Diff Quik and Papanicolaou are complementary and the most utilized types of staining. Diff Quik provides an immediate evaluation of the smears, keeps the background, highlights fibrous connective tissue metachromasia and the orange granules of eosinophils. Papanicolaou provides detailed information on the nucleus and cytoplasm; if necessary, the smear can be distained and used for immunocytochemical stains.
Management of diagnostic material is a crucial point in diagnostic cytology, and mainly in lymph node cytopathology; perfect smears and their immediate evaluation are the first steps of a correct and accurate diagnosis. Ancillary techniques are necessary for accurate diagnosis and indispensable in cases of non-Hodgkin lymphoma (NHL). Nonetheless not all these techniques are available in basic cytology laboratories and each of them has its own specific applications. In fact, FC, which provides extremely useful information for the diagnosis and often the classification NHL, is unsuitable to diagnose Hodgkin lymphoma (HL) and metastases. Conversely, ICC by combining morphological details and immunophenotypic features, is highly contributive in the diagnosis of HL and metastases but may be less effective in light chain evaluation or in the identification of T-rich B-cell NHL. FISH is highly effective in the identification of specific NHL entities but is generally used only when there is substantial diagnostic orientation. These problems, the general scantiness of FNC material and the need to capitalize technical resources have suggested the following algorithm.
Ancillary techniques are indispensable in the diagnosis and classification of most of the lymph node FNC. Each of them may produce specific information and should be used considering the clinical data and cytological features.
The material left in the hub of the needle and/or obtained from a second pass may be suspended in buffered solution at pH 7.4. One or two million cells, easily obtained from one pass, are needed to prepare six or more cytospins to be used for immunocytochemistry. Air dried cytospins can be stored at room temperature for up to 1 week; for longer periods (1-2 years) they need to be stored at -20°. Both immunoalkaline phosphatase and immunoperoxidase can be used on cytospin.
Cell blocks can be prepared by embedding centrifuged cell suspensions in paraffin; Cytoblock cassettes for formalin fixed cell suspensions may be used. The main advantages of cell blocks are the numerous sections they produce, their long term storage, and the same antigenic conditions as formalin fixed histological sections.
Ethanol fixed smears or xylene dewaxed and alcohol rehydrated paraffin sections obtained from cell-blocks can be used for this purpose. Slides are placed in Coplin jars filled with a 0.01 M tri-sodium citrate solution, and microwave-heated three times for 5 minutes. After heating, slides are thoroughly rinsed in cool running water for 5 minutes and then washed in Tris-Buffered Saline (TBS) pH 7.4. After incubation with the primary Ab, slides are covered with biotinylated anti-mouse or anti-rabbit immunoglobulins, followed by peroxidase labelled streptavidine (LSAB); the signal is developed using diaminobenzidine (DAB) as chromogen after incubation for 10 minutes with Horse Peroxidase Enzime (HRP).
The remaining material in the hub of the needle or from additional passes can be suspended in RMPI-1640 or in buffered solution at pH 7.4. One or two million cells, which can be collected in one pass, are distributed into six tubes for a basic panel of fluoresceinated antibodies. The latter are used in groups of three or even four antibodies conjugated with different fluorochromes using double lasers instruments. If possible, it may be useful to keep one tube for further evaluation. Fluoresceinated antibody conjugation has to be performed timely because vital cells may swell in the suspension. After conjugation, cell swelling may be prevented by fixation, adding a drop of buffered formalin; analysis may thus be postponed or repeated. Advantages of FC are: direct reaction antigen-antibody, exact quantification of antigen expression, possible co-expression of two different antibodies on the same cells. Disadvantages are the absence of morphology, loss of large cells, difficulty to identify numerically scanty cell populations.
Fluoresceinated DNA probes are used to visualize specific DNA segments. Chromosomal abnormalities, namely translocations and deletions may be observed and quantified. FISH may be performed on smears or cytospins. The advantage of using cytospins are: high cells concentration, probe sparing and short analysis time.
Aspirated cells may be suspended in RNAlater® for both RNA and DNA preservation and extraction. Cells may be stored at room temperature until DNA extraction. DNA segments are amplified using oligonucleotide probes and polymerase enzymes in repetitive cycles of denaturation, annealing and extension. The amplification products may be analysed by gel electrophoresis or capillary electrophoresis. The JH locus of Ig heavy chains or Tc receptor may be amplified and evaluated to assess monoclonality or polyclonality.