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Automatic volumetric capillary cytometry in the routine of an apheresis unit involved in multicomponent collection
Transfusion Science 21 (1999) 117±121
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Automatic volumetric capillary cytometry in the routine of an apheresis unit involved in multicomponent collection M. Valbonesi *, R. Bruni, G. Florio, G. Lercari, S. Cantarella, F. Torretta Immunohematology Service, San Martino University Hospital, Largo R Benzi 10, 16132 Genoa, Italy Received 20 July 1999; accepted 1 August 1999
Abstract In apheresis, leukodepletion by secondary ®ltration of the platelet components or by the primary use of special high eciency apparatuses is widely used to meet current clinical practice. Leukodepletion of RBC is mandatory for hematooncological patients and new ®lters for plasma are progressively being introduced in the routine of European blood banks. However, since the monitoring of leukodepletion eciency continues to be carried out manually using the Nageotte or the microdroplet ¯uorescence assay (MFA), inaccuracy and labour-intensity of counting will limit the possibility of satisfying the increasing demand for leukodepletion monitoring. Volumetric capillary cytometry (VCC) is a totally automated system that has been shown to correlate well with Nageotte, MFA and ¯ow-cytometric countings of residual leukocytes in platelet and RBC product. In this article we describe the application of VCC in the quality control program of our hemapheresis unit in which all apheresis donations are of the multicomponent collection type. Ó 1999 Elsevier Science Ltd. All rights reserved. Keywords: Volumetric cytometry; Capillary cytometry; Multicomponent collection; Platelet-plasma apheresis
1. Introduction Precise cell counting is becoming exceedingly important in transfusion medicine, both as a part of quality control and for the evaluation/validation of new technology or clinical research trials. Precise cell counting is essential in measuring the pre-apheresis CD34+ count in preparation for PBSC collection or in the separation of progenitor cells from cord blood. It is equally important in the evaluation of WBC contamination of platelet, plasma or RBC products obtained by conventional
* Corresponding author. Tel.: +39-010-352859; fax: +39-0105556679.
systems or by multicomponent collection either to prevent the need for ®ltration or to require the ®ltration of the component. When ®ltration is carried out, precise cell counting is needed to ascertain that the process has been eective. Another ®eld of application is the counting of lymphocyte subsets in HIV+ patients or the evaluation of clinical results of donor lymphocyte infusions. Several methods have been developed and applied to counting residual WBCs in blood components leukodepleted by ®ltration of leukopoor products particularly those produced by the latest generation of cell separators. At our center Nageotte counting was adopted then progressively substituted for by a microdroplet ¯uorochromatic assay [1] and ¯ow cytometry. This (Cytoron,
0955-3886/99/$ - see front matter Ó 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 5 - 3 8 8 6 ( 9 9 ) 0 0 0 8 2 - X
118
M. Valbonesi et al. / Transfusion Science 21 (1999) 117±121
Ortho Diagnostic Systems, Raritan, NJ, USA) was done as part of our search for a practical, accurate, sensitive, standardized method suitable for large scale application with minimal subjectivity or inherent risk of error, and requiring less time compared to other methods. Flow cytometric and polymerase chain reaction based techniques are considered sensitive and speci®c but, presently, are dicult to adapt to routine settings [2,3]. As a consequence we have read with interest the ®rst publications dealing with automatic volumetric capillary cytometry [4±7] in which a precise and sensitive technique was commercially available for counting CD34+ cells prior to PBSC apheresis, for evaluating the WBC contamination in apheresis and WBC reduced platelet or RBC products and for counting T4/T8 lymphocyte subsets in preparation for donor lymphocyte infusion. Volumetric capillary cytometry was introduced in our routine by the beginning of 1997 and since then has replaced Nageotte or MDFA countings and ¯ow cytometry becoming the basic technique for our quality assurance and control program, after a validation period whose results have been partially reported [8]. In this article we describe the results obtained with Imagn 2000 in the quality control of our platelet, plasma and RBC products (after ®ltration) obtained using the most recent apheresis machines for multicomponent collection (MCC). 2. The San Martino University Hospital MCC unit MCC is carried out employing DFC and CFC apparatuses such as the MSC+ (Haemonetics, Braintree, MA, USA) or the TRIMA (Cobe, Lakewood, USA) and the Amicus (Baxter Healthcare, Deer®eld, Ill, USA), the Cobe-Spectra, the AS 204 (Fresenius AG, Bad Homburg, FRG) and the Excel-Pro (Dideco, Mirandola Italy), respectively. Performance of these apparatuses has been extensively studied and reported [9±12]. Donors are randomly assigned to the dierent apparatuses with some preference given to MCS+ or Trima for donors exceeding 70 kg BW. Prior to MCC, donors submit to a medical visit and hematological evaluation that, according to Italian
law, encompasses measurement of platelets, percent hematocrit, prothrombin time and activated partial thromboplastin time. A hematological pro®le is essential also for the operation of the Trima, MCS+ and Amicus, in order to obtain the best platelet yield. For quality control (QC) purposes the hematological pro®le is also obtained after donation and for the MCC products. A Technicon H3 apparatus (Bayer Diagnostics Manufacturing Limited, Chatel Lane, Swords, Dublin, Ireland) is used for these measurements whereas the WBC contamination of the platelet and plasma product is studied using an Imagn 2000 cytometer, operated according to the manufacturerÕs instructions, employing commercial CEQer PRP reagents (Biometric Imaging, Mountainview, CA, USA). The Imagn 2000 with the appropriate reagents (CEQer RBC) is also employed for evaluating WBC contamination of the RBC products after ®ltration, a process which is only on-line only with the MCS+ separator (RC1H, Pall Biomedical Corp., Glenncove, NY, USA, ®lters). Conventional ®ltration is carried out with BIORO2 ®lters (Bio®l srl ± Fresenius Group, Bad Homburg, FRG) within 6 h of RBC collection and after dilution with SAG-M. This is in keeping with our general policy for RBCs which implies that all the products which are not leukodepleted on-line during collection are submitted to ®ltration within 6 h. For platelet concentrates those produced by the MCS+ apparatuses are ®ltered online at the end of the procedure, those produced by AS 204 apparatuses are ®ltered unless submitted for WBC counting by Imagn 2000, whereas the other PC produced by TRIMA, Spectra, Amicus or Excel-Pro are not ®ltered if the machine itself does not suggest the need for ®ltration. These platelets, however, are submitted to weekly quality control with 2±3 PC studied per apparatus. After validation of each apparatus, the WBC contamination of the plasma product is evaluated from time to time. One of the reasons for this is the evaluation of performance of cell separators used for multicomponent collection or plasmapheresis preparative to Cascade ®ltration. Data generated by Imagn 2000 are also used for establishing the ranking of the products when tenders are placed for the acquisition of new machines, ®lters or
M. Valbonesi et al. / Transfusion Science 21 (1999) 117±121
blood bags with ®lters incorporated for prestorage leukoreduction. 3. Results The results of application of the Imagn 2000 have been partially reported by our group [8]. What is new and deserves some attention are the studies on contamination of apheresis plasma. The results of a very preliminary study with 5 of the latest generation cell separators are reported in Table 1. From the data in Table 1 it appears that the dierences among cell separators may be very important and responsible for the choice of the separator itself depending on local needs. The MCS+, which appears to be the worst in terms of WBC contamination of plasma is by far the best in terms of platelet contamination. As to `plateletpheresis applications', the results of our
119
QC in terms of WBC contamination of the platelet products are reported in Table 2. Evaluation of WBC contamination of platelet and plasma products represents approximately 95% of the total application of the Imagn 2000 in MCC. The residual 5% is represented by quality control of RBC concentrates produced, along with platelets, after ®ltration. Filtration is on-line only with the Haemonetics MCS+ system. In this case a Pall ®lter is used immediately after RBC collection. A RC1H ®lter is used (Pall Italia, Milano, Italy). O-line ®ltration with Bio®l ®lters (BioR 02 plus, Bio®l srl±Fresenius Group, Bad Homburg, FRG) is used for RBC components collected along with platelets by the other cell separators. In Table 3 the results of ®ltration are reported: WBC contamination of the PRBC is studied with the Imagn 2000 employing commercial, dedicated reagents. In the table these results are compared with ours employing prestorage on-line ®ltration of whole blood collected with the Macopharma Le-
Table 1 WBC (absolute value and range/lL) and platelet contamination (absolute value and range ´ 103 /lL) of plasma produced during plasmathrombocytapheresis with 5 of the latest generation apparatuses (10 consecutive samples for machine) Machine
Average WBC contamination (absolute value/lL)
Range of WBC
Average platelet contamination (/lL ´ 103 )
Range
MCS3+ (Haemonetics) Excel (Dideco) Trima (Cobe) Amicus (Baxter) AS204 (Fresenius)
4.72 1.21 0.46 1.41 1.36
0.9±17.1 0.2±2.8 0.1±1.0 0.2±6.8 0.1±4.9
9.3 38.1 21.3 33.7 46.9
3±17 9±67 7±38 2±81 16±97
Table 2 Results of QC for WBC contamination of platelet products obtained with dierent cell separators (MCS + platelets after ®ltration) Apparatusa
Sample size
Platelet yield ( ´ 1011 )
Absolute WBC count/lL
Total WBC contam. ´ 105
Apheresis with WBC P 1 ´ 106
Amicus (1)
198 procedures 124 444 64 103 259 137
4.2
1.63 1.1
4.63 1.3
8(%)
3.7 4.1 4.2 4.3 3.7 3.1
3.47 2.5 1.03 0.9 1.31 0.9 0.87 0.8 0.87 0.6 0.57 0.5
9.38 6.1 3.6 1.1 3.98 0.8 1.3 0.9 1.21 0.4 0.81 0.3
47 25b 4 13b 0 0
AS204 PAIP (1) Excel Pro (4) Spectra LRS (1) Trima (1) MCS + LDP (2) Conventional ®ltration of Pooled PLT (PALL PXL2KLE) a
The number of machines employed for thrombocytapheresis is in brackets. Most of the procedures with WBC contamination exceeding 1 ´ 106 were double plateletpheresis carried out in donors with high precounts, exceeding 320 ´ 103 /lL.
b
120
M. Valbonesi et al. / Transfusion Science 21 (1999) 117±121
Table 3 WBC contamination of PRBC produced with dierent cell separators. (only RBC collected with the MCS+ are ®ltered on-line)
a
Cell separator
Sample size
Hb content of the PRBC conc pre ®ltre (g) (post (%))
Total WBC contamination after ®ltration ( ´ 105 )
Amicus AS204 Spectra Trima Excel MCS Conventional PRBC
35 28 32 74 139 61 90 86 28
58.8 2.5 (7%) 61.3 4.7 (7%) 73.3 5.3 (6.8) 58 0.8 (7.3) 87.1 4.1 (5.8) 57.8 0.9 (8.1) 58 0.3 58.6 57.3
3.4 0.6 3.5 0.6 4.3 0.9 3.4 0.3 4.7 0.8 2.85 0.8 0.81 0.28 0.86 0.3 7.9 0.7
A B C
BioR 02 plus O-line PALL on-line BioR 02 o-line Macopharma (on-line) IL a WBF2 (on-line)
WBF 2: ®lters produced for IL by Pall Biomedical Product.
uco¯ex Lst (Macopharma, Tourcoing-Cedex, France) or Instrumentation Laboratory bags (Instrumentation Laboratory, Milano, Italy). The dierences observed in terms of percentage loss of hemoglobin after ®ltration with the same ®lter for the removal of WBC depends on the dierences of the original hemoglobin content in the PRBC concentrates produced with dierent separators which depends, in turn, on the ¯exibility of the collection program that runs the dierent machines. This ¯exibility is maximal with the Excel-Pro and Spectra machines which allow collection of `tailored' units of PRBC depending on the body weight (BW) and hematological status of the donors. A substantial dierence exists, in general, between residual post-®ltration WBC contamination of PRBC collected by apheresis and those collected by the conventional bag system. This seems to depend on the dierent anticoagulants employed which is ACD-A in the ®rst case and CPD in the second, without relation to the ®lter itself which performs properly when used for ®ltration of PRBC collected in the conventional way as shown in Table 3, for the Bio®l BioR 02 ®lter. 4. Comments The Immuhematology Service of the San Martino (IHS) University Hospital has many peculiarities which depend on the hospital complex, on the organization of the Immunohematology Ser-
vice, and on the type of service to Industry that the service oers and delivers. The hospital complex in which the IHS operates is of approximately 3500 beds, is the Regional Hospital of the University and one of the National Cancer Institutes. The hospital complex is a Tertiary Care Referral hospital with 5 hematological units and 4 bone marrow transplantation teams. Solid organ transplantation is very active, and a very busy program of autologous/perioperative blood salvage is carried out directly by the IHS. The hemapheresis unit of the IHS is the heart of the system and is responsible for donation and therapeutic apheresis. Close to 4000 multiple component collections are carried out yearly along with close to 700 PBSC collections, 10% of which are allogenic. Several industries use the Hemapheresis Unit for their experimental, preliminary or validation studies of their machines. As a consequence a very strict quality control program has been set up over the years, which is based on the most sophisticated systems and machines. In terms of WBC contamination of apheresis products, this was represented by Flow Cytometric and Nageotte counting, followed by Microdroplet Assay Fluorescence according to Borzini [1] and presently by Imagn 2000 counting as reported in this article. For concurrent studies such as the ones carried out with apparatuses for true MCC (concurrent collection of PRBC/plasma/SD Platelets), up to 40 reliable counts can be carried out by a single technician in half a day leaving the second half for CD 34+ counting. Most importantly, the technical
M. Valbonesi et al. / Transfusion Science 21 (1999) 117±121
time is minimal since most of the time needed for the procedure involves incubation and reading. Our pratical conclusion after 3 years of experience with the Imagn 2000 is similar to that of Krailadsidi and Seghatchian [13] and can be summarized as follows: 1. Sample preparation is easy; only a single sample is needed. 2. Count and gating are totally automated, which eliminates the need for experienced operators. 3. Automation reduces or eliminates inter-operation/inter-assay/inter-laboratory variation. 4. A batch of 10 samples requires approximately 1 h of technical time (half of which usable for other tasks). 5. The correlation with Nageotte counting is acceptable and almost perfect with MFA [8]. 6. The lower detection limit is approximately 0.4±0.8 WBC /lL, with better limits for platelet concentrates. 7. At present there is no barcoding of the samples and 8. no control of the quality of platelets in terms of membrane activation, but the manufacturer has claimed that this option will be provided taking advantage of a reagent for CD 62. 9. Concurrently a monoclonal reagent will be provided to assess bacterial contamination of the products. 10. Lower limits for WBC detection can be obtained, according to the manufacturerÕs most recent information. 11. Evolution of the system is warranted to include for lymphocytes and PBSC counting. 12. The cost of the test itself may appear high, but at our institution it has been compensated for by reduction in technical time and, most of all, in reduction in the number of ®ltrations carried out for platelet products. 13. This will be extremely remunerative if universal leukodepletion is implemented. In conclusion, since the introduction of leukodepletion by ®ltration or by the apheresis machines has required a considerable testing program for components in order to ensure the ecacy of the system employed, and since it is possible that not only cellular components, but also plasma will need leukodepletion prior to clinical use, we feel
121
that the only way to resolve these problems is with a totally automated system for assessing very low WBC contamination of the products. The Imagn 2000 presently, appears to meet these requirements very well.
References [1] Borzini P, Dumont LJ. Microdroplet ¯uorochromatic assay for the enumeration of white cells (WBCs) in WBCreduced blood components: validation and application for evaluating newly developed WBC-reduction ®lter. Transfusion 1997;37:601±6. [2] Bodensteiner DC. A ¯ow cytometric technique to accurately measure post-®ltration white blood cell counts. Transfusion 1989;29:651±3. [3] Rawal BD, Schwadron R, Busch MP, Endow R, Vyas GN. Evaluation of leukocyte removal ®lters modelled by use of HIV-infected cells and DNA ampli®cation. Blood 1990;76:2159±61. [4] Dictz L, Dubrow R, Messian B, et al. Volumetric Capillary Cytometry: a new method for absolute cell enumeration. Cytometry 1996;23:177. [5] Adams MR, Johnson DK, Busch MP, Schembri A, et al. Automatic volumetric capillary cytometry for counting white cells in white-cell reduced plateletpheresis components. Transfusion 1997;37:29±37. [6] Dumont LJ, Dumont DF, Hamstra A et al. Evaluation of an automated device for counting leukocytes in leukocytereduced platelets. Transfusion 1996;365:11. [7] Moro G, Dabay M, Dubrow RS, Hartz T. Automated counting of leukocytes in apheresis platelets. Transfusion 1996;365:615. [8] Valbonesi M. The Imagn 2000 in the quality control program of an Italian hemopheresis unit. In: Maei LM, editors. Evaluation of New Equipment and Systems. Bethesda MD: AABB, 1997. [9] Valbonesi M, Frisoni R, Florio G et al. Donor thrombocytopheresis with last generation cell separators. Int J Artif Organs. 1993;165:119±24. [10] Valbonesi M, Lercari G, Florio G et al. ErythroThrombocytapheresis and plasma-Thrombocytapheresis with storage in T-Sol of platelets collected by the Amicus cell separator. Int J Antif Organs. 1997;20:272±76. [11] Valbonesi M, De Luigi C, Florio G et al. The Cobe Trima system as a tool for optimizing component collection A further step towards total apheresis. Int J Artif Organs 1999;22:58±59 (letter). [12] Valbonesi M, Bruni R, Florio G et al. Dry platelets with the Dideco Excel separator. Transfusion Sci 1999;20:101± 6. [13] Krailadsidi P, Seghatchian MJ. Evaluation of Imagn 2000: a new system for absolute leucocyte count. Transfusion Sci 1998;19:405±7.
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Automatic volumetric capillary cytometry in the routine of an apheresis unit involved in multicomponent collection M. Valbonesi *, R. Bruni, G. Florio, G. Lercari, S. Cantarella, F. Torretta Immunohematology Service, San Martino University Hospital, Largo R Benzi 10, 16132 Genoa, Italy Received 20 July 1999; accepted 1 August 1999
Abstract In apheresis, leukodepletion by secondary ®ltration of the platelet components or by the primary use of special high eciency apparatuses is widely used to meet current clinical practice. Leukodepletion of RBC is mandatory for hematooncological patients and new ®lters for plasma are progressively being introduced in the routine of European blood banks. However, since the monitoring of leukodepletion eciency continues to be carried out manually using the Nageotte or the microdroplet ¯uorescence assay (MFA), inaccuracy and labour-intensity of counting will limit the possibility of satisfying the increasing demand for leukodepletion monitoring. Volumetric capillary cytometry (VCC) is a totally automated system that has been shown to correlate well with Nageotte, MFA and ¯ow-cytometric countings of residual leukocytes in platelet and RBC product. In this article we describe the application of VCC in the quality control program of our hemapheresis unit in which all apheresis donations are of the multicomponent collection type. Ó 1999 Elsevier Science Ltd. All rights reserved. Keywords: Volumetric cytometry; Capillary cytometry; Multicomponent collection; Platelet-plasma apheresis
1. Introduction Precise cell counting is becoming exceedingly important in transfusion medicine, both as a part of quality control and for the evaluation/validation of new technology or clinical research trials. Precise cell counting is essential in measuring the pre-apheresis CD34+ count in preparation for PBSC collection or in the separation of progenitor cells from cord blood. It is equally important in the evaluation of WBC contamination of platelet, plasma or RBC products obtained by conventional
* Corresponding author. Tel.: +39-010-352859; fax: +39-0105556679.
systems or by multicomponent collection either to prevent the need for ®ltration or to require the ®ltration of the component. When ®ltration is carried out, precise cell counting is needed to ascertain that the process has been eective. Another ®eld of application is the counting of lymphocyte subsets in HIV+ patients or the evaluation of clinical results of donor lymphocyte infusions. Several methods have been developed and applied to counting residual WBCs in blood components leukodepleted by ®ltration of leukopoor products particularly those produced by the latest generation of cell separators. At our center Nageotte counting was adopted then progressively substituted for by a microdroplet ¯uorochromatic assay [1] and ¯ow cytometry. This (Cytoron,
0955-3886/99/$ - see front matter Ó 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 5 - 3 8 8 6 ( 9 9 ) 0 0 0 8 2 - X
118
M. Valbonesi et al. / Transfusion Science 21 (1999) 117±121
Ortho Diagnostic Systems, Raritan, NJ, USA) was done as part of our search for a practical, accurate, sensitive, standardized method suitable for large scale application with minimal subjectivity or inherent risk of error, and requiring less time compared to other methods. Flow cytometric and polymerase chain reaction based techniques are considered sensitive and speci®c but, presently, are dicult to adapt to routine settings [2,3]. As a consequence we have read with interest the ®rst publications dealing with automatic volumetric capillary cytometry [4±7] in which a precise and sensitive technique was commercially available for counting CD34+ cells prior to PBSC apheresis, for evaluating the WBC contamination in apheresis and WBC reduced platelet or RBC products and for counting T4/T8 lymphocyte subsets in preparation for donor lymphocyte infusion. Volumetric capillary cytometry was introduced in our routine by the beginning of 1997 and since then has replaced Nageotte or MDFA countings and ¯ow cytometry becoming the basic technique for our quality assurance and control program, after a validation period whose results have been partially reported [8]. In this article we describe the results obtained with Imagn 2000 in the quality control of our platelet, plasma and RBC products (after ®ltration) obtained using the most recent apheresis machines for multicomponent collection (MCC). 2. The San Martino University Hospital MCC unit MCC is carried out employing DFC and CFC apparatuses such as the MSC+ (Haemonetics, Braintree, MA, USA) or the TRIMA (Cobe, Lakewood, USA) and the Amicus (Baxter Healthcare, Deer®eld, Ill, USA), the Cobe-Spectra, the AS 204 (Fresenius AG, Bad Homburg, FRG) and the Excel-Pro (Dideco, Mirandola Italy), respectively. Performance of these apparatuses has been extensively studied and reported [9±12]. Donors are randomly assigned to the dierent apparatuses with some preference given to MCS+ or Trima for donors exceeding 70 kg BW. Prior to MCC, donors submit to a medical visit and hematological evaluation that, according to Italian
law, encompasses measurement of platelets, percent hematocrit, prothrombin time and activated partial thromboplastin time. A hematological pro®le is essential also for the operation of the Trima, MCS+ and Amicus, in order to obtain the best platelet yield. For quality control (QC) purposes the hematological pro®le is also obtained after donation and for the MCC products. A Technicon H3 apparatus (Bayer Diagnostics Manufacturing Limited, Chatel Lane, Swords, Dublin, Ireland) is used for these measurements whereas the WBC contamination of the platelet and plasma product is studied using an Imagn 2000 cytometer, operated according to the manufacturerÕs instructions, employing commercial CEQer PRP reagents (Biometric Imaging, Mountainview, CA, USA). The Imagn 2000 with the appropriate reagents (CEQer RBC) is also employed for evaluating WBC contamination of the RBC products after ®ltration, a process which is only on-line only with the MCS+ separator (RC1H, Pall Biomedical Corp., Glenncove, NY, USA, ®lters). Conventional ®ltration is carried out with BIORO2 ®lters (Bio®l srl ± Fresenius Group, Bad Homburg, FRG) within 6 h of RBC collection and after dilution with SAG-M. This is in keeping with our general policy for RBCs which implies that all the products which are not leukodepleted on-line during collection are submitted to ®ltration within 6 h. For platelet concentrates those produced by the MCS+ apparatuses are ®ltered online at the end of the procedure, those produced by AS 204 apparatuses are ®ltered unless submitted for WBC counting by Imagn 2000, whereas the other PC produced by TRIMA, Spectra, Amicus or Excel-Pro are not ®ltered if the machine itself does not suggest the need for ®ltration. These platelets, however, are submitted to weekly quality control with 2±3 PC studied per apparatus. After validation of each apparatus, the WBC contamination of the plasma product is evaluated from time to time. One of the reasons for this is the evaluation of performance of cell separators used for multicomponent collection or plasmapheresis preparative to Cascade ®ltration. Data generated by Imagn 2000 are also used for establishing the ranking of the products when tenders are placed for the acquisition of new machines, ®lters or
M. Valbonesi et al. / Transfusion Science 21 (1999) 117±121
blood bags with ®lters incorporated for prestorage leukoreduction. 3. Results The results of application of the Imagn 2000 have been partially reported by our group [8]. What is new and deserves some attention are the studies on contamination of apheresis plasma. The results of a very preliminary study with 5 of the latest generation cell separators are reported in Table 1. From the data in Table 1 it appears that the dierences among cell separators may be very important and responsible for the choice of the separator itself depending on local needs. The MCS+, which appears to be the worst in terms of WBC contamination of plasma is by far the best in terms of platelet contamination. As to `plateletpheresis applications', the results of our
119
QC in terms of WBC contamination of the platelet products are reported in Table 2. Evaluation of WBC contamination of platelet and plasma products represents approximately 95% of the total application of the Imagn 2000 in MCC. The residual 5% is represented by quality control of RBC concentrates produced, along with platelets, after ®ltration. Filtration is on-line only with the Haemonetics MCS+ system. In this case a Pall ®lter is used immediately after RBC collection. A RC1H ®lter is used (Pall Italia, Milano, Italy). O-line ®ltration with Bio®l ®lters (BioR 02 plus, Bio®l srl±Fresenius Group, Bad Homburg, FRG) is used for RBC components collected along with platelets by the other cell separators. In Table 3 the results of ®ltration are reported: WBC contamination of the PRBC is studied with the Imagn 2000 employing commercial, dedicated reagents. In the table these results are compared with ours employing prestorage on-line ®ltration of whole blood collected with the Macopharma Le-
Table 1 WBC (absolute value and range/lL) and platelet contamination (absolute value and range ´ 103 /lL) of plasma produced during plasmathrombocytapheresis with 5 of the latest generation apparatuses (10 consecutive samples for machine) Machine
Average WBC contamination (absolute value/lL)
Range of WBC
Average platelet contamination (/lL ´ 103 )
Range
MCS3+ (Haemonetics) Excel (Dideco) Trima (Cobe) Amicus (Baxter) AS204 (Fresenius)
4.72 1.21 0.46 1.41 1.36
0.9±17.1 0.2±2.8 0.1±1.0 0.2±6.8 0.1±4.9
9.3 38.1 21.3 33.7 46.9
3±17 9±67 7±38 2±81 16±97
Table 2 Results of QC for WBC contamination of platelet products obtained with dierent cell separators (MCS + platelets after ®ltration) Apparatusa
Sample size
Platelet yield ( ´ 1011 )
Absolute WBC count/lL
Total WBC contam. ´ 105
Apheresis with WBC P 1 ´ 106
Amicus (1)
198 procedures 124 444 64 103 259 137
4.2
1.63 1.1
4.63 1.3
8(%)
3.7 4.1 4.2 4.3 3.7 3.1
3.47 2.5 1.03 0.9 1.31 0.9 0.87 0.8 0.87 0.6 0.57 0.5
9.38 6.1 3.6 1.1 3.98 0.8 1.3 0.9 1.21 0.4 0.81 0.3
47 25b 4 13b 0 0
AS204 PAIP (1) Excel Pro (4) Spectra LRS (1) Trima (1) MCS + LDP (2) Conventional ®ltration of Pooled PLT (PALL PXL2KLE) a
The number of machines employed for thrombocytapheresis is in brackets. Most of the procedures with WBC contamination exceeding 1 ´ 106 were double plateletpheresis carried out in donors with high precounts, exceeding 320 ´ 103 /lL.
b
120
M. Valbonesi et al. / Transfusion Science 21 (1999) 117±121
Table 3 WBC contamination of PRBC produced with dierent cell separators. (only RBC collected with the MCS+ are ®ltered on-line)
a
Cell separator
Sample size
Hb content of the PRBC conc pre ®ltre (g) (post (%))
Total WBC contamination after ®ltration ( ´ 105 )
Amicus AS204 Spectra Trima Excel MCS Conventional PRBC
35 28 32 74 139 61 90 86 28
58.8 2.5 (7%) 61.3 4.7 (7%) 73.3 5.3 (6.8) 58 0.8 (7.3) 87.1 4.1 (5.8) 57.8 0.9 (8.1) 58 0.3 58.6 57.3
3.4 0.6 3.5 0.6 4.3 0.9 3.4 0.3 4.7 0.8 2.85 0.8 0.81 0.28 0.86 0.3 7.9 0.7
A B C
BioR 02 plus O-line PALL on-line BioR 02 o-line Macopharma (on-line) IL a WBF2 (on-line)
WBF 2: ®lters produced for IL by Pall Biomedical Product.
uco¯ex Lst (Macopharma, Tourcoing-Cedex, France) or Instrumentation Laboratory bags (Instrumentation Laboratory, Milano, Italy). The dierences observed in terms of percentage loss of hemoglobin after ®ltration with the same ®lter for the removal of WBC depends on the dierences of the original hemoglobin content in the PRBC concentrates produced with dierent separators which depends, in turn, on the ¯exibility of the collection program that runs the dierent machines. This ¯exibility is maximal with the Excel-Pro and Spectra machines which allow collection of `tailored' units of PRBC depending on the body weight (BW) and hematological status of the donors. A substantial dierence exists, in general, between residual post-®ltration WBC contamination of PRBC collected by apheresis and those collected by the conventional bag system. This seems to depend on the dierent anticoagulants employed which is ACD-A in the ®rst case and CPD in the second, without relation to the ®lter itself which performs properly when used for ®ltration of PRBC collected in the conventional way as shown in Table 3, for the Bio®l BioR 02 ®lter. 4. Comments The Immuhematology Service of the San Martino (IHS) University Hospital has many peculiarities which depend on the hospital complex, on the organization of the Immunohematology Ser-
vice, and on the type of service to Industry that the service oers and delivers. The hospital complex in which the IHS operates is of approximately 3500 beds, is the Regional Hospital of the University and one of the National Cancer Institutes. The hospital complex is a Tertiary Care Referral hospital with 5 hematological units and 4 bone marrow transplantation teams. Solid organ transplantation is very active, and a very busy program of autologous/perioperative blood salvage is carried out directly by the IHS. The hemapheresis unit of the IHS is the heart of the system and is responsible for donation and therapeutic apheresis. Close to 4000 multiple component collections are carried out yearly along with close to 700 PBSC collections, 10% of which are allogenic. Several industries use the Hemapheresis Unit for their experimental, preliminary or validation studies of their machines. As a consequence a very strict quality control program has been set up over the years, which is based on the most sophisticated systems and machines. In terms of WBC contamination of apheresis products, this was represented by Flow Cytometric and Nageotte counting, followed by Microdroplet Assay Fluorescence according to Borzini [1] and presently by Imagn 2000 counting as reported in this article. For concurrent studies such as the ones carried out with apparatuses for true MCC (concurrent collection of PRBC/plasma/SD Platelets), up to 40 reliable counts can be carried out by a single technician in half a day leaving the second half for CD 34+ counting. Most importantly, the technical
M. Valbonesi et al. / Transfusion Science 21 (1999) 117±121
time is minimal since most of the time needed for the procedure involves incubation and reading. Our pratical conclusion after 3 years of experience with the Imagn 2000 is similar to that of Krailadsidi and Seghatchian [13] and can be summarized as follows: 1. Sample preparation is easy; only a single sample is needed. 2. Count and gating are totally automated, which eliminates the need for experienced operators. 3. Automation reduces or eliminates inter-operation/inter-assay/inter-laboratory variation. 4. A batch of 10 samples requires approximately 1 h of technical time (half of which usable for other tasks). 5. The correlation with Nageotte counting is acceptable and almost perfect with MFA [8]. 6. The lower detection limit is approximately 0.4±0.8 WBC /lL, with better limits for platelet concentrates. 7. At present there is no barcoding of the samples and 8. no control of the quality of platelets in terms of membrane activation, but the manufacturer has claimed that this option will be provided taking advantage of a reagent for CD 62. 9. Concurrently a monoclonal reagent will be provided to assess bacterial contamination of the products. 10. Lower limits for WBC detection can be obtained, according to the manufacturerÕs most recent information. 11. Evolution of the system is warranted to include for lymphocytes and PBSC counting. 12. The cost of the test itself may appear high, but at our institution it has been compensated for by reduction in technical time and, most of all, in reduction in the number of ®ltrations carried out for platelet products. 13. This will be extremely remunerative if universal leukodepletion is implemented. In conclusion, since the introduction of leukodepletion by ®ltration or by the apheresis machines has required a considerable testing program for components in order to ensure the ecacy of the system employed, and since it is possible that not only cellular components, but also plasma will need leukodepletion prior to clinical use, we feel
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that the only way to resolve these problems is with a totally automated system for assessing very low WBC contamination of the products. The Imagn 2000 presently, appears to meet these requirements very well.
References [1] Borzini P, Dumont LJ. Microdroplet ¯uorochromatic assay for the enumeration of white cells (WBCs) in WBCreduced blood components: validation and application for evaluating newly developed WBC-reduction ®lter. Transfusion 1997;37:601±6. [2] Bodensteiner DC. A ¯ow cytometric technique to accurately measure post-®ltration white blood cell counts. Transfusion 1989;29:651±3. [3] Rawal BD, Schwadron R, Busch MP, Endow R, Vyas GN. Evaluation of leukocyte removal ®lters modelled by use of HIV-infected cells and DNA ampli®cation. Blood 1990;76:2159±61. [4] Dictz L, Dubrow R, Messian B, et al. Volumetric Capillary Cytometry: a new method for absolute cell enumeration. Cytometry 1996;23:177. [5] Adams MR, Johnson DK, Busch MP, Schembri A, et al. Automatic volumetric capillary cytometry for counting white cells in white-cell reduced plateletpheresis components. Transfusion 1997;37:29±37. [6] Dumont LJ, Dumont DF, Hamstra A et al. Evaluation of an automated device for counting leukocytes in leukocytereduced platelets. Transfusion 1996;365:11. [7] Moro G, Dabay M, Dubrow RS, Hartz T. Automated counting of leukocytes in apheresis platelets. Transfusion 1996;365:615. [8] Valbonesi M. The Imagn 2000 in the quality control program of an Italian hemopheresis unit. In: Maei LM, editors. Evaluation of New Equipment and Systems. Bethesda MD: AABB, 1997. [9] Valbonesi M, Frisoni R, Florio G et al. Donor thrombocytopheresis with last generation cell separators. Int J Artif Organs. 1993;165:119±24. [10] Valbonesi M, Lercari G, Florio G et al. ErythroThrombocytapheresis and plasma-Thrombocytapheresis with storage in T-Sol of platelets collected by the Amicus cell separator. Int J Antif Organs. 1997;20:272±76. [11] Valbonesi M, De Luigi C, Florio G et al. The Cobe Trima system as a tool for optimizing component collection A further step towards total apheresis. Int J Artif Organs 1999;22:58±59 (letter). [12] Valbonesi M, Bruni R, Florio G et al. Dry platelets with the Dideco Excel separator. Transfusion Sci 1999;20:101± 6. [13] Krailadsidi P, Seghatchian MJ. Evaluation of Imagn 2000: a new system for absolute leucocyte count. Transfusion Sci 1998;19:405±7.