Fitohemagglütinin konsantrasyonunun Down sendromlu ve sağlıklı bireylerin lenfositlerindeki mitotik indekslerine etkisi

İnsan periferal kan T-lenfositlerinin mitoza yönlendirilmesi ve kromozom analizlerinde birim hacimdeki büyüme ortamına eklenen fıtohemaglütinin (FHE) miktarı bir laboratuardan diğerine göre önemli ölçüde değişmektedir. Bunun nedeni bilinmemekte ve literatürde de bu konuda yeterli bir bilgiye ulaşılamamaktadır. Alışılmış periferal kan kültürü (72 saat) ve kromozom hazırlama işlemi, yaşları 0-8 yıl arasında değişen Down Sendrom (DS) 'lu hasta (N~30) ve sağlıklı kontrollerinden (N=24) alınan kan örneklerinin, aynı fakat konsantrasyonu gittikçe artan şekilde: 0.37 mi, 0.75 mi, 1.48 mi ve 2.21 mi FHE /100 mi Tık ortam 'lora ekilmesi ile yapılmışlardır. FHE 'nin dört farktı konsantrasyonunu içeren dört farklı ortamdan elde edilen her bir preparatta Mitotiklndeks (Mİ) 'ler, birey ve konsantrasyon başına 5000- 6000 uyarılmış lenfositler sayılarak yüzde (%) olarak belirlendi. Hem DS'lıt, hem de* kontrol gruplarının genel ortalamaları alındı. Bireyler arasında önemli farklılıklar görülmesine karşın, kültür ortamında FHE konsantrasyonunun artış yönüne doğru (%0.37, %0.75, %1.48, ve %2.21) lenfositler deki Mİ'lerin genel ortalamaları karşılıklı olarak; 30DSlu'da %4.20±1.73, %4.57±1.31, %4.69±1.19 ve %4.19±1.11 iken 24 kontrolde %4.37±1.67, %5.06±1.60, %5.01±1.47 ve % 4.98±1,43 olarak bulundu. Sonuçta, Biological industries (İsrail) ürünleri kullanıldığında, % 1-1.5 hacim/hacim FHE, hem DS 'lu, hem de kontrollerin kan kültürleri için optimum (hem etkin hem de tutumlu) bulundu.

Effect of the phytohemagglutinin concentration on the mitotic indexes of Dowm syndrome and healthy individuals lymphocytes

The amount of phytohemagglutinin (PHA) amount added to the growing medium in unit volume of human peripheral blood T-lymphocytes induced mitosis and at chromosome analysis varies significantly from one laboratory to another. The reason for this variation is not known, and in literature adequate information on this subject is not available. Conventional peripheral blood culture (72h) and chromosome preparation procedure have been used except that blood samples from Down syndrome (DS) patients (N-30) and healthy controls (N=24), with the age range of 0-8 years, have been cultivated in the same but gradiently increasing PHA concentration containing: 0.37 ml, 0.75 ml, 1.48 ml and 2.21 ml of PHA /100 ml of medium. Mitotic indexes (Mis) of each specimen obtained from four separate media with four different concentrations of PHA were determined as percentage (%) by screening from 5.000 to 6.000 stimulated lymphocytes per concentration per individual. Overall means Mis of both DS and control groups were taken. Although considerable interindividual variations were apparent, overall means of Mis in lymphocytes according to the gradient of PHA concentration (0.37%, 0.75%, 1.48%, and 2.21%) in culture medium were found as 4.20±1.73%, 4.57^.1.31%, 4.69±1.19%, and 4.19±L11% for 30 DS patients and 4.37±1,67%, 5.06±1.60%, 5.01±1.47%, and4.98±1.43% for 24 healthy individuals, respectively. In conclusion, When the Biological Industries (Israel) products are used, 1-1.5 % volume/volume PHA was found optimum (both effective and economic) for the blood cultures of both DS patients and healthy controls.

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1.Morimoto K, Kaneko T, Lijima K, Koizumi A. Proliferative kinetics and chromosome damage in trisomy 21 lymphocyte cultures exposed to y- rays and bleomycin. Cancer Res 1984,44:1499-1504.
2. Park E, Alberti J, Mehta P, Dalton A, Sersen E, et al. Partial impairment of immune functions in peripheral blood leukocytes from aged men with Down syndrome. Clin Immunol 2000, 95:62-69.
3. Anderson D, Jenkinson PC, Dewdney RS, Francis AJ, Godbert P et al. Chromosome aberration, mitogen induced blastogenesis and proliferative rate index in periferal lymphocytes from 106 control individuals of the U.K. population. Mut Res 1988, 204:407-420.
4. Ross G, Dixon JB, Veevers A. Variation in the lymphocyte transformation assay slope analysis of cell dose-response curves. J Immunol Methods 1987, 98:189-193.
5. Tajima M, Fujinaga T, Okamoto Y, Otomo K, Koike T. Relatinship between mitogen receptor in peripheral blood lymphocytes and blastogenic response to mitogen. Res Veter Sci 1990, 48:1-5.
6. Barta O, Barta V, Domermuth CH, Pierson FW. Optimum conditions for the turkey lymphocyte transformation test. Avian D is 1992 a, 36:386-394.
7. Barta O, Barta V, Pierson FW. Optimum conditions for the chiken lymphocyte transformation test. Avian Dis 1992b, 36:945-955.
8.İmamoğlu N, Demirtas H, Donmez-Altuntas H, Hamurcu Z, Üten A. NORs Expression increases on metaphase chromosomes of Down syndrome lymphocytes, in concordance with the mitogen concentration in the culture medium. Cytometry Part B-Clin Cytometry 2005, 66B.36-39.
9.İmamoğlu N, Demirtaş H, İlten A. NOR Expression increases in interphase lymphocytes of Down syndrome babies/children as AgNORs surface, according to the mitogen concentration in the culture medium. Micron 2006, 37:129-133.
10. NagelJE, Chopra RK, Chrest FJ, McCoy MT, Schneider EL, et ai Decreased proliferation, interleukin 2 synthesis and interleukin 2 receptor expression are accompanied by decreased mRNA expression in phytohemagglutininstimulated cells from elderly donors. J Clin Invest 1988, 81:1096-1102.
11. Farrant J, Clark JC, Lee H, Knight SC, O 'Brien J. Condition for measuring DNA synthesis in PHA stimulated lymphocytes in 20 microliters hanging drops with various cell concentrations and period of culture. J Immunol Meth 1980, 33:301-312.
12. İmamoğlu N, Demirtaş H, Dönmez H, Hamurcu Z. Down sendromlu lenfositlerinde Kültür ortamına göre aktif NOR sayısı değişimi. VI. Ulusal Tıbbi Biyoloji Kongresi Özet Kitabı, ss: 173. 2-5 Kasım 2000, Pamukkale Üniversitesi, Denizli.