A Simplified Method for Formalin Fixation and Batch Counting of Leukoreduced Blood Components
By F. Enrique Alvarez and Benjamin Lichtiger
Copyright 1993-2010 The University of Texas MD Anderson Cancer Center, Houston, Texas. All rights reserved.
Leukocyte reduction of cellular blood components has become exceedingly relevant to the current hemotherapy of patients susceptible to leukocyte-related complications (1). A transfused unit of red blood cells (RBCs) contains approximately 2-5 x 109 leukocytes (2). Platelet units drawn by apheresis from donors using older cell separators may each contain up to 5 x 109 leukocytes (3,4), while those processed in more modern cell separators may contain from 1 x 106 to 5 x 108 leukocytes (5,6). Currently, leukoreduction is clearly indicated for patients experiencing febrile nonhemolytic transfusion reactions (FRs), for patients in whom HLA and platelet alloimmunization prevention is desired; and for patients in whom cytomegalovirus infection, reinfection, or reactivation is to be avoided or minimized (1,7,8).
Several methods have been tried for counting residual white blood cells (WBCs) present at low concentrations in WBC-reduced blood components (9). Simple methods using nuclear stains and large-volume counting chambers have been designed to count WBCs in units of packed RBCs (PRBCs) (10-12) or platelet concentrates (PCs) (13-15). In the study reported here, we sought to demonstrate the effectiveness of incorporating the fixative 10% formaldehyde into a program of leukoreduction quality control under the assumption that, if successful, this simple method would allow samples to be either counted in batches at times when technical staff were available or shipped to a reference center for counting when they were not.
Materials and Methods
Filtration of blood components
All blood components were drawn from volunteer blood and PC donors. All single-donor PCs were leukoreduced using Sepacell PL-10A or PLS-10A filters (Fenwal Div., Baxter Corp., Deerfield, IL). Sepacell R-500 filters (Baxter Corp.) were used to reduce leukocytes in PRBCs.
All PCs selected for leukoreduction were issued from our Transfusion Service with an individual filtration device so that the leukoreduction could be performed at bedside in the wards or outpatient clinics. Appropriate aliquots of leukoreduced PCs were obtained from the in-line drip chamber immediately below the filtration device once the transfusion was completed (9).
For the purpose of this study, PRBCs were leukoreduced in our Transfusion Service’s laboratory. In each case, aliquots were extracted from the bag containing the leukoreduced PRBCs.
Representative samples of leukoreduced PCs and PRBCs were collected for counting by both the standard and 10% formaldehyde methods. For the formaldehyde method, PC samples were diluted 1:1 with formaldehyde. The 10% formaldehyde-fixed samples were further aliquoted for counting at 24 hours, 3 days, and 30 days. All samples were stained with Turk’s solution and counted in a Nageotte chamber (16).
The staining process started with the filtration of the Turk’s solution using a Millipore filter with 0.22-µm-diameter pores. A 1:5 dilution of the preserved sample was then made by adding 100 µL of the PC sample to 400 µL of filtered Turk’s solution or, in the case of PRBCs, combining 100 µL of the PRBC sample with 40 µL of lysing agent and 360 µL of Turk’s solution. The mixture was allowed to sit for 15 minutes to allow staining. The Nageotte chamber was then covered with a coverslip and loaded with 300 µL of sample. The chamber was allowed to rest for 15 minutes in a Petri dish containing a gauze sheet soaked with saline to preserve moisture and to permit the cells to settle. The WBCs present on both sides of the chamber were then counted using a bright-field microscope under a low-power objective (10x) for a total magnification of 100x. The use of the Nageotte chamber allowed a sensitivity of 0.1 leukocytes/µL. Prefiltration samples were used as quality controls to confirm the effectiveness of the staining solution.
A total of 30 PC and 30 PRBC samples were examined (Table 1). The formalin-fixation method yielded satisfactory results since 100% of samples had less than 1 x 106 residual WBC. However, 4 PC samples and 5 PRBC samples were not evaluable owing to accidental evaporation that precluded accurate counting.
Table 1. Characteristics of Donor Blood and Platelet Concentrates Before and After Leukocyte Reduction*+
*PRBCs, packed red blood cells; PCs, platelet concentrates.
+Postfiltration means after formalin fixation and storage.
Discussion and Conclusions
The need for readily available leukoreduced cellular blood components in specific patient populations demands simple and cost-effective strategies for quality control. As Friedman et al. (17) and Wenz and Besso (18) have pointed out, the automated cell counters currently available are not capable of counting in very low ranges. Moreover, time is of the essence when depleting blood of leukocytes and getting the leukocyte-reduced blood to the patient. At MD Anderson Cancer Center, filtration is easily carried out at bedside by trained nursing personnel with no major difficulty (9). However, a program of this nature requires a strict quality-control protocol to document its reliability and quality, as the goal is to consistently deliver to the patient leukoreduced cellular blood components with a residual WBC load of less than 1 x 106 WBCs per unit of transfusable concentrates.
The method described herein, a simpler variation of the one previously described by Narvios et al. (19), allows the collection and preservation of samples for quality control and monitoring of bedside leukoreduction at any time during the day or night or on weekends. It permits a more flexible quality-control program without increasing the workload during peak times or reduced-personnel shifts. Samples stored in 10% formaldehyde and refrigerated at 4 degrees C lend themselves to batch counting without significant effects on the reproducibility of results and at an increased savings in materials and personnel time. Furthermore, the samples preserved using the formalin-fixation method can be shipped to other centers where residual WBC counting can be done more cost effectively. In all cases, however, care must be taken in choosing a storage container so as to minimize the effects of evaporation on the batched samples.
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