Hematologics Inc.

 

Hematopathology Services

Questions? Please contact us at clientservices@hematologics.com

Send Hematologics a sample, and we will provide full interpretive reports. We can provide Cytogenetics and Molecular Analysis studies. We have partnered with Diagnostic Cytogenetics for 24 hour turn-around times for karyotyping and fluorescence in situ hybridization studies. Our own state-of-the-art Molecular Analysis laboratory provides a comprehensive test menu for gene rearrangement, chromosomal translocation and point mutation studies with a 24 – 48 hour turn-around time.  Interpretative reports are generated and reviewed by both Drs. Denise A. Wells and Michael R. Loken. Clinical information (patient history, surgical, and/or pathological diagnoses) are essential for workup and evaluation of specimens. Unexpected results clinically significant to patient care are telephoned or e-mailed to the ordering physician or laboratory. Reports are faxed to the client as they are generated usually within 24 hours of receipt of specimen. Final (hard copy) reports are mailed to the client.

Flow Cytometry

1. Immunophenotyping of Peripheral Blood and Bone Marrow Aspirate Specimens

Abnormal cell populations of suspected hematopoietic malignancies (acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogeneous leukemia, myelodysplasia, chronic lymphocytic leukemia, hairy cell leukemia, non-Hodgkin’s B and T cell lymphoma, plasma cell myeloma), and some instances of non-hematopoietic malignancies can be identified in peripheral blood and bone marrow aspirate specimens using High Definition Flow Cytometry™. The diagnostic and prognostic information obtained by flow cytometry supplements clinical, morphological, and cytogenetic parameters.

We have spent several years characterizing the expression of cell antigens on normal hematopoietic cells (1-8). Neoplastic cells are distinguished from their normal counterparts based on the abnormal expression of cellular antigens (9-11, 13-15, 23-24, 26-29). In a reflexive approach, three-color panels of monoclonal antibodies are applied to characterize the neoplastic or reactive process. This multidimensional flow cytometric (MDF) approach to detection of hematological abnormalities based on differences from normal allows identification of aberrant populations even when they constitute a minor proportion of cells (0.5% or less of total nucleated cells). MDF can be used not only for characterization of the cells at diagnosis, but it can be used to detect residual leukemia post therapy or to identify lymphoma present in blood or bone marrow aspirates.

2. Immunophenotyping of Tissue Specimens

Cells for flow cytometric analysis can also be obtained from lymph node biopsies, fine needle aspirates of lymph nodes or tissue masses, or from body fluids (e.g., CSF or pleural effusions). Detection of neoplastic cells of B lineage origin is based on the cellular expression (surface or cytoplasmic) of immunoglobulin restricted to either kappa or lambda light chain. T lineage neoplasms are identified by the aberrant expression of T lymphoid antigens.

It is crucial to distinguish viable from dead cells in these specimens it since poor cell viability may give false positive or uninterpretable results. We use MDF in combination with a viability dye to assess only the intact live cells with two-color immunofluorescence to accurately phenotype the cells in these tissues.

The combination of cell size by light scatter and immunophenotype correlates well with established histological criteria for determining type and grade of non-Hodgkin’s lymphoma while providing sensitive detection techniques for minimal residual disease (32)

3. Clinical Cell Sorting: Tumor Cell Confirmation, Lineage Specific Chimerism, Monoclonality Profiling

When abnormal cells are present at very low levels, flow cytometry, cytogenetics or morphology  often cannot adequately confirm relapse. Therefore, it is useful to  concentrate the abnormal cells via cell sorting for futher analysis and confirmation . We routinely separate cells at frequencies down to 0.2% for analysis by fluorescence in situ hybridization (FISH) or clonality profiling by polymerase chain reaction analysis (PCR), thereby providing an independent assessment of the tumor cells detected at low levels. When appropriate, we will contact the requesting laboratory as to the necessity to sort the cells for genotypic confirmation of relapse.

In a non-myeloablative hematopoietic stem cell transplant situation, it is necessary to assess the chimerism of the T cells and myeloid cells separately. Peripheral blood specimens can be separated into T cell and neutrophil components using fluorescence activated cell sorting. Populations of cells isolated in this manner are >98% pure and can then be assayed for donor/host proportions using either short tandem repeats (STR) analysis or FISH (for opposite sex transplants).

4. Immunocompetancy / Lymphocyte Subset Analysis

Immunocompetancy is measured by identifying and enumerating lymphocyte subpopulations in the peripheral blood and bone marrow using MDF. The percentage of lymphocytes is determined by combining cell surface antigen expression with light scattering characterization. The percentage positive of each monoclonal antibody is taken from the lymphocyte gate, and routine normalization is applied to all ensuing phenotypes. A report is generated with the percentage positive and absolute values for each antibody requested (33).

5. Quantitative Progenitor Cell (CD34) Analysis

The identification of stem cells by MDF permits the detection of these cells as an alternative to time consuming culture systems. In addition to stem cell detection, the enumeration of these cells can be used to define the appropriate timing for harvesting of progenitor cells from peripheral blood following growth factor mobilization. The test quantifies the total percentage of stem cells present by detecting CD34 positive cells while ensuring the exclusion of cell debris, aggregates, and dead cells (34).

The total CD34 events are reported as a percentage of the total nucleated cells. Using the WBC count, the collected volume of the apheresis product, and the patient’s body weight (kg), the number of total CD34 positive cells collected can be determined.

Flow cytometry is also useful in the context of CD34 enrichment procedures to look at both CD34 purity and tumor contamination in the enriched product.

Molecular Analysis

1. Molecular Diagnostic Test Menu

Turn-Around-Time: 24 - 48 hrs

Test Clinical Application Methodology CPT Codes
B-cell Gene Rearrangement (IgH)+ Monoclonality detection and confirmation in B-cell malignancies. Monoclonality profiling for disease monitoring. PCR 83891, 83896, 83900, 83901, 83909, 83912
B-cell Gene Rearrangement (IgK)+ For 10 % of B-cell malignancies not detected by IgH assay (germinal and post-germinal center with somatic hypermutations). PCR 83891, 83896, 83900, 83901, 83909, 83912
T-cell Gene Rearrangement+ Monoclonality detection and confirmation in T-cell malignancies. Monoclonality profiling for disease monitoring. PCR 83891, 83896, 83900, 83901, 83909, 83912
BCL-2 t(14;18)+ Quantitative monitoring of t(14;18) for Follicular Lymphoma. Real-Time Quantitative PCR 83891, 83896, 83900, 83901, 83912
BCL-1 t(11;14)+ Quantitative monitoring of t(11;14) for Mantle Cell Lymphoma. Real-Time Quantitative PCR 83891, 83896, 83900, 83901, 83912
ALL Panel * Detection of BCR-ABL, E2A-PBX1, MLL-AF4 and TEL-AML1 Real-Time Quantitative PCR 83891, 83896, 83900, 83901, 83902, 83912, 83913
E2A-PBX1 t(1;19) * Minimal residual disease (MRD) detection of t(1;19) for ALL. Real-Time Quantitative PCR 83891, 83896, 83900, 83901, 83902, 83912, 83913
MLL-AF4 t(4;11) * MRD detection of t(4;11)and prognostic indicator for ALL. Real-Time Quantitative PCR 83891, 83896, 83900, 83901, 83902, 83912, 83913
TEL-AML1 t(12;21) * MRD detection of t(12;21)and prognostic indicator for ALL. Real-Time Quantitative PCR 83891, 83896, 83900, 83901, 83902, 83912, 83913
BCR-ABL t(9;22) * Detection and quantitative monitoring of t(9;22) for CML and ALL. Real-Time Quantitative PCR 83891, 83896, 83900, 83901, 83902, 83912, 83913
AML Panel * Detection of PML-RARA, AML1-ETO and CBFB-MYH11 Real-Time Quantitative PCR 83891, 83896, 83900, 83901, 83902, 83912, 83913
PML-RARA t(15;17) * Detection and quantitative monitoring of t(15;17) for APL. STAT same day analysis available for diagnostic specimens. Real-Time Quantitative PCR 83891, 83896, 83900, 83901, 83902, 83912, 83913
AML 1-ETO t(8;21) * Detection and monitoring of t(8;21). Favorable prognosis in AML. Real-Time Quantitative PCR 83891, 83896, 83900, 83901, 83902, 83912, 83913
CBFB-MYH11 inv(16) * Detection and monitoring of inv(16). Favorable prognosis in AML. Real-Time Quantitative PCR 83891, 83896, 83900, 83901, 83902, 83912, 83913
FIPILI-PDGFRA del(4q12) * Detection and monitoring of del(4q12) in HES (hypereosinophilic syndrome) and CEL (chronic eosinophilic leukemia). Real-Time Quantitative PCR 83891, 83896, 83900, 83901, 83902, 83912, 83913
JAK2+ V617F JAK2 (Janus kinase 2) point mutation detection for diagnosis of myeloproliferative disorders. High detection sensitivity (0.1 %). Heterozygous versus Homozygous profile detection. Activating Point Mutation Assay 83891, 83892, 83896, 83898, 83909, 83912
c-KIT D816V+ Detection of the D816V c-KIT point mutation as prognostic indicator for core binding factor (CBF) AML, mastocytosis, and systemic mast cell disease (SMCD). Activating Point Mutation Assay 83891, 83892, 83896, 83900, 83901, 83909, 83912
FLT3+ Detection of FLT3 (fms-like tyrosine kinase 3) receptor mutations as prognostic indicator for AML. Activating Point Mutation Assay 83891, 83892, 83896, 83900, 83901, 83909, 83912
MSI+ Microsatellite Instability for Hereditary non-polyposis colorectal cancer (HNPCC) and sporadic colorectal cancers. 88380, 83907 83891, 83900, 83901, 83896, 83909, 83912
CLL
IGHV Mutation Analysis 		The lack of hypermutations in the IGHV gene detected by sequence analysis is predictive of poor prognosis.  

* RNA-based assays = please provide minimum of 5 mL peripheral blood or 3 mL of bone marrow in lavender top tube [EDTA]. Overnight shipping and processing within 24 h required for accurate results.

+DNA-based assays = Fresh or paraffin-embedded specimens. FFPE bone marrow biopsies can result in inadequate DNA due to decalcification treatment [clot sections preferable].

For MSI analysis, please submit normal and tumor tissue

2. B- and T-cell gene rearrangement PCR

HematoLogics Inc. offers B-Cell (IGH & IGK) and T-Cell Receptor (TCRG) Gene rearrangement assays to detect monoclonal cell populations in peripheral blood, bone marrow, body fluids and tissues (fresh or paraffin-embedded).

  • Unsurpassed 24 - 48 h turn-around time.
  • Highly sensitive fluorescence based capillary electrophoresis fragment analysis allows monitoring of individual tumors.
  • Optimized and standardized multiplex PCR protocols for clonal gene rearrangment detection by the BIOMED-2 Concerted Action [van Dongen et al. Leukemia, 17:2257-2317, 2003] with unprecedentedly high detection rate.
  • Immunoglobulin Light Chain Kappa (IGK) available in addition to the Immunoglobulin Heavy Chain (IGH) gene rearrangement assay. IGK analysis can detect B-cell proliferations in which somatic hypermutations [germinal center and postgerminal center B-cell malignancies] may hamper amplification of the IGH loci.
  • Only one specimen for High Resolution Flow Cytometry™ and Molecular Analysis.
  • Integrated results for High Resolution Flow Cytometry™ and Molecular Analysis.
  • Cell Sorting combined with Molecular Analysis can be used for Minimal Residual Disease detection and for confirmatory testing in difficult cases.

PCR graph
Monoclonal Peaks detected with Immunoglobulin Heavy Chain conserved framework (FR) primers at 340 bp for FR1 and 273 bp for FR2. [blue=FR1; black=FR2; green=FR3; red=size standard] in lymph node specimen suspicious for lymphoma.

BCL-2, BCL-1, ALL Panel, BCR-ABL, AML panel, PML-RARA, and FIPILI-PDGFRA

3. Quantitative RQ-PCR assays

HematoLogics Inc. offers a comprehensive test menu of quantitative PCR assays for detection and monitoring of chromosomal translocations.

BCL-2 t(14;18) (monitoring, follicular lymphoma)

Quantitative real-time polymerase chain reaction (PCR) assays with primers for the BCL2 MBR-JH and the BCL2 mcr-JH rearrangement regions are used for specific amplification of the t(14;18) genomic translocations. In approximately 70 % of follicular lymphoma cases the t(14;18) translocation can be detected by PCR and consequently this assay can be used to monitor minimal residual disease.

BCL-1 t(11;14) (monitoring, mantle cell lymphoma)

A quantitative real-time polymerase chain reaction (PCR) assay with primers for the BCL1 MTC-JH rearrangement region are used for specific amplification of the t(11;14) genomic translocation. In approximately 50 % of mantle cell lymphoma cases the t(11;14) translocation can be detected by PCR and consequently this assay can be used to monitor minimal residual disease.

ALL Panel

  • Bcr-Abl t(9;22)
  • E2A-PBX1 t(1;19)
  • MLL-AF4 t(4;11)
  • TEL-AML1 t(12;21)

Quantitative molecular analyses of acute lymphoblastic leukemia can identify chromosomal translocations useful for sensitive disease monitoring and to provide independent prognostic information for treatment strategies. HematoLogics utilizes real-time quantitative polymerase chain reaction (RQ-PCR) assays to detect fusion transcripts which are associated with the presence of the t(9;22), t(1;19), t(4;11) and t(12;21) translocations. All translocation assays can be used individually.

ALL Panel table

Bcr-Abl t(9;22)

Utilizes real-time quantitative polymerase chain reaction (RQ-PCR) to detect BCR/ABL fusion transcripts which are associated with the presence of t(9;22) Philadelphia (Ph) translocation resulting in a small derivative chromosome 22 known as Ph associated with CML, ALL and/or AML.

This test can detect the m-bcr (minor breakpoint cluster region) e1-a2 transcript encoding the 190 kDA (p190) protein and the M-bcr (major breakpoint cluster region) b2a2 (e13a2) and b3a2 (e14a2) transcripts encoding the 210 kDA (p210) chimeric tyrosine kinase protein with a sensitivity level of approximately > 1 in 10e5 transcripts (0.001 %). Quantitative assay units are reported according to the ‘Europe Against Cancer Program’ (EAC) standardized protocol [Gabert J et al. Leukemia 2003 (17): 2318-2357] and can be used for treatment monitoring.

BCR/ABL Expression levels graph

Quantitative Analysis of BCR/ABL expression levels of different K562 cell dilutions.

10e-1 (red)
10e-2 (green)
10e-3 (yellow)
10e-4 (blue)
10e-5 (pink)

AML Panel

  • PML-RARA t(15;17)
  • AML1-ETO t(8;21)
  • CBFB-MYH11 inv(16)

HematoLogics offers quantitative molecular analyses of acute myeloid leukemia to identify chromosomal translocations useful for sensitive disease monitoring and to provide independent prognostic information for treatment strategies. Our laboratory utilizes real-time quantitative polymerase chain reaction (RQ-PCR) assays to detect fusion transcripts which are associated with the presence of the t(15;17) + t(8;21) + inv(16) translocations. All translocation assays can be used individually.

AML table

PML-RARA t(15;17)

Utilizes real-time quantitative polymerase chain reaction (RQ-PCR) to detect PML/RARA fusion transcripts which are the molecular result of the t(15;17) translocation associated with the majority of APL cases, a distinct AML subset with M3 cytomorphology. This test can detect all three possible PML-RARA isoforms, referred to as long (L, or bcr1), variant (V, or bcr2) and short (S, or bcr3) with a sensitivity of at least 1 in 10e4 transcripts (0.01 %). Quantitative assay units are reported according to the ‘Europe Against Cancer Program’ (EAC) standardized protocol [Gabert J et al. Leukemia 2003 (17): 2318-2357] and can be used for treatment monitoring.

APL Patient Graph

FIP1L1-PDGFRA del(4q12)

Utilizes real-time quantitative polymerase chain reaction (RQ-PCR) to detect FIP1L1-PDGFRA fusion transcript, which is associated with the presence of an interstitial deletion on chromosome 4q12.

The identification of the FIP1L1-PDGFRA fusion transcript may assist diagnosis, classification and monitoring of hypereosinophilic syndrome (HES) and chronic eosinophilic leukemia (CEL).

4. Activating Point Mutation Assays

Jak2 (MPD)

The V617F mutation of the JAK2 (Janus kinase 2) gene has been described in 74 – 97 % of polycythemia vera (PV), in 33 – 57 % of essential thrombocythemia (ET) and in 35 – 50 % of idiopathic myelofibrosis (IMF) cases [Baxter et al. The Lancet 2005: 1054 – 1061] [Levine et al. Cancer Cell 2005: 387-397]. The identification of the V617F JAK2 point mutation in myeloproliferative disorders (MPD) is useful to assist diagnosis, classification and monitoring.

c-KIT (Mastocytosis, AML)

The D816V c-Kit point mutation has been associated as a prognostic indicator with shorter event-free survival in core binding factor (CBF) acute myeloid leukemia (AML) [Boissel N et al. Leukemia 2006]. In mastocytosis the c-Kit mutation has been associated with both aggressive systemic disease and increased bone marrow mast cell content [Pardanani et al. Leukemia Research 27 2003: 739-742]. In addition, it has been shown that the D816V c-kit activation loop mutation is highly resistant to Imatinib (Gleevec). Therefore identification of this mutation might be informative for therapeutic decisions in systemic mast cell disease (SMCD) and acute myeloid leukemia (AML) [Krystal GW, Leuk Res 2004: S53-S59; Growney JD et al. Blood 2005 106(2): 721-4].

FLT3 (AML)

HematoLogics utilizes Multiplex PCR amplification in combination fluorescence-based capillary electrophoresis to identify two types of functionally important FLT3 (fms-like tyrosine kinase 3) mutations. The internal tandem duplication (ITD) of the juxtamembrane domain and the missense point mutation of the aspartic acid residue D835 in the activation loop of the kinase domain result in constitutive activation of the FLT3 receptor. Both FLT3 mutations have been described to be an important prognostic factor in AML [Thiede et al. Blood 2002, 99:4326-4335] [Kottaridis et al. Blood 2001, 98:1752-1759] [Boissel et al. Leukemia 2006].

5. Microsatellite Instability (MSI)

MSI detection can be helpful in identifying patients with hereditary non-polyposis colorectal cancer (HNPCC; Lynch syndrome). In addition, microsatellite instability has also been reported for approximately 15 % of sporadic colorectal cancers. Determination of MSI status in sporadic cancers might be useful for establishing prognosis and may predict the benefit from certain chemotherapeutic regimens [Benatti et al. Clin Cancer Res 2005,11(23):-8332-40; Ribic et al. N Engl J Med 2003,349(3):247-57].

6. CLL IGHV Mutation Analysis

The determination of the mutational status of rearranged immunoglobulin heavy chain variable (IGHV) genes in patients with chronic lymphocytic leukemia (CLL) has shown strong and independent prognostic value. The lack of hypermutations in the IGHV gene detected by sequence analysis is predictive of a poor prognosis.

7. Retrospect™ DNA Archiving Service

HematoLogics will archive DNA for future Molecular Analysis purposes for all diagnostic B- and T- cell tumor specimens sent for Flow Cytometry analysis at no extra cost.

Tumor clonality fingerprints can be identified by gene rearrangement analysis from diagnostic DNA specimens. Clonality fingerprints can be used for patient specific analysis of follow-up specimens during treatment monitoring or in the case of a suspected relapse. In addition, patient specific tumor clonality fingerprints allow monitoring at sensitivity levels below 0.01 % by combining flow cytometry cell sorting and gene rearrangement analysis.

Disease Applications

Myelodysplastic Syndrome (MDS)/Myeloproliferative Disorders (MPD)

  1. Detection/enumeration of abnormal myeloblasts
  2. Detection of abnormal maturing myeloid cells and monocytes
  3. JAK2 point mutation detection
  4. Detection of possible myelosuppressive lymphoid abnormalities

Non-Hodgkins Lymphoma

  1. Diagnosis of B,T, or NK cell lymphoma
  2. Staging/monitoirng with molecular fingerprint/cell sorting
  3. Gene rearrangement studies of non-viable specimens not analyzable by flow cytometry

Myeloma

  1. Diagnosis of myeloma/MGUS/lymphoplasmacytoid lymphomas
  2. Clonality light chain restriction/gene rearrangement
  3. Monitoring residual disease with molecular fingerprint

Chronic Myelogenous Leukemia (CML)

  1. Classification of blast crisis by immunophenotyping
  2. Diagnosis/confirmation with cytogenetics
  3. Quantitative PCR for monitoring response to Gleevac™

Acute Leukemia

  1. Diagnosis and classification
  2. Promyelocytic leukemia phenotype triggers STAT with same day molecular analysis for PML-RARA
  3. Identification of AML arising from MDS
  4. Residual disease assessment during and after therapy
  5. Quantitative monitoring for cytogenetic abnormalities including t(15;17), t(8;21), inv(16), t(9;22), t(12;21), t(4;11), and t(1;19)
  6. FLT3 Mutation detection

Paroxysmal Nocturnal Hemaglobinuria

  1. Identification of missing phosphatidylinositol glycan anchored proteins
  2. Differentiation of MDS from aplastic anemia
  3. Detection of genetic polymorphisms