IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 Assessment of Leucocytes Migration Inhibition in Tuberculosis Patients T.S. Khalid , S. K. Al-Agidi, W.Zuhair collage of Medicine, Unive rsity of Baghdad Ibn Al- Baitar Research Center Al-Mansour Technical Institute Re cei ve d in ,31,May,2010 Accepted in , 17, June ,2010 Abstract Leucocy tes M igration Inhibition test in agarose was carried against PPD and Mycobacterium tuberculosisantigen . The p rop ortion of different Kinds of migrating leucocy tes was calculated and exp ressed as a function of the migration distance . Ap p lication of this test to clinical p roblem revealed that with PPD as antigen , a highly significant inhibition was obtained in PPD skin test – p ositive normal individual , as well as, a high significant inhibition was recorded in individuals recovered from tuberculosis . These results demonst rated the suitability of this test for routine detection for cell mediated immunity . Introduction When lemphocytes are activated , either by mitogens or antigen , they p roduce a range of soluble mediators , in addition to antibody , these mediators are known collectively as lymphokines irresp ective of the mode of generation or their subsequent action .The various lymphokines are classified according to their mode of action , as : Lymphotoxin – Kills nucleated cells .M acrop hage activating factor – enhances macrophages mobility and phagocytois. M itogenic factor-stimulates mitosis in lymphocyte pop ulations . M igration-inhibition factor (M I F ) inhibits macrop hages migration. The inhibition of macrophages migration by lymphokines from activated lymphocytes had been st udied in detailed and showed an ap p roximate correlation with invivo immune st atus . [1] Cell mediat ed imm unit y can be detect ed inv it ro by several met hods. In leucocyt es migrat ion inh ibit ion t est ( LMIT ) , a t ot al pop ulat ion of human leucocyt es is incubated in a capillary t ube in t he presence o f Ant igen , an d the size of t heir migrat ion out of t he cap illary is measured . IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 Several modifications were performed , one of them utilizing agarose gel lay er was used to demonst rated the LM IT in p resence of Purified Prot ein Derivative (PPD) in p atients with p ositive PPD skin test [2] . This st udy demonst rates t he phenomenon of leucocytes migration in agarose gel with sp ecial att ention to the nature of the migrating leucocy tes and the dy namic of this ty p e of migration as a function of time . Materials and Methods A t otal of 40 adults were tested . PPD was carried out by intradermal tests . T he reaction was considered as p ositive when erythema was larger than 4mm in diameter [4] . The p atients were divided into four group s : Group -1 : Normal individual with p ositive skin test to tuberculin , and without history of tuberculosis which showed a negative test serologically and bacteriological tests for the p resence of mycobacterium tuberculosis . Group -2 : Normal individual with negative skin test to tuberculin and without history of tuberculosis and showed a negative serological and bacteriological tests for mycobacterium tuberculosis . Group -3 : Individuals with p revious history of tuberculosis ( between 6 – 12 months ) but they were healthy at the time of invest igation which gave negative bacteriological and serological tests for mycobacterium tuberculosis. All individuals of this group had a positive skin test for PPD. Group -4: included p atients with p roven infection with Mycobacterium tuberculosis .All showed a positive skin test for PPD. Mycobacterium was p urified and isolated from sp utum of tuberculosis p atients using a p rocedure that was described by [3],the p rocedure included a p recipition by ammonium sulp hate, filtration on sep hadix G200,electrop horesis and ultracentrifugation on sucrose gradient .Ant igen was then concentrated by vacuum dialy ser in TC medium 199 All exeriments were p erformed under a st erile condition according to [5] . Venous blood was mixed with p reservative-free heparin at a concentration of 12 u of heparin /ml of blood. Leucocy tes were then sep arated by adding 7.0 ml of dexran 250(6%).Aft er one hour of sedimentation 37 ᴏ C , the sup ernatant was taken , and centrifuged at 300 g for min and washed 3x in TC medium 199. The Leucocy tes were counted and the p rop ortion between lymphocytes , neutrop hils and eiosinop hils . was established ,RBCs – contamination was exp ressed as p ercentage of the number of leucocy tes. Leucocy tes (10 x10 5 ) were susp ended in 50 ul of each of the following solutions : - TC 199 and 10% fetal calf serum (FCS ). - TC 199; 10% FCS and 25 ug of PPD. - TC 199; 10% FCS and purified my cobacterium antigen at a final concentration of 1:3 Agaros 30 0 mg was heat ed at 100 0 C in 32 ml of distilled wat er , t hen incubat ed in a water bath at 50 0 C , and mixed wit h 4ml fet al calf serum (FCS) , 4 ml of 10x T C 19 9; 0.5 ml of t issue cult ure T C IBN AL- HAITHAM J . FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 bicarbonat e . T he final pro duct was a gel wit h 0.75% agarose and 10% FCS in T C 19 9 (pH 7.4 ) . T he gel was poured in pet ri dishes , when solidified , 4 wells were punched . Leucocy tes (2 x 10 5 ) were in cubated for 18 hours at 37 0 C in humid atmosp here containin g 2% CO2in order to keep p H constant . The area of migr ation was measured after 8 , 18 and 24 hours of incubation . The migration index (M I) was calculated as : ( Area of migration with Ant igen) M I=_________________________________ X100 [4] ( Area of migration with Ant igen) Results The average number of migrating cells was 108 x 10 2 (st andard deviation 51 x10 2 ). The value rep resented about 4-8% of leucocytes introduced into the well. Table (1) showed the p rop ortion of the different kinds of leucocy tes introduced into well ; into zone of dense migration and in zone of non – dense migration . Figure (1) shows that the p rop oration of the cells within the zone of dense migration varied greatly according to the distance from the wells . This distance was exp ressed as the p ercentage of the longest distance of migration . Eosinophils could not be seen further than 25% of the maximum distance , Lymphocyte-monocyte could be seen as far as 75% of the maximum distance but their p rop ortion was very low when counted so far from the well. The average of the control migration (without Ag) was 155 mm 2 , this corresp onds to a surface of 10 mm 2 . A correlation could be demonst rated between the prop ortion of p olymorph nuclear cells (PM Ns) and size of area of migration (r=0.47 , p =0.001 ) . The contamination by RBCs was on the average 18.3% of total number of leucocy tes and this contamination did not influence the size of migration area (r=0.04) . Table (2) shows t he results of migration inhibition in individuals with negative and p ositive p urified protein derivative (PPD) skin test . The results showed that t he negative PPD skin test group characterized by a migration index greater than 80. In contrast , all (PPD) – p ositive healthy individuals had a migration inhibition less than 80 . Figure (2) the average migration and the average migration inhibition as a function of inoculation time in individuals with p ositive PPD at 18 and 24 hours intervals . Both curves showed that the migration was negligible between 18 th and 24 th hour . Table (3) showed that migration inhibition was higher in control group in comp arison with p atients group . This difference was significant (p <0.05 ) . Discussion: Leucocy tes migration inhibition test is regarded as a rapid economical and simple with reproducible results and used as a tool to reflect the immune resp onse [5]. A good correlation could be obtained between the PPD skin test and the inhibition of leucocy tes migration by IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 Ant igen in the control group and lesser degree in the p atients group . This suggests t hat this test represents a state of delayed ty p e hyp ersensitivity . Such correlation couldnot be obtained during the course of tuberculosis due to depression of lymphocytes activity by the bacteria concerned [6] . The dep ression might p revent migration inhibition by PM N but insufficient to p revent p ositivity of PPD skin test [3] . It was demonst rated that leucocy tes migration inhibition test (LM IT) was happ ened , after 6 hours of incubation . The areaof migration in the p resence of Ant igen remained the same . While the migration in control sp ecimens continued . This might suggest that this p henomenon reflect cell-mediated immunity rather than the effect of cy top hlic Antibody [7] When Mycobacterium antigen was used as an antigen a highly significant inhibition was observed from tuberculosis [7] . The inhibition was indep endent of the p resence of antibody , which suggests that the LM IT detect cell – mediated immunity rather than the effect of cell-bound Ab[5] . In the majority of cases , it can be concluded that lymphocytes inoculate in blood in the p resence of mycobacterium antigen which may saturate their binding cap acity for an antigen , so they are unable to react ( a few hours later ) with weaker concentration of Ant igen . While for PPD , are due to action of bacteria or liberation of inhibitors of lymphokines . [8] Re ferences 1-Oy ey inka-Go;Salimonu-LS;Ladip o-OA and Sshay c-AO.(1995). Leukocytes migration inhibitition studies and neutrop hil cell function during aging in Nigerians .M ech-Agenin g – Dev.85:83. 2-Anderson , ; M unk , M.E.; J .M .Pollock and Doherty T.M .(2000).Sp ecific immune-b ased diagnosis of t uberculosis. The Lancet.1099:356. 3-Richard ,A.G. ; Thomas , J.K.,and Barbara , A.O. (2000)Kuby ” IMMUNOLOGY”4 th ed W.H.Freeman and comp any. NEW YORK. 4-Knechel ,N.A.(2009), T uberculosis: pathop hy siology , Clinical Features , and Diagnosis, Crit Care Nurse 29:34-43. 5-Erad P. (1974) T echnical study of leucocytes migration inhibition test in agaros .Clin .Exp . Immunol.18:439. 6-Agapova – RK; Bogadel’n ikova- I V; Sergeev- -AS; and Perel’man- M I. (1999) Discriminant analysis of clinical laboratory date for diagnosis and treatment of tuberculosis and lun g cancer Vestn-Ross- Akad- M ed-Nauk. 1(1) :47-51. IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 7-Enarson , D.A.; Rieder, H.L.; Arnadottir, T. and Trebucq, A.(1996) . T echnical Guide for sp utum examination for tuberculosis by directs microscopy . In” Tuberculosis Guide for low Income Countries “ .4 th Ed. PP:49-64. 8-Tieman R, de Audreals S, Nihik as , (Aug /7/2007)development of new tuberculosis vaccines : Aglobal p ersp ective on regular issues . p ublic library of science (Pols m edicine). Table (1) Morphol ogical se paration of leucocytes expressed as percentage (± SD) Cell Suspension before mi gration None of migration Dense Non-dense Ne utrophils 45±7.0 89 ±2.0 100 Eosi nophils 7 ±2.2 2± 0.6 0 Lym phomonocytes 28± 3.5 9± 1.0 0 T he results are the mean of 15 experiments Table (2) Leucocytes migration inhi bition test in presence of PPD Group 1 2 3 Group (a+b): Controls 154 158 103 Group b 134 90 68 Group c ( convalescence ) 179 137 73 Group d (patients ) 170 153 93 1-migration in TC199 2-migration in TC199+Myco.Ag 3-migration index Table(3) Leucocytes migration in presence of mycobaterium AG Group 1 2 3 Controls Group (a+b) 130 124 105 Group c 199 126 71 Group q 168 178 103 1-migration in TC199 2-migration in TC199+Myco.Agenl.1500x 3-migration index IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (1) 2011 قیة المجلد للعلوم الصرفة والتطبی 2011) 1( 24مجلة ابن الھیثم تقییم كبح ھجرة خالیا الدم البیض لمرضى السل ورقاءزھیر، سعد خالد العكیدي ،طیبة سعد خالد غدادجامعةب –كلیةالطب مركز ابن البیطار للبحوث المنصور ،المعھدالتقني الفني الخالصة .Mycobacteiumtuberculosis، PPDالسل نفذاختیارمنع ھجرة كریات الدم البیض في االكاروزتجاه مستضد رةخالیا الدم البیض بوصفھا وظیفـة لفـعل مســافة ـ ي وضح تطب. الھجرة حسب نسبة اختالف وتنوع ھـج یق ھذا االختبار ف الجلدي PPD یثبط بشكل معنوي عالیا في االفراد الطبیعیین ذوي فحص ،عالٍ مستضداً PPD المشاكل السریریة باستخدام ــافین من السل الموجب وكذلك تبین ان التثبط عالیاً ـــائج ھذا االختبار مالئمتھ للكشف . معنـویا في االفراد المتع وضـحت نتـ .المناعة تتوسطھا الخالیا الروتیني عن .