By Craig E. Litz, M.D.

 

How Does Yours Stack Up?

Illustrative Case Report

A 67-year-old married female from Louisiana is found to have mild macrocytic anemia and neutropenia on a complete blood cell count (CBC) performed during a physical examination. A blood smear is prepared and a bone marrow biopsy and aspirate are performed.

The blood shows slightly decreased red blood cell and neutrophil counts. There is mild non-specific anisopoikilocytosis. The white cells and platelets are morphologically unremarkable. The bone marrow is normocellular with moderate erythroid hyperplasia and some small uninucleate megakaryocytes with no increase in blasts. Marrow flow cytometry performed by a local laboratory analyzed by technologists using the “gating technique” shows no increase in blasts (2% CD34 + cells) and is interpreted as “no evidence of hematolymphoid malignancy.” Marrow cytogenetic studies sent to an out-of-state national reference lab reports “no growth due to lack of viable cells.” The hematopathologist suggests that, although the morphologic findings might be that of a myelodysplastic syndrome, they are not absolutely diagnostic.

While visiting her sister the next month in Dallas, the patient seeks the opinion of another hematologist. The hematologist suggests a repeat bone marrow biopsy and aspirate. Although flow cytometry using the “cluster method” analysis confirms no increase in marrow blasts, subtle immunophenotypic aberrancies are noted in the myeloblasts. In addition, this analysis reveals that the granulocytes and monocytes aberrantly express CD56. These findings are highly suggestive of a myelodysplastic syndrome.

Conventional cytogenetic evaluation performed at the same Dallas-based lab shows a deletion in the long arm of one chromosome 5 in fifteen of twenty metaphase preparations examined. In light of this additional information, a confident diagnosis of a de-novo myelodysplastic syndrome with a 5qchromosomal abnormality is made.

Over the last two decades, the fields of flow cytometry and cytogenetics have changed from optional ancillary studies to critical adjuncts to morphologic examination in the diagnosis and monitoring of hematolymphoid neoplasia. Not infrequently, the difference in a presumptive and confirmed diagnosis is based on the location and quality of the laboratory performing these studies. Although CAP and CLIA certification is required of virtually all labs associated with patient care, it is not a discriminator of laboratory quality.

KEY QUESTIONS YOU SHOULD ASK OF YOUR LABORATORY ARE:

Flow Cytometry

Does your flow cytometry provider use “gated” or “cluster” analysis and are the results interpreted and analyzed by a well-trained, board-certified hematopathologist?

Typically, technologists at flow cytometry laboratories examine 2 to 3 parameters with “gated” analysis to define a population as “lymphocytes” or “blasts.” This crude, relatively inexpensive technique frequently misses small populations of cells outside the “gates” or cells with aberrant antigen profiles as illustrated in the case above.

In contrast, “cluster analysis” is a sophisticated routine where a total of 6 or more parameters are identified on each cell and all 6 parameters are used to define populations (in 6-dimensional space). In combination with broader panels and the direct involvement of board-certified hematopathologists, this becomes a comprehensive, sensitive and specific tool for detecting and characterizing hematolymphoid malignancies. As a result, tiny homogeneous subpopulations of aberrant lymphoblasts, myeloblasts, or lymphoma cells can be “dissected” out without bias. Neoplastic cells can more reliably be distinguished from normal counterparts and detected at a level lower than with either morphologic or gated techniques. Surveillance and diagnosis are improved giving the hematologist a better idea of a patient’s hematologic status.

Are “no growth” specimens included in the laboratory’s turn-around-time (TAT) calculations?

Turn-around-time (TAT) is an important part of a laboratory diagnostic study, but the oncologist should be aware that in clinical cytogenetics, the fastest TATs are not always the primary criteria for obtaining complete diagnostic results. If specimens are consistently dead or compromised upon arrival and quickly signed out as growth failures, this will artificially reduce the overall TAT if that lab includes them in their TAT average. When comparing TATs from different cytogenetics laboratories, it is reasonable to inquire if specimens that were “no grows” are included in the calculation. A false-negative cytogenetic result or no result at all does not help you or your patients regardless of turn-around-time.

How are specimens transported to the laboratory and how far away is the cytogenetics lab?

Conventional cytogenetic analysis requires living cells capable of division. Exposure to significant time delay, septic conditions, or temperature extremes will all decrease the viability of the cells of interest. The cytogenetics laboratory should be accessible for specimen delivery within 24-hours and the specimen should be delivered to the laboratory by a courier system that will protect specimen integrity. Dead cells equal no results.

What is the experience of the cytogenetics laboratory with fluorescent in situ hybridization (FISH)?

A comprehensive cytogenetics laboratory must have significant and diverse experience with FISH, a molecular testing modality that is often necessary for the accurate diagnosis of complex cases. FISH may also be used at the time of diagnosis to define cost-effective ways to monitor patients for minimal residual disease following treatment or for possible relapse. Laboratory personnel should have facility with FISH for both metaphase and interphase evaluation and should be able to discuss their validation procedures, control methodologies and reference ranges for each FISH probe being used. The oncologist should feel free to inquire about the laboratory’s experience and proficiency with the FISH probes being used for the diagnosis of their patients.