FDA Workshop

Molecular Methods in Immunohematology

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September 26, 2006

Facilitated by Sheryl A. Kochman

Consortium for Blood Group Genes - CBBG
    Marlon E. Reid, PhD, FIBMS
    New York Blood Center

Human Erythrocyte Antigen (HEA) Determinations by DNA Analysis
    Ghazzala Hashmi, PhD
    BioArray Solutions, Ltd.

Application of Genotype Analysis to the Quality Assurance of Reagent RBCs
    Jill R. Storry, PhD, FIBMS
    Lund University

Applications of Blood Group Antigen Expression Systems for Antibody Detection & Identification
    Karina Yazdanbakhsh, PhD
    New York Blood Center

Donor Genotyping
    Sandra J. Nance, MS, MT(ASCP), SBB
    American Red Cross

Proficiency Testing for Molecular Assays
    David B. Bellissimo, PhD, FACMG
    Blood Center of Wisconsin

Overcoming Limitations in Current Pre-Transfusion Compatibility Testing Methods Using Phage Display
    Don L. Siegel, PhD, MD
    University of Pennsylvania Medical Center

Current FDA Processes for Bringing Products to Market
    Sheryl A. Kochman
    FDA/CBER/OBRR/DBA/DRB

Review of Current FDA Guidance
    Sheryl A. Kochman
    FDA/CBER/OBRR/DBA/DRB

Where Do We Go From Here?
    Panel Discussion

Closing Remarks
    Sheryl A. Kochman
    FDA/CBER/OBRR/DBA/DRB

KEYNOTE: "---" denotes inaudible in the transcript.
"*" denotes word was phonetically spelled.

---

P R O C E E D I N G S

(8:40 a.m.)

Opening Remarks
by Sheryl A. Kochman, Facilitator

MS. KOCHMAN: Okay. So we are ready to resume. Yesterday we kept on time really, really well, and I want to thank everybody for that. I do have a couple of announcements to make this morning. If you need a shuttle to either of the airports please see Wanda Dawson by about lunchtime so that she came make those arrangements for you, and also there is an evaluation form. It should be in the lefthand pocket of your handout. We would appreciate it if you could fill that out and give us your feedback on the meeting.

We are going to start today off with a presentation from Marion Reid talking about the Consortium for Blood Genes and how it came into being and what it is all about.

Consortium for Blood Group Genes - CBBG
by Marion E. Reid, PhD, FIBMS

DR. REID: Good morning. So the Consortium for Blood Group Genes or, as I will call it, the CBBG. I want to thank Sheryl for asking me to give this talk because I was getting a little despondent about the whole thing, but I then I realized just how much we have achieved in two years. So it changed my attitude, and everything is about attitude, so I thank her for that.

(Slide.)

So I thought I would tell you how it got started and a little bit about the original name, what the purpose is, what the accomplishments have been, and what the mission and goals are -- not necessarily in that order.

(Slide.)

Actually Greg Denomme reminded me that it came on a wave of a couple of successes. One was the book called Molecular Protocols in Transfusion Medicine, which was coauthored by Greg, Maria Rios* and myself; and it had sold 500-plus copies by 2003, which we thought was a pretty good number considering it was such a niche market -- back then anyway.

We had developed, or I had developed, a proficiency exchange program for DNA samples because I was concerned that we were doing this testing and had no way of doing a proficiency or quality assurance. So it was set up that on the change of the clock once a year I would send DNA samples to all the participants, and then like in the spring and then in the autumn all the participants would send a sample to me so that we would control each other twice a year.

There were and are eight participating labs. Well, to start with actually it was Dan Bellissimo and myself, and then as people became aware of it it expanded. So it now includes Connie Gayle, Jeff in England, Greg in Canada, Jill in Sweden, and Lillian in Brazil, so it is international, and we just send some very simple stuff. It is designed to pass, not fail. So we sent DNA from one or two samples to say which SNPs should be tested. So we would say for FY or for JK or something like that. Then the results are sent back to the lab who sent the DNA, and then they confirm the serology, the typing from serological results so that we have both got documentation. So we are controlling. The testers control the lab that sent and the lab that sent controls the testers. A little strange, but it works and it is very little effort to achieve.

(Slide.)

So how did the CBBG get started? With that as a background, and then in 2004 I was fortunate enough to be speaking at a symposium in Brazil that was about molecule aspects in blood transfusion; and I came up with the idea of having a support group, that was developing and that we could help each other.

(Slide.)

There is me talking and there is Marisa talking. We are very serious and we got into some interesting discussions.

(Slide.)

Then I went on a vacation, relaxed, enjoyed myself, and did usual things in an unusual way. There is me snorkeling, but I am towed behind a boat so I do have to do any of this leg flipping or hard work.

(Slide.)

Then I went back to Campenas and was hosted by Lilian, and for breakfast we had stuff that was just very related to immunohematology. We had --- and --- and ---.

(Laughter.)

So when I mentioned this idea to Lilian she was supportive and encouraged me, so with my usual enthusiasm I continued.

(Slide.)

At that time we thought that America’s Association of Blood Group Genes would be a good name because we knew that the Europeans were already gathering themselves and didn’t need our help. So we called it the AABGG, and then when I returned to the US I contacted Connie and Greg because I knew that they were doing molecular analysis of blood groups for clinical purposes, and they also thought it was a good idea and came onboard.

(Slide.)

So I have put structure, and that is a little constructured. Put me as a coordinator, Lilian as the liaison for Latin America, Greg the liaison for Canada, and Connie the liaison for the USA, and obviously we are volunteers and everybody else who is in the group. We are there to help each other, and it is everybody in it together. It is just somebody has to lead the group. So everything I am going to say from now on is the group that has achieved it. It is not me. I am just the spokesman for CBBG.

(Slide.)

So in the fall of 2004 we contacted people that we could think of who might be interested in molecular analysis for either blood groups or platelets and we asked if they would be interested in joining a consortium and asked them if they knew of anybody else. So we sort of put the network out there trying to get the names of everybody that might be interested and we created an email address of 21 people. I sent out an email outlining the purpose of the group and received a positive response from everybody. It was 100 percent response, which is pretty impressive. We arranged a meeting to be held in Baltimore in October, ad that meeting was attended by 24 people.

(Slide.)

We made some decisions at that meeting. The language would be English, which is fortunate because I don’t speak anything else.

(Laughter.)

The membership is open to -- if membership is the right word, is open to anyone and everyone who is interested in DNA analysis in blood transfusion. Obviously the focus is on red cells, but we are also open to platelets and neutrophils, the AABGG name was changed to Consortium for Blood Group Genes so that there was no implied geographic restrictions to membership. We didn’t want it to be only America’s, but that is our focus. But it doesn’t mean to say that anyone else in the world can’t come and help us and we help them.

So we decided that we should meet where members might be attending meetings anyway so their hotel and travel was already paid for, such as before or during or after the AABB, ASH, or ISBT meetings because we have absolutely no treasury. There is no money. This is all done by individual people paying whatever it is that they need to pay.

The idea was to interact, share knowledge, ideas and concerns, and to identify formats for interacting. There were the obvious ones. We thought of email, newsletters, websites. We did achieve a name. Sergio Taloni paid for and applied the name cbgg.net, and we also thought of the AABB special interest group, or the SIG. The disadvantage of that being that you have to be a member of the AABB, so it could be a little restrictive, but those were some of the thoughts we came up with.

(Slide.)

So at the first meeting we thought it might be useful to write SOPs, standards, and prepare templates of request forms, worksheets, reports, and disclaimers so that were all sort of in the same ballpark and doing something that made sense and weren’t overlooking something obvious. We were to establish and operate a proficiency program, to establish a repository of well-characterized DNA for assays, validation, and for controls, to identify sources of funding, to identify centers of excellence for referring unusual samples for detailed analysis of genes. So if you find something unusual, which lab is good at looking at that particular variant. Like in the good old days if we had something rare we would have sent it to --- Sanger. So to identify those labs that are willing to do that extra work, and we thought we would write a letter to the editors to advertise that we are there and again put out there so that if anybody is interested they can join us. It is not just an elite club.

(Slide.)

The areas of focus are actually pretty much what I just talked about, but there were working parties established of -- you know, this is a small number of people, so you have one or two people in each working party, and there was some overlap. They were working on the disclaimers, the DNA repository, funding, proficiency programs, the forms, the SOPs, standards of practice, structure and bylaws, terminology, and website.

(Slide.)

So then I got brave and I thought, well, you know, if we are going to start this technology we cannot go anywhere with the FDA. They are going to either have to approve it or indicate that it is appropriate. So I spoke to Sheryl and asked whether the FDA would be open to considering or allowing or embracing the molecular methods for testing for blood groups; and she was extremely receptive and encouraging and suggested that there should be a meeting of interested people to discuss the needs, the value and the issues, and here we are. So, thank you, Sheryl.

MS. KOCHMAN: Thank you.

DR. REID: Greg wrote a letter to the editor. It was published in Immunohematology in 2005. The content was describing what happened at first meeting, a little bit of background of why we thought it was needed, an overview of topics that were discussed -- you heard that list, and asked for anybody that was interested to get involved. We heard from nobody, which I was initially very disappointed in, but then I got quite excited because I figured that we had really reached out in that original casting our net and we had captured everybody that really wanted to be involved -- or nobody reads Immunohematology.

(Slide.)

So the second meeting was held in Seattle. There were 19 attendees, and the discussion points included misconceptions about DNA testing, genotyping for blood group genes not being a disease, storage and unlinking DNA samples, the terminology of nucleic acid testing versus DNA testing. A mission statement was prepared. A logo was designed prior to the meeting and voted upon, and working parties broke into small groups to work.

(Slide.)

The mission statement is to establish standards, operate a proficiency program, and provide education for laboratories involved in nucleic acid testing for the determination of blood group and platelet antigens. There is no indication of superiority or inferiority. It is just that is what they were doing.

(Slide.)

Here is the logo, and I thank Lilian and her colleagues in Brazil for playing and tweaking and doing this. So we have indicated the three countries that were initially or are the liaisons, but the DNA is sort of connecting us, but going on around the world indicating that anybody who would like to join is welcome.

(Slide.)

The third meeting will held in Miami Beach on October the 19 th, and we have decided that instead of focusing on too many things, which is overwhelming, that we would focus on a few selected items. So to help us identify which were the most important, we emailed members for their priorities. We listed the working list that you saw earlier, and the majority responded with their preferences, so the winners will be discussed. I haven’t yet analyzed those responses, so I can’t tell you what they are. The agenda will be decided not because of this meeting, but, you know, there is only so much you can do in a day. To date we have 18 -- or it is 19 now. I just heard of one more yesterday. So 19 members plan to attend, and we have had eight regrets.

(Slide.)

So the goals, the primary goals of the CBGG: to establish a DNA bank; to establish and operate and proficiency program; to develop template request forms, worksheets and reports; to develop disclaimers; and to develop standards of practice that we can follow.

(Slide.)

So the CBGG is all about helping each other. It is going between everybody, just an interactive communication. So you might think there is a core expertise in the DNA repository, discovery and research can be one little area. An advisory panel or board, standards or practice, proficiency programs, and participating labs with clinical results and proficiency participation.

I think that is it. That is my usual, you know. DNA is not going to replace hemagglutination soon, but I think the pair of them together are very powerful. Thank you.

(Applause.)

MS. KOCHMAN: I forgot one of my other announcements. A number of the presentations for today are not in your packet, but they are available out at the registration desk. So you can pick those up during the break on your way, however you want to do that, but they are out there.

Our next speaker is Dr. Ghazzala Hashmi from BioArray Solutions, and she is going to talk us about blood cell antigen determination by DNA analysis.

HUMAN ERYTHROCYTE ANTIGEN (HEA) DETERMINATIONS BY DNA ANALYSIS
Ghazzala Hashmi, PhD

DR. HASHMI: Good morning. I am happy to be here this morning. Thank you very much, Sheryl, and FDA for organizing this meeting. So what I am going to do this morning is to start with a brief history of BioArray Solutions. BioArray Solutions came into existence in 1996. For a while it existed as an R&D organization. Platforms were developed and an integrated system was established. In 2001 BioArray Solutions was incorporated, and then we developed several applications on --- platform. Some of our initial applications included the genotyping of inherited disorders such as --- Jewish diseases, cystic fibrosis and others, and also actually typing, actually DNA typing.

We were introduced to blood group DNA analysis by way of scientific collaboration with Dr. Marion Reid. In around 2003 that evolved into the development of our current HEA --- format. Since then we have worked with several leaders in blood centers in immunohematology labs, some of them are present here in the audience, and also in hospitals. So today I will highlight two of those collaborations. One with --- blood center with Dr. Marion Reid on donor site and another one with Montana School of Medicine with Dr. Caroline --- where we looked at the patient population and how the DNA analysis can be used to predict antigen typing.

(Slide.)

After yesterday’s talk I don’t have to tell you how DNA analysis can be used for antigen typing. We had very good discussions about different techniques that have been used, SSPs, PCR, RFLPs, and also microarray analysis. The application of DNA analysis is not new in immunohematology and has been used in various labs for confirmation or for antigen typings and also for difficult cases. But most of these techniques, as we also heard, are manual like manual PCR followed by RFLPs or sequence-specific primer PCR. So now the current understanding is the DNA technology is a useful tool to be used in immunohematology and can be used for used antigen prediction.

So what is the next step what to do? A current practical limitation would be the manual nature of that and if you doing it in donor centers it is hard to do high throughput analysis or larger scale screening of the donors, and also with the patient population it has to be quick assays, quick as a platform where several antigens can be analyzed simultaneously and is in a short period of time.

So that is where we come in, so what we actually

-- we are not really making anything new or developing anything new. What we did is to take that, all that information, basically SSPs, and what we are doing is we are putting it on an array format. The other format that we use is bead-based assays, so these are also standard. Bead formats are the standard format used in molecular diagnostics, and what we are doing, we are putting those beads and assembling it on the microchips that are silicon wafers and doing the multiplex analysis on that.

So --- beadchips are different than the current understanding of microarray. When you think about microarray it is the glass slide where you have several probes that are linked, grown on the surface of the glass slide, and usually the understanding is when you think about microarrays is the expression analysis. We have Affymetrix chips, we have thousands of genes are screened simultaneously, and of course at the end the understanding or the general concept is that at the end there is so much data that analysis is very different. So these microarrays are completely different from that, especially other microarrays or bead arrays. We have our --- on the surface of a three-dimensional structure that is bead, and then beads are going on the surface of the chip, and of course I will describe it in detail.

(Slide.)

So what I intend to do today is to give you an introduction about our technology. So what are the beadchips, method of analysis, and then HEA analysis and how it is done for patient and donor population, and then summarize the results.

(Slide.)

So how do we make the beadchip? So as you can see, we start with a bead that is color-encoded, and then the beads are -- so there are several different colors. We have more than 100 colors of beads now. We synthesize our own beads at BioArray Solutions. We have manufacturing facilities and we also color them in house, so we control all the processes in house. Those beads are then functionalized.

What functionalized means is that when you select an application, say if I want to a cystic fibrosis panel, I have 25 mutations, I will take a number of beads that is enough to do 25 mutations and then for each mutation there will be a different color of bead. So it means that for each probe or each --- there is a specific color, and that color is the address of that probe for downstream analysis. Then these beads are coupled. After coupling these beads are pulled together in another chamber and then assembled on the surface of a silicon wafer. This is a six-inch silicon wafer, and it is microfabricated just by standard methods. That is in the industry using industry right now.

What we do, we microfabricate them in 1.725 millimeter diameter for a chip, but only a small area in the center of this chip is our bead chip or the array, and this area is only 300 microns. So we can make 5,000 chips like that, and each chip, that is 300 microns in diameters, has capacity to hold 4,000 beads in there. So for each application there will be a separate, a different -- so at this point everything is the same. It differs at this, so you will have pools of different applications -- cystic fibrosis, HEA, HLA -- and then the manufacturing is of course a separate process.

(Slide.)

So how these beadchips are used. For using the beadchip I highlight an example of antibody profiling. In this case what we do is that antigen is coupled on the surface of a specific color of the bead. Then these beads are assembled the way I just described. An image is taken of that array. This image is called a decoding image. This image is stored during the manufacturing process and it is unique for each application, so therefore the position of each bead, you know, the color of each bead as it is linked to its --- is already in the database.

What is done after that, the plasma sample from patient or the donor is applied on the surface of the beadchip that will binding of the antibody on the surface of the bead, and then this antibody is detected by a secondary antibody by amplification. Then you will take that.

So that this part is done is done in house. This is the manufacturing process. This is the user part. So what a user is doing, when they are getting these beadchips they are performing the assay and taking just a single image, and this information is already stored in the database that is provided with each beadchip format.

So what will happen in this case is whenever there is a positive signal you will have a fluorescent bead there. If there is a negative signal there is no fluorescent bead. What the software does, that method of analysis is it will take this image off the array assay made and superimpose on the surface of the beadchip. That information is already there. It will find the position of each bead and calculate the signal on each bead type and then give you the result.

This was the formula that was used in one of applications of auto-antibody profiling, and for that we received FDA CDRH clearance in 2001. During that time since our --- system is the same. So the platform that is used for protein typing or for the DNA typing is the same, so we do have a clearance on --- instrument array imaging system.

(Slide.)

So how do we use it for DNA analysis now? For DNA analysis, for example, we are doing antigen analysis. So you will take the donor or patient sample, DNA is extracted from that. We use current protocol, use only 200 microliter of DNA, and we eluded around 50 microliter of volume. Do a multiplex PCR. For HEA I will discuss it later in detail. There are 18 plex PCR, and then there is post-PCR processing. What post-PCR processing is doing is actually making a single-stranded target from the double-stranded one, and then you apply on our beadchip format that has all the probes already assembled on that. So --- if you are doing a 96-sample result for HEA --- 28 antigen result is available.

(Slide.)

The overall protocol is shown here. So this is the multiplex PCR, post-PCR where you produce single-stranded DNA molecules. DNA goes on the surface of the chip, elongate, and you get an assay made. The basic principle is exactly the same. There is a decoding image already present in the assay in the database and the assay

--- that will overlap and give you a signal.

The overall process takes about less than five hours, and in house we did a time motion study with single --- and single instrument, and we found that 200 samples can be done if you start 8:00 in the morning. 5:00 in the afternoon 200 samples can be done completely. So it means at the end of the day you can you have 200 samples with each sample with 28 antigen information. This throughput can be of course increased if there are more instruments or more manpower is available.

(Slide.)

Okay. Now how is it the DNA is actually analyzed? So one of the things that I didn’t mention in the previous slide, how the discrimination, how the allele discrimination is achieved in these cases. In our analysis it is not based on hybridization. That is mostly done in microarray format.

What we do, we basically as I said before in my introduction slide, we are taking the SSP primers, if you will, and putting them on the surface on the bead and actually we are doing a mini PCR on the surface of the chip. So the discrimination is very, very high, so as shown here that is the bead that is linked to each probe is linked here, and the --- assigned target would bind here and elongate if there is a match. The process of deduction, we call it EMAP, or elongation --- analysis of polymorphism, and that means that you are -- the discrimination is very high and it is not based only on the --- of two DNA molecules coming together, but on the specificity of the DNA polymerase. That is very, very specific for any mismatches at the three --- end.

So just in a little bit more detail for each, --- for example in each HEA typing is I am going Fy ab antigen. So there will be one color for Fy ^a and another color for Fy ^b. Te probes are exactly the same in sequence except at the three --- end, and that is decided on the basis of the

SNP that is linked to that antigen expression on the red cell. So when you create your single-stranded target and add it on the surface of the beadchip, a single-stranded target will bind it, an enzyme will bind, and also when the other EMAP makes an elongation it is just like in PCR, includes the DNA polymerase plus DNTPs and --- chloride. You know, the usual PCR mix. Whenever it sees a mismatch there is no elongation. If there is a match, perfect match, it will elongate. And since there is fluorescence present there in those DNTPs, it will fluorescently label that bead and that could be obvious when you take the ---, and when the data is calculated.

Another point I want to make is that redundancy of the probe molecules. So there is another difference between the other microarrays, that in other microarrays it depends how many probes you actually put that are coupled on the glass surface. What we are doing, we using the number of beads. The number of beads is from 50 to 100 beads of each color are present there, and then for each bead type, for each bead, the surface area as compared to the DNA molecule is huge. So each bead can have up to 1,000,000 probes attached to it.

So if you put all that together, there is very high redundancy present there, and that creates very high discrimination for allele discrimination for data analysis. Statistical reliability is already built in the system.

Another thing I didn’t mention is that, you know, with the specific group there will negative, seven negative, and positive controls are also added. That is a different color bead that is added on the pallet. So what happens here is that when you see a signal intensity, this a mean signal intensity on that say 80 beads and each bead of Fy ^a and 80 beads of Fy ^b, and each bead has 1,000,000 in that. So you have a mean intensity of allele A and allele B, and then we do the data analysis by calculating a discrimination ratio.

(Slide.)

So this is how the image is recorded or data is recorded. So what it is doing, we have our array imaging system that is basically a fluorescent molecule that -- excuse me. Fluorescent microscope that has the filters are -- it is modified with filters and it has software of course. So what it is doing is that when you load your chip this is a --- format for a 96 --- plate, and what you have is that each -- this chip carrier has a barcode attached to it.

So the user will after you perform assay you will put this chip carrier on the surface of -- just like on --- focus the first chip. Once the first chip is focused then the whole operation is automatic, and then it goes from chip number one to chip number 96, and each step taking a single snapshot. Since we have --- the size of the array is very small, only 20 microns, you don’t have to obtain the signal intensity separately. So it is just a simple operation where you take the image, a single snapshot of the whole array and the image is analyzed, and then it will produce just like I said before, an assay image; and then for the analysis part it will bring -- the user will get this information for that bead chip carrier which they are receiving, and the analysis will perform by overlapping the signal on these beads on the assay image by overlapping it with a decoding image.

(Slide.)

For data analysis as I said before, that this is a mean intensity on each probe, and you see the two are elongated. This is just the raw data. Two are elongating. It means that it is heterozygous. When only single is elongating it is the homozygous. So for what we do, we calculate the discrimination ratio of allele A and allele B and then a value discrimination ratio or a delta value is created of course by after the background normalization. Then when these --- are generated for each --- or for each SNP, and when they are plotted on a graph it will create tree specific clusters, so this cluster, AA allele A homozygous, allele B homozygous, or AB heterozygous.

There are grey zones, are also present around these clusters. So if any value is something like this fall into these grey zones it will be flagged as an indeterminate call and the user will have to go back and repeat that.

(Slide.)

There is no manual transcription needed here, so once you load your sample and you scan the beadchip barcode, so all the information is in the database. So this is an integrated system as I said. We have several different applications that use the same integrated platform. So it starts with the image’s acquisition with --- imaging system that will do the -- then it goes into the database where you will have the decoding images and image analysis is done, and then genotype or alleles are described on --- in our application server. That is all done in the same application format. Then for HEA typing for mutation analysis a mutation report is generated. For HEA what we do, we convert that genotype or the DNA analysis on each SNP typing into the allele assignment and provide a phenotype report for the user.

(Slide.)

For HEA panel for blood group genotyping this is our panel. This is our current panel that analyze 11 blood group system and 28 antigen in single chip format. We also included one polymorphism for hemoglobin S. So when a sample is analyzed in blood banks or in donor centers or in hospitals that the analysis of whether that donor has hemoglobin S positive or negative is also determined.

(Slide.)

So how the genotype to phenotype or the DNA analysis with the data you are collecting on the basis of SNP is used to determine the phenotype or antigen prediction is just by simple look-up tables. So these look-up tables are part of our software, so I am just describing that to make it, you know, obvious that as it was said yesterday also that most of these antigens are one-to-one. So there is a single SNP as our current knowledge is today, that a single SNP is responsible for single antigen as shown here. For example, for KEL there is a single --- AA. You will get a SNP typing, AA, AB, and BB. So AA means that big K is positive, little k is negative; AB, big K, little k both positive; BB big K negative, little k positive. And it is true for KEL, Kidd, Diego, Colton, Landsiener, Lutheran, and Scianna and also for our hemoglobin as mutation. For hemoglobin S we can say that either sickle trait negative, sickle trait positive, or it is HPS homozygous. So this is a condition when there is only single SNP is responsible for

antithetical antigen, but there are certain cases where more than one SNP is needed.

(Slide.)

For example, in duffy --- case. So in that case the --- present there. When there is a situation the software will go and find any -- you know, on the basis of the DNA typing find that combination and then give a phenotype report. That is also true MNS here. It is actually showing only MNS, but there are silencing mutations that could affect the expression of big S --- antigen, and those polymorphisms are also included.

(Slide.)

The phenotype report that is generated, it looks like this. Just like the phenotype report you are used to seeing by red cell typings with all the antigens are listed on the top. This is our chip ID, meaning that this HEA are a specific, unique number. For a 96 --- plate it will be the same number for all 96, only the position of the well is different, and then there is a field where the user, if like to, add their specific sample number. If you have --- format that could be --- and it will come into this phenotype report; and if there is anything wrong during that assay, say high CV, high background, there is no signal, --- dropout, that will be shown here. As you can see here actually I listed their silence and whether silence is present or not. Now in our current format this field is already integrated in here and that, you know, the software will take care of that and just give a typing on the basis of if the silencing is present or not.

(Slide.)

Okay. So for HEA phenotype by DNA analysis of a current --- we did a large-scale study with Dr. Marion Reid’s group at NYBC and we submitted a manuscript in Transfusion. I will just discuss some of the results. During this study we have New York City donors. There were 2,355 donors all for self-identified ethnicities and they were screened on a beadchip panel. At that panel we did not have silencing mutation and also -- C and E present on that.

In our analysis we had in New York Blood Center they had the phenotype information. These were partially phenotyped, meaning some --- have phenotype available for Duffy, some have available for Kel, Kidd, Dombrock and MNS, and there were 4,534 phenotype information available. We found 4,510 of them are concordant and they are listed here. There were 24 phenotypes that were not concordant. So resolve that we did the sequencing of each amplicon and Megan’s group did the RFLP on those samples, and we found that eight samples out of 15 for big S little s, eight samples had silencing mutations. So that is why there was a discordant there, and since then we added those two mutations on our panel. There were the other ---. They were all resolved in favor or --- and we found that most of them were clerical mistakes or first-time donors that were not really conformed by CEDR* tying at that time.

(Slide.)

S0 in this instance we can that phenotype, that the DNA analysis can predict the phenotype for this set of antigen.

(Slide.)

During this study we also determined the antigen frequencies within these different ethnic groups, and as expected they were different between African-American, Asian, Caucasian and Hispanic populations. The biggest difference was noted in a Duffy and MNS group system.

(Slide.)

So during this study we also as I said there were 4,000-some phenotype information that was available, but by -- there were 19,000, more than 19,000 new phenotypes were also, antigen-negative phenotypes were also identified during that time, and they could be used for further -- you know, by confirmation by serology and could be useful donors to support transfusion of matched units. Numerous examples of a rare configuration but also identified such as S, big S, little s negative, U negative. There were some U -- and U negatives, Lutheran A positive, big K positive, two samples like that, Joseph A negative six, and Colton B positive.

During that time we also did the time motion study as I told you. So now we can say that current protocol in practice, it enhances productivity in a person. A technical person can do more than 200 samples a day, and also this is an assay that is simple to perform that people with not too much molecular background can be trained very easily. We have seen several groups have come to our labs recently for training, and with a week of training in house at BioArray, and then they go and with some practice, a two-week practice, they are proficient to use the system in their regular DNA tying.

(Slide.)

Okay. Now just to see what is the clinical utility of the HEA typing for patients. As I said before, we had a collaborative product project with the Mount Sinai School of Medicine with Dr. Caroline Vincent* and initially what we did was selected a number of cases where we --- the patients with the disease where the disease condition itself or the treatment of the disease might interfere with DNA analysis; and those conditions are listed here, such as cytotoxic drugs that can reduce the number of white blood cell count and also hemoglobin --- several transfusions. So we selected several different conditions, and some of these samples our DNA yield was very, very low, but we were able to amplify the full panel and analyze these samples correctly for as low as around three nanograms of DNA. So a total of around 20, 25 nanograms of DNA that was used in the assay so that our initial collaboration and initial study was very successful.

We feel confident that in the patient in the hospital in the patients that are going through different kinds of therapies it could be used efficiently for this kind of DNA typing. One of the important cases which has been discussed yesterday also in great detail is the multiple transfusion, a patient with multiple transfusions where serotyping is not that -- cannot be done or is not reliable, and also people with autoimmune hemolytic anemia.

(Slide.)

So I am highlighting some of those cases here. There were seven cases where we took the samples pre- and post-transfusion and did the DNA analysis, HEA beadchip analysis on that. Then some of them were very massive transfusions. As you can see here, the liver transplantation has 74 transfusions, and these transfusions are turned very fast and these are whole blood transfusions; and then there were cases where sickle cell crisis, sickle cell anemia, and heart transplant patients that we did the serotyping and genotyping before and after a massive transfusion, and our HEA beadchip typing was able to identify the typing of the patient correctly in all of these cases. Then there are four cases I have highlighted here. Those have serial autoimmune hemolytic anemia that the serotype cannot be determined by phenotype method, and we were able to do that with these patients, too.

(Slide.)

All right. So in summary we can say that critical clinical conditions are so far with critical clinical conditions we have seen the DNA analysis or HEA beadchip analysis was not affected by that, and it was a useful tool to determine the phenotype or predict the phenotype on the basis of DNA analysis and also it permits the analysis on very small aliquots of samples. That goes for both small quantities and quality of the DNA and also a small volume of blood, that as you all know in cases of newborn the blood is a very small volume of blood is available. Even after massive transfusion the valid phenotype could be determined on the basis of this genotype analysis.

(Slide.)

So in summary we can say that our beadchip HEA analysis permits the reliable determinations for extended phenotypes as shown by a large-scale screening for patients and for donors with diverse ethnic backgrounds; and the current protocol in practice, as I said before, enables technical personnel to perform complex tests by minimum training, and enhances productivity because several sample could be analyzed simultaneously in one day.

(Slide.)

So what are our next steps? As I am sure you noticed, yesterday we had a lot of talk about Rh. In our panel this is a minor blood group panel, and we have only two markers. One for CEC, big C, little c, and big E, little e; and as you know there are issues with big C typing, sometime the variant typing, and so we are in the process of developing an Rh variant beadchip that will analyze Ce variant and also D variant. That is most clinically significant. HLA as I mentioned is already being used as already being used a DNA analysis, and we have very well established allele assignment programs that can be used for HLA class one and class two types; and also HPA is our most recent reject that we are working on, and it is in development.

(Slide.)

All right. So if you need more information or a copy of my presentation please write to me at that address, and if you have any questions I would be happy to answer to. I think we will do it at the end. Thank you very much for your attention.

(Applause.)

MS. KOCHMAN: Our next presentation may actually not come off. I am not sure. We were going to try to have Jill Storry give her presentation by phone. I am afraid she may have not been able to stay on the line, but I am going to try to get her right now.

(Attempting phone hookup.)

So we hope this is going to work. The one problem I think is that Jill may not be able to hear. We are going to have to take our questions after her presentation, and she may not be able to hear them, so if there are any I will repeat them. So your first slide is up, Jill, if you can.

Application of Genotype Analysis to the Quality Assurance of Reagent RBCs
by Jill R. Storry, PhD, FIBMS

DR. STORRY: Okay. Great. Well, I would really like to be there, but at least you --- my voice. Thanks to Mark Davis who is --- to phone up, so -- anyway, I am sorry I couldn’t be there, but I thought what I would do really more to get discussion going because it is something that many of us have discussed before is to just review our little study on the application of genotype analysis to the quality assurance of red cells, reagent red cells. This is something I know that many labs have done, but I have just put a few slides together based on our recent paper.

(Slide.)

So the next slide it really describes the aim of our study, and it was to use molecular genotyping methods to examine our in-house test results, and here in Lund we have assorted in-house ---. We have a three-cell antibody screen. We have a four-cell extended antibody screen. The rules definitely seem to be a bit different, and we also have a couple of in-house panels drawn from our local donors.

So we asked the following questions: Are our test red cels what they say they are? Can we miss potentially clinically important antibodies because we are using red cells we think carry double doses of a given antigen? Really I think you will see at the end of the presentation we haven’t --- that. It is very hard to measure. And thirdly, do we need to change any of our test red cells based on the outcome of this study?

(Slide.)

On the next slide I have just shown the test red cell requirements according to the Swedish handbook for transfusion medicine, and as you can see they are very similar to many of the standards described by other European agencies; the UK agency, the German agency, and of course very similar to those described in the US. We are mandated to have as best we know double dose of the commonly encounter Rh antigens, D, C, little c, big E, and little e, Fy ^a, Jk ^a, and big S; and then on our screening cells we are expected to represent CW, big K and little k, Kp ^a, Fy ^b, Jk ^b, and little s, at least in single dose, and then of course MP1 and Le ^a must be represented, but that is the only requirement we have for those.

(Slide.)

On the next slide it is really just information which probably this audience needs no clarification, but I will go through it anyway. Of all the antigen that we encounter in the immunohematology lab, the actions that cannot be categorically determined as carry double dose or determining the dosage at all are A and B actions, D, Fy ^a and Fy ^b, and of course that is because, as shown in the box beneath, a person of group A can be genetically homozygous for the A gene or can be heterozygous for A and assigned O allele. Similarly with D-positive what could be homozygous for a normal RHD allele or one of many other combinations of RHD with a partial D or more commonly RHD ---.

When it comes Duffy A and Duffy B then there are two major silencing or muting alleles in that system. A person that had double doses for Fy ^a can be homozygous for the Fy ^a allele but may also be heterozygous for Fy ^a with a silent allele or Fy ^b with an Fy ^x or even Fy ^b allele, and that is a lot more common certainly in our area of the world. Conversely, you might expect for a person appearing to be double dosed of Duffy B can be homozygous for an Fy ^b allele or heterozygous for F^y or Fy ^x and this is very population-dependent as you all know, so in the European population we are concentrating or expecting perhaps wrongly to see a higher occurrence of Fy ^x. But sort of the migration of the world populations I don’t think we should be too rigid about what we expect to see and not to see.

(Slide.)

On the next slide it just shows can we improve -- titled "Can we improve the profile of our test red cells?" I think the introduction of molecular techniques in the last year or two is we certainly can improve or increase the information by DNA analysis on those antigens for which there are no or few antisera available, and very obvious examples have been described by us and many other people. The antigens of the Dombrock system initiated by the New York Blood Center, and also antigens that are a practical clinical relevance in some countries other than others. For instance, Di a and Di b, and these are antigens where the sera is not readily available commercially and it is really -- it might seem of guilding the lily perhaps, but certainly it has had impact on clinical transfusion medicine.

One thing that is increasingly discussed among many different groups are where genotyping tests are improvement on the serological tests, and I am pretty sure that you have had some discussion about that already. That is particular in the area of the RHD with the identification of weak D alleles and also the identification of D el alleles, and this can have clinical application in patients for transfusion and also of course in pregnancy. So this what we were sort of looking at with our particular cells, how could we improve them.

(Slide.)

So materials and methods of the study was that we simply isolated genomic DNA from frozen red cell samples from our in-house panel; and like many places prepare their in-house panels, we draw whole units from a donor, freeze aliquots in glycerol. And this generally --- are not --- repeated so we can very readily isolate genomic DNA from these samples.

The phenotypes have been deemed most likely based on their serological results, which I think is how we have all done it for long, and we tested 52 samples. We selected 52 samples for RHD zygosity, and the distribution is shown in red. We looked primarily at the R 1R 1. We tested 24 of those samples, 14 R 2R 2 and so on. These are not all panel cells included in our primary panels, but it was really a list of all the cells we had listed as test cells. We selected 59 samples for FY analysis, 33 samples that are currently doubled-dose Duffy Fy a and 26 samples are apparently double-dose Fy b. Then later in our studies we selected 75 samples for DOA and DOB analysis.

(Slide.)

The next slide gives a very simplistic representation of the Pst1 RFLP analysis that we used in our lab, and this is the assay of Wagner and Flegel that was describe in Blood in 2000. Despite all its known limitations now, we have found it very useful as a screening assay, and in this assay we look for the digestion. We amplify fragments of DNA that covers the junction of the hybrid Rhesus, the five-prime Rhesus box and the three-prime Rhesus box that occurs in persons lacking an RHD gene. This is then digested with Pst1.

(Slide.)

You will see on the next slide that is a figure from Wagner and Flegel’s paper in which the amplicon is shown on the top is 1,888 base pairs and then the various digest from the PST enzyme that will give a different banding pattern. In the first, if you click first, you will see that the first three samples are in fact RHD negative samples of varying big C and big CE types; and you can see that there is a definitive pattern notably with a band, a definitive band at 564 base pairs, which is probably the clearest one in this particular group. One click further on and you can see that this group are three samples that are heterozygous for RHD, and they have the characteristic 564 band is actually lighter in this, but quite distinguishable from the deletion types. Then one click further on and you can see that these homozygous samples lack completely that 564 band and therefore you get distinguishable from the others. So we use this assay as a screening assay for those samples in which we are interested in zygosity.

(Slide.)

My next slide shows the results of that assay with the 52 samples that we tested, and fortunately I have got animations on these. If you just want to do two clicks you can show that two of the R1R1 samples were in fact heterozygous for RHD and not homozygous as we had predicted from the red cell serology, and on of the R2R2 samples was also heterozygous for RHD. It carried R double prime gene. What was also interesting was that we included three RO samples in this study, which expected to be R 0r --- to a population which contains a lot of African-Americans where you perhaps expect them to be double dose. We expected ours to be R 0r, and in fact they were. So in this group we show that three panel samples or three reagent samples of 52 gave unexpected results based on their serology, and these are cells that one of the R1R1 samples was a regular donor that we had included in our screening cells. So again, of potential clinical importance.

(Slide.)

On the next slide -- oh, this is just a summary. Sorry. This shows because we know from the studies of --- and --- and others that PstI RFLP analysis is not reliable for detection in non-white populations. We did send the samples to the Sanquin labs and --- in Rotterdam confirmed those by real-time PCR, so we were pretty that our results were what they said they were.

(Slide.)

The Duffy analysis we used an allele-specific approach to --- Fy ^a and Fy ^b stages as shown on this slide, and we incorporated primers ---. So we incorporate primers for the mutation GATA box and also primers for the mutations at the Fy ^a and Fy ^b determining nucleotides.

(Slide.)

So if you look at the next slide you can that for each sample that we run bring on high throughput of assay. Each sample that we run we test four PCR mixes, and so in the first --- you can see the primer panel below. So there is one, two, three, four. One primer, the first primer pair is Fy ^a with the GATA mutated. Two is Fy b plus GATA mutated. Three is Fy ^a plus normal, and four is Fy ^b plus normal. But you can see in the first sample the only amplification --- primer pair three, and this is actually an Fy ^a homozygous. With panel two both alleles three and four have amplicons, and that describes Fy ^a Fy ^b heterozygous. --- the first panel --- first panel. Panel three shows Fy ^b homozygous, and this last panel shows amplification only with primer pair two, which indicates this person is an FY normal, Fy ⁰ Fy ⁰. So there are many methods for determining --- genotyping. This is our method here.

(Slide.)

When we looked at our ASP we showed that of the 33 samples that we thought --- Fy ^a three of those were in fact Fy ^a Fy ^x and surprisingly -- well, surprisingly in our population two out of the 26 samples that we thought we were Duffy -- sorry, double dose of --- were in fact Fy ^b Fy ⁰. So five out of 49 our panel red cells were heterozygous for either Fy ^a or Fy ^b but we expected that they were in fact double dose. I think 8.5 percent represents quite a high number of either falsely determined or falsely called samples, so that is just something to think about.

(Slide.)

Two clicks on gives you the next slide, and this is just a very quick schematic representation of the background of the DOA and DOB polymorphism determined by

--- at nucleotide 793; and we use the allele-specific PCR described by Wu et al in Vox Sanguinis 2001, and amplify at either 162 base pair amplification of DOA or 161 base pair in the case of DOB fragment, so it is very straightforward.

(Slide.)

On the next slide it shows the results of that testing. Of the 75 samples we determined their genotype shown there: 14 apparent DOA are homozygous, 39 DOA DOB heterozygous, and 22 DOB DOB are homozygous. I have given the incidence on the right-hand side, but this not a random sampling. These are our panel cells, and who knows how -- this is not just random donors. These are our cell panels. They had been selected for other reasons. However, the distribution is not too unlike you would expect from the European population. So it was quite nice, it was quite comforting in that way, but the distribution of --- normal.

(Slide.)

So our conclusions from this little study was that the analysis showed that three samples that we thought to be RHD homozygous were heterozygous. One our screening Fy a+b- samples was in fact Fy a Fy x and had been on the panel for a long time, and that is very similar I think to Marion and Christine’s --- in the New York Blood Center. One in seven of our panel of our Asian red cell donors showed a discrepancy between the predicted phenotype and the genotype, and this of course is the essence of the discussion today I think. This in an important issue for quality assurance, particularly when it comes to screening cells. Maybe not so much to panel cells, but then one could argue that the work --- is probably worth the effort.

(Slide.)

So should molecular genotyping be used as part of reagent red cell QC or QA? We now know the molecular basis of 28 out of 29 blood group genes. We know the molecular basis of many of these blood group polymorphisms. In fact, some of the criteria that are demanded by our regulatory agencies can only be met by genotype analysis, so perhaps we have to think that way rather than from our traditional approach of, "Well, should we incorporate it because we can?" Well, maybe we should look at the rules and what is expected of us to begin with. Of course the argument that we used for a long time is that antisera are increasingly more difficult to find and the production cots are rising, and with the introduction of the beadchip in the US and hopefully our blood chip here we are hoping that microarrays are going to be the way of the future. Of course then life will be different because the costs will come down and we can do so much more with one platform.

(Slide.)

The next slide just briefly lists that comprehensive -- as I have discussed, comprehensive profiling of test red cells will aid faster antibody identification. This is certainly true. I think once you have all the information in your test cells certainly for those patients where the antibody specificity -- where the antibody of patients’ serum contain many specificities having a clear idea of what is in your panel cells makes it -- speeds up an investigation considerably and can decrease time delays in obtaining compatible blood; and, as mentioned briefly, high throughput platforms will permit this, will make genotyping affordable.

(Slide.)

So just to end on the people that did the work, because most of this work was done long before I got to Lund. I just put it with words right here. So you can see the Lund people are listed here: Annika, Asa, Elisabet, Pia, and Martin. From Sanquin we would like to thank Martin Tax. Of course thanks to Marion Reid and Greg Halverson who provided Dombrock antisera, and some of the work was sponsored by the Swedish Research Council. So I hope I can participation in the discussion. I want to see how the technology will work. Thanks.

(Applause.)

MS. KOCHMAN: Did anybody have any questions for Jill?

MR. YAZER*: Mark Yazer, University of Pittsburgh. Jill, in spite of the fact that you had some cells on the panel that you thought were double dose DuffyA by really were only single dose, did you find that you were having any clinical consequences from this? Were you missing these antibodies?

DR. STORRY: Sheryl, that is blocking out. I would ask if you could summerize for me.

MS. KOCHMAN: Sure. It was Mark Yazer from University of Pittsburgh. Wanted to know if the fact that because you found one of your screening cells to actually be heterozygous for DuffyA if you had any clinical implications from that.

DR. STORRY: Not that we know of, but then, you know, that is the next step that nobody really wants to take back, to look back. I mean, it is all very well saying, you know, this may have dramatic clinical implications, but I don’t think we or anybody else has actually taken a look at cases that have resolved those particular reagent red cells and noticed whether we have missed anything. So no is the answer to that. We haven’t done that work. We have changed. I mean, it did prompt us to change our reagent red cells from that screening panel, but that is about it so far.

MS. KOCHMAN: Anybody else?

(No response.)

MS. KOCHMAN: Well, thank you again, Jill, for being a good sport in all of this.

DR. STORRY: Well, it has been fun. So, you know, it has been fun. I am sorry about that, but has been quite -- I have quite enjoyed the drama, as usual.

MS. KOCHMAN: Okay. Well, thanks again.

DR. STORRY: Thanks. Bye.

MS. KOCHMAN: Bye.

(Phone call ended with Dr. Storry.)

MS. KOCHMAN: Well, unfortunately today we have gotten ourselves behind, probably partly my fault for getting us started a little late. We are scheduled to have the break following Dr. Karina’s talk, so if you could just be patient and listen to her talk. She is another representative from the New York Blood Center, and I would like to give a little background on why I asked her to make this presentation.

You have heard us mention a few times that the FDA has seen some fatality reports where anti-JKA or anti-JKB have been -- that have been missed have been implicated in the fatality. One of the questions that keeps coming up is isn’t there something we can do to make these antibodies to detect. Can you make super Kidd cells somehow so that we aren’t missing these, and I thought that she might have some interesting perspectives on is there something else we can be doing or should be doing to help us find some of the things we miss.

Applications of Blood Group Antigen Expression Systems for Antibody Detection and Identification
by Karina Yazdanbakhsh, PhD

DR. YAZDANBAKHSH: Thank you to the organizers for inviting me here. So as Sheryl said, the focus of my talk will be slightly different to the other talks given at this workshop. It won’t be on molecular genotyping of a patient and donor samples, but rather applying our knowledge of the molecular basis of blood group antigens in developing reagents that can be used for antibody detection and identification.

(Slide.)

So I don’t need to tell this audience about the definition of a blood group antigen. Suffice to say that these antigenic determinants result from a specific sequence of amino acids that are present in one protein or present in several proteins and/or from those sugar molecules on oligosaccharides that are attached to the red cell surface proteins and lipids.

(Slide.)

As has been said over and over again, the genes encoding the proteins that carry these blood antigens have been commonly sequenced for the most part, and we know their structures as presented on this schematic drawing. It can be single pass proteins like the Kel and the --- or multi-pass proteins such as the Duffy and the Kidd.

(Slide.)

It is not the blood group antigens that cause problems in transfusion medicine, rather the antibodies than can cause the immune haemolysis.

(Slide.)

And to insure safe blood transfusion we have the antigen antibody identification process in place. Current methods rely on using multiple red blood cells, which results in a large number of antigens, and we apply a complex matrix of techniques to identify the clinically significant antibodies in the patient sample.

 

(Slide.)

So again, current methods rely on panels of cells that have got a specific combination of antigens present on them. Again, not for this audience, but we apply different techniques and based on the reactivity panels in a patient’s sample the medical technologist can identify a particular antibody that is present in the sample. In this case it is anti big E, pretty straightforward. However, things can get very hairy when there are a number of other, a number of antibodies that are present such as --- reactivity. All the cells are reactive, and so based just on this pattern you cannot distinguish or identify all the antibodies in this particular sample.

(Slide.)

So the goal of a number of investigators in the field back in the early ‘90s including Marion Reid was can we have system where we express single antigens and --- of our knowledge of the molecular basis of the blood group antigens, and can we develop an object and automated system that can be used for antibody identification. The kind of assays we had in mind was using flow cytometry and some sort of solid base assay such as ELISA for antibody identification. Also since we do know these, the molecular basis of these blood groups, can we use them to make recombinant proteins that can simplify antibody identification processing for such studies as for absorption neutralization studies.

(Slide.)

So this is the basic idea. Here is the red cells, the expressed number of different blood group antigen carrying proteins; and wouldn’t it be great if we could just express a single blood group carrying protein in a given cell line and apply them onto some sort of a solid base assay. In this case it is just showing some sort of an ELISA. So every well will contain a specific line expressing a single blood group carrying protein, and then you come in with your patient sample, add it to these wells, and whatever lights up you can identify that that is your -- that is the particular antibody that is present. Also this doesn’t have to be a cell line expressing recombinant proteins. It could be --- proteins expressed or if we know more and more about the antigenic determinants we can have

--- in these wells. So we have single antigens in each well and then you can just identify the antigens, the underlying antibodies in the patient sample in this way.

(Slide.)

So expression systems are amenable for large-scale production of recombinant proteins right now. The bacterial and the yeast systems are really great. The labs for large-scale production at low cost have a few antigens of interest or your protein or interest requires post-translation or modification such as glycosylation, these two systems are not really the best to go with. Baculovirus allows some limited amount of glycosylation and actually has been used to express soluble forms of blood group carrying proteins. We focused on the mammalian expression system since they do allow glycosylation of the transfected proteins, and there are some cell lines --- that are easy to grow and some even sort of easy to transfect.

The things you need to consider are what kind of a cell line you want to use. Since our interest is red cell antigens, so you want your protein of interest to be -- to look like the naive protein on the red cell membrane. One idea would be to transfect these genes in gene erythroid cell lines that are available, and they are available. However, you run into the problem that for antibody identification you may have some micron reactivity, and that is exactly what we found. So you may want to switch to another species such as mouse or --- cells. However, you have to keep your line if your protein requires glycosylation the mouse and the human are not exactly the same, so you may run into problems there. Also red blood cell lines currently that are available are not that easy to transect. There are some --- lines that are easier to transect, so there are a number of things that you have to keep in mind, and it is always a toss between what is more important and what you are trying to achieve here.

(Slide.)

So what I would like to do today is to show you a couple of examples of the systems that we have used. One is the erythroid expression system and we have used a mouse erythroleukemic cells. These are the MEL cells, and another system is the --- T cells. These are the human embryonic kidney cells. To be able to drive the expression of transfected genes in the case of the MEL we have used this PEV vector which has got human betaglobin locus control region that confers high level expression of the heterologous genes. In the case of the HEK cells, we have used vectors that have the CMV-promoter. These are strong promoters that help to drive the expression of your target gene. --- made both membrane-bound forms of our blood group antigens as well as soluble forms, and in the case of the membrane-bound forms we have used flow cytometry as well as ELISA as the absorption studies to actually show that these can be used and potential used in the clinical lab as well as the soluble forms. We have done antibody neutralization studies.

(Slide.)

So first is the detection by flow cytometry Here is the KEL protein. What we have done is to express the wild type KEL protein in these MEL cells using that EV vector that I told you about. This a transfectant, stable transfectant expressing the wild type protein, and what we are looking at here is by flow cytometry whether the --- expresses the different --- antigens, KPB, JSB, little k. As you can see, the --- is indicative of the reactivity. This is with the red cell, antigen positive red cell, and here is our transfectant being able to detect the anti-KPB JSP and the anti little k at levels that are comparable to the red cell. So actually we have gone, although they are not shown here, but also --- cell lines of the --- antigens to the JPB. Basically JPA, JPC, big J and JSA, so now we have a panel just like our panel of our red cells that we can basically use by flow cytometry to be able to detect the antibodies in a patient sample. We have done those studies.

(Slide.)

Here is the Duffy protein. It has got a pair of

--- antigens, Fy a and Fy b, and we have transfected again. We have got transfectants, stable transfectants expressing the Fy a and the Fy b, and now our Fy a expressing cell line can specifically detect anti-Fy a but not the anti-Fy b antibodies and vice versa. Here again we have a system where we can detect underlying antibodies in a patient’s sample by flow cytometry using these cell lines.

(Slide.)

We have also shown that these transfectants can be detected by ELISA, and this just an example of the kind of readings you can get. So the cell lines have been immobilized on 96 plates and then you come in with the patient sample, and in this case what I am showing is a wild type --- transfectant and the big K transfectant expressing the big K antigen. Basically this the average of the OD values system by ELISA, so it is a columetric assay. You get a reading and it is an average of triplicates where the standard deviation is within 10 percent of each other from well to well and, you know, subtracting the background. These are the kind of numbers you get and then the ratio. Based on the ratio of these values you can make the conclusion that the example has anti big K. Again, as I say, we have done them for a number of transfectants that we have produced in the lab.

(Slide.)

So absorption studies are done in the blood bank to separate mixtures of antibodies, and they aid in the antibody identification process. So red cells that express the specific antigen are incubated with the test serum and the antibodies are absorbed the antigen. However, we have little mutations because red cells carry many antigens and you need multiple rounds of absorptions to be able to phenotypically -- are needed using phenotypically distinct red cells, which could be rare and in short supply.

(Slide.)

So the idea was can we use our transfectants that are expressing single blood carrying proteins for absorption studies, and this is just to show again here is our wild type KEL protein, transfectant --- in our MEL cell lines. What we have here is this particular cell line has completely absorbed that antibody. These are the titers here of the anti little k, Kp b, Js b, and the parental cell line does not, which is just a nice control. Obviously the spectrum doesn’t express the lower incidence antigen and is therefore not capable of absorbing --- anti Kp a. Again here we have our --- tranfectants. They absorb --- specific antibodies in a given sera.

(Slide.)

So neutralization studies are another technique that is used to help in antibody amplification process, and it is specifically used to remove antibodies from antibody mixtures. Basically these inhibition studies are using fluids containing specific soluble blood group antigens. However, there is only a limited number of soluble antigenic substances, which they may dilute our test sera.

(Slide.)

Neutralization studies are usually directed at removing clinically insignificant antibodies, and one of the notoriously difficult antibodies are those against the --- group antigen. These antigens are carried on this protein

--- receptor one, and JoAnn --- and her colleagues nicely showed that recombinant protein --- of this --- receptor one can inhibit and neutralize antibodies in patient serum. So we actually repeated those studies, expressed soluble --- one. In our system we have used a vector that allows the proteins to be tagged so then we can easily purify the proteins and detect them just for quantitation. We have also expressed the different portions of this --- protein, and this a way when we did started these studies. We didn’t know where the antigenic determinants ---, so this was a way of doing --- mapping studies.

(Slide.)

So this is just an example of inhibition studies on patient sera containing one of these anti --- antibodies. A couple of examples of inhibition. Again, soluble --- one can inhibit these two antibodies, so does one of the fragments. This is this long --- repeat D, but not the others, and here is a nice control with anti Yt a where we don’t see any inhibitions, just the control.

(Slide.)

So basically what I have shown today is basically we have expressed several both clinically relevant as well as clinically insignificant blood group antigens at levels comparable to red cells, and these allowed us detect alloantibodies in patient and donor serum by flow cytometry as well as ELISA, and these allow -- they potential for automation, these two techniques. I have also shown that you can do absorption and neutralization of alloantibodies using these recombinant proteins, which will help in antibody detection and identification processes. They will simplify those processes.

(Slide.)

But a word of caution. These are all feasibility studies. We need a lot of work ahead of us. Basically one problem with these cell lines are that after freezing and storing of these expressed cell lines they lose expression. If you keep them a long time in culture they lose expression. So we really need to come up with improve systems, and they are out there to improve -- that would allow stably expressed cell lines in the sense that it is going to be there. It doesn’t matter if you freeze them, thaw them.

It is really important to understand expression requirements for blood group antigens. What we have had problems with is trying to express the RH proteins at levels comparable to red cells. We have been able express the RH antigen D just by using the Rh50 body in the 293 cell line, as well I was talking to Connie. She has expressed them in ---. However to get levels that are comparable to red cells we need to understand what are the requirements there. As I said also, there are a number of groups that also have expressed the --- 562 cell lines, but however the problem there is that, as I said, we have a lot of background using K562 cell lines for antibody detection we wanted to. So that is a problem there.

If we want automation we need to do epitope mapping studies. Really, you know, identify like epitope

--- level what are these antigens so that we can apply them in some sort of a solid base assay. So epitope mapping studies are required, and that is it. Thank you.

(Applause.)

MS. KOCHMAN: As I mentioned before, we are running a bit behind today; s does anybody object to just taking a 15-minute break instead of a 30-minute break to try to catch up a little? Okay. So if you could be back in 15 minutes. Thanks.

(A break was taken at 10:23 a.m.)

MS. KOCHMAN: So we are going to move on to yet another slight shift in gears maybe. We have Sandra Nance from the American Red Cross. She is the Director of the Immunohematology Reference Laboratories and I have learned is also an adjunct faculty at University of Pennsylvania. So we will have Sandra talk to us.

Donor Genotyping
by Sandra J. Nance, MS, MT(ASCP)SBB

DR. NANCE: Okay. So to begin, this is a topic that I am not too familiar with as far as some of the things that I am going to be talking about, but Sheryl asked me to try and cover it, so I will. I just want us to remember that molecular is just a different method as it goes through --- genotyping with an existing sort of test name, a result antigen typing, and with the possible exception of the --- of a testing of single embryo which might be considered differently, and I will cover that at the middle of the talk.

(Slide.)

So Sheryl very nicely gave me some things to cover, and more questions than answered. Will cost provide widespread adoption? What about DNA storage and security? And is one test enough, or will repeat testing be required? I guess I know a lot about the one and the three, but not a whole lot about two, so I had to do a little bit of research.

(Slide.)

I did take a number of quotes that I found in the literature because I thought they represented things that you might want to think about, and I hope to instead of talking science here I hope to really just kind of open your mind about operational aspects and perhaps some other things you might have been wrestling with through our discussions of yesterday and today. So the transfusion medicine specialist of the future may have their disposal molecular techniques to detect red cell genotypes.

(Slide.)

Under the category will cost prevent widespread adoption, I looked at the "as is" situation and I am doing a lot of project management these days, so I always look at the as-is and then look at the "to be" and find out how you are going to get there. So the high cost of labor for testing and labeling in a fading labor market is familiar to all of us.

Many of the labs do perform two types on new donors and compare. If you have a repeat donor you may compare that with antigen typing on previous donations, thus only doing one repeat test. We do know that Rh testing on the ProVue or Galileo may be automated for those facilities who have it, and that could be a test of record, but we do know that all other specificities and methods at least in the US are manual for the test of record.

We also have the ability to have automated prescreening methods with the PK7200 and the ProVue from Ortho, which limits the amount of manual screening need to identify antigen-negative donors. Thus you will be --- a very productive manual testing technique. We also have talked about the limited availability of licensed antisera. Keep in our minds about lows and highs, which are not really commercially available, and we have as the "as is" perform minimal molecular matching for patients with complex serology and inherited mutations.

(Slide.)

What is the labor situation? Well, I have some data from the Red Cross I thought I would share. We have approximately 300 budgeted FTEs across the US in our 36 regional locations. We have approximately a 10 percent vacancy rate, so that means minus 30 people across the whole system. We do know that time to hire is variable in many different labor markets, but one to six months was the average; and this was one point aspect in time in March of ‘06, so really just a point in time. Time to train is three to six months, but to get fully experienced in the IRL most folks would view that as two years.

(Slide.)

I wanted to tell you a little bit more information about the American Rare Donor Program, seeing how molecular typing could really help us out there. In looking at our 35,000 active donors in the American Rare Donor Program, about 90 percent of them phenotype rare, which means that they are negative for one antigen in each of five systems. Under 10 percent are high frequency antigen-negative rare, so obviously that is a place we need to work on; and IgAs are just barely a line here, so there are very few of those. So there is a lot of opportunities here I think.

(Slide.)

Keeping in mind that, you know, we may talk about localizing this molecular testing in different centers and maybe few and maybe a lot, but I wanted to point out that, while these contribute many of the high prevalence antigen-negative rares to the American Rare Donor Program, these are extremely important numbers over at the side as well. So everyone that contributes these high prevalence antigen-negatives are really important to us.

(Slide.)

To show you some data and why I made the comment I did yesterday, data from 2004, ‘4, and ‘5. The total requests that we have gotten into the system, the percent that are filled, really not much of a change here. The number of partially filled means that they asked for four and maybe got two, and we have made a change in that which I am really happy about. However, the percent not filled, and this represents actual patients not getting the blood they need, half of which probably die. This number needs to be zero, so I am really looking for some energy here in what molecular testing may bring to us in that area.

In looking at just data for the last year and a half starting in January of 2005 to June of 2006, I separated out the sickle cell disease patients. So this is a subset of this number here, and just to point out that I don’t think that there are much differences between these percentage numbers as far as whether it is filled, partially filled, or not filled, but we do need to keep our eye on that.

(Slide.)

We do not fill of the requests that we get of course, as you know. So these are the imports for the American Red Donor Program in the last two years, 2004, 2005, that we got from other countries and just to look at the phenotypes to show you what we need.

(Slide.)

So will costs prevent widespread adoption? Well, the "to be" -- which is we looked at the "as is" and now we are looking at the "to be" -- I think we are going to still see continued shrinking of the labor availability. We may have some light at the end of the tunnel that is not an oncoming train with phage display technology, and Dr. Siegel will talk about that later. We will have availability of some semi-automated molecular testing. I think that will help us. We may have the concomitant activity of increased expense for reagents, and instruments and supplies remains to be seen for us in the US, but it does expand our molecular matching possibilities, and electronic antigen-negative labeling will be coming which will decrease the time resources for testing and for labeling.

(Slide.)

It does remain to be seen the cost of the semi-automated molecular testing. Although appropriate pricing might be equal to the current resource that we expend with our manual tests for limited donor testing, so I am looking forward to that. I think it is a given that it won’t be all donors collected unless we are able to have a very inexpensive or we are able to do a single test event on each donor. It means that we don’t repeat them on each next donation, and widespread donor testing can likely only be achieved with totally manual methods if we get some funding. I don’t see that to be widespread donor testing with molecular methods based on the resource that is needed.

(Slide.)

So I designed a little possible workflow. It is the opposite of the approach we heard yesterday, but you may choose whichever you want. What I took was a repeat donor from the South African experience and that they find that it is much more cost efficient to start testing for serology antigen-negative blood with repeat donors because there is a certain number of fallout rate from first-time donors.

So in the event that the serologic antigen typing is known, and here we open up ourselves for look-back. So if it is yes and it is African American -- I divided it between African American and not African American. If it is African American we may want to do DNA looking at the Rh and highs. If it is not African American we might want to do the commons. Here is where we open ourselves up for a look back if the result of DNA may be different that the serologic type we already know. Then my thought is that at the time of the event of the molecular testing on that donation we do a serologic type for negatives and resolve the issues, and the same on the other side.

It is a little bit happier for us in the Blood Center of course is the serologic type is not known because we don’t have look-back opportunities. So we would do DNA on the commons, if it was African American we would DNA, do Rh and highs, and then serologically type the negatives. So that is just my operational look at a potential workflow that we might find achievable.

(Slide.)

David Anstee made a great remark in one of his reviews for Transfusion that I wanted to share with you. Molecular typing for ABO and Rh is not a simple matter of identifying one or two SNPs. We talked about that. The genetic basis of both antigen systems is complex and will require the careful design of multiple reactions before a bullet-proof molecular tying system suitable for all racial groups is achieved. So we could like have combined all two hours that we talked about that into this little slide.

(Slide.)

Okay. Here is what I don’t know about. So what about DNA storage and security, and I returned to the web of course to try to help me with this. But the things I thought about were ethical concerns, and my thoughts are that antigen typing info is the same as serology. So I don’t think we need to be so concerned about that. Storing DNA I think people fear it is going to be used for other things. Potential for distribution for samples of interest and/or proficiencies may be something else we need to think about, and then notification of donor if it is important to the health or genetic planning. The things I thought might fall under there are hemoglobin S and if we HPA 1A screening and then we do an antibody test on the women then that might also be something that would be important to the health or genetic planning.

(Slide.)

So borrowing from some of our friends in HLA, there is something on inspector checklist with regard to storage of the DNA. Just to insure that the samples are stored under conditions that preserve the integrity of the sample for the things that will be tested, and the inspectors are actually supposed to test for -- I mean check for written criteria for short-term and long-term storage of DNA.

(Slide.)

In looking at another review that was in a journal by K. Smith who was a diplomat of philosophy and religion, access to genetic testing should be treated the same way as access to new medical procedures and medications. Namely withheld from the general public until proven safe and effective in larger-scale trials, and I think that is the direction we are going.

(Slide.)

There was a taskforce on genetic testing and there was definitions of that, and it is here for you to read. I think that probably the last part is the most interesting. Such purposes include predicting risk of disease, identifying carriers, establishing prenatal, newborn, and carrier screening, as well as testing high-risk families and individuals. So that is what was determined to be the definition of genetic testing.

(Slide.)

So there are three categories that seem to be defined in the literature. Diagnostic, presymptomatic -- and that is where I think this might come in perhaps -- and reproductive decisions. Hemoglobin S, HPA 1A and the D antigen might fall into those.

(Slide.)

So in scope for us at least under consideration, and I would say this is the patient side, not necessarily donor side, really looking at the patients. For hemoglobin S, paternal testing, and prenatal screening; and I think out of scope, obviously subject to other people’s opinions, donor antigen testing.

(Slide.)

In another journal looking mostly at breast cancers, but I thought it was interesting, the DNA test should safeguard the welfare of the person being tested.

(Slide.)

On the Council of Europe, and this was the latest reference I could find, so I bring it to your attention. It is from 1996, 10 years ago. DNA testing may be performed only for health purposes and subjected to appropriate genetic counseling. So I might look to our European colleagues to see if that is still current or they have heard of that.

(Slide.)

Then from the US Congress, genetic testing was defined as the use of specific assays to determine the genetic status of individuals already suspected to be at high risk -- so that knocks out all the donors -- of a particular inherited condition. The term is genetic test, genetic assay, genetic analysis. They are all used interchangeably to mean the actual laboratory examination of samples.

(Slide.)

Genetic screening, which is different from the first, the slide just before, uses the same assays employed for genetic testing, but is distinguished from genetic testing by its target population. So any of our things that might possibly fall in probably fall in here.

(Slide.)

So what are the thoughts about DNA storage and security? There needs to be some standard development on the rules of engagement here I think. Storage of patient and donor samples may be somewhat different. Certainly we should unlink the samples for interest only. We shouldn’t be sending samples around with patients’ names on them, and I would assume that we would always unlink proficiency samples.

(Slide.)

Next topic. Thank God I am done with that one. Is one test enough, or will repeat testing be required? Great question, and I think it hinges on FDA approval of automated platforms and keeping in mind that new discoveries of mutations would have to be then incorporated into the platform, and then accuracy of testing. Proficiency standards, and we are going to have some discussion about standards of detection.

(Slide.)

In another article that I drew some information form, and it has nothing to do with blood grouping, but looking at microarray platforms in other fields. I think that we will have some differences between the European and US platforms in that the measure their expression of the same gene with different precision on a different scale with a different dynamic range. So this has already been known within the microarray field for a while, and if you do look at the two together, which we might do as, you know, blood banks would do, it might become compromised when applied to data generated by another platform. So maybe the two aren’t the same; maybe it is a little bit more like coagulation assays where each different instrument has a different range and a different proficiency value.

(Slide.)

So why test more than once? Well, I think that we have concerns over accuracy, so that would definitely need to be in consideration, realizing that the techniques we have aren’t perfect either. I think we have a concern that serology is different than molecular, so we may need to marry the two, at least for awhile until we get to the "to be" that is, as Dr. Seigel said, 20 to 30 years from now. We do have a lot of concern over the identification of the specimen if it is not totally automated. People make mistakes. We need to have --- ID throughout the whole course of the test if we want to do one test, and I think concern over changes and new discoveries.

(Slide.)

So my thoughts are about those things: So we would need a validated method. If we have a concern we might want to type once with each method per donor if we are able to have --- ID, and if we don’t have a totally automated system we are going to need to type twice. We are probably going to need to type each donation I think. We need to talk about that a bit, and then new discoveries. We might have to change the platform with each new discovery. I did want to point out, thanks to Marion Reid for bringing this up to me, that we do have an SOP for the American Rare Donor Program which applies to accredited labs by the AABB and ARC. So the American Rare Donor Program SOP allows molecular assay results to be used as historic data. Not for labeling the product, but for informing them. Especially with Dombrock it is has been extremely helpful.

(Slide.)

So what about genomic DNA standards? I think we are going to talk about that later. One of the articles that I brought up did show a fundamental problem in some of the things with microarray analysis which had to do with lack of common standards, spotting efficiency, labeling efficiency, transcript representation, and hybridization. So I think those are things that are probably common to the field that we need to apply as we look for blood group.

(Slide.)

In another article out of NIH, calibration standards need to be stable over time, homogenous, withstand shipping and normal storage, and actually contain a reasonable amount of DNA which would be useful. There is not as I know a standard for blood grouping, but there is a standard which regards DNA, so the laboratory shall check its DNA procedures originally or when changes are made to the protocols against appropriate and available NIST standard reference material or standard traceable to an NIST standard. So hopefully we will be looking to develop that in the future.

(Slide.)

So is one test enough or will repeat testing be required? If the molecular type is wrong is it then, as I alluded to yesterday, a limitation of the technique like with the Olympus PK7200, or is it a recall if you type it wrong? Some of the things you already know about.

(Slide.)

The last thing I would like to leave you with is another quote from David Anstee which I think summarizes all of our thoughts. One can envision kits suitable for molecular typing of individual patients being used at the blood bank and electronic interrogation of the blood center database for selection of the most suitable donations available. So he said that last year.

So in conclusion, I think we ought to think about patient testing maybe in the realm of genetic testing. If it is done under physician order I think that we are probably still covered for the interpretation and the counseling of the patient. I think the rest are potentially genetic screening, and it is no different than the techniques we use right now that yield the same interpretations. Thank you.

(Applause.)

MS. KOCHMAN: Now we are going to move to Dr. Dan Bellissimo who is going to talk to us about proficiency testing. He comes from the Blood Center of Wisconsin.

Proficiency Testing for Molecular Assays
by Daniel B. Bellissimo, PhD, FACMG

DR. BELLISSIMO: Okay. I am going to be talking about proficiency testing for molecular assays, and I do work on the College of American Pathologists Biochemical and Molecular Resource Committee. That is a committee that puts together proficiency surveys for molecular genetic testing, and I also work in the QA Lab Practice Committee in the American College of Medical Genetics, so my comments are likely to reflect the work of those groups.

(Slide.)

First of all, I just wanted to make everyone aware that there are multiple guidelines available for molecular genetics, and I think a lot of these standards and practices would apply to DNA testing regardless of where they occur. First of all, there is CLIA. It is not completely strong on the aspect of genetic testing, but does contain a number of guidelines about performance validation for assay development.

Then there is also the American College of Medical Genetics Standards and Guidelines. These guidelines cover areas --- genetics, biochemical genetics, and molecular genetics. They deal a lot with the different kinds of problems people see in molecular type testing, and a lot of them are directed at problems that have been seen over the years in molecular testing.

There are also multiple guidelines from the National Committee on Clinical Lab Standards. There are standards both for molecular genetic testing, DNA sequencing and molecular pathology. The state of New York also has laboratory standards in regards to molecular testing.

Then there is also the CAP, College of American Pathologists Checklist for Molecular Pathology Labs. This actually a checklist that is used to inspect your laboratory if you are going to be CAP certified, which is the gold standard in laboratories performing molecular testing. These checklist items are very much directed at techniques and the things required to have quality testing in molecular pathology laboratories.

Finally there is the American College of Medical Genetics Disease-Specific Guidelines, and these are guidelines that are written for specific disease diseases where there are specific problems occurring in the testing community where the testing is complicated and requires specific professional direction to insure quality testing. I think from Marion Reid’s comment this morning CBBG has recognized the need for such kind of guidelines in the area of blood group testing also.

So these as a whole, these generate kind of the standard of practice. I said, they have a lot of different comments on techniques and controls necessary for techniques. Those are especially true in the ACMG Disease-Specific Guidelines where for example in the CF guidelines they go through the multitude of different assays platforms being used, what type of controls and things should be considered for each type of assay technique.

I will also mention those that the guidelines in regard to microarrays are probably at an early stage. There is a lot in development going on just because of an assay like cystic fibrosis which run a large screening panel, so a lot of guidelines are being developed, and I will discuss some of those later.

(Slide.)

I also wanted to make everyone aware that the ACMG also has guidelines for prenatal molecular genetic testing, and I think a lot of this again applies to what happens in HDN testing. These are just a summary of some of those guidelines. That the mutation status of one or both parents as appropriate be tested in prenatal testing. I think that is a concern in red cell testing where there are a number of varying alleles and it is particularly important to make sure you test the mother to make she doesn’t have a false-positive Rh variant before testing the fetus.

That laboratories should have some kind of followup program to try to monitor the accuracy of their prenatal testing. That is sometimes difficult to do. That laboratories need to find some way to make sure they are doing accurate diagnosis.

The last two comments relate to the problem with maternal cell contamination that can occur in prenatal samples. The basis of these recommendations is that laboratories need to understand how maternal cell contamination would affect their prenatal result. So labs should have methods for assessing the presence and amount of maternal cell contamination. These are typically by using VNTRs and SCRs to do chimeras* analysis, and that the methods should detect the levels of maternal cell contamination that would lead to a diagnostic error. This is typically done using DNA mixing studies, and I just wanted to illustrate that this is important both to consider for the type of technique being used and also the type of mutation that you are trying to detect.

(Slide.)

So this is an example of a --- muscular dystrophy assay, and most of the mutations in this disorder are deletions, and it is a deletion on the X chromosome. What I am showing here is a multiplex test which tests for a number of different exons in the dystophin* gene. In this --- column here is zero percent or is an affected male, and I think you can just see that these -- well, for simplicity just look that that these top two bands are missing and this patient was deleted. So what we do is then dilute that sample with a female’s DNA mimicking a contamination that might occur in a prenatal sample, and the question is at what level does contamination confound your diagnostic result.

For those who have good eyes can see that around five percent contamination with maternal DNA in this test leads you to detect these two bands from the maternal normal chromosome. It could potentially lead you to an incorrect prenatal result. So this would tell you if you were doing such a deletion test that you need very sensitive methods to detect maternal cell contamination, because things below around five percent or below could confound your result, so that would require very sensitive techniques.

(Slide.)

Then I just want to contrast that with another method in another application, and this is a test that mimics contamination in a prenatal KEL genotyping assay. This is an allele-specific technique which is going to differ because there are two reactions per sample. The first one detects the K1, and there is a K1 homozygote and the K2 reaction is negative, and here is K2 homozygote. The K1 reaction is negative and the K2 is positive.

But in this assay there is a PCR reaction specific to detect the paternal allele that the mother does not have. So what was done in this assay was to take a sample that was heterozygous mimicking a heterozygous fetal and then just do serial dilutions out with maternal DNA. That would be K2K2, and ask the question, well, where do we lose the detection of that paternal K1 allele. Actually at one in 64 this band is still clearly evidence, and if you will look closely at one in 128 you can still pick up this K1 band. So these assays are very insensitive to maternal cell contamination, and we have seen a number of prenatal samples that we know through other testing that they are 95 percent maternal DNA and that we are still able to detect the paternal allele.

So that just contrasts the difference between different methods and different mutation types, but it is under the obligation of the laboratory to understand what level of contamination would affect their assay. Laboratories that don’t have the ability to detect contamination would have to send these out if the laboratory did not have -- you know, if the fetus typed the same as the mother and they couldn’t rule out that possibility.

(Slide.)

So I am going to talk a little bit about the ACMG/CAP proficiency testing program. So this is a program that helps to assure good laboratory performance, and the proficiency survey is just one part of that. The CAP MLG survey is the molecular genetic survey. It includes a number of different genetic disorders, but also includes the RHD gene. The purpose of that program then is to, first of all, assure laboratory performance, but to also look at the problems that we see and the results there. See if they are method based, see what kind of interpretive problems are, and then write participant surveys to help laboratories fix those problems. So it is very much trying not to be a punitive thing, but an educational thing to help laboratory performance.

Also to help that laboratory performance as we see, complications in the proficiency testing. That is sort of what triggers the AMC to start thinking about whether disease-specific guidelines are needed in a certain test. If there is a prevalent error occurring in different assay methods, and they have developed these for cystic fibrosis and --- and Huntington’s diseases because of this.

The final part of that program then is laboratory inspections. I mentioned the CAP laboratory inspection checklist which is used to inspect laboratories, but the CAP and ACMG have done a lot of work to make sure people inspecting molecular laboratories are experts in molecular and they have the ability to see if there are problems there. Some of it is getting the data from the proficiency surveys. If the committees are seeing a problem with proficiency that this information gets back down to our inspection crews that go in so they can look at problems there and try to figure out the problem the laboratory is having.

(Slide.)

So proficiency testing programs ought to assess at least three different components of proficiency. One is the preanalytical, and this is the receipt and processing, and I think we have heard people talking about proficiency surveys and seeing clerical results. In some ways we are kind of glad they are not analytical results, but clerical results are also a problem if it may lead to an incorrect result. People are supposed to treat proficiency samples just the same as they do other laboratory samples, but I am sure most people handle them with the utmost care to make sure there are not errors in those. They even do more than they probably do in regular samples. So the fact that there still errors in handling of these samples is concerning.

The other problem is in processing of the sample, which is a very important part of molecular testing. Especially, you know, we are talking about lots of kits being available to purify DNA. I think the impression is I just have to throw blood on this kit and I will get something good out the other end and I am going forward. I think those of use who work with DNA a lot know that it is not necessarily the case, especially in the type of patients, clinical patients that come in who have had transplants and multiple transfusions, that these samples are not normal blood samples. I expect that we will see that in microarrays, that the quality of DNA going in is of the utmost importance to the performance of the chip.

But it s a problem in molecular testing of how to provide sample. Ideally you would provide the exact same sample coming into the laboratory, as a blood, but for rare genetic disorders this is almost impossible to do. So what we have done is built up a good resource of control cell lines, and DNA is provided is the laboratory, and it is the highest quality DNA that we can provide to make sure people do not have problems with quality DNA as they do the proficiency testing. But ideally it would be a blood sample or whatever sample the laboratory would be analyzing in that test.

The next part is the analytical result. This is just the test result, and I think most of the time people spend most of their time making sure their analytical results are correct. Usually this not as much of a problem in a robust assay format, but certainly there can be analytical problems in certain test methods, especially with variants and polymorphisms that may upset the detection of specific mutations that we are trying to detect.

The last part is the post-analytical, and this is the interpretation reporting. This part has become a lot more important in proficiency testing and our committee has spent a lot more time working on it, because I think in the diagnostic kit assay world what we have happening is a lot of the tests that have become big send-out tests like cystic fibrosis and there are kits available what we find is people are able to a lot of times get an analytical result, but they are not able to accurately interpret the result and what that means. So we have actually started grading both the analytical and the interpretive component of these proficiency surveys, because it does little good to get the correct the analytical result and then interpret it incorrectly which still leads to an incorrect clinical action. So that is a very important aspect, especially in complicated testings that not only can people get the result, but they know what it means. I think what we see, again using the cystic fibrosis as an example, which is a complicated genetic test, that we see a number of laboratories offering this because they are able to get a kit, but some of the complicated genetics involves kind of confounds in the interpretive part of the test.

The other important part of this program then is the is the participant summary that is written each proficiency cycle, which is twice a year, which we try to summarize the problems we have seen. Whether any, you know, method-based or sample switch-based, and if there are any suggestions or recommendations in regard to that performance. Many times, as I said, we see specific problems in the surveys, and that leads to a generation of developing guidelines and everything to help laboratories and us redoing our surveys to try to get at that component of the laboratory error.

(Slide.)

So what are some important resources for a proficiency testing program, and I think people have brought these up also for red cell antigens. The most important part is well-characterized quality control materials, of which we have been better at assembling in genetics. It is really important to be able to have materials that you can send out to laboratories to do these proficiency surveys. We also use laboratories to QC our materials. The materials go through quite a bit of testing to makes sure they are what we think they are before they go out. So when a new cell line or DNA sample is put into use in this survey it gets tested by two independent laboratories.

We call this pretesting, to make sure that the mutations are what they are before they get sent out on surveys; and also the company that produces these cell lines and DNAs, when the prepared a new lot to go out for proficiency survey that sample again is sent out to laboratories to test to confirm that that mutation is as it should be. These were put in place because of past errors in production of some of these lines and different laboratories receiving different materials. So there is a lot of different testing going on to make sure these materials are correct.

Finally, you need experts to select these samples, review the data, assess problems, and write participant summaries. Of course it requires lots of administrative support to get this all done.

(Slide.)

But I do want to emphasize the importance of quality control materials. They are important not only for proficiency testing, but for quality control and test development validation. A lot of times what will come out of recommendations, and I am sure the same will be true here, is that lots of different variants you have to worry about and you have to understand how your test method would react each of those variants. That can only be done if people have access to the type of variants and controls they need to do those tests.

(Slide.)

So what are we doing for quality control materials in genetics right now? A lot of these are just laboratory samples that a lot of them are used. We take samples that are unusual and blind them for controls in the future. We use them to send out and do sample exchanges. There is also a big stored -- a big set of cell line controls at the Coriell Cell Respository, and within that one of their sets of cell lines are called a human genetic cell repository and they contain a number of different materials with different genetic mutations. You can go to that site and search for different things. You do have to be careful in knowing which of these materials have been qualified or not. A lot samples are sent into them, and they immortalize them and make cell lines. Part of the process that is going on with those is that laboratories are qualifying those materials so you can be assured again that the mutations present there are what you think they are.

(Slide.)

The other part of that is something that is called the GTQC, which is a Genetic Testing Quality Materials Program. This was a program done by a number of different people, but Lisa Kalman at the CDC was a coordinator of it. The idea was to help the genetic test communities obtain appropriate and qualified QC materials and to facilitate any kind of information exchange that had to go on between the people to get these materials, which essentially was a patient sample into a laboratory that would immortalize it and then develop cell lines, and to coordinate all the -- you know, so coordinating all those efforts to collect and distribute and test. A lot of testing goes into these materials so they are qualified for use in testing. They have done a really good job --- so far putting together available materials.

(Slide.)

I just wanted to point out what they did in each of these areas. Fragile X and Huntington’s disease are two disease with are caused by tri-nucleotide repeat disorders, so the diagnosis of a disease depends on the size of this repeat within these people’s genes. So it very important to have accurate sizing of these genes in order to give the correct clinical result; and what the GTQC did was find samples that were right at the borderline of people being uneffected and effected, so these would be great controls to be in molecular assays so that the laboratory could assure that what they were testing and measuring against these standards. So they have put together some great size standards for those two disorders.

For cystic fibrosis they pulled together a lot of different rare mutations that are in the panels now for testing, and a lot of those again were very hard to come by. Certain individuals have them. Now they are all available.

They have put together one of the new realms of microarrays that will be coming out, are panels to test for common mutations in the Ashkenazi Jewish population. There are certain disorders that are more common there, and again there was a set of control materials needed so everyone could quality control their assays, and they have also put together pharmacogenomic markers. As I said, I just see this as a program that is growing.

(Slide.)

I think the need for controls in the blood group system is much similar to what is going on here, especially as we move to chips. Those types of control materials can be immortalized cell lines, DNA. I mean, there is also possibilities of even this whole genome amplification, clone controls, and then also synthetic controls which I will mention a little bit more about in a second.

(Slide.)

So controls for a multiplex or chip-based assay I think are a little bit different. Genomic controls are not ideal in this multiplex testing mainly because it is very difficult to use a genomic control for every different polymorphism that would be on a chip. Our recommendation right now in the cystic fibrosis assay in that regard is to rotate the genomic control so each assay we are running a different genomic control, different mutation control through the assay because it really is not possible to run them all at once.

As this complexity continues to increase I think synthetic controls are going to become more important tools for our use, and I just -- these are just some of the synthetic controls that are available for cystic fibrosis. If you are not aware of why this test has become so common in the United States, it had to do with the recommendation made a few years ago by the American College of Medical Genetics and the American College of Obstetrics that women of childbearing age or are pregnant be offered testing for cystic fibrosis. So this lead to huge increases in the screening for mutations. The ACMG came out with a recommendation that, right now we have got 23 different mutations, the most common mutations be tested in the population, but actual test platforms go all the way up to 70 to 90 mutations that different people are testing.

So people have started to try to work on synthetic controls to help control these kind of bead array platforms. One is made by the Maine Molecular Quality Control, Incorporated. I will show you a picture of what they did in just a second. It is interesting because it is a synthetic DNA that contains 38 CF mutations, and they have suspended it in a blood-like matrix. So it looks like a blood, but it is not, but it goes right through your extraction procedure just like a blood sample would. So again, a good property of a control, because your extraction process will be evaluated also when using this type of control. AcroMetrix also has a synthetic DNA that contains CF mutations, and then I will mention the synthetic oligonucleotide mixture that Sacred Heart Medical in Spokane came up with.

(Slide.)

So this is what Maine Molecular did to make a cystic fibrosis control. They basically had this huge plasmid DNA of about 20,000 base pairs, and it has a backbone pretty much of the CF gene. The CF gene has 24 exons, and you can see all those 24 exons are present on this. What they have done is make a synthetic construct that each of these exons and the different mutations in cystic fibrosis are incorporated, and they have a couple of different of these plasmids that put different combinations of all the cystic fibrosis mutations in them. So this is the control that has been put in the blood matrix, and you extract it and test it. It is a way to create homozygotes, heterozygotes, and all different kinds of things by using these things in different combinations.

(Slide.)

The other method that is being out are oligonucleotide base controls, and so basically the way this works is you have a genomic DNA that has a mutation and you create a long synthetic oligonucleotide that spans this region of the mutation all around it. You tag it with two common primers, two common sequences, and this is you would do this for each mutation that you wanted. But they would all have these common sequences at the end, and then you could just take all these sequences and amplify them with these two sets of primers and create more of them. So you creating very little, small DNA segments that all contains these little mutations.

This was a system developed by Dr. Bejjani at Washington State University. The difference between this control is because this is such a short region this would not contain the normal primer binding sites that you would use to amplify this DNA construct. It would be able to be put in your detection system for like an allele-specific extension to bind to your array, but it wouldn’t be an amplification control. But that is the difference, but it would be a way of testing that all your different SNPs on the chips would be detected.

(Slide.)

So proficiency programs for red blood cell antigens right now, there is the CAP survey which includes RHD only so it is very limited. There is the ISBT workshop, and people have talked about the multiple antigen systems that were tested there. There was sample exchange going on, and as I said I have started this program with Marion Reid quite some time ago. I didn’t realize that it had gone international, but that was basically sample exchange between laboratories, and our laboratory also uses our in-house comparison to serology which gets done on all the blood samples.

(Slide.)

The CAP survey, there was very limited participation in this survey. There is only nine or 10 participants. The performance has been excellent, but we haven’t done any challenges on variants. About half the laboratories in there are testing for the pseudo gene, and we need to identify better quality control materials in the RHD area to test people’s performance on variants.

Expansion of the survey has been a little limited because the low participation makes it hard to break this out and expand it, but I have a feeling some of the limited participation is because this is grouped in with a bunch of genetic disorders, and I have a feeling most transfusion labs are not testing for fragile X and cystic fibrosis and all those kinds of things so don’t want to pay for the survey just to do RHD. So that may be something that has to be looked at in the future.

(Slide.)

We have already talked a little bit about the ISBT workshop and what was seen there. I will just point out that it is important that the number of participants has risen quite a bit from 2004 to 2006.

(Slide.)

In regards to performance, I think the first year had an error rate of about five percent, and it dropped to one percent in 2005. Christine talked about the results in 2006, which looked like it went up, but it was mainly due to a couple of laboratories. But I think the important part of that workshop was the recommendation that they made that said use of adequate controls and testing for variant alleles, and I think this just goes back to what we discussed as the importance of quality control materials being available and also standard practice guidelines. That basically I think covers all the types of issues that we are seeing in this workshop.

(Slide.)

So in summary, proficiency testing, it tests the ability to accurate determine and interpret a test result, and the quality control materials are really important for this proficiency test development and validation, but I don’t think they are widely available. I know groups have assembled samples, but it would be good to have some kind of collection fairly easily available, and I think something has to be put together to make that all happen. Finally, the availability of a proficiency testing survey in the US is I think limited by the CAP survey and what Marion has put together. Okay. Thank you very much.

(Applause.)

MS. KOCHMAN: Our last presentation before lunch is Dr. Don Siegel from the University of Pennsylvania, and he -- we are really shifting gears here because we are going to talk about phage display technology and maybe getting back to serology instead of getting away from serology.

Overcoming Limitations in Current Pre-Transfusion Compatibility Testing Methods Using Phage Display
by Don L. Siegel, PhD, MD

DR. SIEGEL: Okay. Thanks very much for inviting me here. What I wanted to start out by saying is one of the issues that came up yesterday which I kind of raised is what we think blood banks will look like in the future, and a number of people have said that some aspects may still require serological technique. One of the questions that I have is not just what serology will continue to contribute, but what would the lab look like in terms of its technology. Would the serology still be done using agglutination? So you would have half of your lab would be a genetic lab, and half would be a conventional serological lab? Or is there some way of combining the platforms together so that whether you are trying to detect genes in a person or proteins in a person with antibodies that the readout could still actually be combined together in the same kind of platform such as a microarray? So this seems kind of strange as to how a microarray could be involved in serology, but that is what I am going to try to get across this morning before lunch.

(Slide.)

So the outline of my talk is first I am going to just review some of the drawbacks of current pre-transfusion testing methods, and then I will give an overview very briefly of phage display and then how phage display can create conventional agglutination-based antibody reagents, and then how I think it can be used to create some novel what I can refer to as genetic-based antibody reagents.

(Slide.)

We weren’t asked to do this as speakers, but I just want to mention that in the effort of full disclosure there is a company called Pheno Tech which was founded by the University of Pennsylvania, and many of the technologies that I will be talking about in my talk have been licensed by this company and I have an equity interest in this company. So I just wanted to mention that.

(Slide.)

So if I put on my medical director hat for second, this is what has been going on at my hospital. This is if you look at the work in the blood bank as a function of the number of types and screens we do, this is from just a few years ago where it was about 30-something-thousand. We are up to 80,000 types and screens last year, and this is a reflection of increased surgery, many other types of programs that require a lot of blood like --- programs, --- programs, and of course the labs and the blood bank don’t really receive any kind of support to deal with this increase.

So this blue line is actually the number of FTE positions that we have, and there is no reason to ask for additional ones because we can’t fill them. So the green line here are the number of filled positions. So this represents the great need for automation and also methods that don’t cost as much. Reagents have doubled their price in recent years twice, and so these are major issues for the hospital side of things.

(Slide.)

So just taking a bird’s eye view and summarizing sort of what we have been saying yesterday and today. Any kind of method is going to need reagents and some kind of method in which to use them. Currently the reagents comprise red cell antibodies, anti-human globulin, reagent red cells, and associated other supplies. The methods currently used, serologically used, agglutination or some variation on that as the readout.

(Slide.)

So as we have talked about over the past couple of days, some of the drawbacks is the expense and in some cases the scarcity of antibody reagents, and that the method isn’t practical for performing extending phenotyping on a routine basis. So sort of the standard of care is that you type units for A, B, and D, you type patients for A, B, and D; and you match them up, and you don’t worry about anything else not matching until there is something showing up as a reaction, as a consequence of not having matched before.

So in other words, we actually have been practicing a reactive type of transfusion medicine rather than a proactive type, and that is basically because of the limitations of the technology. It is impractical to try to completely phenotype serologically all units of blood and all patients and match them up using current methods.

So medically the consequence of this is first delayed hemolytic transfusion reactions, which as many of you know are not typically fatal. They can be, but that is not really the main problem medically. Probably the main consequence is just a gradual destruction of the transfused unit so the patient isn’t getting the benefit of transfusion. They may require another transfusion to make up for it, and that has all the associated issues.

But I think that another medical issue really is that this whole process that we have been practicing, it creates a delay in providing blood to not just a patient who has lots of alloantibodies, but to every patient, because that one patient slows up everything else. So if a patient has because of not getting fully matched blood a patient has a positive screen, that buys another hour or so to perform an antibody identification. It might not be an hour. It could be hours. It could be more than a day. Then you need to identify antigen-negative units on the spot. They are not labeled because they have not been fully phenotyped ahead of time, so you need to pull them out.

Sometimes you can’t find them in your own hospital, so you need to get your blood supplier to find them for you. That takes time. It takes time to ship them to your hospital. Then on top of everything else then you need to perform a full cross match versus an instantaneous computer cross match that would be able to take place if the screen was negative.

I think, you know, the consequence financially of all of this is that when we look at it about 55 percent of all of the testing costs are spent working up about 15 percent of the patients, you know, that wind up having these positive antibody screens. So clearly there is a big financial savings and a medical benefit if you can better match blood to patients.

(Slide.)

So one of the reasons why that is not easily possible is what we talked about yesterday, which is the expense or shortage of many antibodies. So the first thing I want to talk about is just how you can make antibodies using -- for conventional type reactions using alternative methods such as phage display. So many of you are familiar with hybridoma technology where a mouse has been immunized with antigen. The mouse is killed. The spleen is taken out. The spleen cells are immortalized, put into culture, put into many, many plates over a period of a week or two. Each one of 10,000 wells is examined for the presence of a clone making an antibody of interest. Those wells are identified and subcloned, and then you end up having a hybridoma cell line.

So this has been an incredible advance in diagnostic medicine. A couple of people got a Nobel prize for figuring this out, so it is hard to knock it. But in transfusion medicine there are certain limitations. Well, in any application it is labor intensive and expensive. Fairly inefficient because you are screening thousands and thousands of wells to only find a few positive clones.

You get what you get. So if you wanted an IgM antibody or an IgG antibody essentially unless you do something particularly out of the ordinary here you are going to wind up getting cells that make whatever they make, and hopefully you will get what you want.

The other thing is that the antibodies aren’t human. Though we don’t really care about making human antibodies as you would when you wanted to make a therapeutic antibody that you can infuse into a person, as many of you know animals like mice don’t make antibodies to a lot of these clinically-significant antigens such as the Rh antigens and in some of the other clinical-significant alloantigens. The reasons why are probably just because if you immunize a mouse with human red cells and the mouse is looking at the D protein it sees so many differences from itself that the antibodies it makes really can’t differentiate D from C or E for example. It makes more generic type antibodies like an Rh17 or an Rh29 type antibody. That may be the reason. But in any case, it has really been necessary to try to immortalize human lymphocytes from patients who make antibodies to get these monoclonal antibodies, and the process for doing that have certain drawbacks which get into the comments that were made yesterday, which is why the monoclonal antibodies that we have now may not be ideal.

(Slide.)

So one way of immortalizing human lymphocytes is using the Epstein Barr virus transforming approach, which is a fairly inefficient approach. There is no good fusion partner for human lymphocytes, so trying to make hybridomas or what they call heterohybridomas by fusing human cells with mouse myeloma cell lines has a very low fusion frequency. There is a decline in antibody production and growth when you get one of these cell lines, and often they are very unstable and there is a progressive loss of human chromosomes, so it is not a very efficient process. Of course there are many of these cell lines, and they make some of the type reagents that we have, such as the anti-Rh monoclonals are all make in one or a combination of these two methods.

(Slide.)

So what phage display actually allows you to do is do something that gets around a lot of these problems and then actually could allow you to be more selective in the kind of monoclonal you have to get around some of the issues that were mentioned yesterday in terms of the quality of monoclonals.

If you would just thinking for a second sort of a science fiction picture here, if antibodies in serum where actually physically connected to their DNA then making monoclonal antibodies would be pretty simple because if these orange antibodies in the corner -- okay. If you immunized an animal or you had a person and you took their serum and their antibodies were connected to DNA, then if these were antibodies against a specificity you wanted, you could take the serum, absorb out the specificity you wanted, either on the antigen that is in the well of a plate or against a cell like a red cell, elute the antibodies out. Though the elution would destroy the antibody, you would have the DNA here which you could zap into some kind of cell which would see the DNA and start making more of that antibody. If this is the way life was, then if would be very easy to make monoclonal antibodies.

(Slide.)

What phage display attempts to do is recreate this in the laboratory, and the whole thing centers on this guy here named George Smith who in the mid ‘80s had this idea where if he took filamentous bacteriophage, which is an innocuous kind of phage that infects bacteria -- it is filamentous because it is long, and like any other virus particle there is nucleic acid in the middle and proteins around it. He thought, "What if I took DNA that encoded some irrelevant polypeptide and if I cloned it into the DNA or the particle just in front of the DNA that happens to encode this co-protein of the virus? What would happen?" What he found is what he predicted, is that the bacteria and the phage don’t really care if you did that, and what it does is it makes the co-protein a fusion protein of the protein encoded by this exogenous DNA with the DNA for making this protein.

So what he actually accomplished here was the linking together of the phenotype of a protein with the genotype of a protein. So essentially if this was an antibody then he has an antibody that is physically connected to the sequence of DNA that is required to make that antibody. Sort of like having serum physically connected to its DNA.

(Slide.)

Over a period of five or six years what developed was the idea that you could actually take B cells from an animal or a person, peripheral blood lymphocytes from a human, and let’s say this making anti-D or anti-Kel or whatever. You could take this material, extract the RNA, carry out a series of PCR reactions with degenerate primers, and in a couple of weeks of work create a phage display library where each particular expresses a different antibody on its surface but inside has a unique piece of DNA that encodes that particular antibody.

(Slide.)

And so if you take the library and you pan it in a well with antigen you will absorb out the penguins. I guess that is what these things look like, but you absorb out the phage that have an antibody that binds. You elute that out, and although the acid that you might elute with would destroy this antibody here, break this bond, it doesn’t matter because phage itself is resistant to this elusion. So this thing will infect bacteria and this DNA will get in the bacteria, and the bacteria will make more of these particles. This thing is called a round of panning, and you take this stuff and then do it again. After about two or three rounds essentially all of these particles you get have antibodies against the thing you panned against.

(Slide.)

So just to tell you what is actually on the tip of this, of the particle, it can either be -- if this is a regular-looking antibody it can either be just the variable region connected as one protein, the variable region of the light chain and the heavy chain; or it can actually be a Fab fragment where the bacteria actually make the FD fragment of the heavy chain and the complete light chain and assemble a

--- bond in the bacteria itself, and that winds up being expressed on the phage. In either case, inside here is the DNA to encode this or the DNA that encodes this.

(Slide.)

So to summarize this process, the conventional approach for making monoclonals would be to take B cells and try to immortalize these cells in wells. The phage approach is to take the same cells, but break them open and use molecular methods to create these libraries from which you can isolate the relevant antibody and its DNA.

(Slide.)

So some of the advantages is that it doesn’t rely on immortalization. You can adapt it to make antibodies from any species, and these species all you need to know is just what PCR primers to use to amplify antibodies. It is RNA based, so you can get access to all B cell compartments. So plasma cells are rich in RNA, so their antibodies are represented in phage libraries, whereas the hybridoma technique and EBV will not immortalize a plasma cell, which is a consequence of why conventional methods for making monoclonal antibodies in blood typing haven’t been as robust as one would want them to be. You can use just IgG primers if you want to get just IgG antibodies out, which have higher affinity than IgMs. The whole process is very streamlined and rapid.

It can take a month to do one of these experiments, and the antibodies themselves can be stored as bacterial stocks, as phage as particles in the refrigerator, and the phage particles themselves are capable of self-replication. So the antibodies themselves can make more of themselves. When you run out you just take a speck of them, add it to some e. coli, and then by the next morning you have a flask of antibody.

(Slide.)

So the way we have used this --. and I have presented this before. Many of you have probably seen this. We have made antibody phage libraries, in this case from an individual who makes anti-D. It is selected on a Rh positive cell sort of as though you were doing an absorption technique in the blood bank. The process is a little more involved the way we do it. We try to do a negative absorption with Rh negative cells first, or actually at the same time using some procedure I won’t get into, but a way of sort of just having the antibodies against in this case Rh just captured on these cells, having other cells that go about other things. You then elute using the same elution acid glycine you use in the blood bank. You take this stuff, neutralize the acid, and infect e. coli and grow the thing up, and that is how you can do panning on intact cells.

(Slide.)

In a typical experiment, this is from an anti-D, we got over a million anti-D clones from staring with one of these libraries that originate from 28 mils of peripheral blood from an individual. Just looking at 83 out of over a million clones there were 53 different anti-D antibodies.

(Slide.)

How can you work with antibodies or even show that they bind to what you think? So this is your typical indirect agglutination reaction where you would incubate red cells with an antibody wash and then add a --- agent and get agglutination. So similarly what I showed you could do is take red cells and incubate it with these phage particles and then wash, and then add a commercially-available anti-phage antibody and they will agglutinate.

(Slide.)

So this is just in a microplate showing Rh negative cells, Rh positive cells with different dilutions of some of this phage; and this is a positive agglutination reaction, and this is negative. You can see the titers out in this assay quite far. I will say a little more about that in a second.

(Slide.)

But basically this is an electron micrograph of human red cell, and these are the particles I am talking about, and these have anti-RHD on the tip which is a fusion protein to that co-protein which is only located at the tip. You can see that they are actually very large, which provides a lot of ability for secondary reagents to cross-link them, which is why it is a very, very sensitive agglutination reaction.

(Slide.)

So, for example, this is a typical gel card that many of you are familiar with, and if you take one of the gel cards there, there is just a buffer card, and you do what I did, which is to pipette in some anti-phage antibody into the gel card instead of --- reagent. Then you can show that these phage particles will work in the gel card. So this shows the sensitivity of it. If you times 10 ⁷ that this is how many phage are added to -- this impairs and these have Rh negative cells or Rh positive cells, and how many red cells are actually added using the typical recipe with the cards, which is 1.6 times about 10 ⁷ to the red cells. Then you can see that the sensitivity is to about 13 antibodies per red cell. So you need very few antibodies to get a positive reaction in this type of an agglutination reaction. The consequence of this is that one liter of the phage can make enough reagent to do this for to type about 500,000 units of blood, and it would only cost about a dollar or two for the bacterial media. So it is a very inexpensive way of making reagents.

(Slide.)

From that experiment, as you can see from this and some other publications, we got many different D epitope specificities, and you can actually design panning methods so that can select for particular epitope specificities depending on how you design your pan.

(Slide.)

One feature of the system is if what you want to do, you can take the -- here is a phage particle, here is an antibody sequence for an anti-D. What you can do is you can take this sequence out and put it into a different kind of plasmid which would go into a --- cell or a 293 T cell, some kind of --- cell, which would then actually put on a complete FC domain and make it into a bivalent conventional antibody which is in this example I did, and it will function in any conventional agglutination reaction with conventional anti-human globulin. So you can to it in a tube, you can do it in a microplate, and this is with a conventional Coombs gel card just putting this material here with red cells just doing a regular typing. So this stuff you can make is indistinguishable from any other kind of monoclonal.

(Slide.)

So most of the experiments along these lines have been proof of principle studies. So to date there has been very limited number of individuals who have used this to produce red cell phage antibodies. There have been hundreds of other kinds of specificities. These are the ones that you can find in the literature. We recently got funding through an NIH grant, STTR grant, to basically isolate monoclonal --- to a whole bunch of other important alloantigens including these and some others, which will probably take place over the next couple of years. We are funded to do that.

(Slide.)

So to summarize my talk so far, so the process I have described is where you would start with peripheral blood lymphocytes. You would make a library. You would paint it on red cells, and you would get antigen-specific phage particles which you could retain as phage displayed antibodies and use in conventional agglutination assays, but use an anti-N13 type Coombs reagent. Or you can convert them to conventional IgM antibodies and use for direct agglutination or convert to IgG, and then of course if it is IgG you would use it with a conventional Coombs reagent.

So that is sort of how you could use phage display to make conventional reagents to use in conventional assays to get around some of the problems that we have with monoclonals that are currently available. But what I just want to finish off with is sort of a variant on this.

(Slide.)

Which is thee was a paper that came out a few years ago from my institution which I thought was kind of interesting. What they talked about here was taking an antibody and using a gluteraldehyde* chemical coupling a piece of DNA to the antibody. The reason that they did this and what they show in this paper is that you did ELISAs with this if you would add this to a well to detect something instead of adding a secondary enzyme conjugated antibody as in a tradition ELISA, instead you could do some kind of molecular assay that would PCR something off of this. Or in the case of what they did here was to actually use T7 RNA polymerase and make RNA off of this here and detect the nucleic acid material. The reason why they suggested doing this is because the sensitivity of this got down to being equivalent to radioactivity, as if the antibodies were labeled with a radio isotope.

(Slide.)

So what it lead me to realize is that these phage particles have DNA in them and they are physically connected, and I didn’t have to do that. They came out of the bacteria that way. That is actually how they are made. They have the DNA inside of the particle that has an antibody on the tip. So it raised the question in mind as to whether you could detect the binding of one of these phage antibodies to a cell by using a nucleic acid method, and why would you want to do that. So you would imagine it would be very, very sensitive. You would require minute amounts of material. It might be more amenable to automation, plus you could think how it could allow you to actually multiplex serological typing reactions because you could have different specificities, each with a different kind of DNA tag inside the particle.

Now inside the particle are the actual sequences of the antibodies that are displayed, and those are unique. But you could actually put in any kind of tag you wanted into the DNA, the phage, anything that your DNA detection scheme could allow you to discriminate, something that might be on a microarray for example.

(Slide.)

So what I kind of called this was phenotyping by reagent genotyping. So the idea is that you are genotyping the reagent, not the person from whom the red cells came.

(Slide.)

So to show the feasibility of this, here is a phage antibody that has D on the surface, and I put in some arbitrary piece of DNA. I call it a tag here, and here is some. By these arrows indicate PCR primers, and if you just take a particle like this and throw it into a PCR reaction, and using real-time PCR and using cybergreen dye which inter-collates into double stranded DNA as the PCR products form, you see what happens after a certain number of cycles. You begin to get lots of these tags, and then if you tell the machine at this point to lower the temperature down to 64 degrees, which is what the axis here is, temperature, and slowly raise the temperature what happens is at a characteristic temperature based on the melting point of this tag the cybergreen falls out of the PCR product because the PCR product has melted.

So the fluorescence decreases, and if you have a computer replot this data as a negative first derivative of this curve, it just allows you to see a very sharp peak here where the slop is zero basically, and that identifies a very unique melting temperature for this head. So this process takes about 20 cycles, about 10 to 15 minutes to actually complete this process.

(Slide.)

So if you take an anti-D phage particle and you add it to by agglutination it doesn’t agglutinate D negative cells. It does agglutinate D positive cells, and you do this reaction. You see that you get a very strong peak at the characteristic melting temperature of this tag. So in other words, this process can tell you whether the antibody bound to the cells or not. This process in this experiment used about 150 red cells as opposed to -- which is equivalent to 100,000 th of a drop of your typical drop of three-percent red cells. So of course this thing kind of lends itself to being markedly nano technology sized.

(Slide.)

So just to give you a couple of other examples before I finish. So we created three other kinds of phage that had three different tags of different lengths, and this is just showing you if you mix them together you get three peaks corresponding to each of these tags.

(Slide.)

I need to link to a file that allows me to have vertical slides so you can see it. So if we take an anti-D with a short tag and anti-B antibody with a longer tag, and you add it to either O negative cells or positive cells, B negative cells or B positive cells, you can see what you get. Basically these cells don’t agglutinate and all the others agglutinate based on either one or both of these things binding. But then if you do the process I described you either get a peak for the D reagent, you get a peak for a the B reagent, or you get two peaks to show that both of these things bound. So this shows that you can actually multiplex serological reactions using a DNA-based readout.

(Slide.)

But the other possibility is, you know, if this whole thing can take place with only 150 red cells it may not really be necessary in one reaction to do 20 different phenotype reactions. Perhaps you can have in a very tiny kind of a chip that has these liquids in it each having 150 red cells you could use this multiplexing type ability to instead only type for one antigen, but have internal negative and positive controls.

(Slide.)

So for example, we have phage particle here with a short tag with an anti-RH17 antibody on it, and this is something came out of some studies that Marion Reid and I did a number of years ago from a macaque that was immunized with human red cells, and this was isolated using phage display. If you take a particle here that has a slightly longer tag and this is an antibody against something in the skin, so this is totally negative control for red cells, then here is our anti-D with a longer tag. You can see that this reagent will agglutinate D positive and D negative, but not Rh null. This one will agglutinate anything, and this will only agglutinate D positive cells. Individually you can see that they will each give a curve in a different place.

(Slide.)

So if you mix them all three together and incubate it with D negative or D positive cells, you get agglutination in both cases because of at least because of this positive control. But when you look at here the positive controls, positive in both and negative control is negative in both cases, and then only Rh positive cells have a peak in that position. So this allow you in every single individual serological reaction to have a negative and a positive control to tell you that things are working.

(Slide.)

Just for fun, what I did was to take a phage particle and put staph protein A on the tip, and staph protein A is something that belongs to the FC domain of IgG. The question is could you kind of use this method for doing screens or panels, so this is what this particle would look like. It would give you a peak here based on this tag.

(Slide.)

So here are six reagent red cells that are mixed with a patient serum that contains anti-D, and the one, two and the three cells are D positive. These are D negative, and so I have them drawn here with the antibodies on the appropriate cell. If you run them in a gel card this is the typical agglutination reaction you get, and if you instead add a staph A displayed, protein staph A displayed phage particle and used the molecular readout, I am not sure how clear you can see this, but there is a peak here, a peak here, and a peak here and these are negative. So in this case you can use these things for indirect agglutination reactions.

(Slide.)

Here is the same thing with anti-E containing serum, and this is the gel card result and the result you get with this assay.

(Slide.)

So, you know, this is your standard tube, your gel card, and what we are in the process of doing is designing these lab-on-a-chip type devices that have multi-channels in it where you could have many reactions occurring in parallel. They have these --- gates and so forth within the chip that can be used for when you need to wash the cells and so forth to removed unreactive reagent. The PCR reactions, whatever you want to do molecularly, can take place in these chips.

(Slide.)

So, you know, one possibility could be that you could multiplex typing reactions and get a profile of what antibodies bound based on the DNA inside the particle. Or, as I said before, each channel would just tell you about one specificity, but would have internal/external controls.

(Slide.)

So to summarize, what I showed a few slides ago was this idea of using phage display to create either phage antibodies that you could use in agglutination assays, or phage antibodies that you convert to regular antibodies. One thing I didn’t mention is that if you -- you can take monoclonal cell lines that exist right now. Instead of having to start here, you can actually just take the cell line and extract the -- make RNA and PCR at the antibody that is made by them, and then put them into the system if that is what you wanted to do. There wouldn’t be much of a reason to do this unless you wanted to convert an IgG into an IgM or something like that.

But what I want to propose here is that the anti-phage antibodies could be used not only in convention agglutination reactions, but in this reagent genotyping approach. So when we talk about phenotype versus genotyping, one question is what would you do phenotyping or serological reactions for. But separate from that, is there some way of combining the technologies together in terms of the device, the machine, the way things are read out. So really what genotyping is doing is it is starting with DNA, genomic DNA, but essentially what it is doing is creating an array of different pieces of DNA and then asking which ones are there. Which is the same thing as having a bunch of phage display antibodies that had been bound by a cell and eluded off and asking what tags are there that came from those reagents.

So the endpoint really can be the same, whether it be -- regardless of what kind of molecular technique you might be using for seeing what DNAs are there, whether it is a microarray or some other kind of method. So this may be a way technically of at least having the technologies and the readout combine together at the end, even though the input in one case would be genomic DNA and the input in the other case would involve some serological -- some cell antibody reaction initially.

(Slide.)

I would just like to acknowledge some of the funding sources that have lead to this work, from the National Blood Foundation to NIH, to some Pennsylvania-based biotechnology support funds. That is my talk, and it is time for lunch. Thanks.

(Applause.)

MS. KOCHMAN: I had us scheduled to come back -- to leave at 11:30 and come back by 12:30. I don’t want to take a half-hour away from you, so how about if we come back in 45 minutes?

(A luncheon break was taken at 12:07 p.m.)

---

A F T E R N O O N   S E S S I O N

(12:55 p.m.)

Current FDA Processes for Bringing Products to Market
Sheryl A. Kochman

MS. KOCHMAN: I would like to get started again. Before I do, I would like to remind anyone that if you need transportation to one of the airports please get with Rhonda at the registration desk, and I have also been asked to remind you again to fill out your evaluation form. We have also got a pair of sunglasses that were found in a chair in the lobby, so if you know anybody, if they are yours or if you know whose they might be, they are up here.

My first talk is probably going to be primarily of interest to anyone involved or associated with the manufacture of an in vitro diagnostic product, but some people, some of the users might find it useful to understand what FDA does and does not do in terms of premarket review of products.

(Slide.)

So the first question and the thing that I find a lot of people don’t know is that IVD reagents and instruments are medical devices, at least in the United States. They are classified to be medical devices.

(Slide.)

We get that from the FD&C Act, and really all you need to look at on this big slide full of words is instrument and in vitro reagent. But pretty much you will notice that we have thrown everything we can think of into this definition of a device, so if it is a thing it might be a device.

(Slide.)

If it is a think that is also one of three other defining topics, and it is a device, and the reagents and instruments fall in the second class because they are intended for use in diagnosis of disease or other conditions or in cure, mitigation, treatment or prevention of disease in man or other animals. Basically we are trying to prevent hemolytic transfusion reactions, so that is why these are devices.

(Slide.)

There is a whole other part, and basically in plain English this whole paragraph means it is not a drug.

(Slide.)

The other thing is that a lot of people may not realize that IVD reagents can also be biological products.

(Slide.)

Again, here is a whole big, huge paragraph, the last part of which says they may also be biological products subject to section 351 of the PHS Act. This is out of 809.3(A), which is part of the medical device regulations.

(Slide.)

So you might ask when is a device also a biologic. Biologics regs define a biologic product to mean any virus, therapeutic serum, toxin, antitoxin, or analogous product applicable to the prevention, treatment, or cure of diseases or injuries of man. Again we have thrown a lot of things into that definition.

(Slide.)

The next question might be, well, how does FDA regulate medical devices. Interestingly the Public Health Service Act was the first act that came along to regulate things. That was in 1912, and the net act that came along was the Federal Food, Drug, and Cosmetic Act, or the FD&C Act of 1938. Interestingly you will notice that the medical device amendments to the FD&C Act did not come along until May 28 th of 1976. That is important because it is part of the reason some devices are also biologicals. Prior to 1976 the government didn’t have a definition for a device and they weren’t regulating devices. So those products that were available on the market prior to 1976 were either unregulated or they were regulated as a food, drug or cosmetic -- or a biologic. Because of the PHS ACT and because blood grouping reagents and the like are used in testing blood, they were being regulated under the Public Health Service Act.

When the medical device amendments came along, we realized that it fit that definition as well, and so the other medical device acts and amendments have been applied to licensed biologicals that, but IVD since then. There is the Safe Medical Devices Act of 1990, the FDA Modernization Act of 1997, and the Medical Device User Fee and Modernization Act. We affectionately call it MDUFMA. We have to be able to pronounce everything.

(Laughter.)

Of 1992, and that is the act that allows FDA to assess user fees for the premarket review of medical device submissions. The regulations that cover medical devices include 21 CFR part 600 if the IVD is licensed, and 21 part 800 if it is licensed or not.

(Slide.)

The way in which a device is regulated is based on a classification scheme. This classification scheme is to some extent a risk-based scheme. A class one device is a device meeting the lowest level of regulation. It has been determined that general controls are sufficient to provide reasonable assurance of safety and effectiveness for their intended use, and the device is not life supporting or life sustaining, or for a use which is of substantial importance in preventing impairment of human health and which does not present a potential unreasonable risk of illness or injury.

(Slide.)

I mentioned that the second bullet says that general controls are sufficient. General controls are defined in the regulations. They consist of registration and listing by the device manufacturer, adhering to good manufacturing practices. It may include premarket notification or a 510(K) submission. There is a prohibition of alteration, misbranding, or manufacturing banned devices. There is a requirement for record keeping and a requirement for reporting of device failures.

(Slide.)

A class two device is a device that is subject to special controls in addition to the general control requirements in order to be able to provide reasonable assurance of safety and effectiveness for their intended use.

(Slide.)

Special controls that are in addition to those general controls are performance standards. It was the intent when the medical device amendments went into effect that there would be performance standard promulgated for devices as they were classified. This is one of the things that until recently has not really been accomplished. I will touch on that a little bit more later. It also can include post-market surveillance and/or patient registries and/or guidelines and guidances from FDA. It definitely includes design controls and may include tracking requirements.

(Slide.)

A class three device is something for which we believe there is insufficient information to determine that general controls and special controls together are sufficient to provide reasonable assurance of safety and effectiveness, or the device is life supporting or life sustaining, or for use which is of substantial importance in preventing impairment of human health, or the device itself presents a potential unreasonable risk of illness or injury. The risk ran away there.

(Slide.)

In plain English, a class three device is one that has no established predicate. I will explain what a predicate is in a few minutes. The device is associated with some sort of high risk, or because it is a new device and we know little about it, it raises new types of issues of safety and effectiveness.

(Slide.)

I tried to put those three lists together on one chart, and generally what you see is the list of things that apply growing. They are a little bit color coded so that you can see that the top items apply to all of them. Some of these additional standards apply to class three. But the main thing that is important on a class three device is that we require valid scientific evidence, well-controlled studies, and we do allow some use of documented case histories in support of these products.

(Slide.)

Now that you know the classification, you need to know the pathways to market. Not all devices are reviewed by FDA any longer. As part of FDAMA* the Congress asked us to look at our devices and see if there were any that we could potentially stop looking at prior to allowing them to go to market, and so most class one devices are currently exempt from the requirement to submit a 510(K) and some class two devices are exempt from that requirement. So as long as the manufacturer believes that their device is similar to something that is already on the market and they adhere to all of the general controls, they can proceed to market without FDA review.

As I mentioned, a premarket notification is usually referred to as 510(K). It comes from section 510(K) of the FD&C Act. There is also premarket approval or a PMA, and these are for those significant risk devices, the higher-risk devices, and of those higher-risk devices those that are considered significant risk devices require an investigational device exemption before they can be shipped even for use in studies.

Another path to market is the product development protocol. This is sort of a PMA, just submitted in a different format. There is also humanitarian device exemptions. These are like orphan drug submissions. We now have a category of product called analyte specific reagents that you can come to market through that, and then we have the licensure of BLA. With the BLA the submission of BLAs requires an investigational new drug application prior to submission of the BLA. Non-exempt IVDs are those that require it. There are some that are exempt. You might ask why an investigational new drug submission. It is because we were asking for these before we had medical devices. It is analogous to an IDE in many ways.

(Slide.)

The 510(K) process, as I said, comes from section 510(K) of the act. This process requires that the manufacturer demonstrate substantial equivalence or that they are substantially equivalent to another device on the market. The device has the same intended use, similar technological characteristics, and no new issues of safety and effectiveness as compared to something else that is legally on the market. There is a 90-day review clock, and from FDA’s point of view there are lots of limitations in that review. It is basically a paper review. We don’t have any inspection authority. We don’t have any ability to perform hands-on testing. There currently are no performance standards. Most of the products in this area don’t even have a gold standard, and there is a lot of bias in the process.

(Slide.)

The major elements of a submission, the exact criteria for what has be included in a 510(K) submission are at 21 CFR 807.87. But the main thing that the reviewer is looking at is the intended use and indications for use statements, the performance characteristics of the device; and they are comparing those things to the labeling, primarily the package insert.

(Slide.)

Substantial equivalence is similarity of a new device to one that is or was already legally on the market, which we call the predicate device. Note that I say was. It is possible for a manufacturer to come in with a device that was legally on the market but for whatever reason the first manufacturer has decided it was not really a feasible device to stay on the market. It wasn’t profitable for them or for some reason it came off the market voluntarily. A new manufacturer is allowed to use that device as a predicate.

One thing that everyone needs to realize is what substantial equivalence is not. It is not a determination that the new device is exactly the same as the one that is or was already legally on the market, let alone that it is any better than the one that is or was on the market, and it is not an FDA approval. It is simply a FDA review that says this appears to be similar to something that is out there.

(Slide.)

The PMA process we get from Section 515 of the Act. In this case approval is based on reasonable assurance of safety and effectiveness based on valid scientific evidence, and it does say reasonable assurance. It doesn’t say 100 percent assurance. There is a 180-day review clock. We have the same limitations in review -- or similar. Lack of performance standards, lack or gold standards. In this case there is a lack of historical information because there is no predicate, and again we have lack of the ability to test it ourselves.

(Slide.)

What is required for a PMA is described in 21 CDR 814.20. Again we get the intended use and indications for use statements, the performance characteristics and the labeling. But in this case we also will get clinical and/or field trial data, and we also have the opportunity to perform a pre-approval of the manufacturing facility.

(Slide.)

The BLA process comes from the PHS Act. We are looking for safety, purity and potency in this case. A standard application, we are allowed 10 months to review that. A priority application is reviewed in six months, and a priority application is something that both manufacturer and FDA agree is in the best interest of public health to get to the market on a more expedited path. There are supplements to BLAs, and for devices we have between -- some of them are four months, some of them are six months, and some of them are 10 months. In terms of limitations in review for the BLA process we pretty much have very few limitations on our review process, and if there are any it is primarily because they are new, innovative products that we don’t know enough about.

(Slide.)

All of the elements required in the submission are described in 21 CFR 601.2. The major elements again are the intended use or indications for use, performance characteristics, labeling, clinical or field trials. In this case we get conformance lots. We get actually have the product in our hands and do the testing, and we have the opportunity to do a pre-license or pre-approval inspection of the manufacturing facility.

(Slide.)

I tried to condense all those into one slide, and so the bar. I couldn’t think of another word to call it. The bar for 510(K) is substantial equivalence; PMA is reasonable assurance of safety and effectiveness; and BLA is safety, purity, potency, and in some cases also specificity and ---.

I am not going to go over all of this in the interest of time. One of the other key areas, though, that is different is in post-market. Products that go through the 510(K) process are generally only inspected for cause post-marketing. For cause means that there are reports of problems with the device, and the field goes in to follow up to see if the manufacturer has handled those problems appropriately or if there are other problems, if that is just the tip of the iceberg and there are other problems that need to be addressed. But basically the are not on any scheduled basis.

Under a PMA there are periodic inspections. The periodicity is based on the risk associated with the device. It varies, and there is also a requirement for annual reports of changes in the process to come to FDA.

For a BLA we have biennial inspections. We have annual reports just as with the PMA, but we have continued lot release, which means that once we get the conformance lots every lot that a manufacturer makes they must submit it to us. We must okay it before they can distribute it, and we have a requirement for supplements. Which are applications or submissions to FDA for certain kinds of changes, and the manufacturer has to wait for our approval before they can implement those changes.

(Slide.)

Current immunohematology products are either 510(K) regulated or BLA regulated. We don’t currently have anything regulated under the PMA process, and if you look you will see that the 510(K) products are HLA kits, a lot of those accessory kinds of reagents that you are using in a reference laboratory, and some instruments. Automated blood grouping and typing instruments are a 510(K) class two. Centrifuges and cell washers fall under the 510(K), but the reagents, that manufacturer is allowed to put a license number on or the blood grouping reagent red blood cells and anti-human globulin.

(Slide.)

And a word for any manufacturer, we really manufacturers to meet with us before you even start your clinical or field trials. We want to make sure your test plan covers all areas we would want to see covered in a submission. We want to help make sure your pre-market submission is complete. One of the complaints we get is FDA doesn’t move fast enough on anything we submit. The speed at which we can review something is proportional to the amount of work you put into it or don’t put into it. So the better your pre-market submission is coming into us the quicker we are likely to be able to get through it. We also want to make sure that you don’t do anything that we wouldn’t necessarily think you need to do.

In order to request a meeting with us I would refer you to a REG SOPP 8101.1 scheduling and conduct of regulatory review meetings with sponsors and applicants. Our website is there, and then after you have looked at the website and see we spell out exactly what you need to have ready before you can even ask us for a meeting. So once you have that information then you can contact me. I will likely assign it to one of my staff to set up the meeting, and that is that end of that one. I will move quickly to my next presentation.

Review of Current FDA Guidance
by Sheryl A. Kochman

MS. KOCHMAN: The next presentation, review of current FDA guidance. I am not actually going to review guidance. I know that one of the difficulties many people have is finding the information that FDA makes available to the public. The different websites have different ease of use, and I hope to provide you with a little bit of help finding things that might be of interest to you, but of course I have several disclaimers here.

(Slide.)

The information provided on the following pages is not intended to represent an all-inclusive list of guidance documents pertinent to the manufacture and use of molecular methods in immunohematology. I have included some guidances only to provide information regarding FDA’s current considerations in regards to the areas mentioned. Especially I have included some documents that clearly state that they are draft which basically means they are not things that we can rigidly suggest that you follow. It is to let you know which way we are thinking on things, and I may have unintentionally omitted some things that you may find would be helpful. I hope not, but that obviously exists.

(Slide.)

To start off, there are several homepages that I would recommend people be aware of, and each one of you will see something that may be of different use for you depending on the situation you are in, whether you are a manufacturer, whether you are a user, whether you are trying to help a manufacturer by doing field trial testing for them.

So I have got the CBER website; another wealth of information is available from CDRH’s office of In Vitro Diagnostic Device Evaluation and Safety, OIVD for short. They handle all of the in vitro diagnostic reagents that are not related to blood testing. So if there is any clinical implication for an IVD it goes to these people. Their website has links to all of the IVD guidances that are available. They also have links to -- it says IVD standards.

I want to clarify that also part of FDAMA was the requirement that FDA determine what national and international standards are available to manufacturers of devices and determine if any of those provide information and guidance that is acceptable to the FDA in terms of developing and manufacturing a device. So anyone can nominate a consensus standard for recognition by FDA. The process goes through CDRH since they are the primary center for handling devices. They involve us on an as-needed basis. But they look at the standard that has been nominated. They determine whether or not that standard results in testing and documentation that a manufacturer can use to prove that their device is safe and effective or reasonably assures that it is safe and effective, and they can recognize it in whole or in part, or they can determine that they don’t recognize it.

The link to IVD standards does not actually link to the standard itself. It links to a list of standards that FDA has accepted or has recognized, and that way a manufacturer when they make their submission to FDA can state, "I am conforming to the NCCLS guidance on," something or another. If that guidance is one of the ones we have recognized then we have a little bit more of a warm and fuzzy feeling about what that manufacturer is doing.

Another really helpful website for manufacturers’ devices is CFRH’s device advice website. I have you the very, very basics on device classification and on pre-market submissions. This website covers everything except the BLA process. The BLA process is unique to CBER and so you would have to come to us for that. We do have, as I mentioned, medical device user fees now, and so anyone considering bringing a device to market really needs to go and see what there is about the medical device user fees.

(Slide.)

We have a few guidances that are specific for immunohematology reagents. There is no web link here because they are so old they are not on the web, and you will also note that they are all also still draft. I am embarrassed to have to have say that. But we have the recommended methods for blood grouping reagents evaluation. That basically is the document that we encourage manufacturers to consult if they are manufacturing typical blood grouping reagents, and it is the methods that we advise them to use when they are doing their lot release testing. We have a similar document for anti-human globulin reagents.

We also have a document that was developed in conjunction with a 1990 workshop that is called "Points to consider in the design and implementation of field trials for blood grouping reagents and anti-human globulin." I make reference to it because that guidance talks about things like the number of sites we want to see you include in your testing, the kinds of sample conditions you need to consider including, and all sorts of things like that. While these documents are not available on the website, they are available from CBER’s Office of Communications, Training and Manufacturing Assistance or from me or my staff.

(Slide.)

One that is on the web is guidance for industry content and format of chemistry, manufacturing, and controls information and establishment description information for a biological in vitro diagnostic product from March of ‘99. We used to have one establishment license application and then a separate product license application for lots of different kinds of biological products, and in an effort to simplify things we did away with the establishment licensing application and include that information in the biologics license application. Then we have only one form for getting a biologics license application, but to help the manufacturers in completing the form and then building the dossier or the submission we have these guidance for industry on what kinds of information goes in that part of the submission.

(Slide.)

There are a number of documents available on molecular tests, none of which are really specific to this area. I am pointing them out because for one thing I want to show that FDA does recognize that molecular testing are coming or are here, and they may provide some little tidbit of information that could be helpful to you. So there is this one from 2005. I am not going to read all these because I am trying to get everybody caught up here.

(Slide.)

Now this one is interesting because the draft guidance for industry and FDA staff, pharmacogenetic tests and genetic tests for heritable markers, I included this because I thought it might come up. Interestingly Marion indicated that the state of New York has decided that their blood group genotyping is not a test for a heritable marker, so I think that that is interesting. But it is there for you to read if you want information.

(Slide.)

Another area that is really coming on the scene is the whole concept of personalized medicine and matching the drug to the patient, and some of these other guidances are directed at that. I think you will see that in the address you can tell which center issued it. A number of these are from CBOH.

(Slide.)

Here is one on gene mutation detection systems, factor Lieden DNA mutation detection systems. I thought that some of the information about what FDA is looking at in terms of mutations may be helpful to some people.

(Slide.)

In some ways these are a little more dated guidances, but we do still have on the books some guidances for biotechnology products. These ones are out of CBER.

(Slide.)

There is a general IVD guidance for industry and FDA staff, but I actually think that users will find this helpful also. It is for use of symbols on labels and in labeling of in vitro diagnostic devices intended for professional use. This was issued in 2004, and while it is CDRH listed guidance, CBER was also substantially involved in this document.

Because the European Union’s in vitro diagnosis device directive states that if a manufacturer places labeling statements on their label in a give language any member state may require that that labeling statement appear in the language of that member state we -- industry came to FDA and said we are going to have problems with this. We have limited amounts of space on labels and in labeling, and if we have to put labeling information in 14 different languages we are not going to have enough room for people to be able to read the information. So because the current requirements are that there is a specific requirement that states that if a statement is to be included on the labeling it must be included in English there was concern about whether or not we could have kinds of concessions about that.

Because one of the things that the EU recommended was that we incorporate universally acknowledged symbols in the labeling rather than having to have 14 different languages. So this guidance recognizes certain symbols as being universal. They have been tested in an American market to see if they are indeed recognizable, and they may be helpful to users as well as manufacturers.

(Slide.)

There is interesting guidance available on how to use the data that you have gained from your studies. Statistical guidance on reporting results from studies evaluating diagnostics tests. It is still listed as a draft guidance for industry and FDA, but is relatively currently, from 2003, and I think it provides a lot of explanation of why things should be worded certain ways.

(Slide.)

There have been a number of things mentioned here on informed consent. The most recent document that has come out on informed consent, it pertains to the use leftover specimens. For example, when you are in a blood establishment and you have processed all the blood that you have collected, you have pilot tubes left over. Can you use those samples in support of some testing, and if so how do you do that? So this guidance is particularly relevant to anybody who is considering doing testing in support of a manufacturer’s submission. Again, it is on CDRH website, but we had input into it as well.

(Slide.)

These other guidances on informed consent are actually on the webpage of the Office of the Commissioner, so they are at a much higher level, but I would suggest that you take a look at these. The Declaration of Helsinki is used in terms of I have had some people ask questions about whether or not we will accept foreign data. The Declaration of Helsinki deals a little bit with that.

(Slide.)

Here are some other documents that are important for you to have.

(Slide.)

Then there are also some webpages that were available that if you go to these webpages there is are a whole bunch of other links to things that I couldn’t even anticipate whether or not you would be interested; but information is power, so now you have the information.

(Slide.)

We have got a number of guidances on clinical or field trials. Guidance for industry on acceptance of foreign clinical studies. Another equally important guidance for financial disclosure by clinical investigators. Guidance for industry on computerized systems used in clinical trials. I think these three are all pretty important for anyone considering doing studies to support a manufacturer.

(Slide.)

There is one current document that I wanted to include because I anticipate that as any of these technologies come to market there is going to be instrumentation that goes with it. This is included simply because it is one of the most current guidances on instrumentation. I don’t know yet how useful it will be for these products.

(Slide.)

The other thing that is clear is that these products will probably have software associated with them, and we are following this most current guidance for industry and FDA staff on the content of premarket submissions for software contained in medical devices. So this one is equally important.

(Slide.)

This one is of concern. It is actually not a guidance document. It is a compliance policy guide. This is what is made available to FDA investigators when they go out to perform inspections. It is on commercialization of in vitro diagnostic devices labeled for research use only or for investigational use only. Again, this is listed as a draft, but the essence of the guidance is that a path for an IVD to market should have three somewhat distinct phases.

There is the initial phase where you have an in vitro diagnostic and you are trying to determine if it has any potential use, and so you are doing research and you are collecting preliminary data. That is the phase where it is for research use only, and after you have determined that your device -- you believe your device is going to especially useful for a particular intended use.

Then you move to an investigational use study where the intent of the study is to determine "Have I got my intended use current and what are my specific performance characteristics? How well will my device do what I want to say it does?" So this is where you get into sensitivity, specificity, and that sort of thing.

Then the next phase is that once you have done those studies we expect that you are going to bring that product to FDA for premarket review because you want it to be an in vitro diagnostic device that can be used in clinical studies. This guidance gets into some of the explanations of what kinds of labeling are required at the various stages, what kind of labeling on the device is required, and what FDA’s expectations are. I am just going to leave that point at that.

(Slide.)

Thee are some very new draft guidance available on home brew. I think that it probably is a good idea for people to take a look at these, and since they are draft and since they are so new you have the opportunity to provide your input on these. They do I think answer a lot of ambiguities about what exactly are ASRs, when is something an ASR, when is it an IVD and that sort of thing. So I strongly encourage you to look at the ASR guidance.

The second one I would not have had any idea what this guidance was about just by reading the title of it, but this is software that gathers information, analyzes it, and then gives a diagnosis or a result. So this is more related to software products.

(Slide.)

And probably some of you may know about these already, but if you want automatic alerts for new postings of information you can go to the CBER mailing list subscription or to the CDRH mailing list subscription, and you can get daily updates about what has just posted or you can tailor it. The CDRH one you can tailor to get it weekly instead of daily or monthly, and that is a useful thing. The availability of the transcript from this meeting will be announced that way. We can though say that it will available within a couple of weeks, but if you want to know the exact date that it comes out you can subscribe to this list and you will get an email about it, and that is all I have for that.

(Applause.)

Where Do We Go From Here?
by Panel Discussion

MS. KOCHMAN: I think everybody is anxious to have more discussion of where we are going with these things. If I could have today’s speakers all come down if you are still here, and there are some people who have had to leave.

DR. WHITSETT: Sheryl, Carolyn Whitsett over here in the corner. There was a question that I wanted to ask, and it relates to testing of blood donors for hemoglobin S and what we should do about those. At least I believe, Sandra, in your discussion you mentioned that perhaps we should be informing donors that they were S positive. The first thing I would like to do is say that I work with some colleagues who take care of patients with sickle cell anemia, and the feeling in the sickle cell community is that having sickle cell trait is not something that identifies the disease. So they worked very hard to have people who have sickle trait accepted and understood that that is just a normal variant and they are not symptomatic. So given that that is the way many communities visualize having the sickle cell trait, what are we currently doing at the Red Cross in terms of notifying donors if they turn out to be hemoglobin S positive although they are normal individuals otherwise they wouldn’t be donating, and how do you see having molecular testing changing what you are doing?

DR. NANCE: Okay. First of, the Red Cross does not have an organized approach to this, and it varies in what community you are in, so I can’t speak to what your community does. Second, I don’t think the change that I see in just the reading about genetic testing versus genetic screening, we haven’t changed the genetic. We are already doing screening on our donors, and that doesn’t seem to fall into the notification realm. However, with the molecular testing it looks like we will be doing diagnostic testing for hemoglobin S as opposed to just screening for sickle hemoglobin., so I am not sure how that flies. I mean, our community actually had a meeting both in Philadelphia and Washington, DC, and the sickle cell groups that were in attendance did want to know and did want to be notified. So the idea would be that you would have to for them provide some sort of a counseling or a recommendation to go to a place, an d we did have an 800 number to refer to the Sickle Cell Disease Association of America, which has a comprehensive approach to community sort of notification and counseling. So the answer to your question is I don’t know, because we are not changing. We are doing hemoglobin S screening for sickling hemoglobin right now. We are going to change if we do molecular methods to doing hemoglobin S I guess diagnosis or testing that will tell people if they have AS or SS. Obviously probably not SS.

DR. WHITSETT: Well, I guess that was my point, that they are not going to have SS, otherwise they wouldn’t be donating.

DR. NANCE: We would think not.

DR. WHITSETT: They wouldn’t meet the hemoglobin requirements. There are no people walking around with SS who would have a hemoglobin that would get them past the finger stick or whatever they are using. There may be some patients with SE --

DR. NANCE: I think there are. Well, yes. Yes.

MS. KOCHMAN: My one comment would be that in general it is FDA’s expectation that if you obtain clinically relevant information from any of the testing you are doing it is incumbent on you to pass that information along. So I guess it is, you know, your question of is it clinically relevant.

DR. BELLISSIMO: I guess I would argue I know Marion mentioned how the DNA -- the polymorphisms in red blood cell antigens don’t count as genetic disease markers, but I think clearly in this case you are crossing the line in that when you include that test you are doing a genetic disease test and that that would require you to consent to that person you are going to do that. I don’t think you should test them for a genetic sorter without their consent, nor considering what counseling ramifications such a test may have.

DR. NANCE: And I forgot to mention that in many of the articles that I read through hemoglobin S testing was referenced as one of the considerations, but it was really more to the third category, which was reproductive diseases or reproductive -- yeah, decisions, that sort of a thing more than potentially the diagnostic implication, because diagnostic was the most severe one.

DR. WHITSETT: Well, from these comments it sounds to me like we are the very least before the screening is implemented on donors using a genetic-based testing that we will need to have some discussion with the sickle cell community as well as the FDA, because patients with sickle cell trait, individuals with sickle cell trait don’t consider themselves to have an abnormality, and sickle cell trait has not -- with the exception of maybe a form of hematoria* and what happens when you go at very high altitudes, been associated with clinical problems.

So I have some concern about what the impact of this kind of testing may do with our recruitment of minority donors and how this will be perceived. So perhaps some community feedback from the sickle cell community as well as the FDA’s expectations need to be brought to the table to formulate how blood centers would proceed in doing this. Because the information that you are getting is exactly the information you get now from using a test that looks at, you know -- chemically at the presence or absence of S hemoglobin. So you know exactly the same thing, but you have looked for it in a different way, and it sounds like we need to change what we are going to say to donors because we have looked for it in a different way. Because I would guess that blood centers are not notifying normal blood donors that they have sickle cell trait, or is that not correct? It is highly variable. There is no consistent policy.

DR. NANCE: It is variable and it is highly, but I think we are looking at a more precise test now. Instead of just sickling hemoglobin, which primarily will probably be hemoglobin S, and it is listed. The kit manufacturer lists it as a screen assay which needs to be followed up by electrophoresis or other studies. This wouldn’t be. This would seem to be a little more precise to me, but you are right. The interpretation is the same. The label on the blood is the same, and in that way it is the same, but I think it might be different in the patient than for the donor.

DR. STRAUSS: Donna Strauss, New York Blood Center. Just for people’s knowledge, New York Blood Center is not notifying donors when they test for sickle positive trait. In fact, we considered it and we spoke to some clinicians who felt that it wasn’t necessary, that the people in the African American population probably knew their trait and we were truly just testing for labeling of certain products for the safety of the recipients. So we are not notifying them.

DR. MOULDS: JoAnn Moulds, Shreveport. I would like to back up the statement for a new blood center. We following NIH guidelines test all units transfused to sickle cell patients for hemoglobin S by sickle deck screening, and we do not report those results to the donor, nor do we get specific informed consent for that.

DR. SIEGEL: I have a question. So you are talking about things that are done to donors that might be diagnosing a disease, right? That is what we were just talking about, the sickle cell.

MS. : The carrier state.

DR. SIEGEL: Oh, the carrier state. Does that carry over into finding things out in the course of donors that might put them at risk for something? For example, suppose the donor center discovers a very high anti-D titer. So that is not communicated to the donor, but it would be of interest to the donor if it was a female thinking of getting pregnant.

DR. WHITSETT: It is highly likely that most blood donors walking around with a high anti-D titer don’t know about it. It is unlikely.

DR. SIEGEL: I don’t know about that. Or anti-Kel.

DR. NANCE: And I have another example in the talk, was anti-HBA 1A negative with an anti-HBA 1A. I mean, that is clearly, you know, something that might.

DR. WHITSETT: Well, people don’t routinely screen for anti-platelet antibodies unless there is an affected neonate, but obstetricians sort or routinely if a woman is pregnant or anticipating pregnancy will have done a blood type and antibody screen. That is pretty straightforward.

DR. NANCE: I would think that a lot of blood centers are screening for HBA 1A negativity and then following up the negative females with an antibody screen to make sure they don’t have the antibody as well. I saw Sue Johnson shaking her head yes, so I think that is really common across the country is to screen the phoresis donors for HBA 1A.

DR. WHITSETT: So she was shaking her head yes, they do it all the time?

MS. : It is not routine, but if we do run across we would, sure.

DR. WHITSETT: So you inform the donors if you --

MS. : Right. Yeah.

DR. WESTHOFF: I would assume the test could be turned off and not used or at least the results ignored or not interpreted since we are talking about specific test that happens to be on one manufacturer’s chip.

DR. YAZDANBAKHSH: Yes. On the beadchip assay the format right now if you don’t want to look at such that could be turned off. Any antigen including HBA.

DR. NANCE: Then there is the HBA 1A question as well then, isn’t there?

DR. WESTHOFF: I have a question for ---. Are --- able to be processed by your system?

DR. YAZDANBAKHSH: Yes. Well, no. Sorry. Let me back up. I was thinking about poly-transfused patient samples. --- samples we did try to extract DNA, and you were able to extract some DNA, but routinely all our analysis, that was done with donor patient sample. They are not --- samples. They are not --- sample, so these are whole blood samples.

DR. MOULDS: I could address that also because we specifically looked at that, quite unknowingly when I first went to Shreveport, but I tried very hard to extract DNA from segments and couldn’t get a darn thing and found out we were reducing everything. So in our experience, no, you can’t get DNA out of leuko* reduced units. We go to the pilot tubes.

DR. WESTHOFF: My question was specifically because I have been told it is more sensitive, so we certainly know we can’t get enough also out of it per the manual methods, but had anticipated maybe the BioArray or the chip methods would be more sensitive.

DR. YAZDANBAKHSH: Well, in certain cases where they are not really from some of our collaborators where we received the blood they are leuko depleted or maybe they are leuko reduced in some other way. You will be able to extract some DNA, but on a regular basis no.

DR. FIGUEROA: Delores Figuero from --- Systems. We looked at that, too, and I don’t remember exactly our results, but I believe the amount that was detected was really minute also.

MS. : This question is for Dr. Bellissimo. Do you know if the CAP plans to have anymore samples for blood group genotyping available? We are one of those nine to 10 people who subscribe because -- and at the high cost and have petitioned yearly the CAP to at least break the D typing away from the rest. Can you give us a little background of why it was incorporated with genetic testing to begin with and what is the possibility of it moving out in the near future?

DR. BELLISSIMO: I think the reason it is with the genetic testing is just totally historical way back when they started this. It was a marker that people were doing by genetics and probably mainly concerned there that it was being used for prenatal, at least in the type of genetics laboratories. So that is why it is in that survey, and I know we had comments back and I assume a lot of the comments I am hearing about are the ones you made when you called in to CAP.

So, you know, part of what I tried to find out a little bit with Sue Johnson’s help is try to -- you know, the first question they kind of ask is if we did break out such a survey, you know, how many people would participate in it. Because unfortunately that nice process I described I think in proficiency testing with all the quality control materials and administrative support and all that kind of stuff is a costly process, and so there really has to be certain number of people who would participate to make it worthwhile in doing, so maybe. My guess is from what I have seen is that number may be around 20, but I don’t know if you would have a better estimate. Certainly that would help matters.

Then the other question I think now given if some of this other stuff goes forward, would there be a greater need or greater participation because of this other chip work and things. That would require an effort here, and I think, you know, if we had that kind of information we could move forward. Certainly being on that committee I am in a position to help out on that part, and I think now it is a matter of, you know, what kind of numbers would be involved.

Of course, we would have to build the whole quality control stuff in. But, you know, as I said there is a lot of infrastructure there to that. I mean, there are tons of cell lines available and a lot of them by race, so some of it may be just plowing through them like African American samples and things like that to see how many of these we can identify, but they are completely uncharacterized for those kind of gene markers, though there are lots of different population-based controls there.

DR. FLEGEL: Is there an option to internationalize this approach? Because there are many laboratories worldwide to do that if you separate and fractionate it into several different approaches. Then there might not be enough with each proficiency scheme. Flegel from ---.

DR. BELLISSIMO: No, I don’t think there is any reason why it couldn’t be, and I now a lot of people who do participate in our genetics survey are not from the US. A lot from Europe and other places, so there is no reason why such a survey couldn’t be built with international support and even use the -- well, I would have to check to see if they would -- Corriel and --- would be interested in kind of building this thing, but again if people could give me input on what kind of numbers. I am assuming the ISBT had 40 laboratories, and I think certainly we are in a numbers scheme there that would make sense to pursue stuff like this.

DR. MOULDS: Speaking of numbers, I would like to throw this out to maybe Ghazala or Marion or Sheryl also. On the discordant samples that you are finding between serology and DNA what is the acceptable number? I know a lot of you went back and like the U negatives and the Duffy and addressed. But when we are doing our validation what should we consider as acceptable?

DR. REID: Zero.

DR. MOULDS: Yes, but sometimes the serology is wrong.

DR. REID: Well, then you have to look into them and decide what the discrepancy is due to. Otherwise how do you know if it is the tech or the test or the sample?

DR. MOULDS: Well, in our case we are using some of the --- samples, and those are 10, 12, 14 years old, and there is no way to go back and resolve them unless people want to ---.

DR. REID: We know that a lot of those were not characterized that accurately serologically.

DR. MOULDS: Right.

DR. JOHN MOULDS: There will be a lot of scientists of supposed eminence that are going to receive some very nasty letters then.

(Laughter.)

MS. : You are mumbling.

DR. JOHN MOULDS: There will be a lot of scientists of supposed national reputation that will receive a nasty letter then, because many cells we send out we find are not holding up for their characteristics that ---.

DR. REID: The letter is fine, but it doesn’t have to be nasty.

(Laughter.)

MR. : That is the only way you get their attention.

DR. YAZDANBAKHSH: So I think the bottom line is that whatever -- when we start doing the DNA typing. So if we find any discordant samples that need to be resolved as Marion said by whatever method, it is not to say the serology is always right. So we can go back and do what we did in our large-scale study. We did resolve them by sequencing and also by RFLP analysis when those assays were available.

DR. REID: We can’t ignore typographical errors, too.

MS. : Yes, that is true. Sad, but it is true.

DR. MOULDS: It is true. We have already caught our lab in a Duffy.

DR. REID: It is the biggest cause of error.

DR. MOULDS: That is absolutely true. We have already seen that problem.

Closing Remarks
by Sheryl A. Kochman

MS. KOCHMAN: Well, we are getting precariously close to 2:00. I am sure many of you have planes and trains to catch, so I was going to close this out with what is next. At this point the only thing that I know is next is the transcript will come out in about two weeks. I will have to make a report of this workshop to the Blood Products Advisory Committee. They will want to know some of the questions that came up, some of the issues that were identified, and I think I can say it is not going to end there, but I don’t think I can say where it is going. So we just have to all keep at.

I really want to thank everybody for coming because it has really given me a lot to think about. I hope we have given you some things to think about. One of the issues, I should mention this also, I envision that there will probably be -- another reason I listed so many guidance documents on the list is I envision that FDA will perceive there to be a need for guidance for both manufacturers of the test kits and for the users of the kits. As I am sure you are all aware, the guidance writing process can be lengthy. One of the things has happened from time to time with guidances at CDRH is that they actually suggest to the industry that industry present the first draft of a guidance to FDA for FDA to then follow up on.

The thing you need to be aware of is because of the requirement that we follow, what are known as good guidance practices, we can freely discuss the minute issues of something as long as everything is in development, but once we decide it is time to put pen to paper and write that guidance document we can only then talk about it in general terms. We cannot talk publicly about the specifics that are going to be in it. The specifics have to wait until the draft is published, and once the draft is published it is available for public comment at that time.

So it is very helpful to get as much public comment as we can before we actual start the process, and I would ask you to be thinking along those lines so that maybe we can start with a better guidance document to begin with and have less trouble getting it through the public comment period. But there is not a formal process for doing that though.

DR. YAZDANBAKHSH: You said during your presentation that if there is a device or a test is out in the market and the industry is presenting another test and you can show with your data that it is equivalent, then you can use the same criteria? Is that what you said? Like serology for example is licensed by FDA and the DNA analysis come in and you say, okay, you are identifying exactly the same thing but from a different way. What would you say to that?

MS. KOCHMAN: You are speaking about the substantial equivalence process. The quote, unquote, "predicate" in that process has to be either a class one or a class two device, because class three devices are either already classified as class three or if it is a new device it is actually automatically classified as class three because there is no predicate. BLA products are sort of class three devices. A class three device cannot act as a predicate for another device. So someone who wishes to pursue the 510(K) process could not say it is the same as licensed reagents because they are not in the same class.

DR. YAZDANBAKHSH: --- you said is class one, right?

MS. KOCHMAN: Right. Blood grouping reagents are like class three devices, and 510(K) is for class one or class two. It is not as easy. It is not always as easy as it sounds. There are some cases where it is very clear cut, and I can already say that this is an area where there will be a lot of discussion on how we move forward. So I think that is it then.

(Applause.)

DR. REID: I would like to thank Sheryl for being open and putting this meeting on, organizing it and being open to our -- to hearing us. Thank you.

(The meeting adjourned at 2:05 p.m.)

 

September 25, 2006 Transcripts

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