Identifying Optimal Methods for Clinical Quantitative Flow Cytometry
Hyatt Regency Hotel
1800 Presidents Street
Reston, Virginia
April 10, 2003
Proceedings
DR. MARTI: On the record.
DR. STETLER-STEVENS: We're going to get them to open this door and to get more space here. We're going to expand out over there. We have the next room and we're going to have that for a break-out room but I think we have more people than we expected and that is good.
I'm going to start out what precipitated this conference. I'm at NIH, and we are doing a tremendous number of antibody-based therapies. I don't know the new protocol that's come on. It didn't use antibodies as part of its therapy in leukemias and lymphomas. You use it in acute leukemias, lymphomas, chronic leukemias. So it's a big focus.
Every six months a new one comes along. They are finding this to be very complex using antibodies alone, antibodies complex to yttrium, the very rapidly degrading radioisotope, complex to ricin. People in England are afraid of ricin. We have ricin at NIH, a pseudomonas toxin. They've--- had some excellent results. I was involved with one protocol using anti-CD22. I looked at CD22 and CD20 together. I like to look at the two of them. In my hands, normal B-cells are about right here, a nice population. This was a protocol that has looked at the chronic mature B-cell leukemias.
We had three types of cases that came in, hairy cell leukemia. This is where the cells are an expression of CD22 and CD20. CLL, where this is the normal. We had some squanic, marginal zone lymphomas, which are closer to normal. They are treating them with an anti CD22 antibody complex totoxin.
The hairy cell leukemia patients achieve complete response. We detect hairy cell leukemia easily at 0.6 percent of the lymphoid cells which are hairy cell leukemia. You collect enough that they are so abnormal that they pop out. They got to the point where I couldn't detect any leukemic cells. Although we could have acquired a Million events, there was not a distinct populative, excellent response. Their cells normalized. Their immune function came back, excellent response.
CLL had a much poorer response. There was some partial response and the splenic marginal zone was in the middle. It's easy to see that it relates directly to the level of expression of CD22. They are all refractory hairy cell leukemias. A lot of hairy cell variance. CLL patients again are post treatment, all of them are post treatment because this is not a first line treatment. This is an experimental protocol. It was obvious to me that it corresponds to the level of CD22 expression.
Looking at various leukemias and lymphomas there is a spectrum of expression. I think that the level is going to be important in determining responses. The same person that's using CD25 complex of pseudomonas toxin and using it again in CLL when there is dim expression in various leukemias and lymphomas. In adult T-cell leukemia and lymphoma especially with HTLB1, high level of CD25 expression and it responds very well to CD25 therapy of any kind, complex to radioactivity, toxins, whatever. So there is an obvious correlation to the level of the expression of the antigen.
At that point, I decided we should start doing some quantitative studies in these patients. We then went on to some more complex ones like Hu1D10 which is an antibody being used in therapy. It's not as obvious a difference, but there are differences and within the same categories. In CLL, some patients have high levels, some are negative and some patients are low and intermediate. It's a big spectrum. So we started to look at expression of this antigen. We are trying to do some quantitative flow.
It became immediately clear. I'm going to show you all CLL patients. This is all the same disease. This was just in one week's worth of some cases we picked out where we had some funny results. I showed this to Gerry. This was because medical technologists said I can make those values change by how I look at the data. It's already acquired. We were looking at the geometric mean. This is looking at a lymphoid gate in a patient with CLL. He's post treatment so it's not an overwhelming CLL. He's got about 56 percent T-cells. When we look at Hu1D10 which is this variably expressed one in the non T-cells, you get a geometric mean of 349.
DR. SHAPIRO: What is the CD?
DR. STETLER-STEVENS: I'm sorry. This is CD3 versus Hu1D10. This is CD19 versus Hu1D10. The same two. We have 3, 19 and CD14 in with Hu1D10 so that if you look at it, it's expression. Interestingly with this antigen there seem to be toxic reactions in some patients and it has to do with expression of Hu1D10 on other immune cells. T-cells and monocytes might be important in this, they also get stimulated by this antibody. So we are looking at this. But I can find that if I look at the non T-cells I get about 350 as an X geometric mean. If I look at the CD19 positive B-cells, I get 502 and it's in the same two.
DR. SHAPIRO: Say that again please.
DR. STETLER-STEVENS: I'm looking at here CD3 versus Hu1D10, same two. So by looking at it by this way in a lymphoid gate, I drew a scatter gate RA based on the lymphoid cells and I'm looking here. This is CD3. These are the T-cells. These are the non T-cells, lymphoid cells. Basically it should be the patient's tumor. I also run kappa lambda tubes of CD19 so I do look at this another way. I'm going to show you how that leads to problems. So for the non T-cells, I get 350. Someone might use that as a method of looking at it. For the CD19 positive cell same tube, I get a geometric mean of 502, but in the same two.
DR. VOGT: So that's not an inconsistency. That's two different populations.
DR. STETLER-STEVENS: Not necessarily. It is two different populations. There are some differences in there, yes. There could be some NK cells in there expressing. There are various things. Then when I do another 19 specific gate and I look at Hu1D10 and the 19 positive cells, I get with a gate, not all the lymphoid cells. This is gating strategy making my geometric needs. I get 759. Whereas when I was looking at all lymphoid cells, CD19 positive, Hu1D10 I got 502.
DR. MARTI: So no matter how you slice that regardless of whose calibration curve you use in what lab, what instrument, what country, a 200 channel difference in the calibration curve has to give a different antibody finding.
DR. SHAPIRO: Wait a minute. The fundamental problem here is that you have to do the defined population. That is with different gates.
DR. STETLER-STEVENS: Yes, but this is one thing we need to talk about. How do you approach this.
DR. MARTI: Which gate do you want to use?
DR. SHAPIRO: But it seems to me that if you are trying to define and theory and you are looking at some population and you are trying to relate a quantitative antigen expression to tumor response, then however you define it the first thing you have to do before you attempt to do any quantitation is to develop robust criteria to define the population you work with.
DR. BRAYLAN: That's right. What we did with CD34. You have to define a very strict strategy.
DR. STETLER-STEVENS: We've gone with using a template that we do not stray from in CLL. If you are going to compare between patients it has to be exactly the same way, analyzed exactly the same to make it comparable.
*DR. LENKEI: Yes, but I also think specifically in case of some audiences --
DR. STETLER-STEVENS: We have a lot to talk about. Now I have another patient. They are going to be the same gating strategies. This is CLL. As I said I want a tube with CD5, CD19 and kappa in one tube and lambda in another tube, the only thing different between the tubes. Nineteen, Hu1D10 and CD5 and then one tube kappa, the next tube lambda. It's the same fluorochrome kappa in one is FITC and the other one lambda is FITC. Same patient. Same cells with a pipette repeater put out. Everything is the same except one has kappa FITC and the other one has lambda FITC.
When we look at Hu1D10 here there's a lymphoid gate, exact same gate. This actually agrees pretty good. The non T-cells I have about 300 and the CD19 positive cells I have 333. This is with kappa.
Then I look at lambda and it's changed completely. If you look at this versus this, you don't need to look at the numbers. This is a standard setup. The same blood, the same antibodies except one has kappa and one has lambda. There's a big difference. And it's reflected. Look at this. I don't have to tell you that there's a big difference in the geometric. This is with kappa and this is the same patient, same antibodies, with lambda. It's the same patient, the same antibodies. Look at that.
DR. D'HAUTCOURT: Which is the light change of the tumor cells?
DR. STETLER-STEVENS: I have that. Kappa.
DR. FISCHER: It's not the different antibody exposure. These are the same two antibodies with two different tubes. These are infected with a third antibody.
DR. STETLER-STEVENS: This is kappa. The tumor does express kappa.
DR. LENKEI: ** the compensation.**
DR. STETLER-STEVENS: Do you think we have a compensation issue?
DR. LENKEI: Yes.
DR. STETLER-STEVENS: There's the same fluorochrome, etc. Or it could also be interaction between antibodies because the tumor is positive, for one, of the light change. The tumor being positive makes a difference. It's going to shift your values.
DR. LENKEI: I never thought it mostly compensation.
DR. TAMUL: Are the kappa lambda antibodies monomodal or bimodal?
DR. STETLER-STEVENS: They are polymodal in this tube.
DR. BRAYLAN: So your percentage issues are ready to discuss.
DR. STETLER-STEVENS: Yes.
DR. BRAYLAN: Let's not discuss the specifics.
DR. STETLER-STEVENS: This becomes a test. People are going to do this in a lot of laboratories. I believe antibody-based therapy is going to be with us for a long time. It makes sense. What are they going to do when they have a bimodal situation? And this gets to reporting. It goes from all the technical issues of compensation, antibody combinations, gating strategies and reporting.
Having shown this just gleaned together quickly in one week the complex things we saw, I showed to Gerry and Gerry said we have to do something about this. I really believe that this is going to help me stay in flow until I retire. I see this as something that we're going to be able to do and make money doing. I want us to do it right and not have people end up saying well it showed absolutely no correlation with prognosis. Why? Because they just did what I did. They cranked out stuff and it doesn't correlate.
DR. BRAYLAN: Is there a great demand for a quantitative assay for these things or is it just that something semi-complicated will do?
DR. STETLER-STEVENS: For the Hu1D10 they want to know because they want to know the levels of all the cells. They are research people. The one person in the Hu1D10, Wyndham Wilson, is astute and he's aware of these issues. The CD22, I had to explain to this guy about three times what we can do with quantitative flow. He said you mean I can stop doing the radioimmunoassays to determine levels of CD22 expression. He said oh, that's really good. But it takes Millions of cells.
DR. VOGT: That's okay. Actually being able to correlate an RIA result will be a very valuable thing.
DR. STETLER-STEVENS: We're going to try to go and compare our data. He would like to move to us just taking one blood sample that are practically exsanguinating a person to get a large enough sample to run more radioimmunoassays.
DR. SHAPIRO: The fewer the cells the less disease they have remaining. It's like if you try and do blood cultures and you get two units blood.
DR. BRAYLAN: Some people would argue that we really don't have very strong evidence that responds to therapy relates directly to antibody binding sites or expression of the cells. Going back to the example you gave, they will argue the response doesn't necessarily relate to the expression but it relates to the number of cells in the simulation. CLL having the highest marginal and hairy even less so there is no multi-variate analysis for that. That's the agony. On the other hand, you can't prove something that you cannot measure.
DR. STETLER-STEVENS: We get patients that come in actually with high numbers with hairy cell leukemias because these are patients who have failed therapy off the hairy cell variant. I have seen a lot of hairy cell variant. At NIH you see a lot of weird things. I don't see a lot of common things. Every time we get something common, we're overjoyed. So they come in with high numbers. I would say that it's not it, but we don't know until we look. If we look the wrong way, it's going to be like S-phase. People say S-phase is totally worthless. And if you do it bad, it's probably worthless.
DR. MARTI: Mary Alice, my comment to that is that if we use the expression of CD20 on CLL and based on the experience that some patients come into the clinic and they say in Miami they said that my cells didn't express CD20 and therefore the oncologist said I couldn't have Rituxin. But I went to Conti Ries lab in New York and they said that 60 percent of my cells were positive and he would give me Rituxin.
Now my answer to that is don't even look at the CD20. Give the Rituxin and see if it works. That's the clinical approach. The problem with that says is that there is a lot of interrogatory variation.
The second thing I want to make a comment about is Hu1D10, the class two antibody. Mary Alice alluded to it. This appears to be a very effective antibody in indolent lymphomas. Unfortunately in the first 20 patients that were treated, there were three episodes of hemolytic uremic syndrome. That protocol was put on hold and those issues are being worked out.
Those 17 patients that didn't have the hemolytic uremic syndrome, they are beating on the door wanting their next course because it was so effective. Part of the reason for wanting to quantitate is and, not so much on whether or not the tumor cell is positive, but what about the other cells. It looks like the endothelial cells and monocytes could be playing a role in precipitating this HUS.
DR. STETLER-STEVENS: There are differences in the patients who had hemolytic uremic syndrome. They had higher level expression on the non-tumor cells. Another thing in the Ricin toxin therapy they found that patients who had no circulating tumor cells had a lot of toxicity to Ricin. There may be an importance into having too much toxin floating around.
Whereas if you had high affinity and it's quickly taken up, then you won't have too much toxin around. So the dose level may need to be adjusted according to the affinity number of molecules even available in the blood.
DR. WOOD: What I was going to say is that there are a lot of questions that are involved here. The biology is a complex issue. Unfortunately there are a number of serious obfuscations that are going on at the very fundamental level in just detecting light from the instrument. That is how people do these multi-color experiments without batting an eye. The whole issue of the fundamental theory behind the compensation is what does it mean to compensate. That is are you compensating the dyes? Are you compensating dyes on the probes or the actual antibodies themselves? Are the two antibodies, if you are looking at multiple antibodies, are they really truly independent when you are doing the compensation?
Also just something as fundamental as the log display is an enormous confusion factor. In fact, I dare say that the flow community has truly looked at it careful enough to understand how sometimes they are being mislead totally by the presentation and the display. And so, there is at the very basic level a lot of problems, a lot of confusion that's occurring that is totally confounding the biology.
Without going back to the first principles of looking at the light going in to the flow cytometer, the light being converted to the electric signal, there's a lot of electronics that is going on that people just look at as black box and not think about it. Then all of a sudden, magic symbols or magic boxes appear on the screens. And there are a lot of assumptions being made that unfortunately a lot of them are very misleading. That just makes the biology an impossible issue.
DR. MUIRHEAD: I'd just like to second something that Raul just threw up at the end which is you can't ask the question of in which cases is there a real difference in the biology that has implications for treatment strategies or patient management or anything else unless you can get the factors that Jim and everybody else is talking about under control.
In some cases you may only need a semi-quantitative readout. In other cases, the quantitative readout may be really important. But you can't even ask the question unless you can compare data in some common scale across different laboratories.
DR. LENKEI: I should say that today not the instruments are the main concern is really talked about for some reason. They end up mainly to set up instruments. For examples, I refer to the ones with experience. They are so good that when they come and I check with some of my beads and theirs and they are exactly the same. I don't know, I don't even know if the stain is the same. The problem is still that protocols, how we stain our cells, should the compensation when you have an activation. (Inaudible) This is my main concern.
DR. TAMUL: The problem is the different patient cells will behave differently even with the same antibody.
DR. SHAPIRO: Again another point that's come up here is that compensation is the enemy of quantitation. But as Jim McGall can tell you that the cross talk -- what you're trying to compensate out of the signals -- becomes noise on the signal that you have left. If you are trying to do accurate quantitative flow cytometry you should probably have a channel while you are measuring it.
The other thing that we got from the DNA analysis in breast cancer and other things is that if it's not going well, it's not worth doing. This is a complex problem. I think it's fair to say that the instrumentation in the reagent development has progressed to the point where we have a pretty good shot at doing these measurements on the fanciest machines that are out there. What we should aim for is figuring out how to do this well and then figure out how we can do it with the machines that are out there right now.
DR. LAMB: I'm trying to boil the last 15 minutes that I've here down into something practical. I'm thinking that Raul is right about the practicality of it but if we get into a world that is defined more with tumor associated antigens and that sort of thing where quantitative flow is going to be even more important to be able to decide whether and if and how much therapy.
It's already started of course with the Rituxin and Bexar and the anti-CD 22 agents. I think that the methods are going to have to be developed centrally. This stuff is going to be done in a few laboratories in the country like Schiller Psychology Group has two reference laboratories that decided to go on protocol. Then everybody else will catch up. But I think it's probably this group of folks who are trying to put together methods that will work across instruments that will develop the procedure. That procedure will be done in tightly controlled situations and tightly controlled laboratories before it goes off. So you have to crawl before you can walk.
I don't think we can start thinking about exporting this technology to every pathology lab in the country, although the people who make the beast would want to see that happen. At this point, you just have to concentrate on getting the method right and in place in five or six laboratories that can be reference facilities.
DR. FISCHER: It has to be important in the end of make it exportable because I know that at least with our trials at NIH that we also have sites around the country that are part of the overall trial. If they're not doing it the same way we're doing it, we may have the best technology and the best system in the world, but if they are not doing it the same we are we can't compare our results.
DR. STETLER-STEVENS: I think it's not going to be done in a little lab in West Virginia.
DR. PURVIS: I think it's important to go ahead and note at this time though that there's a number of pharmaceutical companies that are now recognizing the utility of doing the quantitation. We have a number of clinical trials that are currently going on with a number of pharma companies where quantitation is of the antibody binding saturation determining dose levels. Doing a BK/BB off of a flow cytometer is one of their primary measurements. Those protocols are being developed and utilized by the pharmaceutical company and as those monoclonals hit the market that is going to be one of the measures that they are going to want.
DR. VOGT: That's the question. I was just going to ask you to comment on that because it's always struck me and Gerry and I have talked about this. I didn't say that because Gerry is not allowed to talk about some things. But I wonder how much data there is in the pharmaceutical industry relevant to this that we don't know about. At least with Rituxin data, it did not look like when it was rolled out that there was an attention paid to this. Is that same mistake going to be made as these others come out?
DR. PURVIS: There are a number of monoclonals that hit the market that quantitation is not done at this level. There is no corollary to should we give it in this disease case or this expression level.
But the new monoclonals, the campass, there's a number of them that we're working on that I know that there is development of corollaries. And those will be in relation that they will provide help to the clinicians. That will be one of the marks that they'll be asking the labs to be able to provide some information back on. You have to establish the protocols now that everybody can agree to. Because here we are. We're doing it in our lab by our protocol and get very good results. If you do it by a different protocol when it hits your lab, it's going to be useless information.
DR. VOGT: Then, Norm, that leads to will the drug company allow you to share your protocol. Is it going to come with a package insert on the drug because there is no sense in reinventing the wheel and certainly no sense in reinventing the flat tire.
DR. PURVIS: I think that's what we're going to discuss today.
DR. VOGT: How about establishing a guideline of this is how we're going to approach quantitation in the future. I'm not going to say that our way is the best way and the only way. It's not. There are a number of different approaches that we can take for quantitation. What we have to do is come up with some kind of guideline so that we can get good results and avoid the mistakes that we're making with DNA that now we're having to go back and trying to correct for. Bad data is going to kill this.
DR. STETLER-STEVENS: This segues into the goals of this meeting which are to identify the problems and identify possible solutions. We're going to meet again in November at the close of the Clinical Cytometry Society Meeting. They are going to sponsor us insofar as they will provide the rooms for us.
Hopefully, in the time between now and then, we will have identified studies that need to be done to look at things across laboratories, perform some studies and come to our final conclusions by next November. That's being optimistic. Or at least have a basic guideline that we can work on.
DR. SHAPIRO: Do you have a statistician report?
DR. STETLER-STEVENS: Anybody?
DR. SHAPIRO: The reason I ask is that if we look back and there's a fairly long experience with both flow cytometry and even quantitative flow cytometry in the AIDS clinical trials group. What we know from that is that it has been verified by physical analysis, is that labs that are doing badly on QC, you can take the data and their information does not have the same prognostic value as data from labs that are doing well. That's one point that we picked up.
The other thing that we picked up from the CD38 studies is that as hard as it was to get labs to agree on simple CD4 counts is that it's that much harder to get them to agree on quantitative analysis. In that framework, we know that we have to start with a few labs and figure out how to do it right and then start spreading it out to good labs. You may never get to the poor labs. That's the way it works.
But from the ground up, the problem is there is this exchange on the cytometry abilities a few months back. How can I implement this vertical for detecting renal residual disease in myeloma. And, in doing that in one haul in a lab is never a good idea. You make a decision with implementation care. You need protocols and you have to figure that out. The bio statisticians and clinical trial statisticians are equal important components of the design of this protocol. So if you are going to play this game, we should get somebody on board for that sooner rather than later.
DR. MARTI: That's a good point, Howard. I certainly will make an effort at our institution both at the MBA and certainly within the Family Study Section at the NCI that they are top heavy with biostatisticians.
DR. SHAPIRO: I used to be a biostatistician at the NCI.
DR. VOGT: In fact if there are clinical trials going on, there are already biostatisticians. The question is have they been engaged in looking at this.
DR. SHAPIRO: We might want to see if we can have some liaison that can go and see the people who are doing the statistics for the NCI. They are up to speed on what that is about.
DR. MARTI: Just an aside comment about the pharmaceutical business, one thing that I can certainly speak about, and this is public information, was one of the things that held up in getting Herceptin antibody to market was the very issue of quantitation. We're not talking about flow. We're talking about one plus the four plus on a histopathological segment color assay. Believe me there was some arm-twisting and torturous discussion about the so-called fourth phase of drug development and that is a post marketing agreement. That's the way that one got out.
For better or worse, I think that the FDA will, in its infinite wisdom, will continue to look even more harshly or strongly at these kind of quantitative issues. I actually think that this problem has emerged. I would call it a problem of quantitative flow in setting of four colors or more. I actually think for those who live and die on the word quantitative flow that this is a gift to figure out this set of problems.
What Jim was saying earlier about the basics -- perhaps in my case I forget the basics. So I have to keep returning to them because the basics just get more complex. One of the examples is that at least with the CyAn and with LSRII, and probably the aria-- I don't know about the LSRII -- but anything that's using digital signal processing guess what? Negatives now become a very important issue.
DR. SHAPIRO: We got that fixed.
DR. MARTI: Certainly enlighten us because now it becomes an issue. Not an issue, it becomes a reality, a possibility. That's just one of the things that jumped out at me recently doing something as trivial as if you thought titrating a reagent so-called QC of the reagents. At a research lab, many of these reagents are made. They are not commercially available. They know QC in a much different way than a microlab does.
DR. FISCHER: It basically doesn't work or not as the QC in essence.
DR. MARTI: For commercially available reagents, it's not a question of whether it works or not, although sometimes it is. It's often a question of overstaining is a very dangerous thing in multi-color worlds. Just think of all the H&E sections or blood films that you've look at that are overstained. How do we ever see anything if something is overstained? It seems to be the same problem with fluorescent dye. It really is relearning the old things in spades over again. They become really important.
One of the things I hope that we walk away from this is compensation controls. I might even be so bold as to say compensation standard. In this illustrious group, we ought to be able to come up with a suggestion of a standard. I know that's a dirty word. Controls are preferred. I just throw that out for thought.
DR. STETLER-STEVENS: Beyond this scope, we have a big problem that we need to work on, kind of clinically. If you have seen the agenda, do you have any additional topics to add? Actually, you can at any point jump in. We're going to talk about everything from sample preparation to --
DR. VOGT: Why don't you run through very quickly the agenda topics?
DR. STETLER-STEVENS: All right. This afternoon, we're going to talk about sample preparation, staining, the analysis. We're going to talk about QC tomorrow for instruments, reagents, sample prep, standardizing data analysis, reporting of results, identifying reporting samples, specific anomalies.
At any point you can jump in with something as it occurs to you. While we need to talk about this, we will probably break into working groups because I thought it would be the techies and the pathologists mainly because there are some things in a techie group I don't want to hear about until it's already decided. Just tell me what works best. With that, why don't we move on to the -- proposed guidelines.
DR. HSI: Is there a way in the context of what we're going to doing today to think of a specific application? If you start generalizing too much, it will be very difficult to do anything. The reality is the things that we are all talking about are rare. Leukemias in a setting when you look at cyto tumors are rare. We may focus on one little thing of which the actual implication is very small versus something that's more generalized. Think of an application that's more full run of flow rather than thinking about the different specific clinical trials.
DR. STETLER-STEVENS: Leukemias, lymphomas, psoriasis.
DR. HOFFMAN: Maybe sepsis is something.
DR. STETLER-STEVENS: Sepsis.
DR. LENKEI: I see quite a bit of this based on one of the antibodies. I see a doctor's continuous development. I have been engaged in projects from these companies for three or four years now. It's a field that's appeared. I can tell you that the pharmaceutical company engaged in my laboratory found another laboratory in Europe really known. At the end of the one year, I was wanting to their papers. They called and asked me to help the other laboratory because it was such that the notes couldn't be used. It's a very serious issue because the patients were called for receptor separation. If you don't have good protocols and standardized methods, they were using the same reagents, but their protocols were different. Then the results after one year couldn't be used by the company. They are very aware what it means.
DR. STETLER-STEVENS: It's not just antibody-based therapy. There are other immune function studies and sepsis groups are going to talk about these studies. There are other applications. My focus is cancer because I'm in the Cancer Institute but there are other applications. Bruce.
DR. DAVIS: Before we get too far, I think that it's important also to agree with what we are trying to accomplish after two days. If I understand the conversations and statements, don't we want to establish principles of quantitative flow, a way to verify the principles. That's what we're going to do between now and November and then establish who is going to do it and how. So is somebody writing? What I'm thinking if we don't have a protocol by the end of tomorrow we would have just gotten together a couple of days of brainstorming.
DR. VOGT: We've asked Howard to be the summarizer and raconteur. But that's a very important point, Bruce. Maybe we should just ask Howard instead to put it up there.
DR. MARTI: I would also make a comment that we are going to make a transcription of this meeting. There are microphones placed in the rooms strategically.
DR. VOGT: By Homeland Security.
DR. MARTI: The transcriptionist has asked two things. One it would be useful, and I haven't done it at all this morning, is to identify yourself as you speak. The second thing is if you can speak from the diaphragm and not this guttural stuff that we normally use. Technically speaking we are obligated by contract to have this available in a public website in ten working days after the meeting. You'll get an opportunity to edit it however you like because I will not edit it before it goes out. I don't think so.
DR. STETLER-STEVENS: But we can certainly identify problems and things that we want to address.
DR. VOGT: Mary Alice, don't you think we could actually start writing some things down up there on one of those two things. That might not be a bad use for a flip chart. I did some of these other applications. Autoimmune disease is a public health problem as opposed to leukemia perhaps.
DR. MUIRHEAD: But I don't think the problem is lack of application. I agree very much with the comment that if you are going to try to put together working groups to generate data to say if we try this as a common protocol for this application, do we get more consistent results among laboratories. I think you're going to shoot yourself in the foot by trying to make a given protocol apply to every kind of application because the critical issues are going to be different for at least some applications than others.
I would second the comment there that we can think about some of the common issues that are going to be found no matter what kind of quantitation you are doing and what kind of reagents. When you come down to putting together the working group plans for what kinds of protocols are going to get saved, I think you should focus them around, I know, pick it, two or three specific applications. Otherwise, I think you do run the risk of being so general that you can't solve the problem in general. It has to be solved in a context of a particular application.
DR. HSI: I think it also reads to other non-flow people what's the utility and why are you doing this. So, if you have some more broad ranging applications than just a single disease, you know, with leukemia, that it makes it more relevant.
DR. SHAPIRO: There's a problem here which I alluded to many times. And that is that, fundamentally, we're all used to the content of offering drugs. What we are dealing here with is talking about diagnostics. If you just look at the economics of clinical flow, automated cytology automation, flow cytometry and hemacytometry really got their start on pap smears of which there were a 100 Million done every year and differential counts of which there are 50 or 100 Million by the end of the year.
If you look at the most common fluorescence flow cytometry diagnostics, and you see the workout, there are probably no more than 1,080,000, probably not 2,000,000 done a year. After you get down to leukemias where you have 10,000 cases a year, this is literally a morpho-diagnostic. If you focus too hard on specific application, somebody is going to decide that they are wasting money.
While it's true that you will have to pay attention to different aspects of the problem as you try to work out the quantitative protocol for one clinical application or another, we're still at the point where we have to define the basics that can go into the formulation of the protocol to be doing with any benefit. We have to say while any particular disease we might be working on is a morpho-diagnostic and when you add them all up, you are dealing with problems that affect large numbers of patients and you could have a substantial effect overall on healthcare, if you get it right.
DR. FISCHER: Doesn't it also come down to the fact that possibly because we haven't done this kind of thing successfully in the past, and I know years ago, Gerry, you organized another one of these meetings, to my knowledge, nothing concrete came out of that. We all got some new ideas. Until that happens, the whole technology is going to be underutilized. There is probably more instances where it could be utilized than it is. To say that only one Million or two Million of these things are done maybe it's because the technology hasn't been recognized because we don't have the kinds of standards that we're talking about.
DR. VOGT: I think that's true. I'll take this opportunity to start my talk now. Part of the problem here is me. We actually did have a very good output from that meeting that Gerry had in that series of meetings that occurred. They were compiled into the special issue of cytometry. More importantly there were two very specific things that came out of that meeting that have just now reached fruition.
One is the NIST program for standard reference materials for this kind of analysis. That is a concrete block that we needed to have in place for some time. We have three folks from NIST here, Dolph Gaigolos who was the leader of this effort overall and his colleague in the lab, Colleen Lange, and Ken Davis. That's one of the concrete things. That came directly out of Gerry's meeting. Gerry met Dolph at that meeting is my recollection and talked to him about that.
The second thing that came out that is specific is the NCCLS subcommittee. That's been held up by me for more than a year. It should have been out a year ago but it's about to come out. It is very important that when we have these meetings and say we're going to do something that we get it out there. I think that point is extremely well made. But those things are going to be out. By the end of this year people will know about them.
Just to add one more little thing. There are trials going on right now in Type I Diabetes using humanized CD3 antibody to try to prevent progression of new onset disease. Two to three in a thousand children born in the United States will get Type I Diabetes. It is probably a preventable disease. We know the risk factors now that will allow us to hone in to a 50 percent predictive value if we did general population survey. If something like that hits, it will change everything in terms of the marketplace for this kind of stuff. I think it will hit. I think Type I Diabetes will become a disease like smallpox used to be.
We need to be very alert to the opportunity and we need to be ready when we strike. I agree with Kathy absolutely that it's still going to be case by case no matter how prepared you think you are. When you get down to that individual case, you're going to find some things that are particular to that case.
DR. MARTI: I would also add to your comment about the outcome of that meeting. In my experience at the FDA, industry refers to that special issue more frequently than we are aware of in the scientific community. The other thing that came out of that meeting which was not really addressed was the total area of positive cell control. An example would be stabilized whole blood. We haven't really specifically addressed that, but at that meeting that was very much encouraged.
I would also point out, just for the record, that prior to that meeting we really only had one or two standards or controls. All of you are old enough to remember finus nuclei. We now have eight or ten products on the market that can be used in this area of standards and controls.
DR. LENKEI: I would also like to say that I agree completely with Howard because after we go to specific applications, it's amazing. When I say basic applications I don't think so much about calibrated. I am talking about other procedures. I agree with them. I agree completely. Now nothing will tell you with specific applications if the basics are wrong.
DR. LAMB: Absolutely. Mary Alice, the other thing is this is a big animal that you are trying to get your arms around here. You can take something like CD34 which we've had 40 zillion consensus conferences on around the world, and I still, after just completing a study with four major core blood centers looking at very methods of measuring CD34 and then taking two manufacturers methods of keeping my hands off of them and just letting them run the way they are supposed to run without tweaking the dot plots, it's still out there. You have published data on quantitative flow cytometry measuring the same thing on the same cell that differs by law.
What it boils down to is the NCCLS and Bob and his Argus group there have to come up with a procedure to where I will trust both the result from the laboratory run by the person sitting next to me and they will trust mine implicitly because we use that technique. We don't get in meetings and say that manufacturer's beads are crap or that manufacturer's beads are no good or we can't use this. We have to do this ourselves because we can't sit at the reference lab because doctors want from us the data. Even above applications and everything else, the first thing that has to happen is you have to be able to do the basic things, to calibrate the instrument and stain the cells correctly which is something that people still aren't doing.
DR. STETLER-STEVENS: We're going to need input from manufacturers of various products to support this. If it's just throwing a dye in, we're going to need everything even up to including software to help people to approach these problems. It's going to be a market for the future. We're going to need to know what we're doing and we're going to also have the right products to be able to do it.
DR. VOGT: Let me run through this. There are a number of people that are participants as members or advisors or observers on this committee. I will be speaking to the choir to some extent. They probably wonder what's happened to the hymn book.
This is my term "quantitative fluorescence calibration." You can also call it "quantitative flow cytometry." You can use the same acronym. Basically we're trying to get toward the direction that Jim was talking about and that is really what are we measuring and how are we measuring it and how do the standards and materials that we're looking at "measure and the word now, how do they relate to the process.
These are some selected slides from something that I put together and gave to Howard that he then improved on and presented last week in Belgium at an international meeting that was hosted by a European group and that our colleagues at NIST had been involved with. The basic idea is trying to get these arbitrary scales or relevant fluorescent intensity scales into some kind of reproducible stoichiometric scale.
Stoichiometry, I actually looked it up as I was writing, basically says that if you start with this set of reactants you wind up with this set of products. All the atoms and electrons that were here have to be over here somewhere. It is the balancing of those reactivities.
As it applies to what we are doing is what I call ligand-binding assays. The NCCLS parent committee that our so committee is out of is the Immunology and Ligand-Finding Assay Committee. Basically what we are talking about here are binding assays. The ligands in our case are the fluorochrome conjugates if you are talking about typing.
As I mentioned the big leg up since 1997 is that NIST scientists have gotten involved in a very big way. The first standard reference material in 1932 which is a fluorescein solution very clearly characterized after being synthesized by Duzon and his colleagues at Molecular Probes is now for notice available March 2003. It was on the NIST website three or four weeks ago with an expiration date and no price. Then a week later the price appeared so now you know that it's real. What is it, Dolph, $100 or $200 per kit?
So one of Dolph's fears is that these things will sit and gather dust. Nobody cares about the money that this brings into the Federal Government but they do care about the indication of interest from the community. I would like to encourage everyone to buy a kit. I'm serious. You think I'm kidding. I'm dead serious. Buy yourself a collector's item because it is my impression that NIST does not intend to do this again.
An SRM is a big effort for them. Future things will be more along the lines of traceable materials. Dolph, is that correct? Where they will use NIST procedures and available NIST standards to make things traceable. That reference solution is now available.
Now the idea of the quantitative fluorescence is that we would use these solutions as the calibrator for our particle measurements. So the companion reference material that we need to go with this is the fluorescein labeled microbead.
By the way, I have this PowerPoint presentation. It was on the computer. I have these little chip things, flash drives, so if people who are giving presentations that have PowerPoint, if you have your laptops with you and the presentation is on it, I'll give you one of these and you can copy it on to it. Conversely I can copy everything that I have and everything that's presented here on to one of these and then you can copy it on to your machine. So we can have a little virtual network.
DR. FISCHER: Can that be made available to the rest of us who aren't giving presentations?
DR. VOGT: Absolutely. That's the intention. We can this posted too. We will certainly make that available. So the companion material is a reference material which is a little easier to get through the system than standard reference material. It's 1933. Dolph and his colleagues are working on that right now.
They are beads that have been custom synthesized by Bangs laboratories and are surface labeled with fluorescein so they have the requisite environmental responsiveness. Dolph and -- now later are working on the exact methods and translation of how you get from the solution to the particle assignment. Dolph, is it fair to say that within in the next year that those beads you think will be available?
DR. GAIGOLOS: That's what we intended to do.
DR. VOGT: I can say that in my years working with the wonderful colleagues that I've had the chance to work with, Dolph has just been extraordinary in his thoroughness and ability to get things through. He never thought too much about the difficulty of all this until he hit the beads most recently now. But we're getting the best possible effort at NIST here. That's a big lift up.
DR. DAVIS: Bob, are you saying that the procedure is still under development?
DR. VOGT: The exact assignments? The exact procedure for assigning?
DR. DAVIS: Yes.
DR. VOGT: Actually thank you for asking that, Bruce. There are a series of papers that are in public domain from the NIST website. I have hard copies here. I have the PDF files with me. Again I can give them to you and they are downloadable from the NIST website. There are three papers, one of which will be, at least one, maybe two, will be an the appendix in the NCCLS document and they describe the procedures both theoretical background, the computations, mathematics of all that and the practical methods. The third paper is called "Practical Methods." Those methods have been published.
Now the exact micro-implementation of those practical methods is again going to be case by case. It's my understanding that's what Dolph and company are working on now. I'll give you an example in just a second -- you know, the devil is in the details. Again case by case you will need to look at that. But the general methods now are in print.
DR. LENKEI: I think you hit one of these on time, to look at the study of the products on the Internet page -- because the terror is so - and you have to look out for the dangers. I had a lot of catastrophes in my other laboratory. Before you assign and you know exactly that this product can be used, you have to have one year to look at the study, and the Internet study.
DR. VOGT: My comment on that would be you are certainly correct and I go back again to what I said that we have to trust each other and that's so important in science. I'm quite serious about that. I do remember a guy named Gorbachev and I think his motto was trust but verify. I don't think we will ever get away from the need for the second part of that.
One of the things in the NCCLS guideline is we do talk about the need for expiration dates, stability and so on but we also say caveat emptor and that kind of stuff. The person who is using the material needs in their own laboratory to also know that it is stable from day to day.
The basis of the assignment of these values, Howard worked this up and then I went back and changed the green to orange since this was mostly Phycoerythrin. If we excite a solution in a cuvette and get fluorescence, we'll have instrument factors that go into the illumination, the energy providing the excitation. Then we have fluorochrome factors and the saline factors or the concentration of fluorochrome. The absorptivity expressed as an extinction coefficient and then the quantum yield.
Then in addition, and this was the big break through to those of us who are not like Jim and Bob Hoffman that are not optical physicists, we always worried about the fact when we have these differing emission spectrum fluorescence which as you all know are broad and tend to vary with respect to environmental conditions and so on. How do we ever assign standards -- whose bandwidth are we going to use? What is the standard? At one point maybe we could come up with that there is going to be a master bandwidth filter that everyone can use.
When this got in the hands of people that knew what they were doing, they said no, you just integrate over the entire emission spectrum. In the assignment of the standards that can be done because you have time to integrate. Now when particles are going through laser beam, you don't have time for that. There's a difference here between this kind of measurement and the analysis on the flow cytometer.
What Dolph is working on - please, Dolph, correct anything I misstate -- the assignment of the standards will not depend on spectral matching. They will depend on the integrated emission energy across the entire emission spectrum.
One of those devils of the detail that I was going to mention. Gerry and I a couple of weeks ago were talking about this and Gerry said how do they know when the emission spectrum begins and ends, anyhow. I thought about that and said I don't know about that either. I asked Dolph. He groaned for a minute.
They look at their noise measurements in the background and they have a mathematical procedure. They actually take their raw data and model the emission spectrum based on this interaction with the noise levels and everything so that they get a defensible amount of energy that is something that is optical physicists like. These are the kinds of things that are going on behind the scenes.
Then, when we go to read the fluorescence, there are a whole lot of instrument factors. They appear in the measurement equations which are developed in this series of papers. In using state-of-the-art detector, we wind up with a relative fluorescence intensity value from this cuvette solution measurement.
We can do the same thing on a suspension of beads. If the devil is in the detail, then this is hell because there is just a tremendous amount of detail in here. But Dolph and colleagues have been hard at work on this and they are tackling the problems one by one to make sure that these assignments can be scientifically authentic.
If we saw that we got the same fluorescence reading in a relative sense whatever our scale is between a one picomolar fluorochrome solution which contains about 600 Million molecules per ML and a fluorochrome labeled bead suspension that contains about 600,000 beads per ML and those two give us the same reading, then we have equivalent fluorescence. Since the fluorescence of 600,000 beads per ML is equivalent to the fluorescence of 600 Million molecules per ML, each bead has a value of 1,000 molecules of equivalent soluble fluorochrome.
Now this is nothing new to most of you all in concept. This is what Abe started doing back in the 1980s. I don't want to say now it's being done right but now it's being very carefully with attention to all the details and so on. What we will find is the values that Abe used for fluorescein 20 years ago are just about the same as the values that come out of this SRM. Fluorescein was always pretty reliable as a standard.
Now from the standpoint of trying to getting antibody-binding capacities, we need to relate this to our fluorochrome ligand conjugate. I've just come up with this FLC thing, because I just like saying it. Once again the whole thing is based on equivalent fluorescence.
So if we have a one picomolar fluorochrome solution and two picomolar fluorochrome ligand conjugate solution, that is to say the molarity of the conjugate molecules is two picomolar -- I bet there are a bunch of devilish details in that -- then, if they give us equivalent fluorescence, each conjugate molecule must be shining with the equivalent of half an MESF per molecule.
This is what we have called the effective F/P ratio because it is the amount of fluorescence that you get from a conjugate. This is how we use the solution as the equalibrator to match the fluorescence between a particle standard measurement and a conjugate label itself. That's the whole thing in theory.
Now these measurements can be made fairly directly in a cuvette. They are not intended with columns of trying to read a micro-particle suspension. There are lots of things about that conjugate measurement that come, up but those at least in theory are solutions with that measurement.
The other thing that came out of the meeting was the NCCLS subcommittee which was convened officially in 1999 or 2000. NCCLS is a long standing clearinghouse for tackling the kinds of problems that we're talking about. A couple of years ago, Gerry, in the official regulatory sense there is now some equivalence of an NCCLS document. The FDA is allowed to use NCCLS approved guidelines as the basis for their evaluation of products. Is that correct, Gerry?
DR. MARTI: Yes.
DR. VOGT: So that was an attempt to streamline the FDA process so that they didn't have to ask their submitters to reinvent the things that NCCLS had gone through. So, there is actually some, I don't want to say exactly teeth, but there is some bite to having an NCCLS guideline.
I looked at our guideline and it said "DRAFT" to be distributed in summer of 2003. I thought I'm right on time and then I remembered that I changed that before I sent it to Howard so that's why it's updated.
It's really true. I have in my hand chapters nine out of ten, chapters one through nine, out of ten, that is actually in the hands of NCCLS now and out of my hands. We will get chapter ten to them and have this out to the Committee members and advisors next week. The voting draft will undergo one quick round of review. I think we will get voted out. It had already been reviewed extensively by the membership. Those comments have been incorporated and it should have been out here about a year ago. It will be to you all who are aware of this process and out for public comment in the summer. We would ask certainly everyone in this room to be part of that public comment process.
Then it will go for three months and then it comes back to the committee. It gets revised based on public comments. Then it spends a year as a proposed guideline and after that becomes the approved guideline. That's the process.
Here is the title page just to prove that it really is. This is the NCCLS boilerplate stuff. These are the ten chapters. There has been some revision. It's actually shorter now than it was before even though more material has been put on it. We try to go through the concepts. We actually do use this as somewhat more of a teaching document than traditional NCCLS documents have been. We fought the battle to get that. I would like to see it used that way.
You can see the major topics that were in discussion here are in the chapters there. I want to go right to chapter nine, Quantitative Ligand-Finding Assays Using Fluorochrome Ligand Conjugates. That's what we are talking about here. What we've just sent in chapter nine a few days to Lois, our liaison at NCCLS, and here is what we sent her in outline form.
The first thing is a short discussion of what we're talking about how we get this equivalent fluorescence and binding values. Then we talk about the molecular properties that influence the binding and the fluorescence measurement. There are the fluorescence properties, of course, and we talked about this effective F/P ratio. There are binding properties in the conjugates.
There is the issue of having micro heterogeneity in most of the conventional fluorochrome ligand conjugates we use. That heterogeneity is with respect to the number of fluorochrome molecules per conjugate molecule. We can get an average value but of course that can vary across a distribution. It could be a Y distribution. Then, that in turn can affect the binding properties of those molecular subspecies. That can be a problem. I suspect that is a problem. I suspect that's one of the things that's causing some of these anomalies.
That led us to go back and look at the recent use and very effective use of unimolar fluorochrome ligand conjugates. Here I'm talking about the one-to-one PE conjugates. We'll hear much more from Lance and from Norm about this and the practical use and we look forward to that.
But it also reminded me going back many years ago to the papers that were published by Bob Ashcroft and Ron Chatalet where they did epidermal growth factor binding. I remember the big deal Bob made or Ron, one of the two, was that none of those experiments worked until they took their FITC EGF and purified it on an HPLC to get unimolar FITC conjugates. Then all of that stuff with their isobarometric titration techniques worked.
So I think the notion of unimolar conjugates, or well defined conjugates which at the moment are not economically justified in the realm of manufacturers, is going to cost more money to make those kinds of conjugates and characterize them and so on. That's one of the market force driving things that we have to get into that cycle of when does it become reasonable for a company to attack that problem. Only when we start buying the reagents is the answer.
Then we talked about exactly how you would go about quantifying the number of conjugates, the binding capacity. You can do it indirectly through an MESF calibration. You can do directly through binding units. In there we mention the fact that when you are looking for binding unit calibrators that there has been some recent success using biologic calibrators. Ultimately we would like to see standards that are easier to use than biologic calibrators.
Sources of variability. This is going to be the bugaboo. You are going to have differences in the fluorochrome ligand conjugates. If we get effective FP values put on them, are they authentic? Are they traceable to reference solutions? If we are using binding values that have been assigned to calibrators, whether they are micro-beads or biologic calibrators such as the CD4 cell, are those values we are presuming correct?
What about the actual authentity and saturability of the binding? There are some misbehaviors in these systems. And what about the multiple interactions between the conjugates and binding targets, particularly when you get to multiple things. That's the shopping list of problems that come up.
And, then, finally, I think some of you alluded to this a little earlier and I was glad to hear it, we might actually want to get QFC to the point where we are measuring affinity constants. One thing you can do at a multiplex setup and there is a lot of stuff about multiplex that you can always analyze. You can also measure one analyte much more carefully if for instance you use microbeads that are labeled with different fluorescent barcodes that have different binding capacities. You do a checkerboard titration where you have those in one dimension and in the other dimension you put differing concentrations of your conjugate using this Ashcroft-Chatalet technique, you can bootstrap a scattered plot and get an affinity constant. If it's a straight line and well-behaved then you are probably dealing with characterizable binding. If it's not, then you are probably not.
I think there's a whole other dimension, literally, that we could apply a quantitative flow to once we get these standards. These things might be as important as the MESF per cell or ABC per cell. That's it. I'm sorry this has taken so long and we're still not quite finished. By the end of the year, this guideline will be on the streets ready to use and I hope that it will be useful in this process.
DR. FISCHER: So the NIST things that you are referring to, just the SRM, is available right now. The RM is coming soon.
DR. VOGT: Yes, and that of course is fluorescein. Fluorescein is still useful but in addition it was a model system to look at process in general and in particular look at a fluorochrome that had a lot of environmental sensitivity to it. The idea was that you would have to have micro particles that were reflective of their environment so that they could probably calibrate the cells within that same environment.
DR. FISCHER: Are there going to ones for the other eight colors?
DR. VOGT: Yes, for the other colors. Dolph, would you want the same thing where you think the NIST program will be competitive with respect to the other fluorochromes?
DR. GAIGOLOS: That pretty much depends on what groups like this say. We're a supporting lab. Do you want a PE standard? I hope not.
DR. VOGT: I would love to see a PE calibration standard come out of this meeting because I think Mary Alice's examples of PE. I changed all my green colors to orange for this meeting. One of the things when we break out and talk about laboratory exercises between now and the fall meeting is we would like to work with folks who are interested in this and headed in that direction.
There are a variety of calibrators out there that can be applied to PE. I don't think we're that far away from getting consistent measurements. But nobody has actually tried to do that in a totally focused way although Lance and the group at UCLA certainly have made great progress. We will see where we stand and where we can go with Phycoerythrin. After that I'll retire.
DR. FISCHER: That actually does become a very key point because as we all know what is the one molecule everybody likes to use for that? And the reason that you use it is because it's so good. That tends to be the one that also is the one that all clinical people want you to use for all of the markers that they're looking at for everything because they can say yes, it's the brightest one. We use FITC but it's only one of nine. The PE is very important for us because of the fact that we do a lot of intercellular staining.
DR. VOGT: Ken, can you give us any little heads-up on where NIST and PE might be?
DR. COLE: Well, we're just starting. We have an interest in assays for Homeland Security. That's what brought me into this. I don't have a lot of experience with flow cytometry. But antibody-based assays are something that we are just starting to work on. So any suggestions we're open to hear.
DR. DAVIS: Along the lines of trying to be practical with this, is this something we can add to the list as part of the protocol which would be to compare existing calibrators that are out there commercially to what NIST has? That kind of thing. So it's a request for the labs to come back and look at these and do we all get the same interrelationship. That could be of value.
DR. FISCHER: Bob, you've actually brought up something while you were talking about NIST traceable standards. Now having spent more than one year in a private company and having had an accrued GOP and GMP and all those kind of regulations, the fact that you have to have the ability to use mistraceable standards for a lot of the equipment that we use when it came down to the flow cytometer, they weren't available.
Here we were trying to follow all the regulations and we asked what do we use for a flow cytometer. I was told that there isn't anything that is the one approved method for building flow for, being something as simple as calibrant. Now we have the CyAn and that's nine colors.
DR. VOGT: Well, we won't have nine colors tomorrow.
DR. FISCHER: I'm a realist. I know that these things take time. The fact I didn't see anything concrete come out of the last meeting was only because the stuff was being done in such small increments. I guess eventually it all comes out. Maybe it isn't publicized well enough for those of us out in the flow community. Look, this was done and you need to go look at these things if you want to do quantitative flow. Maybe that should be one of the keys out of this meeting too. We should get the information out there not just to the 30 of us who are in this room, but to the 3,000 people who are out there who are running flow labs.
DR. VOGT: There was a prolonged ramp-up period. Part of that was just due to me personally. At NIST there's always a ramp-up period. But Dolph and his colleagues were on this solution and everything as quick as could be. So it's my impression that we can move farther faster now. If all 200 of those fluorescein standards were gone in a couple of months, that's going to be the biggest mess. Is that right, Dolph, that the popularity and marketability of that standard would influence the thinking in NIST?
DR. GAIGOLOS: Probably not but if it's not popular, it will.
DR. STETLER-STEVENS: So we have two issue items that have mentioned as actions, one to compare standards and one to publicize what's been done.
DR. VOGT: Right and, Bruce, absolutely that's what we want to do. Between now and the fall, if we can do some scratch studies which are some things between labs that are interested in doing this both levels of cytofluorometry and cuvette fluorometry for those that are interested. There are those here who are.
They can work with Dolph and Ken on this and get them engaged in that initial process, then they will see how bad things are or how good things are. They can take that information and move it into a standardization program. That's a very important output of this meeting.
DR. SHAPIRO: In terms of publicity, the quantitative flow drum gets beaten pretty hard for the condition of flow cytometry and I could explain much of this stuff in English.
DR. FISCHER: Is that done yet?
DR. SHAPIRO: It will be in a couple of months. It's done. It's along side the NCCLS stuff.
DR. VOGT: Right, I was just waiting.
DR. MARTI: One other point that I think that perhaps is useful here is that the use of quantitative flow is somewhat specific in the history of the development of an assay. Where an experiment or experimental finding was just groundbreaking in the basic research lab where they might be the experiment at least twice, on a good day, maybe three to five times, the kind of controls and standards that we need for that experiment are much different from a product that you want to license and sell between states in the U.S. The kind of stringent controls that are placed on that are much different.
Even watch the beginning of the multi-bead multi-analyte. It started out and look like it was going to be really good. Then it slowed down. Now it's picking up. It's getting its second burst. Or use the micro array. Have you ever seen two papers that had same answer yet in micro array? Talk about standardizing that. But it will come if it's going to be used to make decisions about what drug you get or you don't or whether your arm is going to be amputated or not. The kinds of controls that come on that will be much greater.
DR. STETLER-STEVENS: Abe, you're up next. Are you going to tell us what doesn't work? Where the flat tire is.
DR. SCHWARTZ: I might not be invited to any more meetings after I speak today unless you want to make yourself feel good. I can do that for you also. I have no vested interest now, as most of you know. So I would like to make some observations because I am working as a NIST associate researcher. I've been involved with Dolph and the colleagues. I've done the MESFs and ABCs for about 20 years. They have made some significant improvements on what I've done because they have the instrumentation and the knowledge on the formalization of fluorescence.
There are two or three things that have been improved and will make this thing work even better than it has is one, they have the fluorometer that has holographic filters. This allows you to do the whole intubation of the emission spectrum like Bob mentioned. It gets rid of all the background to where two Million beads per Milliliter looks like water in terms of background scatter.
Before that when I started back then it was 500,000 MESFs. It was so bad that I had to make another type of beads that had no scatter, get the assignments and make a calibration curve on the kind of beads they actually commercially use now. That's a major number one improvement they did.
Number two, is that we made a series of beads with different extenders on it. We found out that ones that have sold for 15 years do not match spectrally with antibody-labeled cells. You will see this in the third paper. If you had a longer extension they match almost perfectly with antibody-labeled cells. That's the second improvement. You have to really characterize your calibrators to match what you are measuring.
Back to the title of the thing of what doesn't work, these conferences don't work. How many of you have been to standardization and consensus conferences? Again I've heard it. We walk away with that sounded good and nothing happens. NCCLS documents, I think is going to be a positive thing. The collaboration with NIST is also a positive thing.
The comment that was made that we should really have four or five labs really doing this because they're the only ones who are doing it, how are you going to do it without standards? Because if there's only five people buying them, nobody will spend the effort in making them. They aren't made right yet. Nobody.
For example, who buys BD and Bangs PE calibrators? Do they come up with the same calibration curve? Do they?
DR. LENKEI: Probably not.
DR. SCHWARTZ: Anybody have hard data saying yes or no?
DR. BRAYLAN: The hard data is no.
DR. LENKEI: Univan is working on the second project shows that we can come up with it.
DR. SCHWARTZ: Are they all on top of the same line?
DR. LENKEI: Yes.
DR. SCHWARTZ: They do? Al says no. I haven't bought either of them so I don't know. I suspect that they don't yet.
PARTICIPANT: I haven't seen a lot of them.
DR. SCHWARTZ: And they haven't. These are two companies putting out standards that it doesn't even matter if they're right. They don't even agree.
DR. LENKEI: And speaking about MESF.
DR. SCHWARTZ: Correct.
DR. LENKEI: It's not about ABC.
DR. SCHWARTZ: I didn't say ABC. BD doesn't make ABC.
DR. LENKEI: The correlations?
DR. SCHWARTZ: Correlations of 99.99, but they aren't the same answer.
DR. VOGT: The assignments are definitely not the same.
DR. SCHWARTZ: Why as a community do you allow that especially when you are spending money for it?
DR. FISCHER: That's the point. In several years we haven't bought any of those because they didn't match. Because we couldn't use it from one lab to another, that was why we stopped buying them. Most of our money would talk even when the companies didn't seem to listen to them.
DR. SCHWARTZ: Why didn't they listen? Because they have to survive. I know it more than anything because that was my only products. These people have 100 other products. If people don't buy these, business-wise it doesn't matter. To this group, it is the key. If you do anything, it is to address that issue of being able to have the material that you can start answering these other questions. If you do not, don't waste your time even asking.
DR. FISCHER: Doesn't that go back to the point of making them NIST traceable?
DR. SCHWARTZ: NIST isn't going to do it.
DR. VOGT: I'm going to disagree with that. I think we can come up with Phycoerythrin units that are traceable.
DR. SCHWARTZ: Who's we? Who's going to do it?
DR. VOGT: The people in this room.
DR. SCHWARTZ: So NIST isn't going to it and the companies aren't going to do it. You're going to get your own bead labs and make them.
DR. VOGT: No, the companies and NIST and the people in this room are going to it.
DR. SCHWARTZ: NIST doesn't have the budget. Like you said if you don't buy it, fluorescence is dead.
DR. LENKEI: I want to say something.
DR. SCHWARTZ: If you do buy it, they may try to do it but the amount of energy and effort especially making a PE particle standard is not going to happen.
DR. VOGT: I think it will. I think you're wrong. In three years, we'll have universal PE units.
DR. SCHWARTZ: It's not the units. It's the material. Who's going to make it and who's going to verify it because it's not in the program?
DR. VOGT: BD has had a product for years.
DR. SCHWARTZ: And so did Bangs.
DR. VOGT: Wait. The BD product was tethered to their conjugate. It works. It absolutely works. I don't think it's fair what you are saying.
DR. LENKEI: I want to say something. Conjugation is standardization. So if you speak about selling reagents, to sell them in standardization because I have that I didn't use that because they don't leave us with the exact pairs. But using them in the last ten years, I had a very good standardization. If we tell them, also more is standardization of reagents to get unified results in order to get better significance in all our established teacher grades.
DR. VOGT: It can be done, Abe.
DR. SCHWARTZ: It's part of the question that it can be. I don't see anybody putting the necessary resources to do it correctly. The theory works. Dolph has formalized it and Lily to the point where even I'm convinced that the subjective thing to say is I think this works in proper and it worked for 20 years. People got the kind of the same answer. But in terms of it being really solidly based like we tried to do in North Carolina as a white paper we said his MESF theory, does anybody have a problem with it? That wasn't very satisfactory because the only problem was why are people making money on it.
DR. LAMB: Abe, part of the focus that flow labs are going to buy a standard and use it and that's what's going to drive the thing. It's not going to happen. You're absolutely right. He made an excellent point from Pharma's point of view.
If you are working with tumor-associated antigens which are now just starting to get over the hill as something that's going to be important and in targeted therapy of leukemia which is what my lab works on, when these go to Pharma, what dose of this drug and what antigen density of this cell is going to give you efficacy? Those are the companies that are going to drive this doing thousands and thousands of tumors on clinical trials. Not at a pathology laboratory but in an industry setting.
Then perhaps the technology will get to the point where it will filter down to the individual laboratories. But you say nobody's going to buy it. I'm tell you what. I think the people will buy it.
DR. SCHWARTZ: I'm not saying they wouldn't buy it. I'm saying nobody has the will to put the resources to do to give you the material that you can use. It's a Catch-22. I tried to put as much as I could into it.
DR. MARTI: I think that industry won't pay any attention.
DR. SCHWARTZ: And NIST.
DR. PURVIS: The regulatory agencies are going to be the ones that will drive this as well as the insurance --
DR. MARTI: From my viewpoint, just because a standard doesn't exist in industry, if the market is deemed to be $200 billion, that standard will appear.
DR. SCHWARTZ: It can't until you have proven it's valuable. I've been asking for 20 years how does this save lives. Nobody has said I can't say where it's going to save lives. Now you are coming up with some things that maybe it's important. You are not going to get the two Million assays required to use as a pressure to get somebody to take this stuff seriously to put the effort and energy and resources into it to make a thing that will standardize it.
DR. WOOD: I don't think it's quite as dismal as you have painted it because what you need to look at are the two factors involved here. It's precision and accuracy. If we can shoot for being precisely inaccurate, we can always correct later on. Our problem right now is we need precision. So it doesn't matter whether you get two different numbers. You can argue later on as to which number is more accurate. But, if the precision isn't there you can never argue that.
DR. SCHWARTZ: The precision is getting the same number if it's wrong or right.
DR. WOOD: No, accuracy is getting the same number. Precision is a typical manufacturing problem.
DR. SCHWARTZ: Precision is getting some number that is repeatable by various labs.
DR. WOOD: Right.
DR. SCHWARTZ: It may be right or wrong.
DR. WOOD: Whether it's right or wrong, it's the same number. That's what is important.
DR. SCHWARTZ: That's one number whatever it is.
DR. PURVIS: The same assignments on these beads or you have to have an FP that corrects four of the assignments on the BD.
DR. SCHWARTZ: Because in general we all have to get the same answer whether it's right or wrong. That's what precision is.
DR. WOOD: Correct.
DR. SCHWARTZ: We're not doing that. We have two products out there getting two different answers.
DR. BRAYLAN: That's accuracy.
DR. SCHWARTZ: Both may be wrong but they are different.
DR. BRAYLAN: Right but within each system is repetition.
DR. SCHWARTZ: If you are going to have one disease and you are using one system and they are using another and it's two different answers you can't talk to each other.
DR. VOGT: In these settings a better word than accuracy is bias because it actually is more indicative of what's going on. The difference between precision and accuracy becomes very flaky at some point. What's happened is in flow cytometry generally you have very precise measurements because you are counting so many events. So you can see small differences between things and that is in bias. So there is bias between the PE calibrators, but there is good precision within the use of any particular PE calibrator.
DR. SCHWARTZ: The more manufacturers and standards you have that are getting different answers, it's lucky you only have two that are doing it.
DR. VOGT: That looks like kept work.
DR. SHAPIRO: The guy who shot the red painted bullets is accurate but not precise. The guy who shoot the green painted is precise but not accurate.
DR. SCHWARTZ: But what happens when you have different groups here that gives you a couple of answers. Which one do you believe?
DR. VOGT: Then goes back to what Jim said which is that you can always move to the center if you have a tight cluster anywhere that's in the measurement space.
DR. SCHWARTZ: But it may not even be more important to get to the center.
DR. MARTI: Remember when we started CD34 in U.S., Canada and Western Europe. The variation on a CD34 measurement published was plus or minus 1000 percent. The next thing that happened in the process was let's take a bunch of labs and have a standard method, and you and your lab and your wisdom and your extensive experience, you compare your local method to the standard reference and we're going to do that four times. Guess what happens? They couldn't complete the experiment. After the second mail out, everybody was using the reference method.
DR. VOGT: They broke the code.
DR. MARTI: I don't know about that. But the moral of the story is that these two can move together, or they can move toward the center. The whole idea of standardization is to try to and define what the magnitude of the variation is.
Once you know what that variation is then you can take it apart and see what it is. We generally agree up to this point in time that the variation isn't due to the instrumentation except sometimes we don't know that. We don't know that the instrument's been validated. But when the instrument is validated then the instrument is removed from the equation. I think that's been true in the past. I'm not so sure now with this new generation of instruments whether that's true because it hasn't been tested.
DR. FISCHER: And in the long run don't we need somebody to tell us where the bull's-eye is? That's where NIST or somebody come in because they have to tell you where the bull's-eye because we can all end up outside of the target completely. Yes, we'll be precise and we'll all in some ways be wrong, but until somebody tells us where the right spot is, we'll all think we're right anyway.
DR. MARTI: In retrospect the fact that when we were all getting the wrong answer on CD34, at least on unmobilized peripheral blood two to three or five CD34 positive cells per microliter, it probably was right.
DR. MUIRHEAD: There are two issues. One is the bias issue that Bob brought up. The second is the perception issue. That's an issue in a larger community. In this room or the people that have been thinking about these problems, I think they would agree with a statement that says as long as we know what the biases are, as long as people who are using a given system can get consistent results among the group that's using that system, then we decide there's a way to account for that bias and know what it is. Fine. Then we can start preparing the process.
But to the outside community, then, they are thinking about this, the problem that says I see a group using this system that's getting what looks like a completely different set of answers. That's a perception problem. That's one of the issues that says wait a minute, I don't want to have anything to do with this because the experts can't even get comparable results. So you have to deal with both of those issues.
DR. SCHWARTZ: The biggest concern I have is the material that is certified is not going to be available because there's not the support to do that. Even for NIST, that program is doing these two things but somebody asks what's after that.
DR. MARTI: Standards. It is a big glorious word. But you are never going to get an RO1 funded from NIH on standards. That's the first thing. The second thing is where standards develop and it's supposed to be a big thing at the FDA, I think that every time I write it down on my CD or on my plans for the year, that automatically means negative dollars. That's a reality. I agree with you if there isn't a standard that can't be marketed and properly made on the effort that we need to make it, that's a problem.
DR. SCHWARTZ: That's your biggest problem. The rest of these things don't mean anything unless you have the material to hang your hat on to deal with these other problems. I'm not saying they are not important. They are very important. But you have to have the standards set up first, it's worthless.
DR. MARTI: I guess in order to pull ourselves out of this severe depressive area where you are.
DR. SCHWARTZ: I'm not depressed.
DR. MARTI: The dismal state that you're painting, we can do some things about that with the materials that we have. God forbid even if we have to go back that furaldehyde fixed chicken red cells or thymus nuclei.
DR. HOFFMAN: We do have a lot of resources. We have a lot of capabilities. Let me just say something. I don't think we developed quantiBRITE because we thought we were going to make a lot of money selling quantiBRITE. We developed quantiBRITE because we thought the presence of quantitation was going to be in the future a very useful and hopefully profitable kind of business.
We published how we assigned the values. We published what the molecular weights we assumed from literature values for the PE. We published what we assumed from literature documents the extinction coefficient was. We published all the details about holocene, two inventive methods and we assigned the values and got us some results.
If there are biases there, it's likely coming from the assumption of what the molecular weight was or what the extinction coefficient was. There are resources around the world that know all about hydroproteins. We're in contact with some of those and maybe others. Even if NIST doesn't develop a standard for PE, companies that are interested in developing, manufacturing bead standards have resources at NIST.
BD is interested and we're working with NIST, not necessarily because we expect them to do all the work, but because they are a valued resource. They have this incredible fluorometer. If we can get groups that can agree on things about PE and there are proteins and biologics and there are variations from one kind of bacterium that's making it to another, whatever source of PE that a particular manufacturer is using for their conjugates, we have resources at NIST. We may have other resources in this room. They've been used to come a more complete understanding and assignment of agreed upon values.
Maybe we're not going to have two systems that are going to give exactly the same results, but within a system a PE conjugate, a critical source of PE, we will be able to get consistent results. Then knowing what the biases between those two are, hopefully we can maybe know what it is.
DR. SCHWARTZ: I'm not saying it can't be solved. I'm saying that there's not enough serious focus on getting the resources to the people to be able to do it.
DR. HOFFMAN: I'm just following up with some resources. I'm not suggesting that more than NIST is actually going to want to do it but I certainly hope so.
DR. SHAPIRO: Again the process exists. We know if you have a fluorometer that's appropriately designed you can do these measurements whether they're your beads or anybody's beads. You have a solution. But if you have a label and you have beads and you have the fluorometer set up with the appropriate flow, you can do this. Once there is one machine around, the initial investment is in the first machine. While the filters may be expensive, they're not that expensive. So basically it's fairly easy to implement this process.
DR. SCHWARTZ: NIST's role is to find out the methodology so other people can do it.
DR. SHAPIRO: Right.
DR. SCHWARTZ: And they've done that.
DR. VOGT: The thing is, Abe, even after all that is settled, that's not the biggest problem Mary Alice has. The fluorescence properties and the calibration of the fluorescence scale are tractable problems. The use of phycoerythrin conjugates for staining cells will present perhaps some intractable problems but they will have to be worked out on a case-by-case basis.
That's what I'm more worried about than our ability to get a handle on PE fluorescence. I think we can handle on that, but I'm not sure that we're going to be able to get a handle on every single phycoerythrin conjugate that's made by every single manufacturer. It's still going to be up to the manufacturers to provide conjugates that are characterized in a way that work in their systems.
DR. SCHWARTZ: Let me just part with just one comment with what will work. It is what Norm Purvis`s approach has been for the last half dozen years or so. ABC binding beads will work in the hands of somebody that has the time and energy and effort to do them. That's not most of us, including even myself. I would have to have specialized technicians to be able to do it to get consistent things as best I could do it.
What is practical and again Bob was saying on this effective F/P ratio is if we can develop any MESF beads where everyone agrees no matter what their source they give you the same calibration line because that's what you want and then the manufacturers who make the reagents can put on that bottle because if you have to try to do it yourself, you're in a hell of a mess and it's a waste of your time and you would be willing to pay a few more dollars if that F/P ration is on the bottle. You divide that by whatever you have for MESFs and you will get consistent answers. Whether they are right or wrong, I really don't care and neither do MDs. As long as they have an answer, they can make a decision about it that's consistent. That's all we want in this room.
DR. LENKEI: We can't rely on each company that the figures will be correct because this is a big problem.
DR. SCHWARTZ: It will be correct because hopefully NIST or somebody like that will go into a document like NCCLS and say this is how you assign the number to your antibody. You can do it with a fluorometer. That's the easy one. The problem there is do you know the concentration of the antibody that's going in to get that particular fluorescence. That should be the key push for here in terms of trying to develop something that you can get answers, that you could interpret or misinterpret because of these other additional problems.
DR. PURVIS: This removes the need to also those unimolar antibodies.
DR. VOGT: It might or it might not, Norm.
DR. PURVIS: It depends on how good the conjugate is. As long as the antibodies have not been over-conjugated where we have quenching or have affected the binding.
DR. VOGT: I'm wondering if that's ever going to be possible to insure.
DR. SCHWARTZ: It will because it's tested before they sell it.
DR. VOGT: But again every cell is different. Can you guarantee me that just because it didn't interfere with a complex set of markers in one tumor that it won't interfere with a complex set of markers in another tumor?
DR. SCHWARTZ: No.
DR. VOGT: Nobody in this room would guarantee that.
DR. PURVIS: I think a part of that is going through and doing the proper qualifications on the antibody that you're interested in evaluating. That's simple biology.
DR. VOGT: There's a lot of biology.
DR. SCHWARTZ: And on the bottle it will say that this can be used for such and such a tumor.
DR. PURVIS: They will not put that information on the bottle.
DR. SHAPIRO: Nobody can afford to do that.
DR. SCHWARTZ: That's my two cents.
DR. LENKEI: One point we should look at is FITC or functions specifically to the predicament of fluorocarbon for conjugate.
DR. MARTI: Just go back one step. I may have had a transient temporal lobe seizure there. What was the consensus of the group about how you want a label to read on a vial regarding a fluorescent ligand conjugate?
DR. SCHWARTZ: The effect of that would be how many MESFs per antibodies and divide that number into the MESFs of your cell which is a more stable type of measurement. You get the binding antibodies.
DR. BRAYLAN: So how difficult is it for the manufacturers?
DR. SCHWARTZ: Simple. He does it every week.
DR. BRAYLAN: If that is simple then this should be done.
DR. PURVIS: For the manufacturers, it should be even more simple because at the time that they do the conjugation and antibody verification they know the concentrations.
DR. SCHWARTZ: Right, they know what the concentrations are. It's very hard for them because they put other proteins in there and you can't tell what the real concentration is.
DR. VOGT: I want to emphasize one point that Bob made. This stuff that they did was all published. It's all in public domain. That's really important because if we have a bunch of manufacturers that do their own method of doing this we will have biases. We'll probably have some biases anyhow.
When stuff appears in the open literature and everyone can look and see how it's done, and there some conflict here with the ability and need to make money off of things that may be proprietary. There will always some difficulty in balancing those things.
But the BD methods are published. That's a very important thing. That allows us in the NCCLS guidelines to refer to the BD method which we could never do referring to them as BD methods, but we can certainly refer to the papers that appear in the open literature. One thing that I would like to encourage all the manufacturers to do is publish, publish, publish.
DR. PURVIS: Can we not use what NIST has developed here for procedure and implement that as a consistent way to measure for the manufacturers to actually use this? This is going to be published.
DR. VOGT: I think what Dolph has published for the assignment of values and so on is going to be used in a few laboratories. But the assignment of the effect of FP or whatever you want to call it is trickier. If you look at what was primarily Ken Davis and his group did at BD in working that up, it's a complex thing. It worked real well for one marker and then it took more work to get it to the CD38 zone in time to tell us about it.
DR. MARTI: But it's safe to say that there is a desirability and it would be desirable to have something about what approximates an effective F/P ratio on the label of the antibody ligand conjugate.
DR. VOGT: Absolutely.
DR. MARTI: Going once, going twice, gone. That's something that could be discussed with the manufacturers and get their input to see what they think. Remember that in this room you all take what you read on a label pretty much for granted. But what's on a label from a regulatory standpoint, that's looked at as how much did that label cost. How much research and development went into that label. Did it cost a half a Million dollars to guarantee what was in that bottle?
Industry likes to not to have to put much on that label because the less they have to put on, the less costly it is. As simple as this sounds, I would still keep open to the fact that there might be some dollar amount associated with putting that on a label.
DR. SCHWARTZ: If I had that on a labeled antibody, I would pay extra for it because I wouldn't have to do it.
DR. MARTI: If the cost of an analyzed specific reagent would go up 10 percent, would you pay for this?
DR. McCOY: As long as the Medicare reimbursement goes up by 10 percent.
DR. MARTI: Oh boy, that's another meeting.
DR. BRAYLAN: As long as you get paid either by research or also by clinical policy, I would say that I would be very happy to pay for CD 20 or CD 22 because I know there is clinical application. Otherwise how are we to pay?
DR. FISCHER: I don't think you get it on every reagent. But you want to get it on the main T-cell and the main B-cell quantitative antibodies.
DR. SCHWARTZ: A new classification is what we are talking about.
DR. VOGT: Since we are talking about phycoerythrin reagents dominantly here, I want to go back this unimolar business. If you look at BD papers apparently there is not enough quenching upon conjugation to alter the fluorescence yield of phycoerythrin on an antibody molecule. So the F/P ratio of unimolar conjugates is one. It may be 1.0. It may be 0.9. It may be 1.1 but it's not 1.5 and it's not 0.5.
Gel filtration is easy. I don't know exactly how you purify your antibodies, but when you take something that's 150,000 and add something that's twice as big to it and you need to separate that from something that's 150,000 with two things that are twice as big on it, that's a pretty easy problem in biochemistry.
That's probably easier than figuring out the effective F/P ratio and knowing that you are not altering binding properties by having heterogeneity of your conjugate phycoerythrin ratios. So I actually think unimolar conjugates for phycoerythrin give us both. They give us a constant effect in equivalent fluorescence and they give us constant binding properties.
*DR. SCHWARTZ: But, I don't think that's material.
DR. VOGT: That's the price you may have to pay.
DR. HOFFMAN: We sell some one-to-one conjugates. The price is higher because the yield is less.
DR. LAMB: You have to buy in volume.
DR. VOGT: How much higher is it? Do you know? I actually don't know.
DR. HOFFMAN: I think it's 20 percent or something. It's not twice as much.
DR. LAMB: Bob, the deal is I have bought CD69 one-to-one. You can't order a bottle of it. You have to order a lot.
DR. SCHWARTZ: $5,000 worth.
DR. LAMB: Which they will make for you for about $5,000 and then you have that in your lab.
DR. HSI: CD30 right now is five times as much as the regular CD reagent.
DR. SCHWARTZ: But you don't have to be limited to phycoerythrin if you do it with fluorometry, you can do it on fluorescein or any other fluorochrome.
DR. LENKEI: We can show you the very inch variation and I don't think any company would like to tell you exactly the difference**
DR. SCHWARTZ: Do you mean if they go back to fluorescein?
DR. LENKEI: Not only that you can submit antibodies since they test fluorescein for -- is also part of the company's confidence not to show you exactly.
DR. SCHWARTZ: When I measured these things by the ABC method for fluorescein, I was always coming out 0.7, 0.6 for fluorescein almost across the corporations. So it's not any problem.
DR. LENKEI: I will tell you. I had an amazing range in the beta monoclonal from 6.1 fluorescein protein ratio to 9.0. I almost was going to correct all my figures because it was not as effective.
DR. SCHWARTZ: Was that on the label?
DR. LENKEI: It was a fluorescein label. But I heard from them when I saw this.
DR. SCHWARTZ: That's how much they put in. That doesn't mean how much fixed.
DR. LENKEI: Yes, but it was a bed monoclonal-- it showed because it had nine and inoxidated part of my monoclone.
DR. VOGT: I was going to say you get nine FITCs on a antibody. Let's go back to Ashcroft paper. When they tried to do that experiment with conventional epidermal growth factor fluorescenated ligand, it didn't work. When they purified it and got unimolar, it worked beautifully.
I'm not convinced that the biggest problems here are fluorescence. In fact I'm firmly convinced the biggest problems here are binding. I don't think we're going to get around binding problems by getting effective F/P ratios. We may get around the binding problems by having uniform conjugate reagents.
DR. SCHWARTZ: Not only PE.
DR. SHAPIRO: Probably not PE because as along as we have the work horse laser beam 488 and as long as we want to do seven colors up to 488B then we have PE tandems, quantifying is going to be impossible. If you want to do a quantitative measurement with PE, then you really have to start thinking about gating with antibodies that are excited at other wavelengths, whether its in red or violet or UV where you don't have the spectrum crossed off with PE.
That's giving you maybe four antibodies to gain on it. If you want data on more antibodies than that, then maybe you start thinking about considering the ABC reagent for your quantitation even though it's not going to be as bright as a PE reagent. There is no question that if you are doing PE with six other colors, the cost of it is just ridiculous.
DR. TAMUL: I'd like to second that and also to mention that the manufacturers would like to have the standards just as much as everybody else would. We need something else to go on as well.
DR. MARTI: Mary Alice, before we go on to the next presentation, I would like to make a final comment on Abe's presentation. Abe, one of the really wonderful things that you've done was to use the consensus process to establish a set of performance characteristics of standards. You primarily did that with one of our colleagues who is not here today, Frank Mandy, from Health Canada. That certainly is a big torchlight to have that concept analysis.
DR. SCHWARTZ: That and the window of analysis.
DR. MARTI: I would agree with that too.
DR. LENKEI: It's the biggest thing which we have.
DR. MARTI: That still needs to be tested a little bit further. It needs to be tested in beta setting and not just Western Europe.
DR. LENKEI: We have published the results.
DR. STETLER-STEVENS: I've had a request for a five minute break. Excellent. Have something to drink, go to the bathroom. Five minutes though. Real quick. Off the record.
(Whereupon, the foregoing matter went off the record at 10:15 a.m. and went back on the record at 10:32 a.m.)
DR. VOGT: Well, Jim, there's actually a very good convergence on those measurements over a 15-year period.
DR. WOOD: Well, what I'm saying is to go from one manufacturer to the other, it's going to be important in order to get the precision and reproducibility, as to work within a system. That is you're going to have to have consistency in the antibody, consistency in the dye that you're using and develop a whole system around that for calibration. And then to go from manufacturer to manufacturer, you're going to have to have fudge factors, for these calibration factors to go across.
DR. FISCHER: But you have to go back to having a standard to base all the fudge factors on, right?
DR. WOOD: Well, but then you go back to identifying what CD4 is, for example and say which antibody because you talk about what's a bad antibody. Identifying and making sure that it's really identifying the epitope the way that it should do it.
DR. PURVIS: I'll throw another problem into the mix. If you have the same clone from multiple vendors.
DR. VOGT: You mean the same antibody?
DR. PURVIS: Same clone, same clone.
DR. VOGT: Okay.
DR. PURVIS: If I do a PE conjugation to it or buy them all in PE's, I get all kinds of different results. The F/P's are different and that is where I need that F/P to be able to take care of it. Even if I think, based on what I'm seeing, Prozyme, I went out on their website and I talked about PE's degradation, in the conjugation process itself, even if you gave me unimolar PE conjugates, I'm still going to see differences because the effect of F/P is not one.
DR. WOOD: Just to talk about the issue with phycoerythrin, if we're trying to take different manufacturers with different clones or different versions of PE, and you go to, say, using the Alexa dyes, what you've done is you've now gone to a single manufacturer again in terms of identifying an Alexa dye, that dye, and you identify exactly what it is.
DR. SCHWARTZ: Fluorescein is fluorescein. Alexa is Alexa.
DR. WOOD: What you're doing though is you now reduce the variability of the system.
DR. PURVIS: Yes.
DR. WOOD: And that's what I'd like to see. Naturally when you do that, then your overall precision is going to go up. That was my main point to make in the whole PE thing. Yes, it's great. It's bright. But I -- we are capable of doing it now with the new instrumentation, sensitivities that we have, the resolution, the linearities to be able to use FITC without any problems to quantitate on most of our antigen systems that we're interested in doing our quantitation of. I think the same thing would be true for the Alexas and other dyes.
DR. VOGT: There is another approach to this that I truly believe would work and this is something Abe and I talked about years ago. His original system was get the MESF curve and get the single quantum simply cellular population with a known binding capacity and do your normalization, do your one point, the same as the CD4, except it's quantum simply cellular bead rather than a CD4 cell.
The problem with that system is there is no universal binding capacity that you can assign to the quantum simply cellular beads. Even from lot to lot in the same manufacturer, the same fluorochrome, there's variance. However, it is possible to calibrate those beads on each lot of antibody using, for instance, the isoparametric titration or using Howard's cheap and quick supernatant redepletion method or whatever.
So you actually could provide people with a matched pair of your conjugate and a quantum simply cellular type of bead, a capture bead, which had been calibrated to that conjugate to that lot. You put that on your flow cytometer and read it off in the MESF curve, you get the effect of F/P ratio in your lab for that lot, that bottle of conjugate.
The only thing you have to worry about is whether there had been some degradation in the binding properties in the quantum simply cellular bead between the time that you opened the bottle and ran it from the time of the seal-up to begin with. That system would work but you have to have independent lot by lot quantification of the binding capacity of the capture bead for that particular conjugate. That's the closest thing to a universal system you could get.
DR. LENKEI: Well, do you remember the studies that even now, the most important thing is how you conjugate that antibody on the bead? And it has been a lot of problems and we know about that. The last protocol they were much better because they were taking into consideration the amounts. So we had a lot of problems from the practical point of view.
I want to stress here that what you have, I am talking about clinical applications because the subject of our meeting theoretically, it's very good to go farther for the next case. But for this case, I guess we had problems.
DR. SCHWARTZ: If we took what Bob said to try to make it practical for clinical people where they don't have to do a whole bunch of nonsense to get the quantum simply cellulars pre-stained by the manufacturer - and I didn't have enough money to do it properly and I couldn't get anybody interested in it at that point - and if you pre-stain it and the manufacturer does the assignments and leaves it swimming around in the antibody, then you take a drop out and run it, you will have a calibration curve which will be accurate.
DR. LENKEI: Yes, and I can profess that in our second experiment of the task force, you prepare the reagents last and they were stable for one year. They have exactly the same number, the same intensity when we had all the same application for one year. All the peaks. And it was very good but impractical when it was the monoclonal antibody, they were conjugated by Abe. They were kept in the same conditions in my laboratory. They were distributed to eight laboratories in Europe each month, and so on. Then the variance was very low with quantum simply cellular. But in clinical application in many laboratories, we had a lot of errors.
DR. SCHWARTZ: The clinical people cannot do that work themselves. It has to be done by someone who says all right, I want to tell the manufacturer and sell this thing, that has the right equipment and knows the problems with binding.
Essentially they solved them and sell you something that will give you the same calibration line, just essentially like the quantiBRITE does or the hard dyed beads or whatever but that's a big responsibility for the manufacturers. It really cannot be done by I've given you a protocol and you try to follow it. You do not have the time and experience to do it reproducibly.
DR. FISCHER: We're the hands. I mean, most clinical labs are overworked as it is now.
DR. SCHWARTZ: They shouldn't have to be making the standards.
DR. QUINTANA: In both your method and Bob's neither one is going to come from a bead manufacturer.
DR. SCHWARTZ: It has to come from an antibody.
DR. QUINTANA: And an antibody manufacturer is not going to want to take beads and sell them with antibodies.
DR. SCHWARTZ: Why not?
DR. QUINTANA: The same reason a bead manufacturer doesn't want to buy --
DR. SCHWARTZ: Why not? I mean, they could buy your beads --
DR. D'HAUTCOURT: My opinion is that in my experience are able to validate certain calibrator and this is a good point of quantitation in the future. Before trying to have something that is consistent, first we must focus on the clinical application because they show clearly that the design of the system and this is so obvious that we can validate the stability of the calibrator. This is the work in the opposite sense and this is for me a good move that quantitation is useful in clinical application.
The problem is that if we try to explain everything like biology, measurement and so on, we produce so many complications in the system that most of the clinical labs have no more interest in it.
DR. SCHWARTZ: The clinical labs should not have to deal with any of that. They take something out, they get a calibration curve, they run a cell against it, and they get the same answer.
DR. PURVIS: One calibration material for FITC or PE. This is a very simple process that BD's works by. So is yours if you have a proper F/P ratio. It is a very simple process. It can be reliably implemented in anybody's lab so that it takes a lot of the variability out.
DR. SCHWARTZ: And it shouldn't be done with binding beads. It should be done with a fluorometer and that makes everything straightforward and simple by the manufacturer of the antibodies and write that on the bottle.
DR. MARTI: Kathy is trying to say something.
DR. MUIRHEAD: I'm sure some combination of the manufacturers in here must have thought about it. One of the benefits of the CD4 method is that Mother Nature gives you a reasonably well controlled standard reference material in the form of the CD4 lymph set. Goodness knows we can engineer every other kind of cell. Are there ways of engineering stabilized cellular material that could be used?
DR. SCHWARTZ: Poncelet didn't find out what the CD4 was on cell lines.
DR. MUIRHEAD: Those could then be used across antibodies. It's a different problem but I mean somebody must have either thought about it and tried it and decided not to do it.
DR. VOGT: Kathy, Bill Caldwell did that years ago with the B-cell line. He tried to sell it to Coulter and they weren't interested at the time. This is like early on.
DR. MUIRHEAD: This is where we come back to Jean-Luc's question. This is a chicken and an egg. We can imagine all of these things. We can probably even imagine how to build some of them but it's going to cost either time in the clinical lab, which nobody has, or money on the part of the manufacturer and therefore, on the part of the purchaser. Where's the applications that show that it's worth somebody's time to either manufacture the right reagents because the lab is going to be able to get reimbursed for it? It really is a chicken and egg thing. You have to pick some way to stop it.
DR. LAMB: