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Investigating residual leukemic cells in acute lymphoblastic leukemia: a practical approach using a streamlined
Knarik Karapetyan1, Mane Gizhlaryan2,3, Olga Kalinovskaia1
1Department of Molecular Biology, Hematology Center After Prof. R.H.Yeolyan, Yerevan, Armenia.
Insights
A streamlined interphase fluorescence in situ hybridization (iFISH) method detects leukemic cells in cerebrospinal fluid (CSF) for acute lymphoblastic leukemia (ALL) diagnosis. This fast, accessible technique offers a complementary option for sensitive ALL detection.
Area of Science:
- Hematology
- Molecular Biology
- Cytogenetics
Background:
- Accurate diagnosis of central nervous system (CNS) involvement in acute lymphoblastic leukemia (ALL) is critical.
- Current methods like immunocytochemistry, flow cytometry, PCR, and iFISH on CSF have limitations including sample freshness, cell quantity, and technical complexity.
- A need exists for a rapid, simple, and cost-effective method for detecting leukemic cells in CSF.
Purpose of the Study:
- To develop and propose a streamlined interphase fluorescence in situ hybridization (iFISH) method.
- To detect blast or residual leukemic cells in cerebrospinal fluid (CSF) of ALL patients.
- To offer a complementary diagnostic option for laboratories with limited resources or insufficient samples.
Main Methods:
- Cerebrospinal fluid (CSF) samples were collected in EDTA tubes and processed via centrifugation.
- Pellets were subjected to hypotonic treatment, fixation, hybridization, and washing steps adapted from standard blood sample protocols.
- BCR-ABL1 rearrangements were analyzed in 200 interphase cells using iFISH.
Main Results:
- The streamlined iFISH method successfully detected leukemic cells in CSF.
- 90% of Philadelphia chromosome-positive (Ph(+)) cells were identified in the CSF samples.
Conclusions:
- A significantly streamlined iFISH method for detecting blast/residual leukemic cells in ALL using CSF is proposed.
- This method serves as a valuable complementary test option.
- The technique addresses the need for an accessible and efficient diagnostic tool for CNS ALL.
Introduction:
A precise diagnosis of central nervous system involvement in acute lymphoblastic leukemia (ALL) requires comprehensive knowledge of morphological analysis, with a focus on the quantity and quality of cells being examined. Some research has utilized techniques such as immunocytochemistry, flow cytometry, polymerase chain reaction (PCR), and interphase fluorescence in situ hybridization (iFISH) on cerebrospinal fluid (CSF) cytospin samples to detect any remaining leukemic cells in the CSF. To obtain reliable results using immunocytochemistry and flow cytometry, it is essential to use freshly collected specimens within a limited timeframe. At the same time, PCR requires a sufficient number of cells for DNA extraction. On the other hand, the iFISH procedure on CSF cytospin samples can be challenging and requires practice. Therefore, there is a need for a fast, easy method that will be affordable and marketable in laboratories where the above methods are not available, or the sample is insufficient to use those methods.
Methods:
The samples were prepared by centrifugation of 1 mL aliquots of CSF collected into EDTA tubes. The CSF sample was centrifuged at 3000 rpm for 3 min, the supernatant was removed, and the pellet was placed in KCl hypotonic solution for 5 min at 37 °C. Other steps (fixation, hybridization, wash steps, and analysis) were the same as in the standard protocol for blood samples. The BCR-ABL1 rearrangements were performed and evaluated in 200 interphase cells.
Results:
90% of Ph(+) cells were found in CSF.
Conclusion:
We propose a significantly streamlined iFISH method for detecting blast/residual leukemic cells in acute lymphoblastic leukemia using CSF as a complementary test option.
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