Recommendations for the cytogenetic analysis of lymphoproliferative syndromes specimens

TYPE OF SAMPLE

  • LPDs in general: Bone marrow, peripheral blood (when invaded) and lymph node/ any lymphoid tissue are suitable for the investigation of LPD.
  • MM: Bone marrow is preferred for the investigation of MM.

CULTURE TIMES

  • LPDs in general: > 3 days cultures are recommended using a cell concentration of 12106cells/ml media.
  • CLL: Stimulation is recommended for CLL (using i.e. TPA, LPS, oligo CpG/IL-2).

CHROMOSOME ANALYSIS

In the section below the general rules are outlined concerning the number of mitoses analysed. Important is to look at metaphases with poor chromosome morphology although normal cells with better chromosome morphology might be present in excess.

The laboratory procedures may vary according to whether the sample is diagnostic or follow-up, on the initial cytogenetic findings.

If a normal karyotype is obtained at least 20 metaphases should be analysed in order to exclude a clonal abnormality. FISH analysis maybe required depending on the clinical criteria and results of molecular analysis. Especially in CLL, abnormal B cells have a low mitotic index and therefore, conventional cytogenetic analysis may fail to detect the aberrant clone.

If an abnormal karyotype is obtained a sufficient number of cells should be analysed to establish clonality and identify clonal evolution. It may not be appropriate to characterise all abnormalities if the karyotype is very complex.

FISH ANALYSIS

  • CLL: In CLL, FISH probes should include
  • ATM at 11q22,
  • D13S319, D13S25, and/or D13S272 at 13q14,
  • TP53 at 17p13
  • cen12
  • In addition, FISH for the detection of a 6q21-23 deletion can be performed.
  • In diagnostic uncertainty with respect to other low grade non Hodgkin lymphomas interphase FISH with probes for IGH, BCL2, and CCND1 may be considered .
  • MM: when performed in myeloma, FISH should be done either by concentrating the plasma cells or by employing some means of plasma cell identification so that only these cells are scored. Purification and simultaneous immunostaining and FISH (cIgFISH) are equally valid methods. TP53 at 17p13, t(4;14)(p16;q32) and 13q should be tested (other loci are optional).

References

  • Claudia Haferlach, Harald Rieder, Debra M. Lillington, Nicole Dastugue, Anne Hagemeijer, Jochen Harbott, Stephan Stilgenbauer, Sakari Knuutila, Bertil Johansson, and Christa Fonatsch on behalf of the European Leukemia Net–Workpackage Cytogenetics
    Proposals for Standardized Protocols for Cytogenetic Analyses of Acute Leukemias, Chronic Lymphocytic Leukemia, Chronic Myeloid Leukemia, Chronic Myeloproliferative Disorders, and Myelodysplastic Syndromes
    GCC 46:494–499 (2007)
  • GFCH (Christian Bastard, Evelyne Callet Bauchu, Agnès Daudignon, Dominique Leroux, Hélène Poirel, Pascaline Talmant).
    Recommandations pour la prise en charge cytogénétique des lymphomes malins non hodgkiniens de l’adulte établies par le Groupe Français de Cytogénétique hématologique (GFCH)
    Pathologie Biologie 52: 260–262 (2004)
  • GFCH (HervéAvet-Loiseau, Christian Bastard, Véronique Smadja
    Coordonnateur : Christian Bastard )
    Recommandations pour la prise en charge cytogénétique des myélomes
    multiples établies par le Groupe Français de Cytogénétique
    Hématologique (GFCH)
    Pathologie Biologie 52: 263–264(2004)
  • Ross FM, Avet-Loiseau H, Drach J, Hernandez Rivas JM, and Liebisch P on behalf of the European Myeloma Network FISH Working Party
    European Myeloma Network Recommendations for FISH in Myeloma
    11th International Myeloma Workshop, Kos, 25.06. – 29.06.2007