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ClearLLab LS

ClearLLab LS Lymphoid Screen Reagent

Antibody Combination for Leukemia/Lymphoma Analysis

ClearLLab LS Lymphoid Screen is the first CE-IVD-marked 10-color L&L reagent.  It is intended for the identification of hematolymphoid cell populations by immunophenotyping on a 10-color NAVIOS flow cytometer. The reagent can be used with peripheral whole blood and bone marrow specimens collected in EDTA (K2 and K3), ACD-A and Heparin (Lithium and Sodium) as well as lymph node specimens. The results should be interpreted in conjunction with additional laboratory and clinical findings.

  • Pre-mixed 10-color 12 antibody tube
  • CE-IVD-marked for use with NAVIOS flow cytometers
  • Unitized dry reagent using Beckman Coulter proprietary format
  • 25 tests/package
  • Compatible with WHO 2008-Revised Classification
PART
NUMBER
405 nm
EXCITATION
488 nm
EXCITATION
633 nm
EXCITATION
PB1 KrO2 FITC PE ECD PC5.5 PC7 APC APC-
A7003
APC-
A7504
B74073 CD3 CD45 Kappa/
CD8
Lambda/
CD4
CD19 CD56 CD10 CD34 CD5 CD20

1. Pacific Blue 2. Krome Orange 3. APC Alexa Fluor 700 4. APC Alexa Fluor 750

Sample Data

Normal Bone Marrow

ClearLLab LS Normal Bone Marrow CD45 x Side

This CD45 x Side Scatter dot plot demonstrates typical components of a normal bone marrow, including granulocytes (blue), monocytes (red), lymphocytes (green), myeloblasts (pink) and B-lymphoid progenitors aka “hematogones” (orange).

ClearLLab LS Normal Bone Marrow CD19 x CD10

This CD19 x CD10 dot plot demonstrates characteristic CD19/CD10 co-expression by hematogones (orange). CD19-positive B lymphocytes (green) and CD10-positive granulocytes (blue) are also noted.

ClearLLab LS Normal Bone Marrow CD34 x CD19

This CD34 x CD19 dot plot demonstrates characteristic CD34 expression by a subset of hematogones (orange) as well as by myeloblasts (pink). CD19-positive B lymphocytes (green) are also noted.

Lymph Node with Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma

ClearLLab LS Lymph Node Lymphocytic Leukemia/Small Lymphocytic Lymphoma CD45 x Side

This CD45 x Side Scatter dot plot demonstrates both normal T lymphocytes (green) and a large population of cells displaying slightly lower CD45 expression (red).

ClearLLab LS Lymph Node Lymphocytic Leukemia/Small Lymphocytic Lymphoma CD5 x CD19

This CD5 x CD19 dot plot demonstrates both normal CD5-positive T lymphocytes (green) and CD5/CD19 co-expression characteristic of CLL/SLL. Note that this immunophenotype is not specific for CLL/SLL, and additional testing may be warranted to further characterize this process.

Bone Marrow with B Lymphoblastic Leukemia/Lymphoma

Normal Bone with B Lymphoblastic Leukemia/Lymphoma CD45 x Side

This CD45 x Side Scatter dot plot demonstrates both normal T lymphocytes (green) and a large population of cells displaying lower CD45 expression (red).

Normal Bone with B Lymphoblastic Leukemia/Lymphoma CD34 x CD19

This CD34 x CD19 dot plot demonstrates CD34/CD19 co-expression characteristic of B lymphoblastic leukemia/lymphoma (red).

Normal Bone with B Lymphoblastic Leukemia/Lymphoma CD5 x CD10

This CD5 x CD10 dot plot demonstrates CD10 expression by the B lymphoblastic leukemia/lymphoma (red) and CD5 expression by the normal T lymphocytes (green).

Bone Marrow with Acute Myeloid Leukemia

ClearLLab LS Bone Marrow with Acute Myeloid Leukemia CD45 x Side

This CD45 x Side Scatter dot plot demonstrates typical components of a normal bone marrow, including granulocytes (blue) and lymphocytes (green), as well as a large population of cells displaying lower CD45 expression (pink).

ClearLLab LS Bone Marrow with Acute Myeloid Leukemia CD34 x Side

This CD34 x Side Scatter dot plot demonstrates CD34 expression by the low density CD45 population (pink). Neither the lymphocytes (green) nor the granulocytes (blue) display CD34 expression.

ClearLLab LS Bone Marrow with Acute Myeloid Leukemia CD3 x CD19

This CD3 x CD19 dot plot demonstrates that the normal lymphocytes (green) are split into CD19-positive B-cells and CD3-positive T-cells. The granulocytes (blue) are negative for both CD19 and CD3, consistent with their myeloid origin. The CD34-positive cells (pink) are also negative for both CD19 and CD3, as well as for CD2, CD5, and CD7 (data not shown), suggesting that they may represent acute myeloid leukemia. Additional testing is warranted to further characterize this process.

Symposium

ClearLLab LS – Advantages
Michael Keeney ART FCSMLS(D), Associate Scientist, Lawson health Research Centre, London Health Sciences Centre, Canada

ESCCA 2016 presentation of the ClearLLab LS Lymphoid Screen Reagent. This 15 minute talk includes a description of the clinical workflow as well as immunophenotyping case studies from the clinical trials.

 
0:00 I'm going to talk about ClearLLab LS. I was involved in the study, as Mark has just said, with two other labs and one lab also in Canada, actually in Calgary, and Dr. Kern’s lab over here in Germany.
0:30 One of the biggest challenges we have when doing multi-color flow cytometry, especially with things like Navios 10 color flow cytometry, is reagent preparation. If you think what the lab has to go through for every individual antibody that has to be titrated. Once the antibody is titrated, it's then made into a combination. Once that combination has been made, you have to check to make sure that the interactions with all of the antibodies does not cause a problem. If it does, you go back, you may have to re-titrate some of the antibodies.
1:08 Most labs that are fairly large sized actually have somebody who is dedicated for at least a few days a month, if not a few days a week, just preparing these combinations. Many of us call it the cone of silence, when we actually put somebody in an area and nobody can talk to them while they make the combination. The problem is if there is a mistake when they're adding all these different antibodies, different clones. Then if there is a mistake made it costs a lot of money, first off, because you have to replace that reagent. Number two, if there's an antibody that's wrong and you don't pick up immediately, then there's a potential that you can actually be providing information missing an important marker in a patient group. When you make combinations you always verify them on a group of patients to make sure that doesn't happen. But certainly we have seen in happen before where we have added the wrong antibody.
2:05 Even worse is the documentation that has to go with that. I'm pretty sure it's the same in Europe as it is in North America. Every time you touch an antibody or open a vial you have to date it, you have to put the expiry date on the antibody. If you make a combination you have to date when that combination was made, and then you also have to date when it will expire. When you are using multiple tandem dyes as we do in 10 color flow cytometry analysis, then the tandem that expires the first is the one that's going to be where you decide when you are going to date your product. That can be a real issue because if you are making a big combination of expensive antibodies and then one of them out-dates before you have used that combination, then, again, there's an expense to doing that.
2:59 Beckman Coulter's ClearLLab product is basically a lymphoid screening tube to detect lymphoid malignancies. There are 12 different antibodies and one tube. It can actually be used for any sample type. The samples we have here for peripheral blood for bone marrow and for tissue. So it can be used for lymph node or any other tissue used in the flow cytometry lab. Although it's not yet CE-approved, but we have looked at it in different anticoagulants, so be it in ACD or EDTA or heparin, it works just as well in any of them.
3:40 It uses the culture dry technology. The big advantage of that is actually stability. We used DuraClone for a completely separate product so I can talk about the stability of this product. But we have our own customized tubes and we've used them for over 12 months. The same batch we've been using for a 12 month period.
4:04 The package size, it's a sealed package, so as long as that package isn't opened then it's stable for a very long time. There's 25 test sizes. For a small lab, if you're only doing 25-50 tests a week, that's great. For a big lab, it's not a problem at all. We can actually order in thousands of them.
4:30 The slide says it is compatible with the WHO 2006 guidelines. It's also compatible with their recently released 2016 guidelines, so there has been no real change in recommendations for classification of the lymphoid malignancies as far as by flow cytometry.
4:48 As I mentioned, the intended use is the diagnostics as part of the screening test for hematolymphoid malignancies by immunophenotyping and it was designed for the Navios specifically. It is used in the differential diagnosis of patients with signs or symptoms of hematolymphoid malignancies. The markers are directed toward B, T and NK cell populations. However, there's also CD34 in there and that actually proves to be very, very powerful. I'll show you a few slides later that exemplify that.
5:24 Again the ClearLLab LS is designed as a screening tool so it's not going to answer all of your questions. What it certainly will do is point you in the right direction. Those of you who were in Dr. Kern’s lecture earlier, he mentioned a one stage or a two stage process. For those of us who get many referred examples we prefer to do the two stage which is a screen and then go to something else. In fact, even from the university samples that we received a lot of clinical information on, we still run a screening tool because even if we think it's an acute myeloid leukemia, we would still like to classify the lymphoid populations within that. Se we don't just run acute myeloid, so let’s look for myeloid markers, we would also determine the lymphocyte populations, the NK populations and Kappa Lambda.
6:16 The top shows you what's actually in the lymphoid screen tube, which is CD45. For the T cells, CD3, CD4, CD8, and CD5. For the B cells, CD19 CD20, Kappa and Lambda CD10 for the immature B cells and also the CD5 for example and CLL or a mantel cell that we can pick up on our B cell populations. For NK cells CD56 coupled with CD3 and we can easily identify the NK cells.
6:58 One of the advantages when we are talking about standardization, especially for 10 color is compensation. How do we compensate our instrument. A routine monitor that we can easily reproduce that. 10 color compensation is a lot more complicated than 4 or 5 color compensation. Along with the batch of antibodies we receive, we receive compensation kits. Mostly they are CD4. There is one that says CD8 and one at CD3. You can use whole blood or you can use control cells like CytoComp cells to perform compensation. The beauty of this is the system is so stable you only have to do compensation once a month. That's a real advantage.
7:42 As I mentioned you can use either whole blood or you can use comp cells and the compensation kit, the fluorochromes are matched with the antibody fluorochromes you will be using. Once you've comped it works extremely well with the sample.
8:00 It's probably very hard for you folks to see that in the back, the outline. Basically what you do is you just run a fluorescent bead of FlowSet Pro, it’s a fluorescent bead you run. The kit comes with the target values so you don't need to be concerned about where your negatives fall because the kit has been designed to put your negatives in the first decade and keep them off the axis. That's actually for many labs a big advantage immediately there.
8:28 Once you run the beads you pick the target channels. Use automated software to run the 10 compensation tubes, and then you have an opportunity to use a verify tube to fine tune the comp at that point and save the compensation matrix. You only have to do that once a month. In fact in my own lab, not with this product but with the same dry technology, we actually do not change the compensation until we've had a major event with our flow cytometer or if we see a drift in our monitoring of a system. Daily all you have to do then is run that bead. When you run the bead you make sure the cells fall within the target channels. If they fall with the target channel we monitor Levey-Jennings curve and we don't need to do anything but run the samples. If the target channels are out with your range then adjust the PMTs to bring them back in and then you can use the compensation matrix.
9:27 That's just a workflow diagram. Basically we take one mL of blood, lyse the blood. Let it sit then wash the sample. Resuspend in one mL, add it to the 10 compensation tubes. Leave it incubating, because the antibody's right in the tube. Incubate it for 20 minutes and then just put on the instrument and let it run. That's what we do once a month.
9:59 I just have a few examples here of where it's very useful, the antibody combination in here. This is just showing a bone marrow with CD19, CD10. I'm sorry I don't have a laser pointer, but what I can tell you is that because we have multiplexed Kappa Lambda with CD4 and CD8 you always have to look at Kappa Lambda based on either CD3 expression or CD19 expression. The top right hand graph shows you CD3. It pops out, you see CD4, CD8. Next one you look at Kappa Lambda, but the last population that's negative for both Kappa and Lambda. Because we've CD10 in our tube, we can look at the CD19 versus CD10. That's our CD10 population. You can see CD19 versus CD20. Mature B cells expressing both 19 and 20. There's a beautiful maturation pattern for hematagones. Very early hematogones, bright ten, 20 negative, and then its maturation sequence.
11:11 This is perfect. We know there's although in an immature B cell population, the population is normal. We see here, we can see our 3 perils which is mature lymphs, type 2 hematogones and type 1 hematogones here. It's a very useful tube.
11:30 In contrast for a B acute lymphoblastic leukemia, again if we do our CD3 and CD19, we can split the populations up and we see if either normal within the lymphoid region. However, if we look at the region that's colored blue, it's dim for CD45, and if we look at an ungated plot its CD19 positive, extremely high expression of CD10. Because we CD34 we can the very blue CD34 expression in this population. This is obviously a completely abnormal population with a B ALL.
12:09 This is the next case we had of a pure red cell aplasia. Looking at the bone marrow there were no red cell precursors at all. If you look there are actually very few CD19 cells in it. They are mostly CD3 positive T cells. If we take the CD3 positive T cells and pop them out, we see the expression of CD5, and the variable expression of CD8 from negative to almost normal expression. This was sent for T cell gene arrangement and it can back and rearrange T cells so it was a T cell large granulocytic leukemia.
12:42 Although it's a lymphoid screening tube and it's not designed to classify non-lymphoid malignancies, it will detect non-lymphoid malignancies in almost all cases. The fact again CD34 in it is very, very useful because what we have is we have a large blast region you can see in the top panel left. It's not CD19 positive, its not CD3 positive, so that's our two key markers for T and B cells. Albeit for CD3 we need cytoplasmic CD3 to rule out B and T cell malignancies. But the fact that it’s CD34 positive and it's not expressing 3, 5, 19 or any they other markers in there, would point us in the direction that we are to go the next tube, look for acute myeloid leukemia.
13:27 The other thing in myelodysplastic syndrome, we can quantify the CD34 blast with this. If you think about when we talked about the Ogata score early today, the Ogata score looks at CD45, CD34, CD19 and light scatter. They actually have all of that in this tube. So you can actually apply the Ogata score with this one tube to look at MDS populations. You can see your pre-B cell population, the percent of CD34 positive, the blast that are non-B or T and the light scatter versus your lymphocytes or your granulocytes.
14:06 I'm going to keep it really short because there's many people to talk tonight. Just advantages to the ClearLLab reagents. One lot reagents. Again, we haven't used this specific product over time but the product I use, we buy a years worth or reagents. So one lot, it's already prepared, its ready to go. All that time we spend in reagent validation on lots were we have to throw part of the reagent away because we're using less or more of one or the other is completely gone. It's useful when detecting both lymphoid and non-lymphoid malignancies.

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ClearLLab LS is available in Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, India, Ireland, Italy, Liechtenstein, Luxembourg, Malta, Norway, Poland, Portugal, Romania, Slovenia, Spain, Sweden, Switzerland, Netherlands, and the United Kingdom.
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