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Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking
Published on: October 23, 2019
Albumin enhanced morphometric image analysis in CLL
Matthew A Lunning1, Vincent E Zenger, Ricardo Dreyfuss
1Flow and Image Cytometry Section, Laboratory Stem Cell Biology, Division of Cell and Gene Therapies, Center for Biologics Research and Evaluation, Food and Drug Administration, Bethesda, Maryland 20892, USA.
Insights
Adding bovine serum albumin (BSA) to blood films reduces atypical lymphocytes and smudge cells in chronic lymphocytic leukemia (CLL) samples. This method improves cell morphology analysis for better disease subclassification.
Area of Science:
- Hematology
- Cell Biology
- Medical Diagnostics
Background:
- Chronic lymphocytic leukemia (CLL) presents lymphocyte heterogeneity.
- Blood film artifacts complicate morphologic subclassification of CLL.
Purpose of the Study:
- To evaluate the effect of bovine serum albumin (BSA) on blood film preparation for chronic lymphocytic leukemia (CLL) analysis.
- To determine if BSA improves the accuracy of morphologic subclassification of CLL.
Main Methods:
- Paired blood films from 82 CLL patients were prepared using EDTA with or without BSA.
- Digital photomicroscopy and statistical analysis were performed on selected slides.
- Lymphocyte and nuclear areas, nuclear transmittance, and nuclear:cytoplasmic ratios were measured.
Main Results:
- BSA addition significantly decreased mean cell area and nuclear area.
- BSA significantly increased nuclear transmittance and nuclear:cytoplasmic ratio.
- BSA addition reduced the number of atypical lymphocytes and smudge cells.
Conclusions:
- Bovine serum albumin (BSA) addition to blood films aids in reducing atypical lymphocytes and smudge cells in CLL.
- BSA decreases lymphocyte and nuclear size while increasing nuclear transmittance and nuclear:cytoplasmic ratio.
- Standardized slide preparation with BSA may enhance morphologic subclassification of CLL, improving correlation with cytogenetics and immunophenotyping.
Background:
The heterogeneity of lymphocytes from patients with chronic lymphocytic leukemia (CLL) and blood film artifacts make morphologic subclassification of this disease difficult.
Methods:
We reviewed paired blood films prepared from ethylene-diamine-tetraacetic acid (ETDA) samples with and without bovine serum albumin (BSA) from 82 CLL patients. Group 1 adhered to NCCLS specifications for the preparations of EDTA blood films. Group 2 consisted of blood films containing EDTA and a 1:12 dilution of 22% BSA. Eight patients were selected for digital photomicroscopy and statistical analysis. Approximately 100 lymphocytes from each slide were digitally captured.
Results:
The mean cell area +/- standard error was 127.8 microm(2) +/- 1.42 for (n = 793) for group 1 versus 100.7 microm(2) +/- 1.39 (n = 831) for group 2. The nuclear area was 88.9 microm(2) +/- 0.85 for group 1 versus 76.4 microm(2) +/- 0.83 for group 2. For the nuclear transmittance, the values were 97.6 +/- 0.85 for group 1 and 104.1 +/- 0.83 for group 2. The nuclear:cytoplasmic ratios were 0.71 +/- 0.003 for group 1 and 0.78 +/- 0.003 for group 2. All differences were statistically significant (P < 0.001).
Conclusions:
BSA addition results in the reduction of atypical lymphocytes and a decrease in smudge cells. BSA also decreases the lymphocyte area and nuclear area, whereas nuclear transmittance and nuclear:cytoplasmic ratio are increased. A standardized method of slide preparation would allow accurate interlaboratory comparison. The use of BSA may permit better implementation of the blood film-based subclassification of CLL and lead to a better correlation of morphology with cytogenetics and immunophenotyping. Published 2003 Wiley-Liss, Inc.
