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One of the most fundamentally critical elements of diagnostic histopathology is first the ability to suspend all cellular activity in tissue and prevent egradation, and secondly to process that specimen in a manner that facilitates subsequent steps such as microtomy and staining. When these essential factors are not optimally met, a progressive decline results in the quality of tissue oftentimes resulting in an un-diagnosable specimen and attempts to ‘reprocess’ specimens correctly. There is a cost associated with reprocessing in workload, and more directly in laboratory costs per patient. Moreover, the reprocessed tissue never renders the diagnostic quality of optimal processing, thus compromises the pathologist’s ability to make accurate diagnoses. Every event of reprocessing compromises patient care and must be addressed and resolved to avoid reoccurrence.
- Understand the histochemical process of fixation and processing on tissues
- Illustrate the physical, diagnostic, and monetary cost to reprocessing
- Outline quality assurance and operational imperatives essential and necessary to eliminate the incidence of reprocessing
Skip Brown has 39 years of experience in the field of Histotechnology, over 30 of which being at the supervisory and management levels. He has managed some of the most progressive clinical and research institutions such as Kaiser Permanente of Southern California and Northwestern University Pathology Core Facility.
His passion is teaching and training and he has been one of the premier educators in the U.S. in lab management and histotechnology. He has given workshops and lectures nationally and internationally, and was recently awarded the “Excellence in Teaching Award” at a National Society for Histotechnology Symposium.
Having designed and managed laboratory systems in the past, Skip has always been fascinated at various examples of laboratory workflow and how instrumentation has been integrated into the system to streamline the flow and processes. He is particularly interested in the European models and examples of lab flow, and how the newest technology in instrumentation is being utilized within their systems.
The next slide is our course objectives. The first bulleted objective is to understand the histochemical process of fixation and processing on tissues. To understand what is happening when you need to reprocess, you must understand molecularly what is going on with the tissue and how that is affecting the tissue at a molecular level.
The next bulleted objective is to illustrate the physical, diagnostic, and monetary cost to reprocessing. There is a cost that is involved with physical changes, diagnostic changes, and complications, and the monetary cost to reprocessing. The third bullet is to outline the quality assurance and operational imperatives essential and necessary to eliminate the incidence of reprocessing. If you are having some incidence of reprocessing even occasionally, we will address what operational things you can look at to eliminate or at least minimize your risk or your possibility of having those events occur.
The next slide is a statement I want to make: One of the most fundamentally critical elements of diagnostic histopathology is first the ability to suspend all cellular activity in tissues and prevent degradation, and secondly, to process that specimen in a manner that facilitates subsequent steps such as microtomy and staining. When these essential factors are not optimally met, a progressive, and this is important, a progressive decline results in the quality of tissue, oftentimes resulting in an un-diagnosable specimen and attempts to “reprocess” the specimen correctly. There is a cost associated with reprocessing in workload, and more directly in laboratory cost per patient. This last part is important: Moreover, the reprocessed tissue never renders the diagnostic quality of optimal processing, thus compromising the pathologist’s ability to make accurate diagnoses. Every event of reprocessing compromises patient care and must be addressed and resolved to avoid reoccurrence.
I say this because we are so skilled at what we do as histology professionals, that when we have a tissue that is not optimally processed, we just fix the problem because particularly in patient care, we want to give the doctor something to diagnose because of the patient. In the U.S., there is a 24-hour turnaround time from the time that the patient goes into surgery to diagnosis. If we can get a diagnosable slide to the doctor, even if it is not optimal, so that he can make that diagnosis for the patient, we are going to do that, but this compromises the doctor’s ability to make that diagnosis and compromises patient care.
This next slide exhibits three different areas of comprehensive cost, looking first at diagnostic. This slide shows a block of tissue with a center that looks different. While the pathologists only interpret what they see, the histology professionals shed light and bring revelation to the pathology, to the disease state. If we don’t do our professional job optimally, then the pathologist will never have a clear picture or that light to see. This is diagnostic cost, and the animation represents acute cost, or the immediate cost to the patient. On the lower left is a block showing incomplete penetration where the solutions were not in fixation long enough or they were not in processing cycles long enough, and the solutions for fixation and for processing had not penetrated to the center of the tissue. We will talk about penetration time and penetration rate, or the rate of solutions such as formaldehyde to reach the center of the tissue, which is largely determined by the thickness of the tissue. The tissue on the right is an undifferentiated tumor; this is un-diagnosable because it did not have ample time to penetrate. The block would be very difficult for the doctor to diagnose causing him to go back and do a reprocessing.
The second area of comprehensive cost is financial. The first bullet is operational workload costs, which is the cost in time and manpower allocations. The time refers to the time allocated to go back and reprocess; the manpower refers to allocation of stopping current activities to return the tissues and melt them down. We are not addressing the time involved; we are looking at the cost involved in that patient’s specimen being hung up, to have to go back and be reprocessed.
The second bullet on this slide represents productivity measures, the justification for full-time employees and part-time employees. In productivity and management, we must justify the number of both full-time and part-time employees. One way to look at this number is by the amount of work that you have. The time involved in wasting a lot of our day going back to reprocess could be justified towards having another full-time employee, or maybe not having another full-time employee. It affects our productivity, and it is a long-term cost.
The third bullet represents laboratory operation cost, the cost in reagents, materials, and time: tissue is run back through, and reprocessing costs include reagents being utilized again; the materials; and the time involved. These three are chronic costs which are long-term. We rarely see this cost because we fix the problem and we move on, but the cost to the laboratory is substantial over weeks and months.
The third area of comprehensive cost is in patient care. This is the trust and integrity that we have from the patient to us. The first bullet is confidence in lab testing quality and competency, followed by competence in hospital patient care abilities, and confidence in the patient care ethics of the hospital and health system. Competency in us being able to do basic lab testing and in our staff falls if diagnosis is not optimized. If a hospital is unable to optimize tissue specimens, its competence level is seen as lower and patients may avoid going to that facility. Confidence in patient care ethics can be undermined via word-of-mouth when a healthcare facility falls short of optimizing diagnosis and studies.
The next slide looks at histochemistry. To understand the diagnostic cost, we have to look at how the tissues are compromised at a molecular level. The two methods of fixation in tissues involve a disruption of the secondary and tertiary molecular structures, or a cross-linking of protein groups. Disruption of structure may include a jostling of polar and nonpolar amino acids in aqueous phase; during processing, water molecules migrate out and away from the polar phase. In fixation, particularly using alcohol fixatives, removal of bound water results in brittle tissue. Tissues become overly dehydrated when they are not processed or fixed optimally.
The next slide shows cross-linking of protein groups. Formalin is utilized most often in basic hospital histopathology. Formaldehyde plus reactive hydrogen on tissue results in formation of a reactive hydroxy methyl compound, the additive compound of formalin. Formalin enlarges a tissue molecule. A methylene crosslink bridge is formed, with cross-linking of protein groups and release of water. This process continues: tissue molecules will link on with other tissue molecules in the presence of formalin.
Prior to working in immunohistochemistry, we never had to worry about this cross-linking. But in the early 1980s, we discovered that these cross-linking methylene bridges can block epitope sites and impede them from binding with their antibodies. Thus, false negative results can be seen in histochemistry studies. This is another example of activity at the molecular level, and with most laboratories using formalin, this actually occurs on a day-to-day basis. We must use antigen retrieval steps to break the methylene bridge and free up that epitope site.
From the molecular level, we will address two other topics. The next slide shows penetration rate versus binding time. Penetration rate can significantly affect the infiltration of the tissues, the spreading of the solution or the penetration of the solution into the tissues to create optimal fixation all throughout the tissue, not just on the outer perimeter. With penetration rate, we know that the initial penetration rate is rapid at 1-2 cell layers initially. Following this, penetration rate progressively decreases with time as it continues to sit in a fixative such as formalin in the absence of agitation. Lastly, the variability due to thickness and density of tissues affects the penetration rate. Thus, for good penetration we need to make sure in our laboratories that we have optimal time for fixation or that we do agitation or physical inducement.
Penetration rate is one factor, and the other is binding time to penetrate the tissues. The penetration requires ample time to penetrate the tissues. Yet, we rarely talk about binding time: even when tissues are sliced optimally thin, 3 mm, there is a certain amount of time involved for stabilization of proteins that I just showed you for cross-linking and disruption of secondary and tertiary structures. Events involved in binding time include: stabilization of proteins; formalin is chemically linked to tissue molecule; tertiary structure of the molecule is altered; and a gel-like precipitate is formed. A binding time must occur even with optimally gross tissues. You still need an optimal amount of time for this molecular or chemical reaction to occur at a molecular level.
The next slide shows a quote from the HistoNet net server in 2002, which says that even though thin slices of tissues would be penetrated faster and in thicker slices, it would seem that the binding time is the limiting factor for tissues stabilization. Hewlett is saying that this is more significant than penetration rate, which is equally significant if not more significant.
The next slide is another quote by Hewlett: Failure to recognize the importance of formaldehyde binding time is the leading cause of a tremendous intra- and inter-laboratory variability in performance. This is what led to the College of American Pathologists to develop its CAP safety guidelines in 2005 for ER/PR/