Data Collection

Explore the nuances of leukocyte-rich and leukocyte-poor treatments, and don't forget to consider the role of monocytes. Plus, we'll unravel the mystery of dose calculation and how Patient Reported Outcomes impact registry data. 

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Transcript

Intro

Hey, I'm Dr. Ariana De Mers. I'm an orthopedic sports medicine surgeon, and I have successfully integrated orthobiologics into my busy practice so that I can provide a continuum of care and treat patients who are in the gap. The gap is this gray area in orthopedics where standard conservative treatments have not been effective but surgery may not be warranted and we usually tell our patients, come back when it's worse. What? These are your patients coming to you for help. Orthobiologics is that solution that can fill the gap and help you treat your patients who are in your office looking to you for help. Orthobiologics can also be an excellent treatment for frustrating problems without good surgical outcomes. This podcast will help you create the orthobiologics business that will make you love your job again. We will focus on value of orthobiologics, patient selection, how to talk to your patients about money, office setup, and other logistics. If this is something you've always wanted but don't know where to start, join me in "The Business of Orthobiologics" podcast.

Dr. Ariana

Hi. Welcome to data collection. Here's what we'll cover. So this is how we know how to take care of our patients and what treatments to use. We'll talk about what data we should be collecting, the MIBO criteria, and a primer on cell counting. We'll talk about whether you should get an automatic hemocytometer, a little bit about leukocyte-rich versus leukocyte-poor, and then what about the monocytes. We'll talk about dose calculation and then touch on patient-reported outcomes in registry data. So this is how we know. Data collection in orthobiologic treatments is crucial for several reasons, including assessment of treatment efficacy, ensuring patient safety, advancing our scientific knowledge, and improving patient outcomes. This data encompasses various aspects, including cellular content as well as patient-reported outcomes or PROs. So data collection allows for treatment efficacy assessment, patient safety and adverse event monitoring, scientific advancement, customized treatment plans, regulatory compliance, as well as quality control, patient engagement, and informed decision-making. So a little bit about treatment efficacy assessment. We want to go ahead and look at our cellular content and quantify the cellular composition, including platelets, leukocytes, and red blood cell contamination, as well as any concentration calculations. This allows clinicians and researchers to evaluate the treatment's potential effectiveness and differentiate whether different cell types can have varying effects on the tissue healing and regeneration. Talking about clinical outcomes, collecting data on patient-reported outcomes such as pain reduction and functional improvement over time will help determine whether the treatment that you're using with the cellular content you've recorded is achieving its intended therapeutic goals. Additionally, patient safety and adverse events when we look at monitoring the cellular content of the orthobiologic preparations, such as leukocyte-rich or leukocyte-poor, or monocytes or neutrophils, this is essential for ensuring patient safety. So high leukocyte counts, for example, may lead to excessive inflammation or increase adverse events. So when we talk about adverse events, we want systematic data collection that allows for identification and tracking of these adverse events or complications related to the orthobiologic treatments, including increased pain or swelling or decrease in function, so that we can make sure we are able to discuss this with our patients. When we talk about scientific advancement, we have this research and development, and delayed data collection supports this ongoing research and development efforts in the up-and-coming field of orthobiologics. Researchers can use this data to refine treatment protocols, develop new products, and enhance treatment outcomes. When we talk about publication and peer review, we'll go into this a little bit later but collecting and sharing data with the scientific community through publications and presentations promotes this transparency and peer review, contributing to the advancement of the field as a whole. When we talk about customized treatment plans, looking at patient-specific data is really important, and collecting data on individual patient characteristics and responses helps clinicians tailor orthobiologic treatments to the specific needs and preferences of each patient, as well as the specific diagnoses and patient-specific identification disease processes. This personalized approach can improve treatment outcomes. So continuous data collection allows for long-term monitoring of our patient's progress, and this enables us to adjust the treatment plans as needed. When we have data collection many regulatory bodies require comprehensive data collection for orthobiologic treatments. This data is essential for obtaining and maintaining the regulatory landscape, approvals, and compliance with safety and efficacy standards within the orthobiologic treatment realm. When we talk about quality control and quality assurance, when we look at regularly assessing cellular content, this helps to maintain the quality and consistency of the orthobiologic products that you're producing for injectate monitoring, it ensures that the product meets the predetermined or assumed specifications and then finally, patient outcomes. Monitoring these patient-reported outcomes allows practitioners to gauge the quality and effectiveness of the treatments that we're administering. This information can help lead to improvements in our clinical practice. And then finally, we can help engage our patients in informed and shared decision-making. When we use patient-reported outcomes such as pain, activity limitations, and quality of life assessments, this involves patients in their own care, and this data helps patients and practitioners to make informed decisions about treatment options and adjustments as well as expectations for treatment. So what data should we collect? So there are minimum reporting standards MIBO criteria. So when we talk about PRP, we talk about this checklist of items for study design, including whether the study was conducted in accordance with consort or randomized controlled trial cohort, case-control, cross-sectional, or meta-analyze guidelines. We then make sure and check institutional and ethical approval, demographics including sex and age, comorbidities such as diabetes, blood dyscrasias, inflammatory conditions, preexisting pathology, smoking status, current anti-inflammatory or antiplatelet medications, and injury details. The diagnosis, including the relevant grading system as well as the chronicity results of any preoperative or preinjectate imaging. Next is previous surgical or biologic treatments for this current injury intervention. We then have to list the intervention described sufficiently enough to enable replication. This includes how much blood was drawn, how much was processed, how was it processed, what kind of centrifuge were you using and what kind of kit were you using so that someone could follow along and reproduce or replicate this study specifically. Then we also need to define the intra-treatment findings such as whole blood processing, storage environment, concentration and volume of the anticoagulant, temperature and light exposure, and the whole blood cellular characteristics. Additionally, and we want to talk about the whole blood platelet, the differential leukocyte count, the red cell analysis of all samples. We also want to describe the processing, whether the commercial kits were used in the spin protocols, the platelet recovery rate of the protocol, expected and or calculated, the PRP storage, temperature and light exposure, and the time between the blood draw, the PRP processing, and any activations, and the delivery characteristics. We also need to talk about PRP formats such as liquid, gel, or membrane and then calculate or characterize the number of platelets, the differential leukocyte count, red cell analysis, as well as any activation needs to be described, including volume and concentration of the activating agent. You then also need to describe the point of delivery. Was it intraoperative, postoperative, or serial? Was it one week or three weeks? And then the delivery itself needs to be described sufficiently to enable replication, including volume delivered, concomitant use of other items, details of carriers or scaffolds, and then the post-injection care. So a rehab protocol sufficiently described to enable replication, including immobilization, and physical therapy, and then describe your outcomes assessments, including your functional outcomes, recording any complications, any infections, need for further treatments or surgery, any radiographic outcomes, physical exam finding outcomes, return to activity, satisfaction, radiographic outcomes, physical exam findings and then return to activities and patient satisfaction. So, there's also a cellular therapy MIBO, study design checklist that is very similar to this PRP. I won't go over it in detail, and you can look this up, but basically, what we talk about is that similar to the quality platelet-rich plasma concentrations, all of those things need to be sufficiently described and recorded so that if you desired-- or if anyone desired to be able to replicate your study, they could do so just going off of your data collection, So, next we'll talk about a primer on cell counting. So, cell counting is important because it provides essential information about the cellular composition of the orthobiologic preparation and it helps to tell us what the concentration of the different cell types, including white blood cells or leukocytes, platelets, and any other cell populations like monocytes. So, cell counting methods. Hemocytometer. This is a traditional manual method that includes counting cells using a special microscope slide called a hemocytometer. This method requires staining and careful observation as well as a microscope. We also have automated cell counters. These devices have advanced technology that can automate at providing really accurate and efficient results. And we usually use them in clinical and research settings. So should everyone have an automatic hemocytometer? Well, you can be the deciding factor. There are a couple of ways to decide. Talking about doing a manual count, either you can choose manual count or automated count, or you can send out samples. There are services that have sample send-out calculations. So if you're doing this manually, you will need a hemocytometer report, a microscope, a hemocytometer chamber, a pipette and pipette tips, a centrifuge obviously to create the PRP with blood collection tubes, and a calculator. Remember, please also take your whole blood sample and account that. So the manual counting steps are to assemble the microscope, set up the microscope with the hemocytometer chamber, and ensure that it is clean and free of debris. Dilute your PRP sample, usually a 10:1 dilution. And to count, usually using the isotonic saline, you can use 10:1 or 10:100 depending on your concentration. Load the hemocytometer chamber using a pipette. Load a small volume of the dilute PRP sample and then make sure it fills the chamber evenly without overflowing. Next, count the platelets and then place the loaded hemocytometer underneath the microscope, focus the grid lines, and continue to count several number of squares to get the average count. Or you can get an automatic hemocytometer, which seems to be quite a bit easier. The cost is about $25 per patient to know the starting and final dose of the injectate that you're using. So, in looking at samples of automatic cell counting, we look at the whole blood, the white blood cell count, platelet count, RBC, and then the lymphocyte, monocyte, and granulocyte counts. We then compare it to PRP looking at the white blood cell count, red blood cell count, platelet count, lymphocyte, monocyte, and granulocyte counts. So what is this business about leukocyte-rich and leukocyte-poor? And what about the monocytes? So, to refresh, the cells of interest are leukocytes. These include white blood cells including neutrophils, lymphocytes, and monocytes. The specific types and proportions of the leukocytes can influence biological effects on the orthobiologic product. Additionally, monocytes are a type of white blood cell that can differentiate into macrophages in the tissue, which then play a crucial role in repair and immune response. So monocyte concentrations may be of a significant orthobiologic interest when we're designing preparations for tissue regeneration. So when we talk about leukocyte-rich and leukocyte-poor, I think this is a little bit of an oversimplification and it may not be that simple. So in leukocyte-rich, these preparations usually contain higher leukocyte count, including neutrophils and lymphocytes. The leukocyte-rich PRP may be used or recommended in specific situations where an inflammatory response is desired, such as tissue healing in ligaments or tendons. The leukocyte-poor preparations have a reduced leukocyte concentration primarily to minimize this inflammatory response and are usually preferred in certain applications like osteoarthritis or intraarticular injections to avoid excessive inflammation that can sometimes be counterproductive. But we also have to account for the absolute count of which the leukocytes we are speaking of. As you recall, PRP is created with centrifugation to isolate the platelets based on density, and the leukocytes are also isolated by density with the platelets being the least dense, then the monocytes, and then the lymphocytes and then the neutrophils at the bottom of the leukocyte count and then the RBCs. So there are formulations that are monocyte rich, neutrophil depleted versus leukocyte-poor or leukocyte depleted. So I think we have to be a little bit more specific and that's why data collection and cellular data collection are really important. So, a word on monocytes. Monocytes are a type of white blood cell that plays a crucial role in the body's immune response and tissue repair processes. And while platelet-rich plasma usually focuses on the regenerative potentials of the platelets and the growth factors, monocytes can indirectly influence PRP treatments in several ways, making them really important in the context of PRP therapy. So I wanted to share with you this comparative analysis of M1 and M2 macrophages from Dr. Pradeep Albert, MD. He talks about that macrophages are essential immune cells and they're classified into two major types based on their activation status and functional roles. The M1 macrophage and the M2 macrophage. There's emerging research that emphasizes the significance of this polarization in diverse pathologies like inflammatory diseases, or cancer or autoimmune diseases. He talks about the M1 macrophages being classically activated macrophage that is stimulated by microbial products like lipopolysaccharides or cytokine and they play a crucial role in host defense by producing proinflammatory cytokines like TNF-alpha, interleukin one-beta, and IL6, as well as reactive oxygen species. So to combat these invading pathogens, however, uncontrolled M1 macrophage activation can lead to tissue damage and chronic inflammatory conditions. Now, M2 macrophages also known as the alternatively activated macrophages are stimulated by IL4 or IL13. These macrophages play a role in wound healing, tissue repair, and immune regulation, producing antiinflammatory cytokines like IL10 and TGF-beta. They are characterized by their high production of scavenger, mannose, and galactose receptors which aid in tissue remodeling and repair. So in looking at PRP as an influencer of monocytes and macrophages, they do a couple of things. So the inflammatory response is mediated by the monocytes which are part of the body's innate immune system and involved in the initial inflammatory response to injury tissue damage. When PRP is injected into the damaged area, it can trigger a controlled inflammatory response where the monocytes are recruited to the site of injury or treatment and play a role in clearing the debris and initiating the healing process. So, differentiation into macrophages. So when monocytes enter the tissues, they differentiate into the macrophages. These are specialized immune cells with a wide host of functions including phagocytosis, tissue remodeling, and immune regulation. These are essential for tissue repair and regeneration. So macrophages are key players in tissue repair and remodeling. They help break down the damaged tissues and promote the growth of new healthy tissue. So we also want to remember that immunomodulation is part of the monocyte and differentiated forms of macrophages. They can help modulate this immune response and they help balance the pro-inflammatory and anti-inflammatory processes, ensuring that the healing process is controlled and does not lead to excessive inflammation. Additionally, monocytes and macrophages also contribute to the formation of new blood vessels, angiogenesis, in the area that's injured. And this angiogenesis is essential to delivering nutrients and oxygen to the regenerating tissues to facilitate repair and growth. And then the macrophages also release various cytokines such as growth factors like TGF-beta and VEGF. Now, these signaling molecules play a role in tissue regeneration and can enhance this regenerative potential of PRP. So, while monocytes themselves are not primarily the focus of PRP therapy, their presence and function is quite important and really, I think require a deeper look as to the growth factors and bioactive molecules that can activate monocytes, leading to a coordinated healing response that promotes tissue repair and regeneration. So I hope this has helped clarify why cell counting is critical to be able to define what we're actually using and what can actually affect the tissue where PRP is being used. And maybe we all should have the automatic hemocytometers to really enhance the field going forward and really clarify what we're actually using. So the next part of this discussion is regarding dose calculation. So once we know what the cellular makeup of our injectate is, then we can actually calculate the absolute platelet and white blood cell dose. So let's go through it. So we start with what. Concentrate it to what times the number of cc's equals dose. So the average platelet count in whole blood is 200,000 per ml. And then we calculate the concentration. Now, there's this whole manual process if you'd like to do that. Or you can rely on your automatic hemocytometer. Please remember that there is a conversion factor of microliters to milliliters. And so we have platelets per ml. And then when you compare this to your whole blood number, you can give an x factor so concentrated to 10 times concentration. We then multiply this platelet per ml times the number of mls of your injectate and you can get the total platelet dose in billion. So of note, 200,000 platelets per ml times 60 mls blood draw gives you about 12 billion platelet dose assuming 100% recovery of platelets. So let's go back and look at my example report of a real patient. So her whole blood, white blood cells is one 5.1. Platelet counts 176, which is a little low. That's not the 200,000 we are talking about. RBCs 4.3, lymphocytes 1.7, monocytes at 0.2, and granulocytes 3.2. So we drew 120 ml of blood and made some leukocyte-poor PRP and some leukocyte-rich PRP. And I think this is really helpful to be able to show the difference. So each four ml sample had a 60 ml blood draw. So in the leukocyte-poor, the white blood cell count was 0.8, platelet count was 926, RBC was 0.05, lymphocytes 1.7, monocytes 0.2, and granulocytes 3.2. And so we had an x concentration increase of about 5.3 times concentration and a platelet dose where you calculate-- take the 926 times 4 ml gives us a 3848 or 3.5 billion dose for the 4 ml injection. Now, if we take the leukocyte-rich preparation, which is also from a 60 ml blood draw from her, the white blood cell count is 38.0, platelets are 2025, and RBCs are 0.5. So limited contamination, lymphocytes 17.2, monocytes 2.9, and granulocytes 17.9. Our x concentration increase was 11.5X concentration and our platelet dose is 8100 or 8.1 billion for the 4 ml injectate. So, looking at my patient specifically, if I had 100% recovery from my 60 ml blood draw with her platelets being 179 times 60 ml would be 10,500 or 10.5 billion in an ideal world. Now, obviously, our recovery was not as high and so some of that is because we were not for that leukocyte-poor, we were leaving some of those platelets in the buffy coat with the white blood cells. And so when we are comparing leukocyte-rich to leukocyte-poor, we really have to be clear is it a platelet dose issue or is it actually the leukocytes? And it may not be clear based on how we get these injectates created. So hopefully that was some lovely brilliant PRP math and hopefully, that helps clarify how to calculate our platelet dose. Lastly, let's just touch on patient reporting outcomes from the registry data. So, data reporting is really important. Cell counts are reported in these cells per microliter and then we really have to translate that to cells per ml to get the proper dose and the intended application. So just really, really be clear on this idea that we really have to be clear on platelet dose. Additionally, if you are not currently calculating your platelet dose, there is a way for you to send out a certain number of samples for quality control. It's essential to maintain quality control when you're performing this cell counting to ensure accurate and reproducible results. So we want to make sure that what you're producing, at least from a concentration standpoint is what you think you're producing. But honestly, I think it's probably better because it's so variable and we really need help to drive the field forward that we would like to have that data on every single patient who's getting these orthobiologic injections. Additionally interpreting results. So, interpretation of the cell counts in orthobiologics depends on the intended use and what your desirable injectate may be like lower counts for joint injections, where you wanted elevated counts for tendon or ligament applications. So, as a review of what we've kind of covered, which is quite a lot, data is how we know and how we make decisions going forward for our patients. We've talked about the MIBO criteria in which data we should be collecting. Given you a bit of a primer on cell counting, I've weighed in on whether an automatic hemocytometer would be helpful. We've talked about leukocyte-rich, leukocyte-poor, and monocyte. And maybe it's not as clear as just straight leukocyte-rich or leukocyte-poor. We've given some PRP math for dose calculation, and we've talked about some patient-reported outcomes and registry data. So why do we collect data? So we can customize treatments for our patients based on scientific evaluation so that we can advance this amazing field, as well as quality control, safety, and monitoring for our patients. So in the words of Jeffrey Moore, "Without data, you are blind and deaf and in the middle of a freeway". So thanks so much for listening and stay tuned for our next discussion on patient education and informed consent.

Outro

This has been "The Business of Orthobiologics" podcast. Thank you so much for joining us today. If you want to know more, please join us on the website, "PRP-Now.com" and click on the FREE Masterclass. Also, don't forget to "SUBSCRIBE" to this podcast to get more guidance on integrating PRP in your busy practice. Bye for now.