A Ten-color Tube with Dried Antibody Reagents for the Screening of Hematological Malignancies - Duraclone Screening Tube
By Rodolfo Patussi Correia, Laiz Cameirao Bento and Amr Rajab
The workflow in clinical flow cytometry (FC) laboratories must constantly be reviewed to improve procedures that increase quality and productivity at low costs. Currently, the advances in FC applications have allowed the possibility of increasing the accuracy and standardization in clinical set-up (1, 2). One of the major advances in the line of reagents is the use of dry antibodies cocktail, that unlike liquid reagents requires no refrigeration to store, reduces the time of the analytical phase, as well as the preparation, documentation and quality assurance necessary to validate liquid reagents cocktails. In addition, dry reagents reduce the pipetting errors and the loss of liquid antibodies due to expiration date, eliminates titration, and eliminates the instability of lot-to-lot variation, especially, in tandem dyes (3).
In 2014, we adopted a screening liquid reagent single tube containing 10 colors and 14 fluorochrome-conjugated antibodies, work that is previously published (4). With this tube, we could investigate approximately more than 60% of clinical hypothesis in our FC laboratory. In order to improve our technical FC workflow, we customized this 10-color tube as a dry antibodies cocktail using the Beckman Coulter dry coating technology (named Duraclone), applied to our 10-color screening tube (DST), a study that has recently been published (5). Since 2018, we have used 1,500 DST with success in our clinical routine.
The performance of the DST when compared with liquid reagents provides clinical and numerical data equality even in paucicellular samples such as cerebrospinal fluid (CSF) and fine needle aspirates (FNA). DST tube is able to enumerate the majority of the populations with maximum immunophenotyping information, and to discriminate normal from abnormal population with minimal time and costs (5).
The liquid and dry reagents in the screening tube included 14 fluorochrome-conjugated antibodies as described in Table 1. All the liquid reagents were titrated and validated in order to obtain the ideal concentration for the signal-to-noise ratio, and this same concentration was used for the customized DST.
Table 1. Antibodies used in 10-color screening tube
Liquid reagent and DST were applied in a total of 50 samples, including bone marrow (BM), peripheral blood (PB), lymph node biopsies (LNB), pleural fluid (PF) and low cellularity samples such as cerebrospinal fluids (CSF) and fine-needle aspirates (FNA). All these cases are described in Table 2. The samples were prepared and analyzed at Flow Cytometry Laboratory, Hospital Israelita Albert Einstein, Sao Paulo, Brazil. All the data analysis and cytomorphology were evaluated by two experienced pathologists.
Table 2. Clinical suspicions and samples evaluated by DST.
Flow cytometer settings
The Navios Flow Cytometer setup, compensations, quality control and validation process, both for the liquid reagent and for the DST, have been performed according to our standard operating procedures and are described in detail in our previous publication (4, 5).
Dry reagents Quality Control
New reagent lots are checked against old reagent lots before or concurrently while being placed in service.
The performance of DST was evaluated in 50 samples characterized by heterogeneous types of hematological malignancies, and the comparison with liquid reagent took into account the final diagnosis (clinical validation), the frequency enumeration of normal and abnormal populations (numerical validation), and the mean fluorescence intensity (MFI) of the all markers in the tube.
The clinical validation was concordant in both reagents. Among the 50 samples, 17 had normal immunophenotype and 33 were cases of a hematological neoplasm, including acute myeloid leukemia, B- and T-lymphoblastic leukemia, B- and T-mature cell neoplasm, myelodysplasia, and plasma cell neoplasm.
The comparison of numerical validation was statistically acceptable, and no discrepancies were observed in the enumeration of normal and abnormal populations using liquid reagent or DST (Figure 1).
Click Image to Enlarge.
Figure 1. Comparison of the cell population frequencies identified with the liquid reagents (x-axis) and the DST (y-axis). The concordance between the methods was assessed for each parameter using intraclass correlations (ICC) and confidence intervals (CI). The repeatability of the measurements was also calculated to estimate the differences between the methods. Source: https://doi.org/10.1111/ijlh.12753.
The markers in the DST had a higher MFI and a better signal-to-noise ratio than liquid reagent, with the exception of CD5-PB, CD33 PE-Cy5.5 and CD56-PC7. The difference in MFI could be explained by a concentration effect considering that the volumes used for staining in both strategies are generally different. For example, if 50 ?L of the sample is used for staining with dry reagent, the final volume will be the same, 50 ?L. On the other hand, if 50 ?L of the sample is used for staining with 50 ?L of liquid antibodies mix, the final volume will be 100 ?L. Thus, the concentration of antibody in the dry tube reaction is double that of the liquid tube reaction. These characteristics of DST allowed a better resolution of the cell populations, reduced nonspecific staining and maintained the lineage infidelity in acute leukemia (Figure 2).
Click Image to Enlarge.
Figure 2. Dot plots of the samples stained with the liquid reagents (A-D) and the DST (E-H). (A and E) Representation of the light side scatter vs CD45, which highlight the granulocytes (red), monocytes (green), lymphocytes (purple), and progenitors/precursors (light blue). Figures B and F show a higher mean fluorescence intensity (MFI) for CD3 in the Duraclone sample, with 22.6 MFI for the liquid reagents (B) and 73.3 MFI for the Duraclone (F). The increase in the MFI of the Duraclone markers, which is also presented in Figure G, allowed for a better discrimination between the CD3-negative and CD3-positive populations (F) and improved the gating strategy. Figure C and G presents nonspecific staining in the CD4 and CD8 T cell subpopulations with the liquid reagents (C) and the absence of this population using the DST (G). Figure D and H shows acute myeloid leukemia with aberrant antigen expression and CD19+CD33+ expression using both reagents. Data were acquired from the samples using a Navios Flow Cytometer and analyzed using the Kaluza software (Beckman Coulter, Inc.). Source: https://doi.org/10.1111/ijlh.12753.
We also compared the entire cost of the immunophenotyping procedures using DST and liquid reagent, using the activity-based costing (ABC) method (6), that included fixed costs, variable costs, and surcharges such as: time of the technical procedure, the amount of supplies, fractionations, waste, infrastructure, depreciation and administrative costs. The use of the DST was translated into significant time and cost savings of 15.8% and 12.3%, respectively, compared with the use of the liquid reagent. The cost was reduced by $14.36 per sample (Figure 3).
Figure 3. Comparison of the immunophenotyping time and cost requirements when the assay was performed with the liquid reagents (left x-axis) and DST (right x-axis). The cost in US dollars and percentage of time required for the technical procedure are presented, on the left and right y-axes, respectively. The liquid reagents were considered the initial point of the comparison and correspond to 100% of the time and $15.36 of the cost. Due to confidentiality concerns, the total value of the procedure is not reported. The red line shows a reduction of 15.8% in the time r equired to complete the technical procedure using the DST. The blue line shows a reduction of $14.36 in the cost of the procedure performed using the DST. Source: https://doi.org/10.1111/ijlh.12753.
The cost reduction was not related to the number of tubes pre-ordered from the manufacturer, since the price is not related to the quantity ordered. Initially, we asked for 500 DST because it was in accordance with our routine and also allowed some flexibility with new product trial and error. The expiration date of DST was estimated at one year, but the manufacturer has not established the real stability of our custom-made DST yet.
It is important to remember that any change in the composition of cocktail must be revalidated before manufacturing, that can be time consuming and challenging in the face of rapid technological improvements and advances in the use of new monoclonal antibodies with important clinical value in FC. Another challenge for the use of DST in the clinical routine especially in Brazil, is the delay in the delivery of the products due to the import time, which usually exceed 90 days.
Finally, we investigated the applicability of DST in our clinical routine, and we calculated that more than 60.0% of the samples in our FC laboratory had clinical suspicions that could be investigated by DST, with the exception of acute leukemia, minimal residual disease, and plasma cell neoplasms. In addition, the DST provided a sufficient final interpretation and a conclusive immunophenotype for 43.7% of the cases, without the need for any other marker.
The dry reagent DST was an efficient solution for screening hematological malignancies with improved quality, productivity, standardization, and sustainability. For us, it represented an easiness of storage, rapid training of the technical staff, a reduction in the instability of tandem fluorochromes in liquid cocktails, elimination the pipetting errors, a reduction in the loss of liquid antibodies due to expiration date, and a reduction in the need for reflex additional testing. Together, these improvements could benefit flow cytometry laboratory and patients by enabling faster diagnoses using a higher quality and lower cost procedure.
We thank the Hospital Israelita Albert Einstein (HIAE) and Clinical Pathology Laboratory for providing support. We thank Nydia Strachman Bacal for providing technical and scientific development of the sector flow cytometry laboratory of HIAE. The flow cytometry staff of HIAE (Andressa da Costa Vaz, Daniela Schimidell, Eduardo de Carvalho Pedro, Flavia Arandas de Sousa, Marilia Sandoval Passaro, Nadila Magalhaes Millan, Rodrigo de Souza Barroso), is grateful acknowledged.
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