NLG Guidelines

NORDIC LYMPHOMA GROUP PATHOLOGY GUIDELINES 2000:
HANDLING OF BIOPSIES FROM LYMPHOMA SUSPECTED TISSUE – DIAGNOSTICS OF MALIGNANT LYMPHOMA

The Pathology Working Group, February 2004:

Christer Sundström, Uppsala
Elisabeth Ralfkiær, Copenhagen
Jan Delabie, Oslo
Kaarle Franssila, Helsinki

SAMPLE SELECTION
The diagnosis is preferably based on histologic evaluation of tissue biopsy material. The material can be derived from lymph node, spleen, tonsil, bone marrow or other tissue. In those cases where a fine needle aspiration biopsy has been performed and giving indication of diagnosis and localization, the tissue biopsy should include the lymph node that has been found to be pathological. If a lymph node biopsy is planned and enlarged lymph nodes are available in several locations an axillary or supraclavicular lymph node and secondly a cervical or inguinal lymph node should be prefered. In those cases where lymphoma is suspected in a non accessible area (retroperitoneum, abdomen etc.) a core biopsy can be performed. In special situations where a surgical biopsy is impossible or core biopsy cannot be performed the diagnosis may be based on fine needle aspiration material. If the diagnosis is based on a fine needle aspiration biopsy 2-3 air dried smears should be done for morphological evaluation. Further smears can be used for immunostaining. The best result is however obtained if a cell suspension is done from the aspiration material (the material from several aspirations is put into an EDTA tube with 1 ml buffered saline).

It is of importance that a considerably enlarged lymph node is removed. The larger the material is the better is the chance to come to a conclusive diagnosis. In addition, the more material that is received the more can be done on this material – now and in the future!

Biopsied lymph nodes should be removed intact which means that they must not be cut and that they should be handled cautiously. If the material can be transported to a Department of Pathology within hours it should be put in sterile physiological saline. The specimen should reach the laboratory as soon as possible, preferably the same day or at the latest in the morning the next day. Core biopsies and small gastrointestinal biopsies should be treated cautiously and preferably be put in a tube completely filled with saline.

If the biopsy material can not be sent in fresh the biopsy should be put in neutral, buffered 10% formalin (if the diameter of the lymph node is at least 1 cm it should first be cut in halves and imprints should be made from the cut surface).

REQUESTED ANAMNESTIC INFORMATION
Relevant previous morphological examinations, haematological data, earlier diseases, physical examination.
Clinical evaluation or diagnosis.
Type of biopsy and from where the biopsy has been obtained.
Eventual marking of the specimens.
What has been sent in.

CUTTING RECOMMENDATIONS
On arrival the lymph node should be cut and imprints should be made from the fresh cut surface. The same holds for larger biopsy materials. Relevant material should be distributed for morphological examination, eventually for flow cytometry and freezing. Removed spleens with suspicion of lymphoma should be cut in thin slices (0,5 – 1 cm) before fixation. Obvious infiltrates should be cut out and fixed separately. Lesions suspected for lymphoma in other organs should be cut out and fixed separately. In most cases of skin and gastrointestinal lymphoma renewed biopsies are often required for immunostaining. These biopsies should be sent in fresh.

ANALYSIS
Sections should be cut with 3 ( thickness. Paraffin embedded material should be stained for hematoxylin-eosin, Giemsa and a reticulin stain for evaluation of growth pattern. Fine needle aspirations should be stained with May-Grünewald-Giemsa.

The diagnosis is based on morphological criteria in combination with immunophenotyping. In most cases flow cytometry analysis of antigen expression and immunostainings of histological material should give corresponding results and give information about a tumor’s cell line derivation, its clonality and eventual expression of a phenotype specific for a certain diagnosis. A minimal requirement is that immunostaining should be done on paraffin sections, alternatively on cytological material. The result of the immunophenotyping with flow cytometry should be interpreted in relation to the morphological picture.

In combination with morphology the following panel of antibodies for immunophenotyping should be enough for the diagnosis of most malignant lymphomas (more than 85%): CD3, CD4, CD5, CD8, CD19 or CD 20, CD 23, (/( and MIB-1.

If the analysis is done with flow cytometry doublestaining is recommended with at least the following antibody combinations: (/(, CD5/20, CD19/3 and CD4/8.
If after morphological examination a high grade lymphoma is diagnosed the panel could be reduced to CD3 and CD19 or CD 20.
If HCL is suspected the following antibodies are recommended in addition: CD103, CD11c and CD 25 (not on paraffin sections) alternatively DB44 (on paraffin sections).
If LGLL is suspected the following antibodies are recommended: CD3/CD16+56, eventually CD3/16 and CD3/56 (paraffin sections and flow cytometry).
If mantle cell lymphoma is suspected the following antibody is recommended: DCS6 (a cyclin D1 antibody working on paraffin sections).
Ki-67-staining (MIB-1) is recommended for the differentiation between a low proliferative and a high proliferative lymphoma and for the diagnosis of Burkitt’s lymphoma.
Bcl-2-staining of sections can in some occasions be useful for differentiation between follicular lymphoma and follicular hyperplasia.
In order to confirm a suspected Hodgkin´s disease the following antibodies are recommended: CD3, CD15, CD20, CD30, EMA, (/( and ALK.

With suspicion of myelosarcoma or involvement of AML the following antibodies are recommended: myeloperoxidase and eventually CD34 and CD68 (AML M0 and M5).

T cell lymphomas are often difficult to diagnose; rebiopsy is often needed to secure fresh material. Extensive T cell marker panel often needed. TCR rearrangement analysis sometimes needed.
Send the case for consultation!

REPORT
MACROSCOPICAL DESCRIPTION
The size of the biopsy material is described, especially if it is a spleen or part of an organ with lymphoma involvement.

MICROSCOPICAL REPORT
The diagnosis is given according to the REAL classification (Harris et al., Blood 84:1361-1392, 1994). Non-classifiable non-Hodgkin lymphomas are separated in B- and T-cell lymphomas and into low grade malignant and high grade malignant lymphomas according to the criteria described in the Kiel-classification. In case of a low proliferative NHL evidence for transformation should be stated and in high proliferative NHL the eventual coexistence of a low grade malignant component should be stated and if the high proliferative component in that case is considered as a transformation. The diagnosis should as far as possible be based on examination of histological criterial.

COMPLEMENTARY INFORMATION
Except for a diagnosis according to the REAL classification the result of immunophenotyping should be reported.

FROZEN MATERIAL
If fresh material is sent to the department frozen material should be preserved for future use. Pieces of biopsy material for freezing are put on small pieces of cork and this is then snap-frozen in a mixture of isopentan and dry ice. The pieces are thereafter put in an Ellerman tube with a frozen drop of saline and are thereafter kept in – 70o. If immunophenotyping has been performed with a flow cytometer the remaining cell suspension should be frozen in freeze medium (20% DMSO, 25% fetal calfserum and 55% tissue culture medium). In each aliquot that is frozen cell the cell concentration should be 10-100 x 106 cells/ml.

TUMOUR BANK FOR LYMPHOMA DIAGNOSTICS AND RESEARCH
Preservation of fresh frozen material for future use is strongly recommended but remains to be a local initiative.

As much material as possible should be frozen.
The material should be made available to ’out department’ investigators on request depending on
– the project in question;
– amount of material left;
– funding principles;
– ethical rules/law regulations.

PROPOSED PROCEDURE FOR REEXAMINATION OF LYMPHOMA DIAGNOSES WITHIN STUDIES COORDINATED BY NLG
Patients included in a study are listed by the national study coordinator; the list should include patient name, personal identification number, date of diagnosis, name of pathology department, specimen number/year; an indication should also be given as to whether FCM immunophenotyping and molecular genetics studies have been performed.
Slides and other relevant data to be reexamined are collected by the national reference pathologist.
The NLG pathology group decides how the reexamination procedure is to be performed for each individual project.

REFERENCES
JA Ferry & NL Harris: Atlas of lymphoid hyperplasia and lymphoma. WB Saunders, 1997.
PG Isaacson & AJ Norton: Extranodal lymphomas. Churchill Livingstone, 1994.
AG Stansfeld & AL d´Ardenne: Lymph node biopsy interpretation. 2nd edition. Churchill Livingstone, 1992
R Warnke et al: Tumors of the lymph nodes and spleen. AFIP Atlas of Tumor Pathology, 3rd series, fascicle 14, 1995
LW Weiss: Pathology of lymph nodes. Churchill Livingstone, 1996