Blackwell Science, LtdOxford, UKTRFTransfusion0041-11322005 American Association of Blood BanksJanuary 2005451115118Letters to the EditorLETTERS TO THE EDITORLETTERS TO THE EDITOR
LETTERS TO THE EDITOR Immunoglobulin M in normal human serum is cytotoxic to polymorphonuclear neutrophils Various soluble mediators contained in donor-derived plasma are assumed to cause nonhemolytic transfusion reactions (NHTRs) in recipients.1 In the course of identifying the profiles of these deleterious mediators, we found that sera derived from healthy blood donors displayed notable cytotoxic activity against healthy blood donorderived polymorphonuclear neutrophils (PMNs) in vitro, on the basis of the conventional 4-hour 51Cr-release assay (mean of percentage of specific lysis, 20.2 ± 9.7%; range, 5.6-47.5%; n = 240; each serum sample was tested using PMNs from at least three individuals). The evaluation of correlation between cytotoxic activity against PMNs and the level of various soluble mediators in each serum sample was performed. The analysis revealed a high correlation between cytotoxic activity against PMNs and immunoglobulin (Ig) M level in the serum. The IgM level in each serum sample was determined by ordinal doubledeterminant enzyme-linked immunosorbent assay (ELISA). The correlation was estimated using 20 serum samples from healthy individuals and 60 individually derived PMN samples as targets (a single experiment was performed using 1 serum sample of the 20 samples and 3 different PMN samples out of the 60; total experiment number, 60) (Fig. 1). The cytotoxic activity of serum decreased following incubation with rabbit anti-human IgM monoclonal antibody (MoAb) (Dako, Glostrup, Denmark) at 37∞C for 1 hour, but not with anti-IgA MoAb (Southern Biotechnology, Associates, Inc., Birmingham, AL) or anti-IgG MoAb (Sigma Chemical Co., St Louis, MO) (Fig. 2). Because heat treatment (56∞C for 30 min) did not
decrease cytotoxic activity, the involvement of complements was not likely. Interestingly, these IgM-positive cytotoxic sera appeared to be noncytotoxic against peripheral blood mononuclear cells (mean ± SD of percentage of specific lysis, 5.9 ± 2.4%; range, 0%-14.6%;
Fig. 2. Treatment of serum with anti-immunoglobulin MoAb. Sera from healthy individuals were incubated with diluent control, rabbit anti-human IgM MoAb, mouse anti-human IgG MoAb, or mouse anti-human IgA MoAb at an experimentally determined saturated concentration at 37∞C for 1 hour. 51Crlabeled PMNs (2 ¥ 105) suspended in 50 mL of the serum-free RPMI 1640 were then added to each well and incubated at 37∞C for 4 hours. Percent specific lysis was calculated using the formula given in the legend to Fig. 1. Mean ± SD of percentage specific lysis calculated from the results of six independent identical experiments is shown. **Significant decrease of cytotoxic activity (p < 0.01).
Fig. 1. Correlation between cytotoxic activity and the levels of IgM (left), IgG (middle), and IgA (right). PMNs from healthy individuals were labeled with 51Cr and then challenged with various samples of sera from healthy individuals (1 ¥ 105 PMNs/40 mL of serum). After a 4-hour incubation at 37∞C, radioactivity in cell-free culture supernatants was measured using a gamma counter. Percent specific lysis was calculated using the formula (cpm of culture supernatant of PMNs treated with serum) – cpm of spontaneous release (RPMI 1640 containing 10% FCS)/(cpm of total release [0.1 percent Triton X-100 in RPMI 1640 containing 10% FCS] – cpm of spontaneous release) ¥ 100. The level of IgM in serum was assessed by ordinal double-determinant ELISA. The row data of absorbance at 450 nm in ELISA are indicated. A representative result from 60 independent identical experiments is shown in each anti-immunoglobulin MoAb. Volume 45, January 2005
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n = 80). The mechanisms underlying the present result may be unique, because PMNs did not express Fc receptor for IgM (FcgR: CD7). The identification of the IgMbinding antigen on PMNs by the MoAb-immobilized granulocytes antigen test2,3 and determination of IgM specificity are now under investigation. In conclusion, these results suggest that donor serum–derived IgM exerts its cytotoxic activity against recipients’ PMNs. The injury of PMNs results in the release of reactive oxygen species, several cytokines, and cytokine inhibitors.4,5 We hypothesize that IgM may be responsible for certain NHTRs through release of these detrimental mediators that may injure recipients’ peripheral blood cells and other tissues. This hypothesis requires testing in appropriate patients. Motoko Nishimura, PhD e-mail:
[email protected] Yoshihide Ishikawa, PhD Minoko Takanashi, MD, PhD Masahiro Satake, MD, PhD Research Section Tokyo Metropolitan Red Cross Blood Center 4-1-31 Hir-oo, Shibuya-ku Tokyo 150-0012, Japan
REFERENCES 1. Silvergleid AJ, Haffeigh EB, Harabin MA, Wolf RM, Grumet FC. Clinical value of ashes-platelet concentrations in patients with non-hemolytic transfusion reactions. Transfusion 1997;17:33-7. 2. Ravetch JV. Fc receptors. Curr Opin Immunol 1997;9:121-5. 3. Bux J, Stein EL, Bierling P, et al. Characterization of a new alloantigen (SH) on human neutrophil Fc receptor IIIb. Blood 1997;89:1027-34. 4. Henderson LM, Chappel JB. NADPH oxidase of neutrophils. Biochim Biophys Acta 1996;1273:87-107. 5. Xing L, Remick DG. Relative cytokine and cytokine inhibitor production by mononuclear cells and neutrophils. Shok 2003;20:10-6.
counts, we compared the CD34+ cell content after volume reduction and before freezing, and after thawing and washing, as follows. CD34+ cells were quantified by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA) with monoclonal antibodies conjugated CD45-fluorescein and CD34-phycoerythrin (Becton Dickinson) and 7 aminoactomycin D (7-AAD) as marker of DNA staining. Flow cytometric analysis was performed on 5 ¥ 105 with computer (Cell Quest, Becton Dickinson), gating on 7AAD negative events and excluding debris. Progenitor cell enumeration kits (ProCount, Becton Dickinson) were compared with our standard protocol in 25 cases and gave similar results. The total number of CD34+ cells (¥ 106) was calculated by multiplying the percentage of CD34+ cells determined by cytometry by the total nucleated cell (TNC) count. The results of measuring TNC counts, CD34+ cells, and colony-forming units (CFU) counts before freezing and after thawing-washing are summarized in the Table 1. The mean recoveries for TNC count, CD34+ cells, and CFU counts were 79.8 ± 8.6 percent (range, 59%-110%), 110.7 ± 33.3 percent (range, 45%240%), and 115 ± 53 percent (range, 40%-250%), respectively. Correlation between TNC count before freezing and after thawing-washing was significant (r = 0.964; p < 0.001). Figure 1 shows that this correlation for CD34+ cells was also significant. After thawing-washing, the correlation between CD34+ cells and CFU content was a rho value of 0.665 (p < 0.001), reflecting the functionality of these progenitors (Fig. 2). Based on these results, we conclude that CD34+ cell content before freezing reliably represents the stem cell content after thawing-washing and may be used for selecting cord blood units for transplantation.
Blackwell Science, LtdOxford, UKTRFTransfusion0041-11322005 American Association of Blood BanksJanuary 2005451117118 Letters to the EditorLETTERS TO THE EDITORLETTERS TO THE EDITOR TRANSFUSION 2005;45:000-000.
CD34+ cell content before freezing represents the hematopoietic stem cell content of thawed and washed cord blood units We read with interest the article by Van haute and colleagues1 reporting CD34+ cell counts for frozen but nonwashed cord blood units. Our cord blood bank and many others2,3 wash cord blood units for transplantation after thawing, according to the method of Rubinstein and coworkers.4 To evaluate whether washing cord blood after thawing affected the reliability of prefreezing CD34+ cell 116
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Fig. 1. Relationship between CD34+ cells before freezing and after thawing-washing cord blood units (n = 50; Spearman’s r = 0.857; p < 0.001).
Blackwell Science, LtdOxford, UKTRFTransfusion0041-11322005 American Association of Blood BanksJanuary 2005451118119 Letters to the EditorLETTERS TO THE EDITORLETTERS TO THE EDITOR TRANSFUSION 2005;45:000-000.
LETTERS TO THE EDITOR
TABLE 1. Cell content of cord blood units (n = 50), before freezing and after thawing-washing
Before freezing After thawing-washing
TNC count (¥108) 9.0 ± 3.6 7.2 ± 2.9
Total number of CD34+ cells (¥106) 3.3 ± 3.6 3.5 ± 3.6
CFUs (¥104) 136.4 ± 161.9 145.4 ± 140.5
The above letter was sent to Vandekerckhove et al.; Drs Vandekerckhove and Van haute offered the following reply. Solves and colleagues point out correctly that there is more to cord blood transplantation than measuring CD34 cell content and selecting cord blood
units. A major advantage of cord blood transplantation compared to matched unrelated donor (MUD) peripheral blood progenitor cells or marrow transplantation is the element of “control.” This advantage derives from the fact that cord blood units are stored in cord blood banks and extensively quality controlled before selection of the optimal donor unit for a specific patient. Whereas the expertise for MUD selection is usually located in the HLA laboratory, the combined expertise of the cell processing and HLA laboratory is available for cord blood selection:
1. Selection of the optimal cord blood unit
Fig. 2. Relationship between CD34+ cells and CFU content of cord blood units after thawing-washing (n = 50; Spearman’s r = 0.665; p < 0.001).
Pilar Solves, MD E-mail:
[email protected] Vicente Mirabet, PhD F. Carbonell-Uberos, MD Angeles Soler, MD Valencia Cord Blood Bank Valencia Transfusion Center Avenida del Cid 65-A 46014 Valencia, Spain
REFERENCES 1. Van haute I, Lootens N, De Smet S, et al. Viable CD34+ stem cell content of a cord blood graft: which measurement performed before transplantation is most representative? Transfusion 2004;44:547-54. 2. Armitage S, Warwick R, Fehily D, Navarrete C, Contreras M. Cord blood banking in London: the first 1000 collections. Bone Marrow Transplant 1999;24:139-45. 3. Perotti CG, Del Fante C, Viarengo G, et al. A new automated cell washer device for thawed cord blood units. Transfusion 2004;44:900-6. 4. Rubinstein P, Taylor PE, Scaradavou A, et al. Unrelated placental blood for bone marrow reconstitution: organization of the placental blood program. Blood Cells 1994;20:587-96.
Selection of the optimal cord blood unit should take into consideration all elements defining the quality of the product. The most critical of these are HLA-AB and HLADR matches and total nucleated cell (TNC) and CD34 cell content. Unique to cord blood transplantation, HLA confirmatory typing can be performed on an aliquot attached to the unit (Netcord requirement), thereby assuring the correct HLA type of the graft before conditioning the patient before transplantation. The TNC content measured by the cord blood bank before freezing is automated and standardized; thus the between-laboratory variation is considered too small to be relevant in the clinical setting. Usually CD34 cell content is measured by the cord blood bank only before freezing. This measurement on fresh material has become more reproducible since the introduction of the single platform measurement. What really should be measured, however, is the number of viable CD34 cells that can be recovered from the frozen graft. This measurement incorporates the effect of the freezing process. This measurement is difficult; neither thawing nor flow cytometric analysis is standardized. In addition, the between-laboratory variation is not known. We have shown that the best estimate of the CD34 cell content of the graft is obtained when measured on a sample that was frozen concurrently with the graft and thawed using a standardized thawing technique.
2. Transplantation We agree with Solves and colleagues that cord blood units should be washed before transplantation. Independent of the procedure used, it allows measurement of critical variVolume 45, January 2005
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ables such as cell viability and viable CD34+ cells of the product infused in the patient. We have data (not shown) demonstrating that these variables are stable for hours after washing. The technique for CD34 measurement on frozen and thawed cells, however, should be validated, because such measurements often overestimate the true viable CD34 cell content. In many publications,1,2 including the letter by Solves and coworkers, recoveries of over 200 percent are not unusual. It is our experience that such validation should be carried out by comparing measurements on single cord blood units before and after freezing. Bart Vandekerckhove, MD, PhD e-mail:
[email protected] Inge Van haute, MD
BTCO Dienst voor het Bloed Ottergemsesteenweg 413 B-9000 Gent, Belgium
REFERENCES 1. Itoh T, Minegishi M, Fushimi J, et al. A simple controlledrate freezing method without a rate-controlled programmed freezer provides optimal conditions for both large-scale and small-scale cryopreservation of umbilical cord blood cells. Transfusion 2003;43:1303-8. 2. Perotti CG, Fante CD, Viarengo G, et al. A new automated cell washer device for cord blood units. Transfusion 2004;44: 900-6.
SUBMISSION OF LETTERS Instructions for submission of letters can be found in the Detailed Instructions for Authors published on pages 128 to 133 of the January issue. Submit letters to: S. Gerald Sandler, MD Department of Laboratory Medicine/M-1306 Georgetown University Hospital 3800 Reservoir Road, NW, Washington, DC 20007 fax (202) 444-2440 e-mail:
[email protected] EDITOR’S NOTE: To permit timely publication of correspondence, the references have not been verified as they are for articles appearing in TRANSFUSION, and, therefore, the accuracy of references cited in Letters to the Editor is the sole responsibility of the authors. Payment is not required for submission of Letters to the Editor.
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