I wrote recently about the talk I was going to give at the TOBI conference. And in this post I would like to introduce you to a new strategy I presented during that talk on our efforts to better understand the role of the various cellular components present in PRP (Platelet-rich Plasma) and BMC (Bone Marrow Concentrate); the two most frequently used patient-derived materials in regenerative medicine. Let’s start with what I mean by “cellular components”. Both PRP and BMC preparations contain varying levels of platelets, white blood cells, and red blood cells, which are the main categories of components. Of course, there are several different types of white blood cells: lymphocytes, monocytes, neutrophils, basophils and eosinophils. While whole blood contains a low level of precursor or stem cells, bone marrow contains much higher levels and a greater variety of precursor cell types. For example, there are mesenchymal and hematopoietic stem cells (MSCs, HSCs) in bone marrow, which are absent (MSCs) or circulate at low levels (HSCs) in peripheral blood. The complexity of PRP and BMC presents a challenge to clinicians in trying to figure out if the preparation they are about to inject will have a therapeutic impact. Enter the method of ratios. But first, I will provide some background to aid in orienting you to the world of hemoanalytic data.
The standard approach to quantifying components in blood is to use a hemoanalyzer. The hemoanalyzer Greyledge uses is validated to provide a 27-parameter analysis of components found in blood, like RBCs, Platelets, WBCs and so on, which means that the instrumentation to measure the important components in blood is well established. However, challenges still exist when considering accurate measurements of concentrated preparations. One challenge is the need for an extended linear range for counting the number of platelets and total nucleated cells (WBCs), since these components are concentrated in the final PRP and BMC preparations. Another challenge is the need to have a strong Quality program in place to make sure that the hemoanalytic results are accurate. Still another challenge is that while hemoanalyzers are validated for characterizing the components in PRP, there are no validated instruments for analyzing BMC. I will deal with this issue in a future guest post.
Unfortunately, it also isn’t yet possible to obtain a quick count of the number of mesenchymal and hematopoietic stem cells present in the patient’s preparation, since there are no rapid and reliable counting assays for these stem cells, and for other precursor cells (e.g., endothelial progenitor cell). Consequently, the bulk of my talk was focused on the hemoanalytic data that I receive from the Greyledge processor who creates the PRP and BMC preparations I use to treat my patients. To review the Greyledge platform in more detail, read about our process.
A point I made in the talk was: “You have hemoanalytic data, now what?” Obviously, I don’t routinely review a patient’s mean corpuscular volume (MCV) or their hematocrit when I scan the results listed on the analytic report prior to my injecting the patient with their PRP or BMC preparation. But I do want to know the levels of RBCs, WBCs, Platelets, Neutrophils, and Monocytes (especially for BMC—where we can mathematically estimate an MSC count), as well as the total number of platelets to be injected. Having an opportunity to review these critical components gives me a chance to customize the preparation. For example, I have noticed on many occasions that a BMC preparation will have a low platelet count, or that the total number of platelets is low for a PRP preparation. In such cases, I have asked that additional whole blood be processed to produce an RBC/WBC-reduced PRP preparation to be combined with the BMC or PRP preparation. Yes, this takes time, but the orthobiologics lab is located in my clinic and I believe it provides a better therapeutic treatment. Obviously, the adjustments I make can only be achieved by performing a hemoanalytic assessment.
Since I have been looking at hemoanalytic data for more than seven years, I am comfortable with the numbers I usually check. Over time, I have established a framework for quickly forming an opinion on the therapeutic potential of a PRP or BMC preparation. My informal approach has served me well, but in an effort to continually improve my clinical capability, I currently am adding an outcomes tracking software package to provide more concrete feedback on patient benefit.
I realize that a physician who is just starting to work with hemoanalytic data might feel overwhelmed with integrating all of this on busy practice days. For example, I presented the following hemoanalytic data during my talk:
PRP Sample Composition:
RBC: 0.003 x 106/µL
WBC: 0.213 x 103/µL
Neu: 0.103 x 103/µL
Mono: 0.019 x 103/µL
Plt: 998 x 103/µL
The other piece of data that is helpful is the total number of platelets being injected in the treatment. In this case, the final volume of the PRP was 10 mL, so there were 9980 million platelets injected. Before you contact me to tell me that I made a mistake, you need to keep in mind that most hemoanalyzers report platelet levels on a “per microliter” basis, but PRP and BMC volumes are measured in milliliters. So, you need to multiply the value by 1000 to go from microliters to mL and then multiply by the volume of the preparation. See what I mean about the data being cumbersome when there are 20 more patients with an appointment?
One objective when thinking about hemoanalytic data is to balance out the six parameters mentioned above to try to assess a preparation’s therapeutic potential. Complicating matters for PRP is the fact that while platelets are the obvious component that contributes to the positive therapeutic value of PRP, there are opinions that RBCs, WBCs, and Neutrophils have a negative impact on the treatment. For example, there are reports in the literature that pain and inflammation are associated with PRP that contains RBCs and “leukocytes”.
In view of the complexity of dealing with hemoanalytic data, I have worked with Greyledge’s chief scientist, Ted Sand, PhD, to try to make the hemoanalytic data set less cumbersome and more immediately informative. In the next post, I will cover existing strategies for categorizing PRP preparations in terms of their cellular components, and introduce the concept we are calling the method of ratios.