DISCUSSION. Sj Slichter: I have no experience with these procedures.

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1 DISCUSSION P.e. Das and S. Murphy P Rebulla (Milan): Dr. Slichter, how do you standardize the amount of plasma which is left: on the platelet concentrate, I mean in routine. Can you comment on the practical aspects of separating platelet concentrates from the buffy coat. Sj Slichter (Seattle): The amount of plasma transferred is standardized by putting a wedge in each corner of a plasma expressor. The thickness of the wedge has been determined to leave the desired residual plasma volume on the platelet concentrate. However, to prevent plasma siphoning once the expressor plate has met the wedges, the plasma hag must be placed above the leyel of the platelet concentrate bag. P Rebulla: Well, if I understood properly in The Netherlands there are several centers where the platelet concentrates are separated from buffy coats. I think they make buffy coats and then they make centrifugations in order to sediment red cells and white cells. Do you have any experience? Sj Slichter: I have no experience with these procedures. JC Bakker (Amsterdam): Dr. Slichter, you mentioned five methods by which we could measure platelet function or predict platelet function in vivo. You did not comment which method is your method of choice. A lot of investigators in the field of platelet preservation, prefer morphology as seen under the microscope, but it is not an objective method. Which would be your method of choice? Sj Stichter: I am one of those who have predominantly relied on in vivo measurements. I think all of us would love to have a standardized way that we could all agree on to do in vitro measurements. Sofar the one we do agree on is ph extremes. There is no good evidence from studies like dr. Murphy has done, that some of the lesions (either morphological changes, aggregation changes, hypotonic shock response, or whatever in vitro parameter you have been evaluating and have found to be abnormal), may well reverse either simply after taking off the plasma, in which the platelets have been stored adding fresh plasma in vitro and then showing that the abnormal parameter improves or in addition by simply putting the platelet in vivo into the patient's plasma. There is increasing evidence, however, that when the disc-sphere transformation measured by whatever technique is irreversible, there is a non-vital cell.

2 238 One of the major problems is that we cannot be exactly sure without in vivo transfusion experiments. So, I really believe that the in vitro measurements help us to exclude, but cannot necessarily help us to include platelets in terms of changes and storage to ensure we are delivering a quality product to the patient. Ie. Bakker: Does dr. Murphy have any further comments? S. Murphy (Philadelphia): Well, to summarize the situation we have a variety of measurements, which I think often correlate with each other. I think the safest and best thing to do in developmental work is to do a panel of in vitro studies. As a generalization cells that have good maintenance of morphology and therefore good maintenance of results in the more objective reflections in morphology, invariably do well when labelled with chromium. The problem is that some of the changes are more or less slowly reversible. One may have preparations after storage which will do well in vivo, but will not have done so well in the in vitro tests. So I think that as long as the in vitro tests are giving good results, you can feel relatively confident that the in vivo results will be good. But some poor results may still be compatible with good in vivo viability. I fundamentally agree with dr. Slichter that any method in storage has to be confirmed in vivo. je. "Bakker: Dr. Arnaud, the propylene glycol as a cryoprotectant seems promising as far as toxicity is concerned. In the beginning of your paper you mentioned a toxicity of dimethyl sulphoxide and glycerol. It would have been nice if you could compare the toxicity of glycerol and dimethyl sulphoxide to propylene glycol. FC. Arnaud (Cambridge): I did not do the experiments that way. If you look at the results quoted in the literature they are very difficult to compare because no one uses the same technique of preparation and assay of the cells. It is very difficult to correlate all the results.'so, what we can say is just that: propylene glycol seems much less toxic thandmso, as 2.5'PG is tolerated by. platelets. The advantage of propylene glycol is that, compared to glycerol for instance, it has to be intubated with platelets for a shorter time. One just has to incubate propylene glycol and platelets for 40 or 50 seconds and the propylene glycol will penetrate the cells. So it is much less toxic in the sense that the period of contatt is reduced. Ie. Bakker:IDDes, DMSO' penetrate the cell more slowly? FC. Arnaud: No, faster. Propylene glycol penetrates faster than glycerol. Propylene glycol. permeation is somewhere in between glycerol and DMSo. Ie. Bakker: Propylene glycol is not a normal compound of the body, so you would expect to have to remove it to zero levels. Is that possible?

3 239 FC. Arnaud: Well yes, I have checked this by measuring the osmolarity of the final solution, when the cells are resuspended in stored plasma and I have obtained the same osmolarity as platelets suspended in fresh plasma. So, there might be still some propylene glycol, but this is not significant. C.B. Humphrey (Croningen): Is there an arcadian rythm to circulating peripheral blood stem cells under normal circumstances or during the period of chemotherapy induced overshoot. Are there times when harvesting would be more ideal as a function of the time of the day? M. Korbling (Heidelberg): Yes there is, but what we do is using this induction chemotherapy treatment just to mobilize the stem cells, that means we can reconstitute hemopoiesis. I think the problem is the pretreatment of the patient himself. If the patient is heavily pretreated we can not mobilize the stem cells at all, for example in the end-stage Hodgkin patients. If the treatment is less it is very well possible to mobilize the stem cells. C. Th. Smit Sibinga (Croningen): Dr. Brozovic came at the end of his presentation with the bedside practice of leukocyte removal. We now know how crucial it is to have good control of the optimal depletion of white cells from, specifically, red cell concentrates, which are the major transfusion commodity, as well as from platelet concentrates. How do you implement quality control of the bedside practice? B. Brozovic (London): In experimental trials one can take samples at various points of the giving set. We have done that at the bedside and found that blood which comes through the filter is almost leukocyte free. However, you can simulate this exercise in the laboratory. If you adjust the flow of the blood through the filter to 30 drops per minute, collect samples continuously, and count the white cells, you will find that filters perform well. So it seems that the efficiency of the filter to remove white cells is not dependant on the flow of blood through the filter. C. Th. Smit Sibinga: I see your point. However, we are now shifting major responsibility from the experts in the field of tissue preservation to the bedside practitioner. Not that I do not have confidence in my clinical colleagues and the nursing staff, but we should realize that there is a danger in shifting this responsibility from the Blood Bank laboratory to the bedside. B. Brozovic: You are quite right. There arc two aspects of this problem. One is the responsibility for the quality of the product and the criteria used for it. The other perhaps more important question is who pays for the filter. Once the filters are used at the bedside, the financing or purchasing of the filters will fall entirely on the hospital. C. Th. Smit Sibinga: That immediately brings you to the next danger in this practice, because now we rely on the policy of a hospital for buying whatever there is on the market. There are major experiences that not always the

4 hospital buys the best and the most efficient material for optimal clinical use, because of economical impact. B. Brozovic: You are quite right on that. My personal view is that filtration techniques ought to remain in Blood Transfusion Centers. There is no reason why that cannot be done and it is really just convenience to place them at the bedside. M.K. Elias (Groningen): Dr. Brozovic, I have a comment on the efficiency of the filter to retain the leukocytes, especially the specific filters. Although called selective for leukocytes they also retain red cells and platelets, but this is not valid for all the products. If I take for example the Imugard filter, the recovery of platelets is 60 or 70% when filtering a unit of whole blood, while the recovery is 90%, when filtering leukocyte-poor blood. We have done a comparative study in Groningen using different leukocyte contamination gradients to determine the capacity of the filter to retain the leukocytes. So we have used the leukocyte-poor platelet concentrates and a pool of standard platelet concentates and a pool of buffy coats. We found that the more the product is contaminated with white and red cells, the less is the recovery of platelets. B. Brozovic: Well, this is certainly a vary interesting study. We do not know cxactly the mechanism of retention of leukocytes and platelets on the filters. There must be a good explanation for your observations, but I can not say. P Rebulla: I would like to make a comment on the bedside filtration point. Thc best quality control of this procedure could be monitoring the febrile transfusion reaction rate in a reactive patient population. We have been quite happy during the last year transferring the whole filtration procedure to the wards with two diherent filters: the Pall and the Sepacell. The overall reaction rate remained unchanged in thalassemia patients, when it was compared with units filtered in the Blood Bank. So, we are quite happy with the bedside procedure and no problems were seen. S. Murphy: Dr. K6rbling, about the collection of stem cells from the peripheral blood, do you need to do CFU-C and CFU-GM measurements to know whether you have an adequate dose or could you not just count the number of cells which look like lymphocytes to know whether you have had an adequate collection. As I understand it, there is no known necessary correlation between the colony forming cells and the cells that rescue the patient. M. Korbling: You are totally right. The CFU-C or CFU-GM assay is the best indicator, but what we have learned especially from purging is that the relationship shifts completely. If we do a pharmocological purging, we transplant marrow grafts for example without any evidence for CFU-GM and the patient reconstitntes very well.

5 241 C. Th. Smit Sibinga: Dr. Korbling, you see for the future an indication in peripheral stem cell collection for those who are at risk for continuous exposure to irradiation. We see now what comes of Tsernobyl and all know that these disasters might occur anywhere in the world. Do you see this indication limited to only workers in nuclear plants or do you see this broader. How about the mobility of people and how do you see then bone marrow following them around the world. M. Korbling: We made such a proposal to the German government. First of all the procedure is very expensive, per person about 10,000 DM. Now, that is not the real problem. The real problem is a political one. Nobody would like to admit, especially in Germany, that nuclear power plants are not 100% safe. That is the reason that we cannot proceed with that approach. Even the workers in the plant would not admit that their plant is not 100% safe. The people we would like to apherese and collect stem cells from, are mainly people who have to do with repairment. That is the most risky thing to do in atomic power plants. The government of Baden Wurtenberg West Germany, has some 200 names on a list. That means a realistic number of 200 people from whom blood stem cells are to be collected and preserved as a preventive measure.