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In the use of diagnostic methods for malaria parasites, based on polymerase chain reac-
tion (PCR) in whole blood, leucocyte presence frequently causes interference which can
completely mask resuks.'ln order to avoid this problem, different methods were tested to
eliminate leucocytes from whole blood, based on the density and size of blood compo-
nents. This elimination procedure was controlled by DNA polymerase chain reaction us-
ing arbitraty primers (RAPD). This method's high sensitivity allowed us to accurately de-
fine that whole blood's filtration through a cellulose-sephadex G-25 column allgws the
best elimination, compared to other matrices or separation methods such as ficoll, dext-
ran or ficoll-dextran.
98
Biorndica 1996:16:98-104 EVALUACION DE METODOS PARA LA SEPARACION DE LEUCOCITOS
Figura l. Un mL de sangre
completa y fresca se prepar
como se describe en materiales
v mtodos. Se oas a travs de
os diferentes 'colchones y se
lis con saponina. Un microlitro
de este lisado se someti a
amplificacin por RAPD (1-2),
sangre completa sin pasar por el
colchn, utilizada como control
(3-4), sangre pasada por
colchn de ficoll (5-6),sangre
pasada por dextrn (7-E),
sangre pasada por ficall-
dextrn (9), 3,5 k g DNA
humano usado control positivo
(lo),500 ng DNA Ladder 1 kb
usado como marcador de
tamatio.
-~
Fiours 2.
~ - .
Dos mL de sanore m m ~ .., . .
l e t a ~ sDreDararon
e como se describe en materiales v mtodos: 1 mLse pas a travs
oe jna columna de 4 m~-oeagodoh'en rama y el otro por una a>lumna.de 4 ml oe algodon comercial. Despus de la
f.lrracin. os er trocios se saron con saponina y de este lisaao sc lomaron a ic~otaspara la amplilicacion. Cada punto
se hizo par duplicado. (1-6),sangre fipaba,a travs de algodn en rama; (1-2),1 $-del l i d o ; (54),5 @del lisado y
(5-6)lo pL del Iisado, (7) 3,5 pg DNA humano usado control positivo; (9-12),sangre filtrada a travs de algodn
comercial, (9-lo), 5 WLdel lisado y (11-12),1 @Ldel lisado. (8) y (13)500 ng DNA Ladder 1 kb utilizado como marcador
de tamao
f
DE CASTROJ.,ROJAS M.O., WASSERMAN M.
nuclear leukocyies from human blood by the hypaque- 9. Steneker 1, van L u y n MJA, van Wachem PB,
ficoll method. J lmmunol Meth 1980;36:109. Biewenga J. Electronmicroscopic examination of
white cell reduction by four white cell-reduction filters.
.
5. Best CL. Pudnev J.. Anderson DJ, Hill JA. Modula-
Transfusion 1992;32:450.
tion of human granulosa cell steroid production in vitro
bv tumor necrosis factor aipha: implications of white 10. Nakao M, Nakayama T, Kankura T. A new method
biood cells in cuiture. Obstet Gynecol 1994;84:121 for separation of human blood components. Natural
New Biol 1973;246:94.
6. Ramos RR, Curtis BR, Dufiy BF, Chaplin H. Low re-
tention of white cell fragments by polyester fiber white 11. Goman M, Langstey G, Hyde JE, Yankovsky NK,
reduction platelet filters. Transfusion 1994;34:31 Zolg JW, Scaife JG. The establishment of genomic
7. Best CL, Grlfin PM, Hill JA. lnterferon gamma inhib- DNA libraries for the human malaria Parasite Plasmo-
its lutelnized human granulosa cell steroid production dium falciparum and identification of individual clones
in vitro. Am J Obstet Gynecol 1995;172:1505. by hybridisation. Mol Biochem Parasitol 1982;5:391.
8. Beutler E, West C, Blume KG. The removal of leuko- 12. Fotino M, Merson EJ, Allen FM. Micromethod for
cytes and platelets from whole blood. J Lab Clin Med rapid separation of lymphocytes from perlpheral blood.
1976;88:328. Ann Clin Lab Sci 1971;1:131.