved in supporting this work. Article Versions (1) version 1 Published: 21 Jul 2025, 14:712 https://doi.org/10.12688/f1000research.163436.1 Copyright © 2025 Njau DG et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Njau DG, Muge EK, Kinyanjui PW et al. Effects of Flunitrazepam Drug on the Decomposition Process of Pigs Liver in PMI Determination: A Histopathology Perspective [version 1; peer review: 1 approved] . F1000Research 2025, 14 :712 ( https://doi.org/10.12688/f1000research.163436.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 21 Jul 2025 Views 0 Cite How to cite this report: Julita U. Reviewer Report For: Effects of Flunitrazepam Drug on the Decomposition Process of Pigs Liver in PMI Determination: A Histopathology Perspective [version 1; peer review: 1 approved] . F1000Research 2025, 14 :712 ( https://doi.org/10.5256/f1000research.179788.r408362 ) The direct URL for this report is: https://f1000research.com/articles/14-712/v1#referee-response-408362 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 19 Sep 2025 Ucu Julita , UIN Sunan Gunung Djati Bandung, Bandung, Indonesia Approved VIEWS 0 https://doi.org/10.5256/f1000research.179788.r408362 This manuscript addresses a crucial forensic question regarding the impact of flunitrazepam on postmortem liver decomposition. The topic is novel and relevant; however, the study has several methodological and reporting limitations that require revision. The abstract ... Continue reading READ ALL This manuscript addresses a crucial forensic question regarding the impact of flunitrazepam on postmortem liver decomposition. The topic is novel and relevant; however, the study has several methodological and reporting limitations that require revision. The abstract provides a clear overview but lacks quantitative results. Terms such as “significant impact” are misleading without statistical support. Including percentages of preserved tissue and acknowledging the small sample size (n = 2) would enhance transparency. The introduction outlines autolysis and liver anatomy, but the research gap is insufficiently emphasized. The rationale for focusing on flunitrazepam compared to other drugs, and the justification for using pigs as human analogues, should be strengthened. Methods are described in detail, particularly histological processing; however, environmental variables (temperature, humidity, and insect activity) are not controlled or reported, which weakens the forensic applicability. Significantly, the absence of quantitative histological scoring and blinded assessment raises concerns about subjectivity. Results are comprehensive but overly narrative. The lengthy day-by-day descriptions should be condensed into comparative tables or graphs, with figures including scale bars and annotations. Without statistical analysis, claims of delayed decomposition must remain descriptive rather than conclusive. The discussion largely repeats results instead of analyzing mechanisms. Greater focus should be placed on pharmacological explanations (e.g., inhibition of proteolytic enzymes), explicit acknowledgment of limitations, and clearer forensic implications. Overall, the study provides preliminary evidence that flunitrazepam delays the early decomposition of the body. However, revisions should focus on concise data presentation, the incorporation of quantitative measures, an expanded mechanistic discussion, and a stronger recognition of limitations to increase the manuscript’s scientific rigor and forensic value. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? No Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: entomology, animal physiology, and behavior I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Julita U. Reviewer Report For: Effects of Flunitrazepam Drug on the Decomposition Process of Pigs Liver in PMI Determination: A Histopathology Perspective [version 1; peer review: 1 approved] . F1000Research 2025, 14 :712 ( https://doi.org/10.5256/f1000research.179788.r408362 ) The direct URL for this report is: https://f1000research.com/articles/14-712/v1#referee-response-408362 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Comments on this article Comments (0) Version 1 VERSION 1 PUBLISHED 21 Jul 2025 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 Version 1 21 Jul 25 read Ucu Julita , UIN Sunan Gunung Djati Bandung, Bandung, Indonesia Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Julita U. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 19 Sep 2025 | for Version 1 Ucu Julita , UIN Sunan Gunung Djati Bandung, Bandung, Indonesia 0 Views copyright © 2025 Julita U. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This manuscript addresses a crucial forensic question regarding the impact of flunitrazepam on postmortem liver decomposition. The topic is novel and relevant; however, the study has several methodological and reporting limitations that require revision. The abstract provides a clear overview but lacks quantitative results. Terms such as “significant impact” are misleading without statistical support. Including percentages of preserved tissue and acknowledging the small sample size (n = 2) would enhance transparency. The introduction outlines autolysis and liver anatomy, but the research gap is insufficiently emphasized. The rationale for focusing on flunitrazepam compared to other drugs, and the justification for using pigs as human analogues, should be strengthened. Methods are described in detail, particularly histological processing; however, environmental variables (temperature, humidity, and insect activity) are not controlled or reported, which weakens the forensic applicability. Significantly, the absence of quantitative histological scoring and blinded assessment raises concerns about subjectivity. Results are comprehensive but overly narrative. The lengthy day-by-day descriptions should be condensed into comparative tables or graphs, with figures including scale bars and annotations. Without statistical analysis, claims of delayed decomposition must remain descriptive rather than conclusive. The discussion largely repeats results instead of analyzing mechanisms. Greater focus should be placed on pharmacological explanations (e.g., inhibition of proteolytic enzymes), explicit acknowledgment of limitations, and clearer forensic implications. Overall, the study provides preliminary evidence that flunitrazepam delays the early decomposition of the body. However, revisions should focus on concise data presentation, the incorporation of quantitative measures, an expanded mechanistic discussion, and a stronger recognition of limitations to increase the manuscript’s scientific rigor and forensic value. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? No Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise entomology, animal physiology, and behavior I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Julita U. Peer Review Report For: Effects of Flunitrazepam Drug on the Decomposition Process of Pigs Liver in PMI Determination: A Histopathology Perspective [version 1; peer review: 1 approved] . F1000Research 2025, 14 :712 ( https://doi.org/10.5256/f1000research.179788.r408362) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-712/v1#referee-response-408362 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. 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