Making a science out of preanalytics: An analytical method to determine optimal tissue fixation in real-time

Modern histopathology is built on the cornerstone principle of tissue fixation, however there are currently no analytical methods of detecting fixation and as a result, in clinical practice fixation is highly variable and a persistent source of error. We have previously shown that immersion in cold formalin followed by heated formalin is beneficial for preservation of histomorphology and have combined two-temperature fixation with ultra-sensitive acoustic monitoring technology that can actively detect formalin diffusing into a tissue. Here we expand on our previous work by developing a predictive statistical model to determine when a tissue is properly diffused based on the real-time acoustic signal. We trained the model based on the morphology and characteristic diffusion curves of 30 tonsil cores. To test our model, a set of 87 different tonsil samples were fixed with four different protocols: dynamic fixation according to our predictive algorithm (C/H:Dynamic, N = 18), gold-standard 24 hour room temperature (RT:24hr, N = 24), 6 hours in cold formalin followed by 1 hour in heated formalin (C/H:6+1, N = 21), and 2 hours in cold formalin followed by 1 hour in heated formalin (C/H:2+1, N = 24). Digital pathology analysis revealed that the C/H:Dynamic samples had FOXP3 staining that was spatially uniform and statistically equivalent to RT:24hr and C/H:6+1 fixation protocols. For comparison, the intentionally underfixed C/H:2+1 samples had significantly suppressed FOXP3 staining (p<0.002). Furthermore, our dynamic fixation protocol produced bcl-2 staining concordant with standard fixation techniques. The dynamically fixed samples were on average only submerged in cold formalin for 4.2 hours, representing a significant workflow improvement. We have successfully demonstrated a first-of-its-kind analytical method to assess the quality of fixation in real-time and have confirmed its performance with quantitative analysis of downstream staining. This innovative technology could be used to ensure high-quality and standardized staining as part of an expedited and fully documented preanalytical workflow.

THE ROLE AND SIGNIFICANCE OF MORPHOLOGICAL CHANGES IN INTRAORGAN VESSELS IN ANIMALS DURING PATHOGENESIS OF BABESIOSIS

The aim of this study is to investigate morphological changes in intraorgan vessels and their role in susceptible animals in the pathogenesis of babesiosis. The material of the study included the intraorgan vessels of domestic dogs with clinically and laboratory confirmed babesiosis and wild mouse-like rodents from latent foci of babesiosis in Volyn, Zhitomir, Kiev, Poltava, Sumy, Kharkov, Chernigov regions. For histological study, we used standard fixation in a 12% aqueous formalin solution in phosphate buffer (pH = 7.0-7.2). After the post-fixation and dehydration, the samples were placed into paraffin blocks; then a series of histological slices (5 μm) were prepared. The preparations were stained with hematoxylin and eosin, according to Van Gieson technique. All test animals, regardless of the species taxonomy, were divided into two groups. The control group included clinically healthy animals (∑ = 36). The group under clinical study consisted of animals with clinically and laboratory confirmed babesiosis (∑ = 91). Histological changes in intraorgan vessels in the control group corresponded to the anatomical and functional parameters in the health. The preparations taken from the group with basesiosis demonstrate that microvessels are branched, different in size, spasmodic; their walls have signs of delamination and through lesions. Delamination developed gradually, started with the detachment of a small horizontal fragment of the outer layer. Defects in the integrity of microvessels resulted in penetrations; there was an active diapedesis of erythrocytes into the periovascular space. The accumulation of erythrocytes directed to the areas of connective tissue, branching of blood vessels, and fatty fragments. Hemorrhages were local in nature, concentrated in the form of small blurred foci of irregular shape. In the pathogenesis of babesiosis, the defects in the walls of blood vessels with their further penetration and dissection, hemorrhages, microvesiculation of the endothelium, and its desquamation play a critical role. Other manifestations included the development of intravascular blood coagulation, thrombosis, stasis, microcirculation disorders followed by ischemia, destructive and degenerative changes in the walls of blood vessels.

Variable fixation promotes callus formation: an experimental study on transverse tibial osteotomies stabilized with locking plates

Background A new locking screw technology, named variable fixation, has been developed aiming at promoting bone callus formation providing initial rigid fixation followed by progressive fracture gap dynamisation. In this study, we compared bone callus formation in osteotomies stabilized with standard locking fixation against that of osteotomies stabilized with variable fixation in an established tibia ovine model. Methods A 3 mm tibial transverse osteotomy gap was stabilized in three groups of six female sheep each with a locking plate and either 1) standard fixation in both segments (group LS) or 2) variable fixation in the proximal and standard fixation in the distal bone segment (group VFLS3) or 3) variable fixation in both segments (group VFLS6). The implantation site and fracture healing were compared between groups by means of radiologic, micro tomographic, biomechanical, and histological investigations. Results Compared to LS callus, VFLS3 callus was 40% larger and about 3% denser, while VFLS6 callus was 93% larger and its density about 7.2% lower. VFLS3 showed 65% and VFLS6 163% larger amount of callus at the cis-cortex. There wasn’t a significant difference in the amount of callus at the cis and trans-cortex in groups featuring variable fixation only. Investigated biomechanical variables were not significantly different among groups and histology showed comparable good healing in all groups. Tissues adjacent to the implants did not show any alteration of the normal structure in all groups. Conclusions Variable fixation promoted the formation of a larger amount of bone callus, equally distributed at the cis and trans cortices. The histological and biomechanical properties of the variable fixation callus were equivalent to those of the standard fixation callus. The magnitude of variable fixation had a biological effect on the formation of bone callus. At the implantation site, the usage of variable fixation did not raise additional concerns with respect to standard fixation. The formation of a larger amount of mature callus suggests that fractures treated with variable fixation might have a higher probability to bridge the fracture gap. The conditions where its usage can be most beneficial for patients needs to be clinically defined.

A novel temporary cranial fixation device for awake cranial surgery: Technical report of 14 cases

Background: Awake craniotomy has become the gold standard in various cranial procedures. As part of the awake technique, three-point pin fixation of the patient’s head is important. One of the issues we encountered is the problem of matching the scalp infiltration site with the final pin position. To overcome this problem, we developed a flat plunger type fixator that adapts to the Mayfield holder. Methods: Our fixator has a 2.5 cm metallic shaft that articulates in a ball and socket joint to allow its concave surfaces to adapt to the patient’s scalp. After placing the patient in the desired position, the head is fixed with the three plungers, circles are drawn around each plunger, and they are then removed for the circles to be infiltrated with bupivacaine. Standard fixation pins are then placed in the Mayfield holder and aimed at the center of the circles. Results: So far, we have operated on 14 patients with this technique. No patient experienced pain during temporary fixation, and the drawn circles ensured that there were no mismatches between the local anesthetic and pin locations. The technique was particularly useful on hairy scalps, where infiltration sites were hidden. We also used only 22.5 mg bupivacaine at the pin sites, freeing a dose for the field block around the scalp incision. Conclusion: The temporary plunger type fixator provided a simple method to economize on local anesthetic use, check the patient’s head position before final fixation, and ensure that the Mayfield pins matched with the anesthetized area.

Cementless versus Cemented Fixation in Total Knee Arthroplasty

Despite the success of total knee arthroplasty (TKA), more than 20,000 revision TKA procedures are performed annually. In an effort to decrease failures due to loosening in the past, cementless fixation of TKA was suggested. The preliminary results of cementless fixation for TKA proved to be discouraging, with midterm results linking the use of uncemented components to early aseptic loosening. While cemented TKA has remained the gold standard fixation technique, the changing demographics of the average TKA patient have led some surgeons to revisit cementless fixation as an option.

Constitutional Features of the Histological Structure of the Human Knee Meniscus

The purpose of the study was to determine some constitutional features of the histological structure of the human knee menisci. Material and methods. Internal and external menisci of both knee joints of 84 corpses of men aged from 32 to 55 years were studied. After a standard fixation of the biological material, meniscus sections were prepared at the level of their anterior and posterior horns, and of the body. Then pre-stained hematoxylin-eosin prepared histological specimens were subjected to a survey microscopy, description and morphometry of the cuts of knee joint meniscus specimens. For the individualizing the revealed features, anthropometric research was carried out with all the investigated objects, followed by self-typing using the W.L.Rees-N.J.Eysenck method. Results. As a result of the study, the presence of age-specific features of the histological structure of the meniscus was established. In addition, it is proved that menisci of knee joints of men of picnic type of constitution, in contrast to representatives of other types of physique, are subject to structural changes in the histological pattern at the level of the hindbust. For men of asthenic type of physique, such changes at the level of the anterior horn of the meniscus are characteristic in comparison with other sites of the meniscus. Conclusion. Traumatologists and sports physicians can use the revealed features of the histological structure of meniscus knee joints in men of different body types at the stages of diagnosis and treatment of diseases of the knee joints, as well as in the development of measures to prevent the development of destructive changes in them. In addition, research data may influence the choice of certain rehabilitation exercises that help strengthen specific areas of the ligamentous apparatus of the knee joints of patients who have damaged menisci.

Evaluation of the Area of the Lisfranc Ligament Damaged by Screw Fixation

Background. A cannulated screw is currently the standard fixation method to reduce and stabilize diastasis at the lisfranc joint following injury. Currently, there is no literature examining the area of the lisfranc ligament damaged by screw placement. The objective of this investigation is to define the area of the ligament damaged by fixation with a 3.5-mm cannulated screw. Materials and Methods. Twelve cadaveric feet were dissected to identify the Lisfranc ligament metatarsal insertion site and origin on the medial cuneiform. A 3.5-mm cannulated screw was then passed over the course of the ligament and removed. The Lisfranc joint was then dissected to measure the dimensions of the ligament and damage from screw passage at the origin and insertion using imaging software. Results. Mean injury area on the metatarsal was 3.49 mm2 and the area of injury for the cuneiform was 3.33 mm2. The mean percent of the area damaged was calculated to be 1.75% and 2.43% at the cuneiform and metatarsal, respectively. Conclusion. A proportionally small area of the Lisfranc ligament is disrupted following screw fixation, but the implications of this on ligament healing and outcomes remain to be elucidated. Levels of Evidence: Level V: Cadaveric study

Technical note: A histochemical approach in diagnosing hanging mechanical asphyxia on cadavers undergoing advanced putrefactive phenomena

The authors used a particular protocol on putrefied corpses to highlight the cutaneous furrow. Two groups of cadavers were selected: in the first group (suicide by hanging mechanical asphyxia), the authors sampled cutaneous lozenges on the furrow still macroscopically recognisable, while in the second group (corpses deceased by different means), we sampled cutaneous lozenges in the hypostatic leaning. All specimens were divided in two: one underwent standard fixation while the other, previously rehydrated in Sandison’s solution, was then fixed in formalin. All specimens were stained with hematoxylin and eosin and Resorcin-Fuchsin. Samples processed with formalin and the H&E staining underlined various artifacts; vice versa, the Sandison solution associated with Resorcin-Fuchsin staining demonstrated the compression of the elastic fibres, with focal positivity in hanged corpses, and diffuse positivity in the entire hypostatic leaning. Sandison’s rehydrating solution and Resorcin-Fuchsin staining exalt the supposed application of an asphyctic mean even on putrefied corpses in those cases burdened by a diagnostic doubt.

Percutaneous Transpedicular Fixation in Traumatic Spondylolisthesis of L5 Vertebra

The use of percutaneous transpedicular fixation in traumatic spondylolisthesis of L5 vertebra without neurologic complications in 37 years old patient is presented. Surgical intervention, technical capability and peculiarities of surgery when using low invasive technique for vertebral body reduction are described. In cases of non-complicated injury percutaneous transpedicular fixation could be a prospective alternative to a standard fixation technique. However it should not be recommended to wide clinical application as it requires special skills and experience in use of low invasive systems and appropriate instrumentation.