Turkish Journal of Hematology Volume: 35 - Issue: 4

Olcay L, et al: Hematopoiesis in Families with Congenital Neutropenia
Turk J Hematol 2018;35:229-259
Table 2. CD95, CD95 ligand, and annexin levels on the lymphocytes, granulocytes, and monocytes of the patients and their parents (%).
Dead cells***
Granulocyte
Necrotic
cells**
Granulocyte
Apoptotic
cells*
Granulocyte
CD95 ligand
Granulocyte
CD95
Granulocyte
Dead cells***
Monocyte
Necrotic
cells**
Monocyte
Apoptotic
cells*
Monocyte
CD95
ligand
Monocyte
CD95
Monocyte
Dead cells***
Lymphocyte
Necrotic
cells**
Lymphocyte
Apoptotic
cells*
Lymphocyte
CD95 ligand
Lymphocyte
CD95
Lymphocyte
67.70±27.11
(24.1-100.0)
56.04±31.60
(2.60-98.40)
11.65±12.64
43.96±31.19
40.76±31.52
1.65±2.15
(0.4-7.9)
96.69±4.84
(86.5-99.8)
69.29±41.22
(1.80-
100.00)
21.34±33.82
47.95±37.81
(0.10-33.50)
2.34±2.06
(0.0-6.6)
81.57±32.28
(7.9-99.6)
84.71±29.31
(20.10-
100.00)
(0.50-92.40)
(1.0-70.70)
(0.00-98.00)
(0.0-96.9)
0.54±0.31
(0.16-1.3)
Patient 34.65±15.66
n=11 † /8 †† (12.6-69.0)
52.63±45.83
(0.00-100.00)
49.94±46.81
(0.00-
100.00)
2.69±3.43
76.26±37.75
62.97±33.63
13.29±12.17
2.19±2.79
(0.0-9.5)
97.38±2.03
(92.8-99.7)
57.89±41.71
(0.00-
100.00)
7.59±12.55
50.30±40.98
(0.00-12.90)
0.99±0.78
(0.4-2.8)
91.97±22.77
(16.4-99.8)
(0.0-97.5)
(0.00-37.00)
(0.00-36.10)
(0.0-99.8)
0.5±0.31
(0.1-1.2)
(0.00-
100.00)
Parent 49.88±8.56
n=13 † /13 †† (40.90-
71.40)
0.85±0.88
0.77±0.81
0.08±0.10
0.94±1.82
0.87±1.65
0.06±0.21
0.68±0.61
(0.0-1.46)
85.87±12.59
(63.76-98.3)
0.18±0.18
(0.00-0.40)
0.07±0.08
0.11±0.10
(0.00-2.60)
(0.00-2.30)
(0.00-0.30)
0.19±0.14
(0.0-0.48)
94.60±6.17
(79.69-99.65)
(0.00-5.40)
(0.0-4.70)
(0.00-0.70)
(0.00-0.20)
(0.0-0.20)
0.28±0.31
(0.0-0.99)
Control 27.07±8.42
n=12 † /11 †† (11.07-38.75)
0.001*
0.003*
0.015*
0.002*
0.236
0.002*
0.0001*
0.0001*
0.365
0.001*
0.0001*
0.021*
0.016*
0.015*
P p a
0.002*
0.003*
0.0001*
0.0001*
0.236
0.0001*
0.024*
0.006*
0.365
0.003*
0.003*
0.019*
0.004*
0.016*
0.0001*
0.454
0.651
0.504
0.0001*
0.236
0.874
0.545
0.355
0.365
0.685
0.335
0.789
0.975
0.325
0.006*
0.123
p b
p c
*Apoptotic Cells: Those with only annexin positivity; **Necrotic Cells: Those with both annexin and PI positivity; ***Dead Cells: Apoptotic and necrotic cells; p a : Comparison between the patients and the control group (Kruskal-Wallis test);
p b : Comparison between the parents and the control group (Kruskal-Wallis test); p c : Comparison between the patients and their parents (Kruskal-Wallis test).
† For CD95 and CD95 ligand.
†† For annexin and propidium iodide.
G1 arrest and apoptosis were established in one patient’s
lymphocytes (ZG) and those of her parents while the cell cycle of
lymphocytes in the sibling of ZG (patient AG) was normal. The cell
cycles of the parents’ granulocytes were normal (Figures 3A-3F).
Rapid Cell Senescence
Eight patients and eight parents were evaluated. The leukocytes of
only 2 patients (siblings AG and ZG) and their mother were stained
with SA-β-gal by 88%, 76%, and 94%, respectively (Figures 3G, 3H,
3I). These patients were members of a family and were evaluated
for cell cycles at the same time (Figures 3A-3F).
Cell Morphology
Neutrophils
The incidences of bizarre nuclei (34.0±17.4% vs. 15.2±4.7%,
p=0.015), pseudo Pelger-Huet (PPH) and PPH-like cells (15.7±9.3
vs. 4.1±3.3, p=0.003), striking chromatin clumping (38.1±27.7%
vs. 11.0±9.7%, p=0.036), macropolycyte percentage (diameter ≥15
µm) (38.71±27.46% vs. 6.44±6.00%, p=0.0001) of neutrophils, and
neutrophil diameter (13.46±1.76 vs. 9.79±1.73, p=0.0001) were
significantly higher in the neutrophils of the parents than those of
the control group by light microscope.
The few neutrophils of the patients in their peripheral blood and
bone marrow also revealed the same abnormalities, but no scoring
could be done due to the low number.
Ultrastructural study of the patients and parents revealed that
secondary granules of neutrophils were low in number, primary
granules were heterogeneous in shape and size, and chromatin
clumping and apoptosis were striking (Figures 4D, 4E, 4L, 5D, 5E,
and 5H).
Lymphocytes
The peripheral lymphocytes of both patients and parents revealed
few lymphocytes with tiny cytoplasmic protrusions. Ultrastructural
study of peripheral blood of patients and parents showed that the
lymphocytes were abnormal or active (Figures 4G, 5F, and 5G).
Monocytes, Macrophages, Histiocytes, and Other Phagocytes
The peripheral blood of patients and parents revealed monocytes
with features of apoptosis, abnormal nucleus, necrosis, or
pseudopod formation under light microscope and abnormal
mononuclear cells under TEM with or without features of apoptosis
(Figures 4F and 5H).
The bone marrow examination of the patients revealed many
monocytes, macrophages, histiocytes, neutrophils, bands,
eosinophils, and eosinophil myelocytes undergoing phagocytosis.
Sea blue-like and Gaucher-like histiocytes in the bone marrow of
the patients were striking. The phagocytosed cells were lymphocytes,
234