Table 2.

Technical Recommendations for Chromosome Banding Analysis in CLL

Item	Recommendations	Remarks
Materials
Anticoagulants	Heparin	EDTA is not suitable for cytogenetic cultures. However, in such case, it is worth trying to wash the sample twice with sterile RPMI medium or 10× PBS and set up cultures (if there is no other option)
Cells/tissue	Peripheral blood	Bone marrow or lymph node biopsies may be an option in those cases with few circulating clonal cells
Conditions	Set up cultures before 24 h sample obtention	The transport time is highly relevant for samples with high WBC counts 20–22
Methodsa
WBC count	Adjust cultures to 2 × 106 leucocytes/mL medium
Mitogens	Set up 2 parallel cultures: Culture A: 50 µL TPA Culture B: 500 µL IL2 + 100 µL (10 nmol) DSP30b	Following this strategy, more cases with abnormal karyotypes are identified, as 5% to 20% of cases are found carrying aberration(s) only with 1 of the 2 mitogens8
Culture times and Colcemidc	Culture A: incubate 72 h at 37°C, add 50 µL Colcemid, incubate 2 h, harvest Culture B: incubate 48 h at 37°C, add 100 µL Colcemid, incubate 16–24 h, harvest
Analysis
Number of metaphases	20, fully analyzed	Recommended to avoid overlooking subclonal aberrations and complexity underestimation49
Interpretation-clinical   significance		HC (≥5 structural/numerical abnormalities in the same clone) is generally associated with unfavorable prognosis, excepting patients with a CK harboring +12, +19. The predictive significance of high-CK in the era of novel agents is still unclear

^aHarvesting, slides preparation, aging, and staining are performed following standard cytogenetic procedures.⁵⁰

^bDSP30: sequence: 5′-TsCsgsTsCsgsCsTsgsTsCsTsCsCsgsCsTsTsCsTsTsCsTsTsgsCsC.

^cColcemid concentration 0.15 g/mL.

CK = complex karyotype; CLL = chronic lymphocytic leukemia; EDTA = ethylenediaminetetraacetic acid; HC = high-complexity; PBS = phosphate-buffered saline; RPMI = Roswell Park Memorial Institute; WBC = white blood cell; TPA = 12-O-tetradecanoly-phorpol-13-acetate.