Screen Hematological

Screen Hematological Diseases via Flow Cytometry

EuroFlow™ Screening Tubes

In response to the clear need for a more efficient and standardized approach to classifying hematological malignancies, the EuroFlow™ Consortium spent several years validating multicolor antibody combinations, resulting in the EuroFlow™ product line of pre-selected and lyophilized antibody panels.1 The EuroFlow™ panels offer a comprehensive method for the proficient screening of suspected leukemia, myeloma and lymphoma samples. A cytometrist also has access to online SOPs for guidance on instrument set up, calibration, compensation, sample preparation and staining to maintain consistency across testing.  In addition, the affiliated Infinicyt™ software is available for intuitive integration and multidimensional analysis of resulting flow cytometry data.

ALPCO has partnered with Cytognos, S.A. to offer the EuroFlow™ panels and Infinicyt™ software program to flow cytometry researchers in the U.S.

Flow Cytometric Immunophenotyping

Flow cytometric immunophenotyping is a process used to identify cells, based on types of antigens or markers on a cell’s surface. In 2006, the EuroFlow™ Consortium, a group of international scientific experts, met to determine which clinical situations flow cytometric immunophenotyping should be indicated for.2  The consensus was that it is specified for several clinical situations, including the presence of atypical cells or blasts in peripheral blood, bone marrow or bodily fluids. Not only can the presence or absence of disease be established through flow cytometric immunophenotyping, but it can also be used to stage neoplasms and monitor response or relapse.

Immunophenotyping hematological malignancies using flow cytometry has led to vast advancements in the field. However, many of today’s leukemia, lymphoma and myeloma antibody panels have not been validated. As a result, screening and diagnosis has not been standardized, hindering reproducibility in multicenter studies. The lyophilized EuroFlow™ antibody panels provide the opportunity for centers to standardize their approach in screening hematological malignancies.

Markers for Immunophenotyping Hematological Malignancies

A table outlining Markers for Immunophenotyping Hematological Malignancies

EuroFlow™ System

A chart outlining the uses for each type of EuroFlow™ screening tube.

EuroFlow™ Screening Tubes

  • Lyophilized for stability and reproducibility
  • Pre-mixed antibody combinations for accuracy and efficiency
  • Optimized standard operating procedures for easy integration into workflow

Acute Leukemia Orientation Tube (ALOT)A product image of the Acute Leukemia Orientation Tubes (ALOT)

  • Pre-mixed, lyophilized combination of 8 antibodies
  • Optimized for recognition of BCP-ALL, T-ALL, AML and acute leukemias of ambiguous lineage

The ALOT kit contains a combination of 8 antibodies designed for initial assessment of the nature of immature populations of hematopoietic cells in acute leukemia samples to allow appropriate orientation towards a full BCP-ALL, T-ALL and/or AML/MDS characterization panel.

Technicians can compensate using the reagents provided with the ALOT screening kit without needing to cocktail antibodies or validate fluorochrome combinations. Simply reconstitute the two vials included in the kit, one containing 5 extracellular antibodies and one containing 3 intracellular/cytoplasmic antibodies, then stain the samples and acquire data for analysis.

Results obtained with the ALOT screening tube will help guide the user toward the appropriate next step in testing.

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ALOT Fluorochrome-Marker Combinations

A table outlining ALOT Fluorochrome-Marker Combinations

 

Lymphoid Screening Tube (LST) and Small Sample Tube (SST)A product image of the Lymphoid Screening Tube (LST) and Small Sample Tube (SST)

The typical workflow for a suspected lymphoma sample may involve the analysis of up to 16 different 8-color tubes with ≥8 antibodies in each tube, which is not only time consuming but leaves room for high variation across samples. Instead, the LST and SST panels offer a straightforward screening method with reconstitution of one premixed antibody cocktail for staining samples and orientation towards further testing with previously validated B-CLPD, T-CLPD, NK-CLPD or PCD characterization tubes.

The LST kit is a complete 8-color screening tube for lymphocytosis studies. This 12-marker combination of antibodies is aimed at detecting phenotypically aberrant populations of mature B-, T- and NK-cells including lymphocytosis, lymph node enlargement, splenomegaly, monoclonal serum components and unexplained cytopenias.

The SST panel is an enhanced version of the LST panel with 11 conjugated antibodies optimized for processing low cellularity samples such as cerebrospinal fluid, vitreous humour, and fine needle aspirates.

 

Lymphold Screening Tube (LST)

  • Pre-mixed, lyophilized combination of 12 antibodies
  • Optimized for the identification of lymphoid populations with aberrant or clonal phenotypes

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LST Fluorochrome-Marker Combinations

A chart outlining LST Fluorochrome-Marker Combinations
*TCRƔδ is optional

Small Sample Tube (SST)

  • Pre-mixed, lyophilized combination of 11 antibodies
  • Currently the most complete flow cytometry assay for clinically suspect CSF and vitreous samples

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SST Fluorochrome-Marker Combinations

A chart outlining SST Fluorochrome-Marker Combinations

 

Plasma Cell Screening Tube (PCST)

  • A product image of the Plasma Cell Screening Tube (PCST)Pre-mixed, lyophilized combination of 8 antibodies
  • Optimized for the specific identification, enumeration and discrimination between normal/reactive and aberrant plasma cells

The PCST kit contains 8 conjugated antibodies designed for identification, discrimination and enumeration of coexisting normal/reactive and aberrant plasma cell populations. It is recommended for the initial screening of Plasma Cell Dyscrasias (PCD) to differentiate between monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM).

Indications for PCD are bone pain, recurrent infections, anemia and/or lytic lesions.3,4 PCD is apparent with the presence of an abnormal free light chain (FLC) ratio where the FLC ratio is determined using quantitative nephelometry or immunofixation electrophoresis (IFE).5 Next, the plasma cells are classified as normal/abnormal using immunophenotyping by flow cytometry and then tested for the presence of common MM markers. To determine the tumor burden, the sample is subjected to beta-2 microglobulin testing by quantitative immunoturbidimetry. Some instances may occur where B-CLPD presents as MM but differentiation can be obtained through analysis of CD19 expression because lymphomas express this marker but myelomas rarely do.3,4

By replacing current testing standards for MM and MGUS with the PCST assay, only one technique is needed to determine the FLC ratio, state of the plasma cells, likelihood of presence of MM or B-CLPD and tumor burden.

 

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PCST Fuorochrome-Marker Combinations

A chart outlining PCST Fuorochrome-Marker Combinations

Infinicyt™ Flow Cytometry Software

Infinicyt™ was developed and validated within the EuroFlow™ venture as a specialized software program for addressing the needs of any basic or advanced flow cytometrist. Infinicyt™ has the ability to read files from any flow cytometer and provides an exclusive set of tools for advanced identification and description of cell populations with faster, easier and more sensitive data analysis.

An image of the Infinicyt™ Flow Cytometry Software.

Basic Software Package

  • Analysis of multiple parameters simultaneously (File Merge)
  • Automated gating and analysis strategies (Batch Analysis)

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Advanced Software Package

  • Automated n-dimensional separation of clusters (APS)
  • Comparison of new samples with previous events (Reference Image)
  • Identification of similarities and difference between samples (Compass)
  • Advanced evaluation of maturation patterns (Maturation Tool)

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Resources and References

Webinar

This webinar presented by Roberto Juanes Juanes from Cytognos S.L. discusses the EuroFlow™ screening tubes and the rest of the EuroFlow™ product line.

Works Cited

1. Van Dongen JJM, et al. EuroFlow™ antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia. 2012; 26:1908–1975.
2. Craig, FE and Foon, KA. Flow cytometric immunophenotyping for hematologic neoplasms. Blood. 2008; 111:3941-3967.
3. ARUP Consult, Topics. Plasma Cell Dyscrasias. 2006. Accessed 10NOV13. <http://www.arupconsult.com/
Topics/PlasmaCellDyscrasias.html>
4. ARUP Consult, Algorithms. Plasma Cell Dyscrasias. 2006. Accessed 10NOV13. <http://www.arupconsult.com/Algorithms/PlasmaCellDyscrasias.pdf>
5. Kyle, RA and Rajkumar, SV. Criteria for diagnosis, staging, risk stratification and response assessment of multiple myeloma. Leukemia. 2009; 23 (1) : 3-9.Additional References

Additional References

6. Van Dongen JJM, Orfao A. EuroFlow™: Resetting leukemia and lymphoma immunophenotyping. Basis for Companion Diagnostics in personalized medicine. Leukemia. 2012; 26: 1899–1907.
7. Kalina T, et al. EuroFlow™ standardization of flow cytometer instrument settings and immunophenotyping protocols. Leukemia. 2012; 26:1986–2010.
8. Pedreira CE, et al. Generation of flow cytometry data files with a potentially infinite number of dimensions. Cytometry, Part A. 2008; 73A: 834−846.
9. Pedreira CE, et al. A probabilistic approach for the evaluation of minimal residual disease by multiparameter flow cytometry in leukemic B-cell chronic lymphoproliferative disorders. Cytometry, Part A. 2008; 73A:1141−1150.
10. Costa ES, et al. Harmonization of light scatter and fluorescence flow cytometry profiles obtained after staining peripheral blood leucocytes for cell surface-only versus intracellular antigens with the Fix & Perm™ reagents. Cytometry, Part B. 2010; 78B: 11−20.

Additional publications related to EuroFlow™ Consortium and EuroFlow™ product line can be found here:
http://www.euroflow.org/publications.php
http://www.infinicyt.com/index.php/publications

The EuroFlow™ panels and Infinicyt™ software are for Research Use Only.
ALPCO is a distributor of the EuroFlow™ panels and Infinicyt™ software in the United States.
Infinicyt™ is a trademark product of Cytognos, Salamanca, Spain.
EuroFlow™ is a trademark of the EuroFlow Consortium.