Flow cytometry immunophenotyping (FCI) of fine needle aspirates (FNAs) of lymph nodes [Imunofenotipizacija uzoraka dobivenih citološkom punkcijom limfnog čvora metodom protočne citometrije]

Kardum Paro, Mirjana Mariana and Šiftar, Zoran and Kardum-Skelin, Ika and Šušterčić, Dunja and Nazor, Aida and Flegar-Meštrić, Zlata and Jakšić, Branimir (2010) Flow cytometry immunophenotyping (FCI) of fine needle aspirates (FNAs) of lymph nodes [Imunofenotipizacija uzoraka dobivenih citološkom punkcijom limfnog čvora metodom protočne citometrije]. Collegium Antropologicum, 34 (2). pp. 359-365. ISSN 0350-6134

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Abstract

Flow cytometry immunophenotyping (FCI) has an important role in the clinic work-up of fine needle aspirates (FNAs) of lymph nodes. Its standardization has been defined by proposed analytical protocols and procedures used to assure proper analytical results also in those non-routine samples. In Institute of Clinical Chemistry, "Merkur" University Hospital, FCI is accredited method according to laboratory accreditation standard ISO 15189. According to this laboratory accreditation standard, participation in external quality assessment (EQA) programs is a prerequisite for assuring integrity and quality of the entire laboratory process. A critical analysis of our institutional experience in the feasibility of FCI of the material obtained by FNA of lymph nodes with suspected lymphoma represented the purpose of the study. During an eight-year period in Institute of Clinical Chemistry, "Merkur" University Hospital, a total of 1295 FNA analysis was done, 245 of them with a possible diagnosis of B-cell Non-Hodgkin lymphomas (B-NHL) formed the basis of the study. Lymphocytes were isolated on density gradient according to Boyum et al. The average feasibility of FNAs for FCI analysis was 86% (ranged 78-93%). An acceptable total cell number in FNAs for FCI analysis (4257) was established. In total population of respondents statistical significances in expressions of cellular antigens CD3, CD5, CD22, CD23, CD19 and CD5 on B-cells (CD5+CD19+) between patient's with final diagnosis of benign, reactive lymphoid proliferations and patient's with diagnosis of B-NHL were found. EQA results analysis showed that all results were either inside target values (X +/- 1SD) or inside accepted values (X +/- 2SD). Compatibility of the restriction of immunoglobulins light chains determinated by FCI and cytomorphology diagnosis depends on the choice of criterion values of the light chains ratio which determine the monoclonality. According to the matrix of shares of all classified data of retained neural network, ranges of diagnostic sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and prevalency of 82%, 72%, 93%, 48%, and 72% were produced. As a conclusion, FCI is a reliable methodology for phenotyping FNAs of lymph nodes with suspected B-NHLs detecting their clonality easily.

Abstract in Croatian

Imunofenotipizacija metodom protočne citometrije ima važnu ulogu u diferencijalnoj dijagnostici limfadenopatija. Standardizacija metode protočne citometrije podrazumijeva korištenje predloženih analitičkih protokola i procedura koji osiguravaju pouzdanost analitičkih rezultata i u uzoraka koji nisu rutinski. U Zavodu za kliničku kemiju Kliničke bolnice »Merkur« imunofenotipizacija metodom protočne citometrije je akreditirana metoda prema HRN EN ISO 15189 »Medicinski laboratoriji – Posebni zahtjevi za kvalitetu i osposobljenost«. Svrha ovog rada je kritička analiza imunofenotipizacije ne-rutinskih uzoraka dobivenih citološkom punkcijom limfnog čvora metodom protočne citometrije, a u kojih je postojala sumnja na B staničnu neoplazmu (B-NHL). Tijekom osmogodišnjeg razdoblja u Kliničkoj bolnici »Merkur« ukupno je učinjeno 1295 citoloških punkcija limfnog čvora, od toga je 245 uzoraka punktata limfnog čvora u kojih je postavljena sumnja na B-NHL imunofenotipizirano metodom protočne citometrije. Limfociti su izdvojeni na gradijentu gustoće (metoda Boyuma i sur.). Određena je prosječna prihvatljivost uzoraka punktata limfnog čvora za imunofenotipizaciju metodom protočne citometrije (86%) koja je tijekom razdoblja iznosila od 78 do 93%, kao i prihvatljiv broj stanica u uzorku punktata limfnog čvora (4257 stanica). Analizom ekspresija staničnih površinskih biljega CD3, CD22, CD23, CD19 i CD5 pozitivnih limfocita B (CD5+CD19+) utvrđena je statistički značajna razlika u ekspresiji ovih biljega u benignih, reaktivnih limfadenopatija nepoznatog uzroka i u B-NHL. Podudarnost citomorfološke i imunofenotipizacijske dijagnoze ovisila je o odabiru graničnih vrijednosti omjera lakih lanaca imunoglobulina koji određuju monoklonalnost. Prema matrici udjela svih klasificiranih podataka zadržane neuronske mreže izračunate su dijagnostička osjetljivost (82%), specifičnost (72%), pozitivna prediktivna vrijednost (PPV, 93%), negativna prediktivna vrijednost (NPV, 48%) i prevalencija (72%) metode protočne citometrije. U zaključku, metoda protočne citometrije je pouzdana metoda za imunofenotipizaciju uzoraka punktata limfnog čvora, a u kojih je postavljena sumnja na B staničnu neoplazmu (B-NHL).

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