Hematopathology Diagnostic Testing:

The area of molecular diagnostics is rapidly evolving. The UW hematopathology laboratory has been involved in molecular testing since 1987, and we are continuing to develop and implement new methodology. Because of the rapid pace of development, the following list of tests is likely to be incomplete fairly soon after the production of this brochure. Informational mailings will be sent out as new tests become available. Please call the hematopathology laboratory DNA section at (206) 548-6215 with any questions or special requests.

Detection of Clonality

Lymphocyte Gene Rearrangement Panel (To detect T cell/B cell lineage and clonality)

The diagnosis of neoplastic lymphocytic processes has relied on histologic and immunocytochemical methods to determine clonality and cell lineage based on the detection of T and B cell antigens on the surface of cells. Clonality often can be clearly assessed in B cell malignancies with the presence of either kappa or lambda light chains on the surface of the neoplastic cells. In T cell malignancies clonality is less easily determined. Occasionally, the presence of mixed cell populations or lack of detectable surface antigens may lead to non-diagnostic or ambiguous results. Since the T cell receptor or immunoglobulin genes rearrange in a unique way for each lymphocyte during lymphocyte development, molecular biologic techniques can be used for the detection of clonal populations of T or B cells.

DNA is extracted from frozen tissue or liquid specimens (peripheral blood, bone marrow aspirate, body fluids), digested with endonucleases, separated by agarose gel electrophoresis, transferred to a membrane (the Southern blot technique), and then hybridized with P-32 labeled probes. Clonal rearrangements are detected as DNA fragments migrating differently from unrearranged DNA. This technique is able to detect a clonal population comprising as few as 1-5% of the total cells in the specimen. The laboratory director will issue a written report and interpretation for each case.

Specimen requirements: EDTA anticoagulated peripheral blood (lavender top tube) or bone marrow, fresh or frozen tissue specimens, body fluids

B Cell Clonality Analysis by the Polymerase Chain Reaction (IgH PCR)

When the immunoglobulin heavy chain gene rearranges, a V (variable), D (diversity), and J (joining) region are brought together to form a functional unit. While V-D-J joining occurs, random numbers of DNA basepairs are inserted between the V-D and D-J junctions, forming the third complementarity determining region (CDRIII) that is involved in antigen specificity. Consequently, all non-neoplastic B cells have slightly different distances between the V and J segments. The polymerase chain reaction allows one to produce large amounts of DNA, the ends of which are determined by the primers one uses. For B cell clonality analysis, we use two PCR primers: one that corresponds to a consensus sequence common to most V segments, the other a consensus sequence common to most J segments. When the DNA is amplified, the length of the PCR product is determined by the number of random nucleotides added at the time of VDJ joining. In a non-neoplastic population of B cells, each PCR product is slightly different in size. When these products are separated by electrophoresis, a smear is seen on the gel. In the case of B cell lymphoma, all the neoplastic cells have the same VDJ rearrangement. Therefore, the neoplastic cells all produce a PCR product of the same size, resulting in the appearance of a discrete band on the gel.

The PCR method has a number of advantages over Southern blot technology including the ability to use less DNA, shorter turnaround time, and the ability to analyze paraffin-embedded specimens. B cell clonality analysis can be performed on small specimens such as endoscopic biopsies that often yield insufficient DNA for Southern blot analysis. The ability to perform gene rearrangement studies on fixed tissue can result in substantial cost savings for patients who in the past would have had repeat biopsies to obtain fresh or frozen tissue for gene rearrangement studies when frozen tissue was not saved initially. The laboratory director will issue a written report and interpretation for each case.

Specimen requirements: EDTA anticoagulated peripheral blood or bone marrow, other body fluids, tissue specimens including fresh, frozen, or fixed/paraffin embedded. Note: some fixatives such as B5 or Bouin's damage DNA and make it unsuitable for PCR amplification.

For more information please see PCR Testing in Hematopathology

T Cell Clonality Analysis by the Polymerase Chain Reaction

The T cell clonality PCR assay is based on the same principle as the B cell clonality PCR assay. Consensus primers for the T cell receptor g chain V and J regions are used to amplify across the V-J join N region. As with the B cell clonality assay, a polyclonal T cell population will produce a smear on a gel, while a clonal process will produce a discrete band.

Considerably less DNA is needed for the PCR assay compared with the Southern blot method. Therefore, small biopsies, such as punch biopsies of the skin or endoscopic biopsies of GI tissue, which would have been insufficient for Southern blot analysis, yield enough DNA for PCR analysis. Furthermore, the assay can be performed on paraffin-embedded specimens, allowing for analysis of archival material and avoiding the necessity for repeat biopsies in cases where fresh or frozen material was not obtained initially. The laboratory director will issue a written report and interpretation for each case.

Specimen requirements: EDTA anticoagulated peripheral blood or bone marrow, other body fluids, tissue specimens including fresh, frozen, or fixed/paraffin embedded. Note: some fixatives such as B-5 or Bouin's damage DNA and make it unsuitable for PCR amplification.

Detection of Translocations

BCR-ABL Reverse Transcriptase PCR

A majority (90%) of patients with chronic myelogenous leukemia (CML) have a characteristic reciprocal translocation of DNA between chromosomes 9 and 22, termed the Philadelphia chromosome (Ph'). As a result of this translocation, the proto-oncogene c-ABL, normally present on chromosome 9, is translocated to chromosome 22 where it is fused with a gene designated BCR (for breakpoint cluster region). A subset of patients with acute lymphoblastic leukemia also have a Philadelphia chromosome. The breakpoints in the BCR gene are different in Ph'-positive CML and ALL, resulting in the production of different RNA molecules and proteins. Importantly, the BCR-ABL fusion protein produced from the ALL-type breakpoint has stronger transforming ability than the CML-type BCR-ABL protein, resulting in the more aggressive disease. Reverse transcriptase-PCR (RT-PCR) enables detection of the BCR-ABL fusion RNAs, and the type of breakpoint can be determined by the size of the PCR product.

RNA is extracted from cells in the peripheral blood or bone marrow, converted to cDNA using the enzyme reverse transcriptase, and then the DNA is detected using a nested PCR technique. This technique is positive if as few as 0.1% of the total cells in the specimen have a BCR rearrangement. The laboratory director will issue a written report and interpretation for each case.

Specimen requirements: EDTA anticoagulated peripheral blood (lavender top tube) or bone marrow

BCL-2 Translocation Analysis by the Polymerase Chain Reaction (BCL-2 PCR)

Greater than 85% of follicular lymphomas have a translocation in which the BCL-2 gene on chromosome 18 is translocated into the immunoglobulin heavy chain locus on chromosome 14. This translocation can be detected by PCR using primers specific for both major and minor translocation breakpoints on chromosome 18, and a second primer specific for chromosome 14 sequences. If t(14;18) is present, a PCR product is produced. If the translocation is not present, no product is produced. Since a negative result could indicate DNA that cannot be amplified by PCR, control reactions amplifying the cystic fibrosis transmembrane regulator (CFTR ) gene or IgH CDRIII region are performed to verify the quality of the DNA. This technique is capable of detecting as few as one in 100,000 cells, and thus can be used to detect the presence of minimal residual disease in blood or bone marrow. The test can also be performed to confirm the diagnosis of follicular lymphoma in cases where classification is difficult. As with IgH PCR, this technique can be performed both on fresh/frozen or fixed tissue specimens. The laboratory director will issue a written report and interpretation for each case.

Specimen requirements: EDTA anticoagulated peripheral blood or bone marrow, other body fluids, tissue specimens including fresh, frozen, or fixed/paraffin embedded. Note: some fixatives such as B5 or Bouin's damage DNA and make it unsuitable for PCR amplification.

For more information please see PCR Testing in Hematopathology.

Molecular Diagnostics - Billing Information:

Billing information available in downloadable PDF format

Last updated: 6/22/00