Supervisors info:
Ε. Λιανίδου, Καθηγήτρια, Τμήμα Χημείας, Εθνικό και
Καποδιστριακό Πανεπιστήμιο Αθηνών (ΕΚΠΑ)
Α.Ψαρρά, Χημικός MSc PhD, Τμήμα Ανοσολογίας
– Ιστοσυμβατότητας, Γ.Ν.Α. «Ο Ευαγγελισμός
Summary:
Purpose of the study: Chronic myelomonocytic leukemia (CMML) is a
myelodysplastic syndrome/ myeloproliferative neoplasm whose diagnosis is
currently based on the elevation of peripheral blood monocytes to >1 × 109
/L,
measured for ≥3 months. In the peripheral blood of humans, the expression of CD14
and CD16 distinguishes CD14+CD16- (classical), CD14+CD16+ (intermediate) and
CD14+lowCD16+ (non-classical) monocytes. In healthy conditions, up to 94% of
monocytes are classical monocytes. In healthy donors, the proportions of classical
monocytes in the circulation are approximately 85%. Studies have shown that an
increase in the percentage (> 94.0%) of classical monocytes in peripheral blood
specimens is a specific marker for rapid, accurate and easy diagnosis of CMML,
whereas in bone marrow the corresponding percentage may be lower. The main
objectives of this thesis were the study of monocyte subsets and the correlation
between the immunophenotype of monocytes with the diagnosis of CMML.
Materials and methods: Peripheral blood and bone marrow samples from patients
with absolute monocytosis and probability of CMmL were analyzed by flow
cytometry, in Beckman Coulter's Navios EX by Beckman- Coulter and BD FACS
CANTO II by BD Biosciences. The monoclonal antibodies CD7- A700, CD24- PE,
CD16- PB, CD14- PC7, CD45- Krm, CD56- PC5,5 (method BC) and CD7- FITC,
CD24- PE, CD16- BV421, CD14- PECY7, CD56- APC, CD45-V500 (method BD)
were used, respectively, for the two above cell counts. Older samples analyzed by
monoclonal combinations: CD7- FITC, CD24- PE, CD16- ECD, CD14- PC5, CD45-
PC7 and CD56- PC7 or CD36- FITC, CD64- PE, CD16- ECD, CD14- PC5, CD45 -
PC7, included in the study. Results were analyzed with Kaluza (BeckmanCoulter,
method BC) and Infinicyt (Cytognos, method BD) software and the three monocyte
subsets were studied.
Results: Of the 52 peripheral blood samples analyzed with Kaluza software, the
immunophenotype was in agreement with the WHO diagnosis of the disease in 48
of them (p <0.001). In the case of analysis with Infinicyt software, agreement was
found for 35 of the 37 samples (p <0.001). For the case of the 19 bone marrow
samples analyzed with Kaluza software, the immunophenotype were in agreement
with with the WHO diagnosis for 15 of them (p = 0.023 <0.05). Compared with
healthy donors and patients with reactive monocytosis or another hematologic
malignancy, CMML patients demonstrate a characteristic increase in the fraction of
classical monocytes (95,14 ± 7,28 ως 83,78 ± 13,35 for Kaluza and 95,86 ± 1,19 to
78,70 ± 13,19 for Infinicyt, p<0,05). This increase appears also in the bone marrow
of patients (95.14 ± 7.28 to 83.78 ± 13.35 for Kaluza, p = 0.086)
ROC curves for peripheral blood samples showed that classical monocytes had
higher AUC (AUC = 0.896 and AUC = 0.936 for Kaluza and Infinicyt, respectively,
p<0.001) than the other two monocyte subsets. The same image was observed in
the bone marrow with AUC = 0.733, p = 0.086.
Peripheral blood and bone marrow samples, from 10 patients, were analyzed
simultaneous with Kaluza software. There was agreement in the diagnosis between
peripheral blood and marrow for 8 of the 10 samples (p = 0.3).
Finally, 37 peripheral blood samples were analyzed with both of the above methods.
Comparison of results showed agreement between the two methods for all 37
samples (p <0.001). Specifically, 15 were designated as CMML and 22 showed
proportion of classical monocytes <94%. There were no significant differences in
the percentages of classicalal monocytes between the two methods.
Conclusions: Patients with CMML show an increased proportion of classical
monocytes, both in peripheral blood and marrow, compared to other monocyte
subsets and patients with monocytosis or other hematologic malignancies.
However, there is no statistically significant association between the diagnosis in
peripheral blood and diagnosis in bone marrow. Also, 94% for classical monocytes
is a cutoff value with high sensitivity and specificity in the diagnosis of CMML,
whereas for bone marrow this value is lower (≈ 91%). Analysis of larger number of
samples from patients with CMML and comparison with the immunophenotype of patients with monocytosis and other MDS/ MPN will help to more accurate diagnosis
of CMML with flow cytometry as well as to monitor the therapeutic approach.
Keywords:
flow cytometry, immunophenotype, monocytes, leukemia, myelodysplastic syndrome, myeloproliferative neoplasm, chronic myelomonocytic leukemia, CMML
