January-April 2004

Fresh Frozen Plasma Is Not a Cellular Blood Component: Transfusion Practice in Bone Marrow and Peripheral Blood Stem Cell Transplant Recipients

Transmission of CMV via transfusion of red blood cells and platelets has been well documented (6-8). These blood components may play a significant role in the transmission of CMV via transfusion, especially in immunocompromised patients. Therefore, it has been suggested that leukoreduction of such blood components can prevent CMV infection (9,10). However, reports in the medical literature have provided clinical evidence that transfusion of FFP may not be an important factor in the transmission of CMV (11- 17). In light of these findings, the purpose of our study was to determine whether the number of WBCs present in units of FFP warrants leukoreduction of FFP prior to transfusion to prevent CMV infection.

Materials and Methods

Production of FFP

Fifty units of FFP were produced in routine fashion at The University of Texas M. D. Anderson Cancer Center Blood Bank in accordance with regulatory standards and in compliance with the standard operating procedures of the blood bank. Briefly, FFP was prepared from units of whole blood and frozen at -18°C within the required time frame for the anticoagulant or collection process (18,19).

Of the 50 units of FFP produced for this study, 20 were used for enumeration of leukocytes before and after filtration via a manual method with a Nageotte chamber. Ten units were used for determination of the WBC populations present after filtration via flow cytometry. Samples of the remaining 20 units were analyzed via cell culture to determine whether CMV was present.

Leukoreduction of FFP

Twenty units of FFP were filtered through standard leukoreduction red blood cell filters (Sepacell 500; Baxter, Deerfield, IL). There were no FFP-specific leukoreduction filters available in our blood bank. Leukoreduction was performed at room temperature without any priming of the filter. The filtration period for each unit of FFP with an average volume of 220 ml was 5-10 minutes. Each filter was used for filtration of four units of FFP.

Counting of WBCs

WBCs from the 20 units of FFP samples were counted before and after filtration. This was done manually with the aid of a Nageotte chamber. Turk's solution was added to the samples for visualization of the nuclei of the WBCs with a standard microscope with a 10x and 40x ocular as described previously (20).

Flow cytometry

Ten units of unfiltered FFP were analyzed via flow cytometry after being counted with a Z1 Series Coulter Counter (Beckman Coulter, Miami, FL). Immunophenotypic analysis was conducted with the use of a FACScan (BD Biosciences, San Jose, CA) with different antibodies, such as Leu4 (CD3), Leu3 (CD4), Leu2 (CD8), Leu12 (CD19), Leu1 (CD5), LeuM1 (CD15), CD41, CD13, CD33, CD45, and LeuM3 (CD14).

The samples were incubated with 10 microliters of each antibody at 20-80°C for 15 minutes in the dark and washed twice with a solution of buffered phosphate. The samples were then resuspended, fixed in a 1% solution of paraformaldehyde, and analyzed with the CellQuest software program (BD Biosciences). Lymphocytes, monocytes, and granulocytes were discriminated based on the density of CD45 and characteristic pattern of scattering on the cell surface.

Culture and antigen detection of CMV in FFP

One sample from each of the 20 units of unfiltered FFP was analyzed to determine the presence of CMV. This was performed via direct fluorescent antigen detection with the use of a monoclonal antibody directed against the CMVpp65 structural protein and culture-based methods. Briefly, FFP was thawed rapidly in a 35°C water bath, and the cellular component was recovered via centrifugation at 200g for 10 minutes at 40°C. Antigen detection was performed with a CMVpp65 antigenemia assay kit (Chemicon International, Temecula, CA). Appropriate positive and negative controls were used. The cells were washed in phosphate-buffered saline, and erythrocytes were lysed with cold ammonium chloride. The typical yield of 450 ml of FFP was 1 x 10⁹ cells. Slides were stained and interpreted as per antigen detection of CMV. For the culture-based methods, 2 ml of residual supernatant was used to inoculate two MRC5 shell vials (0.5 ml of inoculum each) and two diploid human foreskin fibroblast cells lines (SF cells inoculated with 0.5 ml each). The shell vials were incubated for 18-24 hours and read with the use of the Bartels CMV stain (Chemicon International, Temecula, CA), and the SF cells were interpreted for cytopathic effect for 21 days (21).

 *SD, standard deviation.

Table 1 shows the mean WBC counts before and after filtration of FFP as determined manually with the Nageotte chamber. The mean number of WBCs in the prefiltration samples was 1.57 x 10⁶ (range, 0.11-3.25 x 10⁶). This was below the number required by regulatory standards for leukoreduced products but was clinically significant enough for our bone marrow transplant recipients receiving CMV- unscreened blood products. The mean number of residual WBCs after filtration was 0.00246 x 10⁶ (range, 0.001-0.010 x 10⁶). In addition, the mean percentage removal of WBCs through leukoreduction was 98.52% (range, 96.75%-99.99%). This showed highly efficient removal of WBCs.

Table 2 shows the results of our postfiltration sample analysis via flow cytometry. The mean number of residual WBCs after leukoreduction was 0.19 x 10⁶ (range, 0.03-0.29 x 10⁶ ). In the 10 leukoreduced FFP units, we found that the residual WBCs were composed of a mean of 10.9% lymphocytes (range, 2%-25%), 3.8% granulocytes (range, 1%-10%), and 5% monocytes (range, 1%-10%). Flow cytometric analysis showed that the mean concentration of CD4, CD8, and CD19 was 4.2%, 2.2%, and 2.7%, respectively. Dead cells were also seen, which could have accounted for the remaining cells. Furthermore, the mean number of platelets was 16.57 x 10⁶ (range, 14.0-21.7 x 10⁶). There was no evidence of CMV in any of the samples.

Discussion

Several authors have reported finding large numbers of WBCs in units of FFP. For example, Willis et al. (4) reported a contamination rate of more than 1 x 10⁶ WBCs per unit of FFP. Similarly, Bernvil et al. (2) reported a mean of 2 x 10⁶ WBCs per unit of FFP, and Wieding et al. (3) found a mean of 3.2 x 10⁶ WBCs per unit.

The findings of the present study are quite similar to those reported by the above-mentioned authors. Furthermore, we were able to quantitate the platelets in the FFP units. Although the number of platelets in FFP appears to be low, their presence may play an important role in the development of platelet refractoriness. Lymphocytes present in FFP may also play a role in the development of posttransfusion GVHD. Another important finding in our study was that the presence of CMV could not be ascertained despite the use of various methods of detecting it.

Conclusions

The results of this study demonstrate that FFP is not acellular but is contaminated with a substantial number of WBCs and platelets. The presence of these cells may lead to serious adverse effects in susceptible immunocompromised hosts who receive a transfusion of FFP. In light of these findings, bone marrow and peripheral blood stem cell transplant recipients who are supposed to receive leukoreduced cellular blood components could benefit from transfusion of FFP that has been subjected to the manipulation described herein. At M. D. Anderson Cancer Center, units of FFP that are destined for transfusion in patients who are on a leukoreduction protocol, including bone marrow and peripheral blood stem cell transplant recipients, are administered after filtration with a red blood cell leukoreduction filter (four units of FFP per filter). This is performed at the patient's bedside. Although we are aware that plasma filters are commercially available, we found the red blood cell leukoreduction filter to be economical and practical for this purpose.

Acknowledgement

We thank Dr. Elpidio Pena for assisting us in this study.

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