When we receive fresh hearts for research, we attempt to perfusion fix each heart so that they are preserved in an end-diastolic state, which is the state when the heart is filled with blood immediately before contraction. To initiate the specimen preservation process, four of the great vessels are cannulated tubes placed inside so that there is a cannula connected to each chamber of the heart.
The remaining vessels are plugged or closed. The heart is then placed in the formalin-filled lower chamber and its cannulated vessels are connected to the upper formalin-filled chamber as seen in the figure to the right. The upper chamber is set to elicit a head pressure of approximately 50 mmHg. In this set-up, formalin flows antegrade through the aorta into the coronary arteries. In terms of outcomes, the available evidence suggests that perfusion fixation probably leads to equivalent or improved subjective histology quality compared to immersion fixation of relatively large volumes of brain tissue, in a shorter amount of time.
During the review process, there were several changes made between the original protocol and the methods we employed. The database search strategies include a combination of subject headings and keywords. To identify additional publications that are missed by these searches, we screened the references and citing articles as identified by Scopus of all included articles.
Any scholarly publication such as a journal article or textbook chapter that describes methods for perfusion fixation of the human brain was included. To be included, a study only needs to report on the perfusion fixation of human brain tissue and describe the methods for doing so; it does not need to be primarily about the process of perfusion fixation of the human brain. Fixation was defined as the use of a chemical substance or mixture of chemicals designed to preserve the tissue architecture and molecules in their lifelike state.
Perfusion fixation was defined as using the vascular system in order to distribute fixatives throughout brain tissue. Studies on human brain tissue of any age were included. Studies were included if they perfuse the whole brain, only part of the brain such as a hemisphere, or a particular brain region.
Studies that are performed by the same investigators and describe the same methods without substantive changes were considered together as one study, referred to by the study with the most detailed description of the methods.
Studies written in any language were considered. If not written in English, studies were translated with the help of online tools such as Google Translate and Yandex Translate. Although our focus is on the use of perfusion fixation for brain banking, our search strategy allowed us to identify articles that used perfusion fixation of postmortem human brain tissue for any type of research study, rather than only brain banking in particular.
We used this approach to try to increase the pool of studies using perfusion fixation on human brain tissue from which we could learn and draw conclusions. Using the online software Covidence, one reviewer A. Subsequently, two individuals W. Disagreements were resolved by a consensus meeting. For all included studies, at least two reviewers H. In the case that there was disagreement between these reviewers that could not be addressed by further assessment of the manuscript by one of the reviewers A.
The data variables that were extracted are: number of perfusion- and immersion-fixed brains; exclusion criteria that would prevent the use of perfusion fixation for fixing brain tissue, for example long postmortem interval or vascular disease; tissue processing prior to vascular access; vessels accessed for perfusion; prefixative infusion; fixative mixture and buffer; time for perfusion; amount of fluid perfused; perfusion pressure; tissue processing before postfixation; postfixation procedure for perfusion fixed brains; tissue processing and storage procedure for perfusion fixed brains; metric s for fixation quality; downstream assays used or suggested; metric s for comparison to immersion fixation; and outcomes in comparison to immersion fixation.
In the case that the variables were likely performed, known, or measured by the study authors but not reported, we attempted to contact the corresponding author s of the study via email and inquire about the variables. To harmonize the study appraisal tool with the downstream Cochrane tool for grading outcomes by the risk of bias of the studies included, we made one change to this checklist: we added an explicit question about the use of blinding by each study in the outcome assessment Additional file 3.
The study quality metrics were assessed by at least two reviewers H. In the case that there was a disagreement between these reviewers, an additional reviewer W. The study quality metrics were taken into account when considering the strength of the evidence in the outcomes that they report. A qualitative survey of the different methods that have been reported for perfusion fixation in human brain banking was performed.
Where possible, comparisons were made between the reported outcomes of immersion compared to perfusion fixation for brain banking. Because the studies were not expected to measure or report quantitative data on fixation quality, we performed a qualitative synthesis rather than a quantitative meta-analysis.
Each outcome between perfusion and immersion fixation was considered separately and had its own row in the summary of findings table.
There were two outcomes assessed: 1 the subjective histology quality following either immersion or perfusion fixation and 2 the subjective histology quality following either immersion or perfusion fixation and after long-term storage in fixative.
There are four possible levels for outcome quality in the GRADE method: high, moderate, low, and very low. In the GRADE method, all results derived from randomized trials start with a grade of high, while results derived from non-randomized studies start with a grade of low. Next, these grades were downgraded by one level for serious concerns or two levels for very serious concerns about risk of bias, inconsistency, indirectness, imprecision, and publication bias.
They were upgraded by one level for large magnitudes of effect, for a dose-response relationship, or when the effects of all plausible confounds would go against the effect seen. The risk of bias for each study was assessed as a part of the JBI critical appraisal checklist.
For example, confounding bias was assessed by the JBI checklist question about whether the participants in any comparisons were similar. Two reviewers W.
We note the following changes from the preregistered protocol. First, to grade the outcomes identified in the studies between perfusion and immersion fixation, we added these components to the questionnaire and methods.
Critical appraisal of studies was only performed for studies that included a comparison between perfusion and immersion fixation, as the other studies were descriptive. In order to maintain the same appraisal criteria consistently across randomized and non-randomized experimental studies, all of the studies that compared perfusion fixation to immersion fixation or compared methods of perfusion fixation were critically appraised using the JBI checklist for quasi-experimental studies.
Because it was not possible to adequately appraise studies that made only an implicit comparison between perfusion and immersion fixation, we changed the protocol so that only studies that made an explicit comparison were included in this section of the review.
Therefore, we attempted to identify brain donor exclusion criteria that were particular to the use of perfusion fixation. After the data extraction process, we decided that the studies, methods, and outcomes for the comparisons between methods of perfusion fixation identified were too few and heterogeneous to provide any meaningful qualitative synthesis across studies.
Therefore, we did not perform outcomes grading for comparisons between methods of perfusion fixation. We also did not identify any studies that compared how the perfusion fixation and immersion fixation approaches differed in fixation quality based on the brain tissue characteristics, so this was also not addressed.
The outcomes selected for comparison between immersion and perfusion fixation were determined after the data extraction stage on the basis of the available data, and were not included in the original protocol. The first part of this review section will list the methods for perfusion fixation used by the included studies, while the second part will summarize any outcomes of comparisons between perfusion and immersion fixation.
We screened abstracts, full-text publications, and identified 35 studies that met our inclusion criteria, which collectively reported on the perfusion fixation of human brains Fig. Reasons for full-text exclusion decisions were that: no humans were studied i. The studies were classified into three types: histology, e. Of the articles focused on histology, there was an additional distinction between studies focused primarily on blood vessels e. By plotting the methods used and the number of brains reported as perfused in each study, it is possible to examine qualitative trends over time, such as a relative decrease in the use of the in situ approach for histology studies Fig.
Characteristics of human brain perfusion fixation methods employed over time. Studies that had unclear approaches or did not report the number of perfusion-fixed brains are not drawn in the figure. This chart was prepared using R v. A major difference among studies that emerged was whether the investigators performed the perfusion fixation while the brain was still in the skull i.
There were two major subcategories for each approach. For the in situ approach, vessels were accessed either after making surgical incisions in the neck or thorax or after separating the head. For the ex situ approach, vessels were accessed either in the whole brain or in one isolated brain hemisphere. Two studies reported on multiple approaches. Istomin [ 43 ] reported methods for both ex situ whole brain and in situ neck dissection approaches, whereas Waldvogel et al. Kalimo et al. This problem appears to be mitigated by using the ex situ approach.
For example, Sharma et al. They found adequate or high-quality histology results when they did perfusion fixation on these brain samples.
Another challenge with the in situ approach is that it is more difficult to monitor perfusion fixation. Because the brain should harden during fixation, in an ex situ approach, it is possible to directly monitor fixation by applying pressure to the brain and noting resistance. In the in situ approach, the best monitoring method is likely fixation of the eyeball, which Donckaster et al. However, fixation of the eye may not always be completely reliable, due in part to the anastomosis between the external carotid and internal carotid through the ophthalmic artery.
Finally, a practical downside of the in situ approach is that it can interfere with funeral and embalming practices. For example, Istomin [ 43 ] noted that it was necessary to prepare the face of the cadaver prior to beginning the perfusion fixation, such as closing the eyes. The in situ separated head approach was reported by 3 studies, all of which had the primary goal of surgical training. One consideration for the in situ separated head approach is the spinal level at which the head separation should be performed.
Benet et al. For the ex situ approaches, one of the challenges described is the mechanical damage and deformation that occurs while the organ is removed from its regular location in the skull. In the animal literature, mechanical postmortem trauma has been found to result in histological artifacts such as dark neurons [ 44 ]. Investigators described several different approaches to minimize trauma. One approach is to suspend the brain in cloth; for example, Istomin [ 43 ] reported using a hammock of dense fabric for holding the brain in place.
Another approach is to bathe the brain in liquid; for example, Beach et al. Beach et al. Another challenge with the ex situ approach is that the arteries can be easily damaged while handling the brain, which will make subsequent perfusion more challenging or impossible.
Regarding the ex situ one hemisphere approach, there are some special considerations. The process of cutting the brain introduces additional mechanical trauma that causes damage to the unfixed brain tissue and severs the arteries that supply the contralateral hemisphere, requiring additional artery ligations to prevent leakage of washout and fixative solution.
Furthermore, the absence of collateral circulation from the contralateral circulation is likely to lead to worse overall fixation quality compared to the whole brain approach. In the process of cutting one hemisphere, it is also necessary to cut off the brainstem and cerebellum, with the result that these brain regions will not be perfusion-fixed because they are detached from the rest of the brain where the fixative is being perfused [ 95 ].
As a result of these problems, the ex situ one hemisphere approach is typically performed only in cases where the other hemisphere needs to remain unfixed, to preserve the tissue for biomolecular or biochemical studies.
Taken together, there were four major approaches to brain perfusion fixation reported, each of which have reported benefits and downsides, although there is very little data on comparisons among them.
Many of the studies listed criteria for the inclusion of brain tissue in their studies; however, it was almost always unclear whether these exclusion criteria were specific to the perfusion fixation preservation procedure rather than overall inclusion in the study. The one exception is Adickes et al.
In these cases, the investigators used immersion fixation. These exclusion criteria make biological sense, as these conditions are likely to interfere with flow through the cerebrovascular tree and therefore prevent adequate fixation. While we did not identify any study that specifically noted that an extended postmortem interval PMI was an exclusion criterion for perfusion fixation, many of the studies reported the PMI range of the brain tissue used in their studies.
The PMI range tolerated appeared to be associated with the goals of the investigators. On one extreme, Latini et al. At the other extreme, Kalimo et al. Another study of ultrastructure, by Suzuki et al. Somewhere in the middle of these extremes fell the majority of the light microscopy-based immunohistochemistry studies. For example, Beach et al. As another immunohistochemistry example, Halliday et al.
In summary, cerebral vessel thrombosis or large intracerebral hemorrhages were the only exclusion criteria specific to perfusion fixation. Several studies also suggested that a short PMI was preferred, with the PMI range tolerated depending on the type of the downstream study.
Among the studies that we evaluated, there were many different choices in the vessels that they accessed for subsequent perfusion steps, which depended on the overall approach that they employed Table 2. A key trade-off is ease of vascular access and technical perfusion quality versus the degree of dependence on intact collateral circulation for reaching more distant brain regions. All of the included studies attempted to perfuse the anterior circulation of the brain via the carotid artery distribution in some form; either via the common carotid artery or arteries, internal carotid artery or arteries, or the aortic arch.
Waldvogel et al. If only one side of the two carotid arteries is cannulated for perfusion, then interhemispheric collateral circulation will likely provide some fixative to the other hemisphere via the anterior communicating artery [ 55 ].
However, the perfusion quality in that hemisphere will be limited, especially if the anterior communicating artery is absent or hypoplastic [ 78 ]. In the in situ approach, if the internal carotid was cannulated, several of the investigators Table 2 also clamped the external carotid to prevent shunting of perfusate to the often lower-pressure external carotid circulatory distribution, as opposed to the brain.
The remainder of the studies either did not focus on brain regions supplied by the posterior circulation or relied on collateral circulation from the anterior to the posterior circulatory system.
Collateral circulation via the posterior communication arteries is not intact in approximately one-fifth of people [ ], although some degree of leptomeningeal collateral circulation may still be present [ 73 ]. Notably, the ability to visualize the posterior communicating arteries directly is an advantage of the ex situ approach, as the likely amount of collateral circulation through the circle of Willis can be visually assessed and the vessels to perfuse chosen accordingly performed by Insausti et al.
For obvious reasons, it is technically easier to cannulate fewer arteries, and this also decreases the time interval for tissue degradation prior to the initiation of washout and fixation. Cannulating more arteries also potentially affects perfusion quality within each one of the arteries when using a perfusion setup with a tube splitter to distribute the perfusate, as was used in Beach et al. This is because perfusion flow will distribute to the lowest pressure arteries, and cannulating a low-pressure artery that distributes fixative to a less important region of the brain may lead to worse quality fixation in a more important region of the brain.
Finally, one of the advantages of the ex situ approach is that it is easier to access more blood vessels on the ventral surface of the brain without requiring more extensive neck dissection to access the vertebral artery. Relatively more of the studies using the ex situ than the in situ neck dissection approach reported consistently cannulating at least one artery in the posterior circulatory system Table 2. One study, Sharma et al. This method likely allowed for improved fixation of periventricular brain structures such as the hypothalamus.
The lateral ventricular perfusion method was also used with good reported results by Toga et al. This study found that their intraventricular delivery system led to better and more uniform fixation preservation quality than perfusion of fixatives through the carotid and vertebral arteries. They speculated that this was due to erratic blood clot formation during the postmortem interval. Torack et al. They first perfused through the internal carotid arteries and the basilar artery.
Next, they clamped the middle cerebral artery distal to the anterior choroidal artery and the posterior cerebral artery distal to the posterior choroidal arteries.
Following these occlusions, the perfusion fixation should have been more targeted to the hippocampus. The main goal of vascular access points in perfusion fixation is to perfuse a large portion of the brain with little damage to the tissue. The studies that were able to successfully cannulate the anterior circulation as well as the posterior circulation would likely perfuse the largest amount of brain tissue.
We are unable to determine if the quality of the tissue isolated from brains with different perfusion access protocols is significantly different. This step aims to remove clots, blood cells, and other intravascular debris to improve flow of fixative, although it comes at the cost of increased procedural complexity and a longer delay prior to fixation.
Adickes et al. Donckaster et al. Of the studies that employed a washout step, saline or phosphate-buffered saline were the most common base washout solutions used, while two of the studies used mannitol, and one study used Ringer solution. Published perfusion fixation methods for laboratory animals often start while the animal is anesthetized [ 30 ]. This protocol prevents substantial premortem and postmortem clot formation [ 36 ], which means that the major purpose of the washout solution is to remove blood cells from the vessels.
On the other hand, in postmortem human brain perfusion fixation, there is frequently an abundance of blood clots that limit perfusion quality [ 22 ]. This means that in addition to washing out the cells, the washout step is often used by investigators to also decrease the clot burden by driving them out with pressure.
In addition to mechanically removing blood clots via perfusion pressure, another approach is to degrade or inhibit clots enzymatically. Four of the studies added the anticoagulant heparin to their washout solution, which may help to limit the spread of blood clots Table 3.
Two of the studies, Halliday et al. Sodium nitrite may help to dilate blood vessels and has been found to improve perfusion fixation quality in animals [ 71 ]. Several of the studies also reported performing the washout step until the venous outflow was clear of blood, clots, or debris. One potential problem with the use of a washout solution in brain perfusion fixation is that it may induce brain edema.
In animal studies it has been shown that perfusing too much saline into the brain e. The edema induced may be related to the osmotic concentration of the washout solution. Consistent with this, Benet et al.
Grinberg et al. The additives used and the precise procedure reported differed widely, and there were few comparisons between methods. Consistent with its widespread use throughout pathology and histology, formaldehyde was a component of the fixative used in almost all studies.
The only exceptions were one condition in Grinberg et al. Some studies used paraformaldehyde, which is a polymerized storage form of formaldehyde, while others used formalin, which is a form of formaldehyde that includes methanol to inhibit polymerization. The addition of methanol in formalin keeps the formaldehyde depolymerized and avoids its precipitation.
Twelve of the studies employed glutaraldehyde in the perfusion solution, at various concentrations ranging from 0. In general, adding glutaraldehyde to the fixative solution allows for improved tissue morphology preservation for electron microscopy [ 67 ], at the cost of decreased immunogenicity of antigens for immunohistochemistry [ 47 ].
However, at lower concentrations of glutaraldehyde, such as the 0. In addition to formaldehyde and glutaraldehyde, some investigators have used other fixatives. Picric acid, also known as 2,4,6-trinitrophenol, was used by Halliday et al. Picric acid has been found to improve preservation of immunogenicity compared to aldehyde fixation alone [ 82 ], although safety concerns make this fixative less desirable due to its explosive properties.
Pakkenberg et al. This is consistent with the dehydrating effect of alcohol fixatives [ 39 ]. Other studies that used alcohol in their fixative solutions included Feekes et al. Two of the studies used sucrose as a component of their perfused fixative solution, Shinkai et al.
The addition of ammonium bromide is thought to facilitate silver staining of neural cells [ 52 ]. The addition of potassium dichromate has been found to aid in the fixation of lipids [ 38 ], which is consistent with the focus of von Keyserlingk et al.
They concluded that the custom fixative was superior for surgical simulation, in part because it caused less hardening and therefore allowed for more realistic tissue retraction. The fixative vehicle or buffer can also have important effects on tissue preservation [ 16 ]. The most common buffer in the studies we identified was phosphate buffer, which was reported in 19 of the studies.
Although there is some controversy on this point, aldehyde fixatives themselves are generally not considered major drivers of the osmotic concentration, as they easily cross semipermeable cell membranes, and therefore do not exert a sustained osmotic force [ 37 ].
As a result, the osmotic concentration of the fixative vehicle is called the effective osmotic concentration. Hypertonic fixative solutions can cause grossly shrunken brain tissue and cell shrinkage, whereas hypotonic solutions can cause edema and resistance to flow in the perfusion procedure [ 77 ]. It would be convenient to be able to identify the optimal vehicle osmotic concentration that would minimize osmotic tissue changes.
Several of the included studies manipulated the temperature of their fixative solution prior to perfusion. Lower temperatures can help to inhibit metabolism and thereby mitigate tissue degradation, although it has also been reported to cause vasoconstriction [ 29 ].
One study, Kalimo et al. The most important determinants of the fixative are the assay of interest and the tissue or cell type of interest e. The choice of fixative buffer is an important way to balance tissue shrinkage and swelling while the fixative is being perfused and can affect fixation quality. The three major methods for driving the flow of solution during perfusion are syringes, gravity, and perfusion pumps.
All three methods were reported by the included studies: 2 studies reported using a syringe, 8 studies reported using gravity, and 4 studies reported using a pump Table 4. The majority of studies did not report their drive method. Upsides of a syringe are that it is easier to inject a specific amount of fluid in each vessel, while it is more difficult to control flow rate and pressure. From the perspective of a perfusion circuit, the included studies were open-circuit in that they did not describe using a method for re-introducing the outflow of the perfusate back into the vessels.
A major trade-off in setting the perfusion pressure is that too high of a perfusion pressure may lead to a higher risk of vessel rupture [ 76 ], while too low of perfusion pressure may lead to incomplete perfusion, decreased clot removal, and decreased tissue penetration of the fixative [ 17 ]. In laboratory animals, investigators often suggest that perfusion pressure should be maintained at roughly the same pressure that it was during life, which is called physiologic pressure [ 25 , 30 ].
Consistent with this, Halliday et al. However, Latini et al. Techniques using syringes, gravity, and perfusion pumps have all been employed to drive perfusion flow at a variety of different pressures. However, there were no studies that made comparisons between these alternative methods or identified an optimal perfusion pressure range for a particular application. The procedure for postfixation depends on whether the perfusion fixation was perfused in situ or ex situ Table 5.
If in situ, then the brain was often left in the skull for some amount of time to allow for fixative diffusion prior to removal. Many of the studies reported cutting the brain prior to additional postfixation; for example, in Nakamura et al.
Perfusion-fixed tissue is harder and therefore easier to cut than fresh tissue. Cutting the tissue makes the subsequent immersion fixation process faster because there is a shorter distance for the fixatives to diffuse, with the obvious issue of damaging tissue at the cut interfaces. How long investigators chose to postfix for may depend in part on their perception of the quality of their perfusion fixation. One major advantage of postfixation is that it will allow for fixation even in regions of the brain where perfusion has been minimal or absent, for example as a result of persistent blood clots.
A key trade-off in the length of postfixation is that longer amounts of time will lead to better fixative penetration of deeper regions of the brain or tissue block, while it may also lead to over-fixation and decreased antigenicity in the outer regions of the brain i.
As a result, a significant disadvantage of a long period of postfixation is that immunohistochemical staining and quantification will result in variable gradients across the tissue section. However, these gradients can be minimized by pre-processing steps that cut the tissue into smaller sections prior to postfixation. For example, Shinkai et al. The majority of the studies used the same fixative for perfusion fixation and postfixation.
One exception is glutaraldehyde fixation studies, which typically omitted it from the postfixative, likely in order to mitigate further antigen masking. Another exception is three studies that prepared tissue samples for electron microscopy, Tanaka et al. In summary, postfixation is used commonly and it allows investigators to compensate for the possibility of poor perfusion quality.
There was a wide range of postfixation procedures reported, ranging in time from a few hours to several weeks.
Storing the brain in formaldehyde for the long-term prior to use is an economical and convenient way to prevent microbial and autolytic degradation. It is especially convenient for gross tissue preservation for surgical training, as was performed in Alvernia et al. However, for histology purposes, storage in formaldehyde has been found to lead to a decrease in antigenicity over time. Lyck et al. Similarly, McGeer et al. An alternative method for long-term storage for subsequent histology is to store tissues at sub-zero temperatures.
However, this method requires the distribution of cryoprotectant throughout the tissue to prevent ice damage. Four studies reported using this method for long-term storage Table 6.
Notably, the glycerol-dimethylsulfoxide cryoprotectant method used by Insausti et al. For 6 studies, at least two reviewers agreed that the study made an explicit comparison between immersion and perfusion fixation. For one of these studies, Adickes et al. Sharma et al. These are both considered optimal methodologies that were considered equivalent to a randomized study.
The other 3 studies did not describe their methods for allocating donor brains to different interventions and were classified as non-randomized experimental studies.
The outcome described by the 4 of the studies, Adickes et al. Because the Sharma et al. The outcome of Lyck et al. For the outcome of immediate subjective histology quality, Adickes et al. Notably, the immersion fixation protocol was performed on the whole brain in Grinberg et al. For Adickes et al.
When we mention time in this context, we are referring to the total time for the brain tissue to bathe in fixative during immersion fixation or postfixation before it is ready for downstream studies.
In contrast, the time required for a trained worker to perform the procedure will almost certainly be longer for the perfusion-based methods.
For the outcome of immunostaining in samples stored in fixative long-term, Lyck et al. During the data extraction process, at least two independent reviewers appraised the included studies on the JBI quality metrics Fig.
Three of the studies reported blinding of the histology quality assessors, while this was not mentioned for the other two studies. For the confounding question, Beach et al. Risk of bias assessment for the studies comparing perfusion to immersion fixation.
For the outcome of subjective histology quality immediately following the procedures, we assigned an evidence grade of moderate quality Table 8.
Because of the study methodologies of Adickes et al. The reason for downgrading this to moderate was imprecision, which came in two forms. First, the sample sizes were relatively small, especially in Adickes et al. Second, while experts such as neuropathologists assessed the histology quality grades, these scores are semiquantitative. Future work that identifies and quantifies particular features present in each of the histology images would allow for more precise testing of differences in fixation quality between the different methods.
Aside from the time required to perform perfusion fixation and possible osmotic or hydrostatic effects on tissue resulting from the perfusion process, the main difference between perfusion fixation and immersion fixation is in the time needed for postfixation. Therefore, perfusion fixation can be thought of as a shift along the fixation time-fixation quality curve, such that there is an improvement of histology quality following a given duration of immersion fixation or postfixation.
This strength of this shift will vary based on the quality of the perfusion fixation. In the extreme case of ideal perfusion fixation, postfixation may not be necessary, but human brain tissue quality is often compromised by the time it reaches a brain bank, for example by a long PMI, which will typically prevent ideal perfusion fixation. For the outcome of long-term immunostaining quality in initially perfusion-fixed or immersion-fixed brain tissue stored in fixative, the one study identified, Lyck et al.
The studies that did not make a formal comparison between immersion and perfusion fixation or were assessed as having a low-quality study design regarding this comparison, often remarked on differences between these two fixation methods. Insausti et al. Von Keyserlingk et al. These informal comparisons support the use of perfusion fixation for the most complete fixation of brain tissue, although the purpose and aims of the study should be evaluated individually while determining the fixation method.
To the best of our knowledge, there has not been a previous systematic review focused on the topic of perfusion fixation in human brain tissue. There has been a previous systematic review of perfusion techniques for surgical training [ 8 ]; however, it did not focus on perfusion fixation and histology quality in particular. A response to Adickes et al. Another narrative review that was not specific to brain tissue noted that the literature contains conflicting evidence about whether perfusion fixation yields improved morphologic quality when compared to immersion fixation [ 4 ].
One book chapter by Connolly et al. However, corroborating one of the critiques of Miller [ 66 ], they note that perfusion fixation can occasionally cause irregular white matter pallor on hematoxylin and eosin stain that is likely artifactual. Connolly et al.
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