Importance of intraoperative histology in the diagnosis of infection in long bone nonunions - an experimental prospective study

Importance of intraoperative histology in the diagnosis of infection in long bone nonunions - an experimental prospective study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Importance of intraoperative histology in the diagnosis of infection in long bone nonunions - an experimental prospective study Tomáš Zídek, Michaela Doležalová Hrubá, Ondřej Nikolov, Martin Kloub This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8351891/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction Diagnosis of infection during revision surgeries for nonunion of long bone fractures is challenging and remains a clinical issue. Paraffin histology is recognized as a confirmatory criterion for fracture-related infection (FRI); however, its results are not available during surgery. Cryohistology (frozen section) offers rapid intraoperative evaluation, but its diagnostic accuracy has not yet been fully validated. The aim of this study was to assess the incidence of infection in patients undergoing surgery for fracture nonunion and to compare the diagnostic yield of cryohistology with the conventional paraffin method. Method In the prospective study, two specimens of soft tissues that surrounded the nonunion were collected intraoperatively from patients. The two specimens were processed by both cryohistology and paraffin embedding methods and analysed for the presence of polymorphonuclear neutrophils (PMNs) per high power field (HPF) of view. The obtained data were analysed using descriptive and non-parametric statistics. Results The study included 36 patients with nonunion of the long bones. Infection was microbiologically confirmed in 16 cases (44%). Cryohistology and paraffin histology detected infection with the same level of sensitivity (18.8%) and specificity (95.0%). Most samples showed no presence of neutrophils; positive findings were recorded in only four patients. Statistical analysis did not show a significant difference between the methods (p > 0.05), suggesting comparable diagnostic yield. Conclusion Cryohistology provides comparable results to paraffin histology while allowing for rapid intraoperative diagnosis. However, due to its low sensitivity, it cannot be used as a standalone diagnostic tool for detecting fracture-related infections. Therefore, histological findings must always be interpreted in conjunction with clinical findings, microbiological results, and other laboratory indicators. Its use may be beneficial as a supplementary method within a multimodal diagnostic approach. However, further validation in larger and more homogeneous patient groups is necessary for its broader implementation in clinical practice. Orthopedics cryohistology (frozen section) paraffin histology nonunion FRI (fracture-related infection) intraoperative diagnosis Figures Figure 1 Figure 2 Figure 3 1. Introduction Impaired fracture healing, resulting in nonunion, is a complex process influenced by a wide range of factors and represents a significant complication in orthopedics and traumatology. According to current research, one of the key negative factors for fracture healing is the presence of infection, often caused by low-virulent bacterial strains (Bowers and Anderson, 2024 ; Mills et al., 2016 ). The presence of infection fundamentally changes the course of treatment and usually requires a combination of surgical revision and targeted, ideally also local, antibiotic therapy (Simpson et al., 2002 ). Histological examination, especially the conventional paraffin embedding technique, is one of the confirmatory criteria in the diagnosis of fracture-related infection (FRI) according to expert recommendations (Athanasou et al., 1995 ; Fehring and McAlister, 1994 ; Li et al., 2023 ; Metsemakers et al., 2018 , 2020a , b ; Morgenstern et al., 2018b ). However, histology is rarely used in routine clinical practice in long-bone nonunion surgery. Diagnosis of infection during revision surgeries for nonunions is usually based on the result of microbiological examination of soft tissue samples taken during surgery from around the nonunion. Although both paraffin histology and microbiological examination provide high diagnostic accuracy, their time-consuming nature limits their use for intraoperative decision making. A quicker alternative to assess the presence of inflammatory changes is cryohistology, a method of frozen section analysis, which is used, for example, in the diagnosis of periprosthetic joint infection (PJI). It allows the surgeon to adjust the treatment procedure directly during the operation (Athanasou et al., 1995 ; Fehring and McAlister, 1994 ; Li et al., 2023 ; Metsemakers et al., 2018 , 2020a ; Morgenstern et al., 2018b ). Although some studies show that the frozen section procedure has a lower sensitivity compared to paraffin sections, its complementary use is generally supported (Egol et al., 2002 ; Li et al., 2023 ). However, the diagnostic yield of cryohistology in the surgical management of non-unions has not yet been sufficiently verified. Thus, it is not entirely clear whether it could play a standard role in the FRI diagnostic algorithm in the future. For this reason, the objective of our study was to compare intraoperative cryohistology with conventional paraffin histology in the diagnosis of infection in long-bone nonunions. Our hypothesis was that cryohistology would yield comparable results to paraffin histology and could be used as a practical diagnostic tool during surgery. The aim was not limited to analyzing septic nonunions, but also to establish a clinically relevant model reflecting common surgical situation in which infection is not always clearly identifiable preoperatively. In such cases, intraoperative findings often inform critical decisions regarding the surgical strategy, such as whether to proceed with a one- or two-stage procedure. The findings could help to decide whether it makes sense to include this method among the routine diagnostic tools in surgical procedures in patients with fracture healing disorders. 2. Materials and methods This prospective experimental monocentric study was conducted to evaluate the role of intraoperative histology in the diagnosis of infection in patients with impaired healing of long bone fractures (nonunions) indicated for surgical revision. The study included patients aged 18 years or older who underwent surgical treatment for long bone nonunion at a Level I Trauma Centre between September 2023 and January 2025. Patients with nonunions of any long bone (clavicle, humerus, radius, ulna, femur, tibia or fibula) were included, regardless of preoperative signs of infection. Nonunion was defined as a clinically and radiologically confirmed failure of fracture healing without spontaneous consolidation. The time interval between the initial fracture and surgical revision was not a limiting criterion. Patients with chronic conditions who underwent surgery after a prolonged waiting period due to an initial absence of symptoms, refusal of surgery or delayed transfer from another medical centre were also included in the study. Criteria for eligibility included the ability to undergo general or regional anaesthesia and the signed provision of informed consent. Patients who did not provide consent or were unable to do so were excluded from the study. The study was approved by the local Ethics Committee on 22 September 2023 (ref. 114/23). All patients underwent standard preoperative clinical and laboratory evaluations, including C-reactive protein (CRP), leukocyte count, and assessment of clinical signs of infection (swelling, redness, discharge, tenderness). Clinical, laboratory, and radiological parameters indicative of infection were systematically evaluated in accordance with the international diagnostic criteria for fracture-related infection (FRI) as defined by Metsemakers et al. (Metsemakers 2018). Additional monitored parameters included epidemiological data (age, sex), the location and type of nonunion. The type of nonunion was classified based on preoperative X-ray and CT findings and intraoperative observations, according to the Weber and Čech classification into vital (hypertrophic, oligotrophic) and avital (atrophic) types (Cech and Segmüller, 1973; Fischer, 2020; Rupp et al., 2018). 2.1. Specimen collection and histological analysis. Two soft tissue specimens were taken from the area of the nonunion for histopathological processing. As standard, the specimens were collected using separate sterile instruments from the instruments intended for culture collection to avoid possible contamination from the surgical wound area (Hellebrekers et al., 2019). Both specimens were placed in tubes without fixative fluid and immediately sent to the laboratory of the hospital´s Pathology Department for processing. In the pathological laboratory, both specimens were processed by both methods. 1) Cryohistology: both specimens were frozen in a cryochamber (-26 °C), then processed into sections, stained with hematoxylin-eosin (H&E) and evaluated by a histopathologist. 2) Paraffin method: both samples were fixed in formaldehyde for more than 24 h, then dehydrated with ethanol, embedded in paraffin, processed into sections, stained with H&E and evaluated by a histopathologist. Histological evaluation included analysis of the number of polymorphonuclear neutrophils (PMNs) per high power field of view at 400x magnification (HPF), with a minimum of five randomly selected fields from of view evaluated per specimen, in accordance with international consensus guidelines (Parvizi et al., 2018). Based on the number of neutrophils, PMNs infiltration was classified into three categories on the number of neutrophils, using a bimodal threshold of ≥5 PMNs/HPF as the cut-off value, as proposed by Morgenstern et al.: (Morgenstern 2018) 1) 0 PMNs/HPF 2) 1-5 PMNs/HPF 3) ≥5 PMNs/HPF This classification enabled a comparison of the two histological methods in terms of their ability to detect inflammatory infiltration. The histological findings were recorded in the hospital information system and were not disclosed to the operating surgeon. Additionally, five separate soft tissue samples were collected from each patient for microbiological analysis. In accordance with the definition by Metsemakers et al. (Metsemakers 2018), infection (FRI) was considered confirmed if two or more cultures from independently obtained samples yielded the same pathogen. Furthermore, any osteosynthetic material present was removed during surgery and sent for sonication. The result of the sonication was evaluated as a separate microbiological finding. Empirical antibiotic prophylaxis was initiated only after all samples had been collected to avoid influencing the culture results. All patients underwent surgical debridement and, depending on the indication, either implant exchange or removal. Bone defects were managed as needed, for example by using an antibiotic-loaded spacer. 2.2. Data evaluation and analysis In the postoperative period, the quality of histological image interpretation was evaluated. The data were analysed using descriptive statistics. As each patient provided two samples analysed by two methods, the dataset was paired in nature. Also, the analysed data did not follow a normal distribution. Therefore, to compare the results of the two methods, we used the non- parametric Wilcoxon signed-rank test for paired values. Specifically, it tests whether the differences between results of both methods are symmetrically distributed around zero. The p-values were considered statistically significant at a significance level of more than 95% (p-value < 0.05). The statistical calculations were performed using Microsoft Excel software and the Python programming language. 3. Results A total of 36 patients were included in the study, of whom 27 were men (75%) and 9 were women (25%). The average age of the patients was 53 years (median 52 years, range 19–83 years, standard deviation 15.12). The obtained data shows that men were on average younger than women, who showed a higher age range and the average age (p = 0,011). The average interval from primary surgery to surgical solution of the nonunion was 12 months (median 9 months, range 3–60 months, standard deviation 11.25). In terms of the non-union typology according to Weber and Čech classification, avital forms of non-unions predominated, diagnosed in 22 patients (61%). The most common subtype was avital necrotic nonunion (n = 7; 32% of avital nonunions). The hypertrophic type was predominant among the vital nonunions in 14 patients (39%). An interesting finding was that avital nonunions were more common in women than in men (p = 0,062). The cohort characteristics are shown in Table 1 . Table 1 Characteristics of the cohort Characteristic, n (%) total men women p-value Number of patients 36 27 (75%) 9 (25%) - Average age years (SD, range) 53 (± 15.12; 19–83) 47 (± 13.53; 19–70) 67 (± 10.29; 51–83) 0,011* Non-union type - vital 14 (39%) 13 (48%) 1 (11%) avital 22 (61%) 14 (52%) 8 (89%) 0,062** *Mann-Whitney U test **Fisher’s exact test For the analysis of PMN/HPF presence, all collected samples were subjected to both cryohistological and paraffin processing (Table 2 ). The cryohistology method revealed the complete absence of PMNs/HPF in 70 out of 72 samples (Fig. 1 ). In specimens processed by the paraffin method, PMNs/HPF were not present in 67 samples (Fig. 2 ). Positive findings were recorded in four patients. In one patient, all specimens collected were found positive by both histological methods (Table 3 , Fig. 3 ). In three additional patients, positivity was detected only in paraffin histology, with a maximum infiltration density of 2 PMNs/HPF. All patients reported preoperative pain in the area of the nonunion. Mechanical failure of osteosynthesis (implant breakage or loosening) was observed in 8 patients (22%). Clinical signs of infection, such as redness, discharge, or fistula, were present in 3 patients (8%). In all three cases, infection was confirmed microbiologically by the presence of ≥ 2 positive cultures of the same pathogen, thus meeting the diagnostic criteria for fracture-related infection (FRI). Histological positivity was detected in only one of these cases by both methods; in the remaining two, it was observed solely in the paraffin sections and at low intensity (maximum two PMNs/HPF). The histological results were compared to the microbiological analysis, which was used as the reference method to confirm infection. Of the 36 patients, 16 (44%) met the FRI criteria (≥ 2 positive cultures of the same pathogen), while 20 (56%) had negative microbiological findings. Diagnostic parameters for both histological methods were then calculated based on these data. In paraffin histology, three patients were positive by both methods (true positives), one patient was positive by histology but negative by microbiology (false positive), 13 patients were positive by microbiology but negative by histology (false negatives) and 19 patients were negative by both methods (true negatives). The sensitivity of paraffin histology was therefore found to be 18,8%, the specificity 95.0%, the positive predictive value (PPV) 75.0%, and the negative predictive value (NPV) 59,4%. Frozen section histology achieved identical results, confirming its comparable diagnostic value (Table 4 ). Statistical comparison of the two methods was performed using the Wilcoxon paired test, which showed no statistically significant difference between frozen and paraffin histology (p > 0.05), indicating comparable diagnostic yield. Test results, including the p-value, are presented in Table 5 . Table 2 Summary of PMN/HPF analysis results for individual samples and both methods Histology method number of samples 0 PMNs/HPF number of samples 1–5 PMNs/HPF number of samples > 5 PMNs/HPF max. number of PMNs/HPF Cryohistology (n = 72) Specimen 1 35 1 0 3 Specimen 2 35 1 0 2 Paraffin method (n = 72) Specimen 1 34 2 0 5 Specimen 2 33 3 0 3 PMNs - neutrophilic polymorphonuclears, HPF - high power field Table 3 Positive histological findings in all samples in one patient Cryohistology specimen 1 Cryohistology specimen 2 Paraffin method specimen 1 Paraffin method specimen 2 3 2 5 3 PMNs - neutrophilic polymorphonuclears, HPF - high power field Table 4 Diagnostic yield of both methods Method Sensitivity (%) Specificity (%) PPV (%) NPV (%) Paraffin method 18.8 95.0 75.0 59.4 Cryohistology 18.8 95.0 75.0 59.4 PPV – positive predictive value, NVP – negative predictive value Table 5 Statistical comparison of histological methods using the Wilcoxon signed-rank test Wilcoxon test Specimen 1 result 297.5 p-value 0.532 Specimen 2 result 280.5 p-value 0.361 4. Discussion The aim of this study was to compare the efficacy of frozen section histology and conventional paraffin histology in detecting infection in patients with long-bone nonunions. The results showed that there was no statistically significant difference between the two methods (p > 0.05), thus confirming their comparable diagnostic yield. This conclusion is consistent with previous studies, which have demonstrated the reliability of cryohistology in detecting inflammatory cells in specimens collected during revision surgeries in patients with suspected FRI (Athanasou et al., 1995 ; Fehring and McAlister, 1994 ; Martínez et al., 2025 ; Morgenstern et al., 2018b ). Similar findings have also been published in the field of periprosthetic joint infection (PJI) diagnosis, where paraffin histology remains an important diagnostic tool due to its high accuracy, while cryohistology offers the advantage of rapid intraoperative availability of results (Izakovicova et al., 2019 ; Sangoi et al., 2009 ; Trenkwalder et al., 2024 ) Our results confirm the comparable ability of both methods to detect PMNs, which also corresponds to the findings of studies focusing on PJI (Fehring and McAlister, 1994 ). In the study Martínez et al. ( 2025 ) confirmed a high level of concordance between frozen and paraffin sections during revision surgeries for humeral nonunions. This highlights the advantage of frozen section histology in intraoperative decision-making. The authors reported a high sensitivity (88%) and specificity (96%) for detecting infection with frozen sections, and a positive predictive value of 78% and a negative predictive value of up to 98%. These results suggest the strong diagnostic reliability of negative findings, enabling reliable intraoperative exclusion of infection. In contrast, our prospective analysis, which included patients with nonunions of both upper and lower limbs and an overall lower infection prevalence (44%), demonstrated significantly lower sensitivity for both methods (18.8%), while maintaining high specificity (95.0%). This discrepancy can be attributed to several factors. Our study encompassed a broader patient population with various types of nonunion, without prior selection based on clinical suspicion of infection. This resulted in a lower positive predictive value (75%) and particularly a reduced negative predictive value (59.4%). These findings confirm that the diagnostic value of intraoperative histology, particularly with regard to negative results, is highly influenced by the prevalence of infection within the study population. Unlike the findings of Martínez et al., our data do not support the reliable exclusion of infection based solely on negative histology. However, a positive histological finding, especially with a higher density of PMNs, still showed strong diagnostic significance in our cohort and can serve as a confirmatory criterion within a comprehensive diagnostic approach (Martínez et al., 2025 ). Our findings are partially consistent with those of the study by Athanasou et al. ( 1995 ), which demonstrated that frozen sections can provide useful information about infection during revision procedures. The authors reported lower sensitivity for frozen section histology, but high specificity. In our cohort, both histological methods achieved identical sensitivities (18.8%) and specificities (95.0%), which supports their comparable diagnostic value. Although the sensitivity of both methods was low, their high specificity indicates that a positive histological finding is of significant diagnostic importance, particularly when used as a confirmatory criterion within a comprehensive diagnostic algorithm (Athanasou 1995). It is important to emphasize, however, that neither method is suitable as a standalone diagnostic tool. Histological findings must be interpreted in conjunction with microbiological and clinical data. In our cohort, the values of positive and negative predictive value (PPV 75%, NPV 59.4%) were significantly influenced by the relatively low prevalence of infection (44%). Therefore, these indicators reflect the prevalence of infection rather than the performance of the test, and must be interpreted with caution. In settings with higher or lower infection rates, the PPV and NPV could differ substantially. The majority of our samples showed no polymorphonuclear neutrophils (PMNs) per high-power field (HPF) in both frozen section and paraffin histology. This finding is consistent with the recommended threshold of ≥ 5 PMNs per HPF proposed by Morgenstern et al. (2018) as the histological criterion for fracture-related infection (FRI) (Morgenstern et al., 2018a ). The low neutrophil detection rate may also be influenced by patient selection; most participants did not exhibit preoperative clinical signs of infection. As our study did not include patients based on suspected infection, it enabled an objective assessment of the true incidence of infectious complications and the diagnostic value of histology in routine surgical practice. However, this approach also explains the lower sensitivity observed in our results. Similarly, Sigmund et al. ( 2020 ) pointed out that PMNs are minimal in aseptic complications and more often in septic cases there is an increase in PMN infiltration (Sigmund et al., 2020 ). The low detection of PMNs may be related to patient selection; if the majority of nonunions were without evidence of infection, it is logical that histological signs of infection were rare. Our study was limited by the relatively small number of patients (n = 36; 72 specimens) and the specific focus on patients with long-bone non-unions. Statistical comparisons may also have been affected by the high proportion of zero PMN values, which reduces the interpretive power of the results. Given that our data showed a statistically insignificant difference between the methods, we can reflect on several factors that may have influenced our results. While our study focused on histological evaluation of PMNs, other approaches combine histology with microbiology, PCR, or serological markers, which may increase diagnostic sensitivity. False negative results may also be caused by technical factors such as insufficient number of specimens or their collection outside the area of factual infection or incorrect specimen evaluation. This may contribute to the lower sensitivity of cryohistology in some cases. Metsemakers et al. (2020) therefore recommend the validation of diagnostic methods in larger cohorts of patients with FRI (Metsemakers et al., 2020b ). Although our study did not demonstrate the statistical superiority of one method over another, frozen section histology is a practical tool that can be used for rapid intraoperative decision-making. Due to its availability during surgery, it can complement conventional paraffin histology effectively. Based on our findings and those of previous studies, we recommend using histological analysis alongside other diagnostic methods, particularly microbiology, PCR or immunohistochemistry, as part of a multimodal approach to increase diagnostic sensitivity for fracture-related infection (FRI) (Govaert et al., 2020 ; Sangoi et al., 2009 ). 5. Conclusion Our study confirms that frozen section and paraffin histology provide comparable diagnostic results for infection in long bone nonunions. Frozen section histology has the advantage of being available rapidly during surgery and can complement standard diagnostic methods. However, due to their low sensitivity (18.8%) and negative predictive value (59.4%), these methods cannot be recommended as standalone tools for ruling out infection. They must always be interpreted alongside microbiological findings, the clinical presentation and other diagnostic criteria for fracture-related infection (FRI). Further validation through larger patient cohorts is required before broader implementation in clinical practice can be considered. References Athanasou NA, Pandey R, de Steiger R, Crook D, Smith PM (1995) Diagnosis of infection by frozen section during revision arthroplasty. J bone Jt Surg Br Vol 77:28–33 Bowers KM, Anderson DE (2024) Delayed Union and Nonunion: Current Concepts, Prevention, and Correction: A Review, Bioengineering, 11, 525. https://doi.org/10.3390/bioengineering11060525 Cech O, Segmüller G (1973) [Physiopathology of pseudoarthrosis]. Helv Chir acta 40:259–262 Egol KA, Karunakar MA, Marroum M-C, Sims SH, Kellam JF, Bosse MJ (2002) Detection of Indolent Infection at the Time of Revision Fracture Surgery. J Trauma: Inj Infect Crit Care 52:1198–1201. https://doi.org/10.1097/00005373-200206000-00030 Fehring TK, McAlister JA (1994) Frozen Histologic Section as a Guide to Sepsis in Revision Joint Arthroplasty. Clin Orthop Relat Res 304:229–237. https://doi.org/10.1097/00003086-199407000-00036 Fischer C (2020) Diagnostik und Klassifikation von Pseudarthrosen. Unfallchirurg 123:671–678. https://doi.org/10.1007/s00113-020-00844-0 Govaert GAM, Kuehl R, Atkins BL, Trampuz A, Morgenstern M, Obremskey WT, Verhofstad MHJ, McNally MA, Metsemakers W-J, Group F-R (2020) I. (FRI) C.: Diagnosing Fracture-Related Infection: Current Concepts and Recommendations. J Orthop Trauma 34:8–17. https://doi.org/10.1097/bot.0000000000001614 Hellebrekers P, Rentenaar RJ, McNally MA, Hietbrink F, Houwert RM, Leenen LPH, Govaert GAM (2019) Getting it right first time: The importance of a structured tissue sampling protocol for diagnosing fracture-related infections. Inj 50:1649–1655. https://doi.org/10.1016/j.injury.2019.05.014 Izakovicova P, Borens O, Trampuz A (2019) Periprosthetic joint infection: current concepts and outlook. EFORT Open Rev 4:482–494. https://doi.org/10.1302/2058-5241.4.180092 Li C, Margaryan D, Perka C, Trampuz A (2023) The role of biopsy in diagnosing infection after hip and knee arthroplasty: a meta-analysis. Arch Orthop Trauma Surg 143:1779–1792. https://doi.org/10.1007/s00402-021-04323-y Marsh D: Concepts of Fracture Union, Delayed Union, and, Nonunion (1998) Clin. Orthop. Relat. Res., 355, S22–S30. https://doi.org/10.1097/00003086-199810001-00004 Martínez EF, Holc F, Victorica PB, Gallucci GL, Abrego MO, Carli PD, Roitman PD, Boretto JG (2025) Intraoperative frozen section analysis for detection of fracture-related infection in nonunion of the upper limb. Diagnostic accuracy study. Injury 56:112139. https://doi.org/10.1016/j.injury.2024.112139 Metsemakers WJ, Kortram K, Morgenstern M, Moriarty TF, Meex I, Kuehl R, Nijs S, Richards RG, Raschke M, Borens O, Kates SL, Zalavras C, Giannoudis PV, Verhofstad MHJ (2018) Definition of infection after fracture fixation: A systematic review of randomized controlled trials to evaluate current practice. Inj 49:497–504. https://doi.org/10.1016/j.injury.2017.02.010 Metsemakers W-J, Fragomen AT, Moriarty TF, Morgenstern M, Egol KA, Zalavras C, Obremskey WT, Raschke M, McNally MA (2020a) Evidence-Based Recommendations for Local Antimicrobial Strategies and Dead Space Management in Fracture-Related Infection. J Orthop Trauma 34:18–29. https://doi.org/10.1097/bot.0000000000001615 Metsemakers W-J, Morgenstern M, Senneville E, Borens O, Govaert GAM, Onsea J, Depypere M, Richards RG, Trampuz A, Verhofstad MHJ, Kates SL, Raschke M, McNally MA, Obremskey WT, Metsemakers W-J, Obremskey WT, McNally MA, Athanasou N, Atkins BL, Borens O, Depypere M, Eckardt H, Egol KA, Foster W, Fragomen AT, Govaert GAM, Hungerer S, Kates SL, Kuehl R, Marais L, Mcfadyen I, Morgenstern M, Moriarty TF, Ochsner P, Ramsden A, Raschke M, Richards RG, Sancineto C, Zalavras C, Senneville E, Trampuz A, Verhofstad MHJ, Zimmerli W (2020b) General treatment principles for fracture-related infection: recommendations from an international expert group. Arch Orthop Trauma Surg 140:1013–1027. https://doi.org/10.1007/s00402-019-03287-4 Mills L, Tsang J, Hopper G, Keenan G, Simpson AHRW (2016) The multifactorial aetiology of fracture nonunion and the importance of searching for latent infection. Bone Jt Res 5:512–519. https://doi.org/10.1302/2046-3758.510.bjr-2016-0138 Morgenstern M, Kühl R, Eckardt H, Acklin Y, Stanic B, Garcia M, Baumhoer D, Metsemakers W-J (2018a) Diagnostic challenges and future perspectives in fracture-related infection. Inj 49:S83–S90. https://doi.org/10.1016/s0020-1383(18)30310-3 Morgenstern M, Athanasou NA, Ferguson JY, Metsemakers W-J, Atkins BL, McNally MA (2018b) The value of quantitative histology in the diagnosis of fracture-related infection. Bone Jt J 100–B:966–972. https://doi.org/10.1302/0301-620x.100b7.bjj-2018-0052.r1 Parvizi J, Tan TL, Goswami K, Higuera C, Valle CD, Chen AF, Shohat N (2018) The 2018 Definition of Periprosthetic Hip and Knee Infection: An Evidence-Based and Validated Criteria. J Arthroplast 33:1309–1314e2. https://doi.org/10.1016/j.arth.2018.02.078 Rupp M, Biehl C, Budak M, Thormann U, Heiss C, Alt V (2018) Diaphyseal long bone nonunions — types, aetiology, economics, and treatment recommendations. Int Orthop 42:247–258. https://doi.org/10.1007/s00264-017-3734-5 Sangoi AR, Rogers WM, Longacre TA, Montoya JG, Baron EJ, Banaei N (2009) Challenges and pitfalls of morphologic identification of fungal infections in histologic and cytologic specimens: a ten-year retrospective review at a single institution. Am J Clin Pathol 131:364–375. https://doi.org/10.1309/ajcp99ooozsniscz Sigmund IK, Dudareva M, Watts D, Morgenstern M, Athanasou NA, McNally MA (2020) Limited diagnostic value of serum inflammatory biomarkers in the diagnosis of fracture-related infections, Bone Jt. 102–B:904–911. https://doi.org/10.1302/0301-620x.102b7.bjj-2019-1739.r1 Simpson AHRW (2017) The forgotten phase of fracture healing. Bone Jt Res 6:610–611. https://doi.org/10.1302/2046-3758.610.bjr-2017-0301 Simpson AHRW, Wood MK, Athanasou NA (2002) Histological assessment of the presence or absence of infection in fracture non-union. Injury 33:151–155. https://doi.org/10.1016/s0020-1383(01)00078-x Trenkwalder K, Hackl S, Weisemann F, Augat P (2024) The value of current diagnostic techniques in the diagnosis of fracture-related infections: Serum markers, histology, and cultures. Injury 55:111862. https://doi.org/10.1016/j.injury.2024.111862 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8351891","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":559760600,"identity":"a1de5df1-4bbe-4caa-8aaf-51011c055d43","order_by":0,"name":"Tomáš Zídek","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYNACA2YGCfYGIhXzwLXwHAAyEojWwgDUIpFApBZ79u4Exh8F1nKSMx8/fFz4417i2gbmxx/w2sJzdgODhEG6sbR0mrHxjITixG0H2AwM8GqRyN3AYGBwOHGedA6bNE9CAlALD373gbUkGByunyd5hv03TMsBgloOGBxOkJbgYWOGamFswKvlzNkNBxsM0g1n9qQZS/OkJRhvO8xmjE8HA3t778aHP/5Yy0scP/zwM49Nguy24834QwwE0FzOTEj9KBgFo2AUjAKCAABerEQUEfhC9gAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0001-5746-6122","institution":"Hospital České Budějovice, Faculty Hospital Pilsen","correspondingAuthor":true,"prefix":"","firstName":"Tomáš","middleName":"","lastName":"Zídek","suffix":""},{"id":559760601,"identity":"2a94430f-1627-409e-93be-f9fdd4ce2e3b","order_by":1,"name":"Michaela Doležalová Hrubá","email":"","orcid":"https://orcid.org/0009-0004-9241-0302","institution":"Hospital České Budějovice, Faculty Hospital Pilsen","correspondingAuthor":false,"prefix":"","firstName":"Michaela","middleName":"Doležalová","lastName":"Hrubá","suffix":""},{"id":559760602,"identity":"c7ac78dc-24fe-41f4-977a-b7bd40aa1722","order_by":2,"name":"Ondřej Nikolov","email":"","orcid":"","institution":"Hospital České Budějovice","correspondingAuthor":false,"prefix":"","firstName":"Ondřej","middleName":"","lastName":"Nikolov","suffix":""},{"id":559760603,"identity":"094ee0b5-ecdc-4ea1-8343-19a8aebdb60e","order_by":3,"name":"Martin Kloub","email":"","orcid":"https://orcid.org/0000-0003-0442-2879","institution":"Hospital České Budějovice, Faculty Hospital Pilsen","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Kloub","suffix":""}],"badges":[],"createdAt":"2025-12-13 10:06:56","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":true,"humanSubjectCaseReport":true,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8351891/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8351891/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98284082,"identity":"9dc96787-166b-4014-b077-fa7cc44b4ce4","added_by":"auto","created_at":"2025-12-16 06:27:06","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4033905,"visible":true,"origin":"","legend":"","description":"","filename":"Importanceofintraoperativehistologyinthediagnosisofinfectioninlongbonenonunions.docx","url":"https://assets-eu.researchsquare.com/files/rs-8351891/v1/bbfbdd6fcb93bdca22ed73cd.docx"},{"id":98284077,"identity":"988d3f93-8599-451c-895e-a4a94917fd3b","added_by":"auto","created_at":"2025-12-16 06:27:06","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":342,"visible":true,"origin":"","legend":"","description":"","filename":"rs8351891.json","url":"https://assets-eu.researchsquare.com/files/rs-8351891/v1/7ebf69122b6361f24ad85c19.json"},{"id":98284079,"identity":"911b438e-83ce-4a8a-9ca0-d75e6f7b0dbd","added_by":"auto","created_at":"2025-12-16 06:27:06","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":86397,"visible":true,"origin":"","legend":"","description":"","filename":"rs83518910enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8351891/v1/0705c5839bdaaf762e9c0c17.xml"},{"id":98284087,"identity":"385b8379-b5f5-45a0-a237-fb10e1268982","added_by":"auto","created_at":"2025-12-16 06:27:06","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1909814,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8351891/v1/7932a651b54791c41f3590e3.jpeg"},{"id":98435085,"identity":"a1f8ef95-991e-44b9-98a7-08fc2df15a1c","added_by":"auto","created_at":"2025-12-17 16:53:06","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1944934,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8351891/v1/594d84975e78dae3574ab020.jpeg"},{"id":98435111,"identity":"8b9a8d36-6293-46df-83eb-94e153dc2752","added_by":"auto","created_at":"2025-12-17 16:53:08","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127450,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8351891/v1/3ca14914db3cf72f93bacfee.jpeg"},{"id":98436224,"identity":"e6b39b00-7fe4-4472-98c3-9868433497f7","added_by":"auto","created_at":"2025-12-17 16:55:07","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":122226,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8351891/v1/a8f2bcfdddccda3e9bcbe557.png"},{"id":98435053,"identity":"05a98119-7901-4685-a26b-8d9d7333dc0c","added_by":"auto","created_at":"2025-12-17 16:53:01","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":96249,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8351891/v1/afe339e3aeb2bbb6267e9ca2.png"},{"id":98436401,"identity":"e657fd6e-0259-4537-a628-74c19297a84b","added_by":"auto","created_at":"2025-12-17 16:55:38","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":92587,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8351891/v1/0222ccacc347def05777a762.png"},{"id":98284090,"identity":"7d5fe3ee-5682-4ef0-93d3-5a0a30979ffd","added_by":"auto","created_at":"2025-12-16 06:27:06","extension":"xml","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":84724,"visible":true,"origin":"","legend":"","description":"","filename":"rs83518910structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8351891/v1/fcd3c0653c04076e71d7da56.xml"},{"id":98436050,"identity":"0d57c265-ef03-43cf-bbc4-d9c8d269e91b","added_by":"auto","created_at":"2025-12-17 16:54:49","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":91196,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8351891/v1/9c20ed69ff2911b4ecfbbf84.html"},{"id":98435419,"identity":"33a8926b-5452-4d3c-b0ee-e0aacce58bbe","added_by":"auto","created_at":"2025-12-17 16:53:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":259363,"visible":true,"origin":"","legend":"\u003cp\u003eCryohistology (frozen section) – 0 PMN/HPF category,\u003c/p\u003e\n\u003cp\u003ehaematoxylin-eosin (H\u0026amp;E)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8351891/v1/599058be553f9e486517d5ef.png"},{"id":98284080,"identity":"a56f8de3-24b1-43b1-a4ef-76b95047d000","added_by":"auto","created_at":"2025-12-16 06:27:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":327991,"visible":true,"origin":"","legend":"\u003cp\u003eParaffin histology – 0 PMN/HPF category,\u003c/p\u003e\n\u003cp\u003ehaematoxylin-eosin (H\u0026amp;E)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8351891/v1/c114b8778d4ea6cdc04d52e7.png"},{"id":98435450,"identity":"06f3fe65-3058-46c2-835a-55b4b49b3864","added_by":"auto","created_at":"2025-12-17 16:53:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":278296,"visible":true,"origin":"","legend":"\u003cp\u003eParaffin histology - 5 PMNs/HPF, hematoxylin-eosin (H\u0026amp;E)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8351891/v1/270dfacb7f8cf4dc01860aea.png"},{"id":98445418,"identity":"a7885074-c7b6-4d72-b0ff-376383aec890","added_by":"auto","created_at":"2025-12-17 17:19:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1757954,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8351891/v1/c2ab922c-25d7-4dd3-b916-4056d8ca7351.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eImportance of intraoperative histology in the diagnosis of infection in long bone nonunions - an experimental prospective study\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eImpaired fracture healing, resulting in nonunion, is a complex process influenced by a wide range of factors and represents a significant complication in orthopedics and traumatology. According to current research, one of the key negative factors for fracture healing is the presence of infection, often caused by low-virulent bacterial strains (Bowers and Anderson, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Mills et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The presence of infection fundamentally changes the course of treatment and usually requires a combination of surgical revision and targeted, ideally also local, antibiotic therapy (Simpson et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Histological examination, especially the conventional paraffin embedding technique, is one of the confirmatory criteria in the diagnosis of fracture-related infection (FRI) according to expert recommendations (Athanasou et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Fehring and McAlister, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Metsemakers et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Morgenstern et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e). However, histology is rarely used in routine clinical practice in long-bone nonunion surgery. Diagnosis of infection during revision surgeries for nonunions is usually based on the result of microbiological examination of soft tissue samples taken during surgery from around the nonunion. Although both paraffin histology and microbiological examination provide high diagnostic accuracy, their time-consuming nature limits their use for intraoperative decision making. A quicker alternative to assess the presence of inflammatory changes is cryohistology, a method of frozen section analysis, which is used, for example, in the diagnosis of periprosthetic joint infection (PJI). It allows the surgeon to adjust the treatment procedure directly during the operation (Athanasou et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Fehring and McAlister, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Metsemakers et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Morgenstern et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e). Although some studies show that the frozen section procedure has a lower sensitivity compared to paraffin sections, its complementary use is generally supported (Egol et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the diagnostic yield of cryohistology in the surgical management of non-unions has not yet been sufficiently verified. Thus, it is not entirely clear whether it could play a standard role in the FRI diagnostic algorithm in the future. For this reason, the objective of our study was to compare intraoperative cryohistology with conventional paraffin histology in the diagnosis of infection in long-bone nonunions. Our hypothesis was that cryohistology would yield comparable results to paraffin histology and could be used as a practical diagnostic tool during surgery. The aim was not limited to analyzing septic nonunions, but also to establish a clinically relevant model reflecting common surgical situation in which infection is not always clearly identifiable preoperatively. In such cases, intraoperative findings often inform critical decisions regarding the surgical strategy, such as whether to proceed with a one- or two-stage procedure. The findings could help to decide whether it makes sense to include this method among the routine diagnostic tools in surgical procedures in patients with fracture healing disorders.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003eThis prospective experimental monocentric study was conducted to evaluate the role of intraoperative histology in the diagnosis of infection in patients with impaired healing of long bone fractures (nonunions) indicated for surgical revision. The study included patients aged 18 years or older who underwent surgical treatment for long bone nonunion at a Level I Trauma Centre between September 2023 and January 2025. Patients with nonunions of any long bone (clavicle, humerus, radius, ulna, femur, tibia or fibula) were included, regardless of preoperative signs of infection. Nonunion was defined as a clinically and radiologically confirmed failure of fracture healing without spontaneous consolidation. The time interval between the initial fracture and surgical revision was not a limiting criterion. Patients with chronic conditions who underwent surgery after a prolonged waiting period due to an initial absence of symptoms, refusal of surgery or delayed transfer from another medical centre were also included in the study. Criteria for eligibility included the ability to undergo general or regional anaesthesia and the signed provision of informed consent. Patients who did not provide consent or were unable to do so were excluded from the study. The study was approved by the local Ethics Committee on 22 September 2023 (ref. 114/23).\u003c/p\u003e\n\u003cp\u003eAll patients underwent standard preoperative clinical and laboratory evaluations, including C-reactive protein (CRP), leukocyte count, and assessment of clinical signs of infection (swelling, redness, discharge, tenderness). Clinical, laboratory, and radiological parameters indicative of infection were systematically evaluated in accordance with the international diagnostic criteria for fracture-related infection (FRI) as defined by Metsemakers et al. (Metsemakers 2018). Additional monitored parameters included epidemiological data (age, sex), the location and type of nonunion. The type of nonunion was classified based on preoperative X-ray and CT findings and intraoperative observations, according to the Weber and Čech classification into vital (hypertrophic, oligotrophic) and avital (atrophic) types\u0026nbsp;(Cech and Segmüller, 1973; Fischer, 2020; Rupp et al., 2018).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.\u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSpecimen collection and histological analysis.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo soft tissue specimens were taken from the area of the nonunion for histopathological processing. As standard, the specimens were collected using separate sterile instruments from the instruments intended for culture collection to avoid possible contamination from the surgical wound area (Hellebrekers et al., 2019). Both specimens were placed in tubes without fixative fluid and immediately sent to the laboratory of the hospital´s Pathology Department for processing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the pathological laboratory, both specimens were processed by both methods.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e1)\u0026nbsp; \u0026nbsp;\u0026nbsp;Cryohistology: both specimens were frozen in a cryochamber (-26 °C), then processed into sections, stained with hematoxylin-eosin (H\u0026amp;E) and evaluated by a histopathologist.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2)\u0026nbsp; \u0026nbsp;\u0026nbsp;Paraffin method: both samples were fixed in formaldehyde for more than 24 h, then dehydrated with ethanol, embedded in paraffin, processed into sections, stained with H\u0026amp;E and evaluated by a histopathologist.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHistological evaluation included analysis of the number of polymorphonuclear neutrophils (PMNs) per high power field of view at 400x magnification (HPF), with a minimum of five randomly selected fields from of view evaluated per specimen, in accordance with international consensus guidelines (Parvizi et al., 2018). Based on the number of neutrophils, PMNs infiltration was classified into three categories on the number of neutrophils, using a bimodal threshold of ≥5 PMNs/HPF as the cut-off value, as proposed by Morgenstern et al.: (Morgenstern 2018)\u003c/p\u003e\n\u003cp\u003e1)\u0026nbsp; \u0026nbsp;\u0026nbsp;0 PMNs/HPF\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2)\u0026nbsp; \u0026nbsp;\u0026nbsp;1-5 PMNs/HPF\u003c/p\u003e\n\u003cp\u003e3) ≥5 PMNs/HPF\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis classification enabled a comparison of the two histological methods in terms of their ability to detect inflammatory infiltration. The histological findings were recorded in the hospital information system and were not disclosed to the operating surgeon. Additionally, five separate soft tissue samples were collected from each patient for microbiological analysis. In accordance with the definition by Metsemakers et al.\u0026nbsp;(Metsemakers 2018), infection (FRI) was considered confirmed if two or more cultures from independently obtained samples yielded the same pathogen. Furthermore, any osteosynthetic material present was removed during surgery and sent for sonication. The result of the sonication was evaluated as a separate microbiological finding. Empirical antibiotic prophylaxis was initiated only after all samples had been collected to avoid influencing the culture results. All patients underwent surgical debridement and, depending on the indication, either implant exchange or removal. Bone defects were managed as needed, for example by using an antibiotic-loaded spacer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.\u0026nbsp; \u0026nbsp;Data evaluation and analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the postoperative period, the quality of histological image interpretation was evaluated. The data were analysed using descriptive statistics. As each patient provided two samples analysed by two methods, the dataset was paired in nature. Also, the analysed data did not follow a normal distribution. Therefore, to compare the results of the two methods, we used the non- parametric Wilcoxon signed-rank test for paired values. Specifically, it tests whether the differences between results of both methods are symmetrically distributed around zero. The p-values were considered statistically significant at a significance level of more than 95% (p-value \u0026lt; 0.05). The statistical calculations were performed using Microsoft Excel software and the Python programming language.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003eA total of 36 patients were included in the study, of whom 27 were men (75%) and 9 were women (25%). The average age of the patients was 53 years (median 52 years, range 19\u0026ndash;83 years, standard deviation 15.12). The obtained data shows that men were on average younger than women, who showed a higher age range and the average age (p\u0026thinsp;=\u0026thinsp;0,011). The average interval from primary surgery to surgical solution of the nonunion was 12 months (median 9 months, range 3\u0026ndash;60 months, standard deviation 11.25). In terms of the non-union typology according to Weber and Čech classification, avital forms of non-unions predominated, diagnosed in 22 patients (61%). The most common subtype was avital necrotic nonunion (n\u0026thinsp;=\u0026thinsp;7; 32% of avital nonunions). The hypertrophic type was predominant among the vital nonunions in 14 patients (39%). An interesting finding was that avital nonunions were more common in women than in men (p\u0026thinsp;=\u0026thinsp;0,062). The cohort characteristics are shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCharacteristics of the cohort\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCharacteristic, n (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003etotal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003emen\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ewomen\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of patients\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27 (75%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9 (25%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAverage age years\u003c/p\u003e\n \u003cp\u003e(SD, range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003cp\u003e(\u0026plusmn;\u0026thinsp;15.12; 19\u0026ndash;83)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003cp\u003e(\u0026plusmn;\u0026thinsp;13.53; 19\u0026ndash;70)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003cp\u003e(\u0026plusmn;\u0026thinsp;10.29; 51\u0026ndash;83)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,011*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNon-union type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evital\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14 (39%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13 (48%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (11%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eavital\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22 (61%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14 (52%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (89%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,062**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e*Mann-Whitney U test **Fisher\u0026rsquo;s exact test\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eFor the analysis of PMN/HPF presence, all collected samples were subjected to both cryohistological and paraffin processing (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The cryohistology method revealed the complete absence of PMNs/HPF in 70 out of 72 samples (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In specimens processed by the paraffin method, PMNs/HPF were not present in 67 samples (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Positive findings were recorded in four patients. In one patient, all specimens collected were found positive by both histological methods (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In three additional patients, positivity was detected only in paraffin histology, with a maximum infiltration density of 2 PMNs/HPF. All patients reported preoperative pain in the area of the nonunion. Mechanical failure of osteosynthesis (implant breakage or loosening) was observed in 8 patients (22%). Clinical signs of infection, such as redness, discharge, or fistula, were present in 3 patients (8%). In all three cases, infection was confirmed microbiologically by the presence of \u0026ge;\u0026thinsp;2 positive cultures of the same pathogen, thus meeting the diagnostic criteria for fracture-related infection (FRI). Histological positivity was detected in only one of these cases by both methods; in the remaining two, it was observed solely in the paraffin sections and at low intensity (maximum two PMNs/HPF). The histological results were compared to the microbiological analysis, which was used as the reference method to confirm infection. Of the 36 patients, 16 (44%) met the FRI criteria (\u0026ge;\u0026thinsp;2 positive cultures of the same pathogen), while 20 (56%) had negative microbiological findings. Diagnostic parameters for both histological methods were then calculated based on these data. In paraffin histology, three patients were positive by both methods (true positives), one patient was positive by histology but negative by microbiology (false positive), 13 patients were positive by microbiology but negative by histology (false negatives) and 19 patients were negative by both methods (true negatives). The sensitivity of paraffin histology was therefore found to be 18,8%, the specificity 95.0%, the positive predictive value (PPV) 75.0%, and the negative predictive value (NPV) 59,4%. Frozen section histology achieved identical results, confirming its comparable diagnostic value (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Statistical comparison of the two methods was performed using the Wilcoxon paired test, which showed no statistically significant difference between frozen and paraffin histology (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating comparable diagnostic yield. Test results, including the p-value, are presented in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSummary of PMN/HPF analysis results for individual samples and both methods\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHistology method\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003enumber of samples\u003c/p\u003e\n \u003cp\u003e0 PMNs/HPF\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003enumber of samples\u003c/p\u003e\n \u003cp\u003e1\u0026ndash;5 PMNs/HPF\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003enumber of samples\u003c/p\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;5 PMNs/HPF\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003emax. number of PMNs/HPF\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCryohistology\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;72)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpecimen 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpecimen 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eParaffin method\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;72)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpecimen 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpecimen 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003ePMNs - neutrophilic polymorphonuclears, HPF - high power field\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePositive histological findings in all samples in one patient\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCryohistology specimen 1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCryohistology specimen 2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParaffin method specimen 1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParaffin method specimen 2\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003ePMNs - neutrophilic polymorphonuclears, HPF - high power field\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDiagnostic yield of both methods\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMethod\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSensitivity (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecificity (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePPV (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNPV (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eParaffin method\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e75.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e59.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCryohistology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e75.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e59.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003ePPV \u0026ndash; positive predictive value, NVP \u0026ndash; negative predictive value\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"char\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eStatistical comparison of histological methods using the Wilcoxon signed-rank test\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWilcoxon test\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSpecimen 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eresult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e297.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep-value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.532\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSpecimen 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eresult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e280.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep-value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.361\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe aim of this study was to compare the efficacy of frozen section histology and conventional paraffin histology in detecting infection in patients with long-bone nonunions. The results showed that there was no statistically significant difference between the two methods (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), thus confirming their comparable diagnostic yield. This conclusion is consistent with previous studies, which have demonstrated the reliability of cryohistology in detecting inflammatory cells in specimens collected during revision surgeries in patients with suspected FRI (Athanasou et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Fehring and McAlister, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Mart\u0026iacute;nez et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Morgenstern et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSimilar findings have also been published in the field of periprosthetic joint infection (PJI) diagnosis, where paraffin histology remains an important diagnostic tool due to its high accuracy, while cryohistology offers the advantage of rapid intraoperative availability of results (Izakovicova et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sangoi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Trenkwalder et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) Our results confirm the comparable ability of both methods to detect PMNs, which also corresponds to the findings of studies focusing on PJI (Fehring and McAlister, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1994\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the study Mart\u0026iacute;nez et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) confirmed a high level of concordance between frozen and paraffin sections during revision surgeries for humeral nonunions. This highlights the advantage of frozen section histology in intraoperative decision-making. The authors reported a high sensitivity (88%) and specificity (96%) for detecting infection with frozen sections, and a positive predictive value of 78% and a negative predictive value of up to 98%. These results suggest the strong diagnostic reliability of negative findings, enabling reliable intraoperative exclusion of infection. In contrast, our prospective analysis, which included patients with nonunions of both upper and lower limbs and an overall lower infection prevalence (44%), demonstrated significantly lower sensitivity for both methods (18.8%), while maintaining high specificity (95.0%). This discrepancy can be attributed to several factors. Our study encompassed a broader patient population with various types of nonunion, without prior selection based on clinical suspicion of infection. This resulted in a lower positive predictive value (75%) and particularly a reduced negative predictive value (59.4%). These findings confirm that the diagnostic value of intraoperative histology, particularly with regard to negative results, is highly influenced by the prevalence of infection within the study population. Unlike the findings of Mart\u0026iacute;nez et al., our data do not support the reliable exclusion of infection based solely on negative histology. However, a positive histological finding, especially with a higher density of PMNs, still showed strong diagnostic significance in our cohort and can serve as a confirmatory criterion within a comprehensive diagnostic approach (Mart\u0026iacute;nez et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur findings are partially consistent with those of the study by Athanasou et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), which demonstrated that frozen sections can provide useful information about infection during revision procedures. The authors reported lower sensitivity for frozen section histology, but high specificity. In our cohort, both histological methods achieved identical sensitivities (18.8%) and specificities (95.0%), which supports their comparable diagnostic value. Although the sensitivity of both methods was low, their high specificity indicates that a positive histological finding is of significant diagnostic importance, particularly when used as a confirmatory criterion within a comprehensive diagnostic algorithm (Athanasou 1995).\u003c/p\u003e \u003cp\u003eIt is important to emphasize, however, that neither method is suitable as a standalone diagnostic tool. Histological findings must be interpreted in conjunction with microbiological and clinical data. In our cohort, the values of positive and negative predictive value (PPV 75%, NPV 59.4%) were significantly influenced by the relatively low prevalence of infection (44%). Therefore, these indicators reflect the prevalence of infection rather than the performance of the test, and must be interpreted with caution. In settings with higher or lower infection rates, the PPV and NPV could differ substantially.\u003c/p\u003e \u003cp\u003eThe majority of our samples showed no polymorphonuclear neutrophils (PMNs) per high-power field (HPF) in both frozen section and paraffin histology. This finding is consistent with the recommended threshold of \u0026ge;\u0026thinsp;5 PMNs per HPF proposed by Morgenstern et al. (2018) as the histological criterion for fracture-related infection (FRI) (Morgenstern et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe low neutrophil detection rate may also be influenced by patient selection; most participants did not exhibit preoperative clinical signs of infection. As our study did not include patients based on suspected infection, it enabled an objective assessment of the true incidence of infectious complications and the diagnostic value of histology in routine surgical practice. However, this approach also explains the lower sensitivity observed in our results. Similarly, Sigmund et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) pointed out that PMNs are minimal in aseptic complications and more often in septic cases there is an increase in PMN infiltration (Sigmund et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe low detection of PMNs may be related to patient selection; if the majority of nonunions were without evidence of infection, it is logical that histological signs of infection were rare. Our study was limited by the relatively small number of patients (n\u0026thinsp;=\u0026thinsp;36; 72 specimens) and the specific focus on patients with long-bone non-unions. Statistical comparisons may also have been affected by the high proportion of zero PMN values, which reduces the interpretive power of the results.\u003c/p\u003e \u003cp\u003eGiven that our data showed a statistically insignificant difference between the methods, we can reflect on several factors that may have influenced our results. While our study focused on histological evaluation of PMNs, other approaches combine histology with microbiology, PCR, or serological markers, which may increase diagnostic sensitivity. False negative results may also be caused by technical factors such as insufficient number of specimens or their collection outside the area of factual infection or incorrect specimen evaluation. This may contribute to the lower sensitivity of cryohistology in some cases. Metsemakers et al. (2020) therefore recommend the validation of diagnostic methods in larger cohorts of patients with FRI (Metsemakers et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough our study did not demonstrate the statistical superiority of one method over another, frozen section histology is a practical tool that can be used for rapid intraoperative decision-making. Due to its availability during surgery, it can complement conventional paraffin histology effectively. Based on our findings and those of previous studies, we recommend using histological analysis alongside other diagnostic methods, particularly microbiology, PCR or immunohistochemistry, as part of a multimodal approach to increase diagnostic sensitivity for fracture-related infection (FRI) (Govaert et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sangoi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur study confirms that frozen section and paraffin histology provide comparable diagnostic results for infection in long bone nonunions. Frozen section histology has the advantage of being available rapidly during surgery and can complement standard diagnostic methods. However, due to their low sensitivity (18.8%) and negative predictive value (59.4%), these methods cannot be recommended as standalone tools for ruling out infection. They must always be interpreted alongside microbiological findings, the clinical presentation and other diagnostic criteria for fracture-related infection (FRI). Further validation through larger patient cohorts is required before broader implementation in clinical practice can be considered.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAthanasou NA, Pandey R, de Steiger R, Crook D, Smith PM (1995) Diagnosis of infection by frozen section during revision arthroplasty. J bone Jt Surg Br Vol 77:28\u0026ndash;33\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBowers KM, Anderson DE (2024) Delayed Union and Nonunion: Current Concepts, Prevention, and Correction: A Review, Bioengineering, 11, 525. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/bioengineering11060525\u003c/span\u003e\u003cspan address=\"10.3390/bioengineering11060525\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCech O, Segm\u0026uuml;ller G (1973) [Physiopathology of pseudoarthrosis]. Helv Chir acta 40:259\u0026ndash;262\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEgol KA, Karunakar MA, Marroum M-C, Sims SH, Kellam JF, Bosse MJ (2002) Detection of Indolent Infection at the Time of Revision Fracture Surgery. J Trauma: Inj Infect Crit Care 52:1198\u0026ndash;1201. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/00005373-200206000-00030\u003c/span\u003e\u003cspan address=\"10.1097/00005373-200206000-00030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFehring TK, McAlister JA (1994) Frozen Histologic Section as a Guide to Sepsis in Revision Joint Arthroplasty. Clin Orthop Relat Res 304:229\u0026ndash;237. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/00003086-199407000-00036\u003c/span\u003e\u003cspan address=\"10.1097/00003086-199407000-00036\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFischer C (2020) Diagnostik und Klassifikation von Pseudarthrosen. Unfallchirurg 123:671\u0026ndash;678. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00113-020-00844-0\u003c/span\u003e\u003cspan address=\"10.1007/s00113-020-00844-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGovaert GAM, Kuehl R, Atkins BL, Trampuz A, Morgenstern M, Obremskey WT, Verhofstad MHJ, McNally MA, Metsemakers W-J, Group F-R (2020) I. (FRI) C.: Diagnosing Fracture-Related Infection: Current Concepts and Recommendations. J Orthop Trauma 34:8\u0026ndash;17. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/bot.0000000000001614\u003c/span\u003e\u003cspan address=\"10.1097/bot.0000000000001614\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHellebrekers P, Rentenaar RJ, McNally MA, Hietbrink F, Houwert RM, Leenen LPH, Govaert GAM (2019) Getting it right first time: The importance of a structured tissue sampling protocol for diagnosing fracture-related infections. Inj 50:1649\u0026ndash;1655. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.injury.2019.05.014\u003c/span\u003e\u003cspan address=\"10.1016/j.injury.2019.05.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIzakovicova P, Borens O, Trampuz A (2019) Periprosthetic joint infection: current concepts and outlook. EFORT Open Rev 4:482\u0026ndash;494. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1302/2058-5241.4.180092\u003c/span\u003e\u003cspan address=\"10.1302/2058-5241.4.180092\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C, Margaryan D, Perka C, Trampuz A (2023) The role of biopsy in diagnosing infection after hip and knee arthroplasty: a meta-analysis. Arch Orthop Trauma Surg 143:1779\u0026ndash;1792. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00402-021-04323-y\u003c/span\u003e\u003cspan address=\"10.1007/s00402-021-04323-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarsh D: Concepts of Fracture Union, Delayed Union, and, Nonunion (1998) Clin. Orthop. Relat. Res., 355, S22\u0026ndash;S30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/00003086-199810001-00004\u003c/span\u003e\u003cspan address=\"10.1097/00003086-199810001-00004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMart\u0026iacute;nez EF, Holc F, Victorica PB, Gallucci GL, Abrego MO, Carli PD, Roitman PD, Boretto JG (2025) Intraoperative frozen section analysis for detection of fracture-related infection in nonunion of the upper limb. Diagnostic accuracy study. Injury 56:112139. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.injury.2024.112139\u003c/span\u003e\u003cspan address=\"10.1016/j.injury.2024.112139\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMetsemakers WJ, Kortram K, Morgenstern M, Moriarty TF, Meex I, Kuehl R, Nijs S, Richards RG, Raschke M, Borens O, Kates SL, Zalavras C, Giannoudis PV, Verhofstad MHJ (2018) Definition of infection after fracture fixation: A systematic review of randomized controlled trials to evaluate current practice. Inj 49:497\u0026ndash;504. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.injury.2017.02.010\u003c/span\u003e\u003cspan address=\"10.1016/j.injury.2017.02.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMetsemakers W-J, Fragomen AT, Moriarty TF, Morgenstern M, Egol KA, Zalavras C, Obremskey WT, Raschke M, McNally MA (2020a) Evidence-Based Recommendations for Local Antimicrobial Strategies and Dead Space Management in Fracture-Related Infection. J Orthop Trauma 34:18\u0026ndash;29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/bot.0000000000001615\u003c/span\u003e\u003cspan address=\"10.1097/bot.0000000000001615\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMetsemakers W-J, Morgenstern M, Senneville E, Borens O, Govaert GAM, Onsea J, Depypere M, Richards RG, Trampuz A, Verhofstad MHJ, Kates SL, Raschke M, McNally MA, Obremskey WT, Metsemakers W-J, Obremskey WT, McNally MA, Athanasou N, Atkins BL, Borens O, Depypere M, Eckardt H, Egol KA, Foster W, Fragomen AT, Govaert GAM, Hungerer S, Kates SL, Kuehl R, Marais L, Mcfadyen I, Morgenstern M, Moriarty TF, Ochsner P, Ramsden A, Raschke M, Richards RG, Sancineto C, Zalavras C, Senneville E, Trampuz A, Verhofstad MHJ, Zimmerli W (2020b) General treatment principles for fracture-related infection: recommendations from an international expert group. Arch Orthop Trauma Surg 140:1013\u0026ndash;1027. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00402-019-03287-4\u003c/span\u003e\u003cspan address=\"10.1007/s00402-019-03287-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMills L, Tsang J, Hopper G, Keenan G, Simpson AHRW (2016) The multifactorial aetiology of fracture nonunion and the importance of searching for latent infection. Bone Jt Res 5:512\u0026ndash;519. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1302/2046-3758.510.bjr-2016-0138\u003c/span\u003e\u003cspan address=\"10.1302/2046-3758.510.bjr-2016-0138\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorgenstern M, K\u0026uuml;hl R, Eckardt H, Acklin Y, Stanic B, Garcia M, Baumhoer D, Metsemakers W-J (2018a) Diagnostic challenges and future perspectives in fracture-related infection. Inj 49:S83\u0026ndash;S90. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0020-1383(18)30310-3\u003c/span\u003e\u003cspan address=\"10.1016/s0020-1383(18)30310-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorgenstern M, Athanasou NA, Ferguson JY, Metsemakers W-J, Atkins BL, McNally MA (2018b) The value of quantitative histology in the diagnosis of fracture-related infection. Bone Jt J 100\u0026ndash;B:966\u0026ndash;972. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1302/0301-620x.100b7.bjj-2018-0052.r1\u003c/span\u003e\u003cspan address=\"10.1302/0301-620x.100b7.bjj-2018-0052.r1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParvizi J, Tan TL, Goswami K, Higuera C, Valle CD, Chen AF, Shohat N (2018) The 2018 Definition of Periprosthetic Hip and Knee Infection: An Evidence-Based and Validated Criteria. J Arthroplast 33:1309\u0026ndash;1314e2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.arth.2018.02.078\u003c/span\u003e\u003cspan address=\"10.1016/j.arth.2018.02.078\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRupp M, Biehl C, Budak M, Thormann U, Heiss C, Alt V (2018) Diaphyseal long bone nonunions \u0026mdash; types, aetiology, economics, and treatment recommendations. Int Orthop 42:247\u0026ndash;258. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00264-017-3734-5\u003c/span\u003e\u003cspan address=\"10.1007/s00264-017-3734-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSangoi AR, Rogers WM, Longacre TA, Montoya JG, Baron EJ, Banaei N (2009) Challenges and pitfalls of morphologic identification of fungal infections in histologic and cytologic specimens: a ten-year retrospective review at a single institution. Am J Clin Pathol 131:364\u0026ndash;375. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1309/ajcp99ooozsniscz\u003c/span\u003e\u003cspan address=\"10.1309/ajcp99ooozsniscz\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSigmund IK, Dudareva M, Watts D, Morgenstern M, Athanasou NA, McNally MA (2020) Limited diagnostic value of serum inflammatory biomarkers in the diagnosis of fracture-related infections, Bone Jt. 102\u0026ndash;B:904\u0026ndash;911. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1302/0301-620x.102b7.bjj-2019-1739.r1\u003c/span\u003e\u003cspan address=\"10.1302/0301-620x.102b7.bjj-2019-1739.r1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimpson AHRW (2017) The forgotten phase of fracture healing. Bone Jt Res 6:610\u0026ndash;611. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1302/2046-3758.610.bjr-2017-0301\u003c/span\u003e\u003cspan address=\"10.1302/2046-3758.610.bjr-2017-0301\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimpson AHRW, Wood MK, Athanasou NA (2002) Histological assessment of the presence or absence of infection in fracture non-union. Injury 33:151\u0026ndash;155. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0020-1383(01)00078-x\u003c/span\u003e\u003cspan address=\"10.1016/s0020-1383(01)00078-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrenkwalder K, Hackl S, Weisemann F, Augat P (2024) The value of current diagnostic techniques in the diagnosis of fracture-related infections: Serum markers, histology, and cultures. Injury 55:111862. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.injury.2024.111862\u003c/span\u003e\u003cspan address=\"10.1016/j.injury.2024.111862\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Nemocnice České Budějovice","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cryohistology (frozen section), paraffin histology, nonunion, FRI (fracture-related infection), intraoperative diagnosis","lastPublishedDoi":"10.21203/rs.3.rs-8351891/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8351891/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntroduction\u003c/p\u003e\n\u003cp\u003eDiagnosis of infection during revision surgeries for nonunion of long bone fractures is challenging and remains a clinical issue. Paraffin histology is recognized as a confirmatory criterion for fracture-related infection (FRI); however, its results are not available during surgery. Cryohistology (frozen section) offers rapid intraoperative evaluation, but its diagnostic accuracy has not yet been fully validated. The aim of this study was to assess the incidence of infection in patients undergoing surgery for fracture nonunion and to compare the diagnostic yield of cryohistology with the conventional paraffin method.\u003c/p\u003e\n\u003cp\u003eMethod\u003c/p\u003e\n\u003cp\u003eIn the prospective study, two specimens of soft tissues that surrounded the nonunion were collected intraoperatively from patients. The two specimens were processed by both cryohistology and paraffin embedding methods and analysed for the presence of polymorphonuclear neutrophils (PMNs) per high power field (HPF) of view. The obtained data were analysed using descriptive and non-parametric statistics.\u003c/p\u003e\n\u003cp\u003eResults\u003c/p\u003e\n\u003cp\u003eThe study included 36 patients with nonunion of the long bones. Infection was microbiologically confirmed in 16 cases (44%). Cryohistology and paraffin histology detected infection with the same level of sensitivity (18.8%) and specificity (95.0%). Most samples showed no presence of neutrophils; positive findings were recorded in only four patients. Statistical analysis did not show a significant difference between the methods (p \u0026gt; 0.05), suggesting comparable diagnostic yield.\u003c/p\u003e\n\u003cp\u003eConclusion\u003c/p\u003e\n\u003cp\u003eCryohistology provides comparable results to paraffin histology while allowing for rapid intraoperative diagnosis. However, due to its low sensitivity, it cannot be used as a standalone diagnostic tool for detecting fracture-related infections. Therefore, histological findings must always be interpreted in conjunction with clinical findings, microbiological results, and other laboratory indicators. Its use may be beneficial as a supplementary method within a multimodal diagnostic approach. However, further validation in larger and more homogeneous patient groups is necessary for its broader implementation in clinical practice.\u003c/p\u003e","manuscriptTitle":"Importance of intraoperative histology in the diagnosis of infection in long bone nonunions - an experimental prospective study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-16 06:27:02","doi":"10.21203/rs.3.rs-8351891/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ecbb2f58-534d-4de3-9caf-fb3225e43c2d","owner":[],"postedDate":"December 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59597176,"name":"Orthopedics"}],"tags":[],"updatedAt":"2025-12-16T06:27:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-16 06:27:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8351891","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8351891","identity":"rs-8351891","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}