ISSN: 1300-7777 E-ISSN: 1308-5263
Both Granulocytic and Non-Granulocytic Blood Cells Are Affected in Patients with Severe Congenital Neutropenia and Their Non- Neutropenic Family Members: An Evaluation of Morphology, Function, and Cell Death [Turk J Hematol]
Turk J Hematol. 2018; 35(4): 229-259 | DOI: 10.4274/tjh.2017.0160  

Both Granulocytic and Non-Granulocytic Blood Cells Are Affected in Patients with Severe Congenital Neutropenia and Their Non- Neutropenic Family Members: An Evaluation of Morphology, Function, and Cell Death

Lale Olcay1, Şule Ünal2, Hüseyin Onay3, Esra Erdemli4, Ayşenur Öztürk5, Deniz Billur4, Ayşe Metin6, Hamza Okur2, Yıldız Yıldırmak7, Yahya Büyükaşık8, Aydan İkincioğulları9, Mesude Yılmaz Falay10, Gülsüm Özet11, Sevgi Yetgin2
1Ankara Oncology Training and Research Hospital, Clinic of Pediatric Hematology, Ankara, Turkey
2Hacettepe University Faculty of Medicine, İhsan Doğramacı Children’s Hospital, Clinic of Pediatric Hematology, Ankara, Turkey
3Ege University Faculty of Medicine, Department of Medical Genetics, İzmir, Turkey
4Ankara University Faculty of Medicine, Department of Histology Embryology, Ankara, Turkey
5Ankara University Faculty of Medicine, Department of Pediatric Molecular Genetics, Ankara, Turkey
6Ankara Children’s Hematology Oncology Training and Research Hospital, Clinic of Pediatric Immunology, Ankara, Turkey
7Şişli Etfal Children’s Training and Research Hospital, Clinic of Pediatric Hematology, İstanbul, Turkey
8Hacettepe University Faculty of Medicine, Department of Internal Medicine, Unit of Hematology, Ankara, Turkey
9Ankara University Faculty of Medicine, Department of Pediatric Immunology and Allergy and Pediatric Molecular Genetics, Ankara, Turkey
10Ankara Numune Training and Research Hospital, Clinic of Hematology, Ankara, Turkey
11Ankara Numune Training and Research Hospital, Clinic of Hematology, Ankara, Turkey; Yıldırım Beyazıt University Faculty of Medicine, Department of Internal Medicine, Clinic of Hematology, Ankara, Turkey

Objective: To examine granulocytic and non-granulocytic cells in children with severe congenital neutropenia (SCN) and their nonneutropenic parents.
Materials and Methods: Fifteen patients with SCN and 21 nonneutropenic parents were evaluated for a) CD95, CD95 ligand, annexin V, propidium iodide, cell cycle, and lymphocyte subsets by flow cytometry; b) rapid cell senescence (of leukocytes) by senescenceassociated β-galactosidase stain; c) aggregation tests by aggregometer; d) in vitro bleeding time by PFA-100 instrument; e) mepacrine-labeled dense granule number of thrombocytes by fluorescence microscope; and f) hematomorphology by light and electron microscope. HAX1, ELANE, G6PC3, CSF3R, and JAGN1 mutations associated with SCN were studied in patients and several parents.
Results: Significant increase in apoptosis and secondary necrosis in monocytes, lymphocytes, and granulocytes of the patients and parents was detected, irrespective of the mutation type. CD95 and CD95 ligand results implied that apoptosis was non-CD95- mediated. Leukocytes of 25%, 12.5%, and 0% of patients, parents, and controls showed rapid cell senescence. The cell cycle analysis testable in four cases showed G1 arrest and apoptosis in lymphocytes of three. The patients had HAX1 (n=6), ELANE (n=2), G6PC3 (n=2), and unidentified (n=5) mutations. The CD3, CD4, and NK lymphocytes were below normal levels in 16.6%, 8.3%, and 36.4% of the patients and in 0%, 0%, and 15.4% of the parents (controls: 0%, 0%, 5.6%). The thrombocytes aggregated at low rates, dense granule number/ thrombocyte ratio was low, and in vitro bleeding time was prolonged in 37.5%-66.6% of patients and 33.3%-63.2% of parents (vs. 0% in controls). Under electron and/or light microscope, the neutrophils, monocytes, lymphocytes, and thrombocytes in the peripheral blood of both patients and parents were dysplastic and the bone marrow of patients revealed increased phagocytic activity, dysmegakaryopoiesis, and necrotic and apoptotic cells. Ultrastructurally, thrombocyte adhesion, aggregation, and release were inadequate.
Conclusion: In cases of SCN, patients’ pluripotent hematopoietic stem cells and their non-neutropenic parents are both affected irrespective of the genetic defect.

Keywords: Severe congenital neutropenia, Monocytes, Lymphocytes, NK cells, Thrombocytes, Phagocytes, Apoptosis, Senescence, Parents, Family


Ciddi Konjenital Nötropenisi Olan Hastalarda ve Nötropenik Olmayan Ebeveynlerinde Hem Granülositik Hem Granülositik Olmayan Kan Hücreleri Etkilenir: Morfoloji, Fonksiyon ve Hücre Ölümü Yönünden Bir Değerlendirme

Lale Olcay1, Şule Ünal2, Hüseyin Onay3, Esra Erdemli4, Ayşenur Öztürk5, Deniz Billur4, Ayşe Metin6, Hamza Okur2, Yıldız Yıldırmak7, Yahya Büyükaşık8, Aydan İkincioğulları9, Mesude Yılmaz Falay10, Gülsüm Özet11, Sevgi Yetgin2
1Ankara Oncology Training and Research Hospital, Clinic of Pediatric Hematology, Ankara, Turkey
2Hacettepe University Faculty of Medicine, İhsan Doğramacı Children’s Hospital, Clinic of Pediatric Hematology, Ankara, Turkey
3Ege University Faculty of Medicine, Department of Medical Genetics, İzmir, Turkey
4Ankara University Faculty of Medicine, Department of Histology Embryology, Ankara, Turkey
5Ankara University Faculty of Medicine, Department of Pediatric Molecular Genetics, Ankara, Turkey
6Ankara Children’s Hematology Oncology Training and Research Hospital, Clinic of Pediatric Immunology, Ankara, Turkey
7Şişli Etfal Children’s Training and Research Hospital, Clinic of Pediatric Hematology, İstanbul, Turkey
8Hacettepe University Faculty of Medicine, Department of Internal Medicine, Unit of Hematology, Ankara, Turkey
9Ankara University Faculty of Medicine, Department of Pediatric Immunology and Allergy and Pediatric Molecular Genetics, Ankara, Turkey
10Ankara Numune Training and Research Hospital, Clinic of Hematology, Ankara, Turkey
11Ankara Numune Training and Research Hospital, Clinic of Hematology, Ankara, Turkey; Yıldırım Beyazıt University Faculty of Medicine, Department of Internal Medicine, Clinic of Hematology, Ankara, Turkey

Amaç: Ciddi konjenital nötropenisi (CKN) olan hastalar ve nötropenik olmayan ebeveynlerindeki granülositik ve granülositik olmayan kan hücrelerini incelemektir.
Gereç ve Yöntem: CKN’si olan 15 çocuk ve nötropenik olmayan 21 ebeveynin lenfosit, granülosit ve monositlerinde CD95, CD95 ligand, annexin V, hücre siklusu (periferik lenfositler, granülosiler +/- monositlerde) ve lenfosit alt grupları akım sitometri ile, b) hızlı hücre yaşlanması (lökositlerde) yaşlanma-ilişkili β-galaktozidaz boyası SA- β-galaktosidaz boyası ile, c) agregasyon testleri agregometre ile, d) in vitro kanama zamanı, PFA-100 aleti ile, e) trombositlerde mepakrin işaretli kaba granül sayısı floresan mikroskopu ile, f) hematomorfoloji ışık ve elektron mikroskopu ile değerlendirildi. Hastalarda ve bazı ebeveynlerde CKN ile ilişkili olarak HAX1, ELANE, G6PC3, CSF3R, JAGN1 mutasyonları çalışıldı.
Bulgular: Akım sitometri ile, hasta ve ebeveynlerinin monosit, lenfosit ve granülositlerinde apoptoz ve sekonder nekrozda belirgin artış olduğu ve bunun konjenital nötropeni mutasyonunun cinsi ile ilişkili olmadığı gösterildi. CD95 ve CD95 ligand sonuçları, apoptozun CD95 yolu ile olmadığını gösteriyordu. Hasta, ebeveyn ve kontrol olgularının lökositlerinin %25, %12,5 ve %0’ı SA-β-gal boyası ile boyandı. Dört olguda yapılabilen hücre siklusu analizinde üç olgunun lenfositlerinde G1 arresti ve apoptoz görüldü. Hastalarda HAX1 (n=6); ELANE (n=2); G6PC3 (n=2) ve belirlenemeyen (n=5) mutasyonlar saptandı. CD3, CD4 ve NK lenfositleri sırasıyla hastaların %16,6; %8,3; %36,4’ünde, ebeveynlerin %0, %0, %15,4’ünde, kontrolün %0, %0, %5,6’sında yaşa göre normal aralığın altında idi. Hasta ve ebeveynlerin trombositleri düşük oranda agrege oluyordu (olguların sırasıyla %66,6 ve %63,2’sinde, kontrolün %0’ında), kaba granül sayısı/trombosit oranı düşük (hasta, ebeveyn ve kontrolün %50, %35 ve %0’ında); in vitro kanama zamanı uzun (farklı kartuşlarla olguların %37,5 ve %33,3’ünde ve ebeveynlerin %18,8 ve %12,5’inde) idi. Işık ve elektron mikroskopta hasta ve ebeveynlerin periferik kanlarındaki nötrofil, monosit, lenfosit ve trombositleri displastik idi; hastaların kemik iliğinde fagosit aktivitesinde artış, dismegakaryopoez, nekrotik ve apoptotik hücreler bulunuyordu. İnce yapısal olarak trombositlerde adezyon, agregasyon, salınım yetersiz idi.
Sonuç: CKN’de, pluripotent hematopoietik kök hücreler ve nötropenik olmayan ebeveynleri genetik bozukluktan bağımsız olarak etkilenirler.

Anahtar Kelimeler: Ciddi kongenital nötropeni, Monositler, Lenfositler, Trombositler, Fagositler, Apoptoz, Yaşlanma, Ebeveyn, Aile


Lale Olcay, Şule Ünal, Hüseyin Onay, Esra Erdemli, Ayşenur Öztürk, Deniz Billur, Ayşe Metin, Hamza Okur, Yıldız Yıldırmak, Yahya Büyükaşık, Aydan İkincioğulları, Mesude Yılmaz Falay, Gülsüm Özet, Sevgi Yetgin. Both Granulocytic and Non-Granulocytic Blood Cells Are Affected in Patients with Severe Congenital Neutropenia and Their Non- Neutropenic Family Members: An Evaluation of Morphology, Function, and Cell Death. Turk J Hematol. 2018; 35(4): 229-259

Corresponding Author: Lale Olcay, Türkiye


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