RE: anti-malarial antibodiesI haven't followed the details of this thread but I remember a study years ago that thiazole orange (the TO component of many of the DNA binding dyes from Molecular Probes) was used successfully for labeling Plasmodium in red cells. A quick look brought up the following that may be useful.
Dave
========================================
David M. Coder, Ph.D.
Consultant in Cytometry
email: d_coder@msn.com or dcoder1@hotmail.com
tel./messages: 206-499-3446
Title
Development and application of a modified flow cytometric procedure for rapid in vitro quantitation of malaria parasitaemia
Author(s)
Schulze DLC, Makgatho EM, Coetzer TL, Louw AI, Van Rensburg CEJ, Visser L
Source
S.Afr.J.Sci. 93 4 (1997 Apr)
Page(s)
156-158
Document
Article
Abstract
The most lethal form of human malaria, caused by the parasite Plasmodium falciparum, adversely affects the lives of millions of people each year In order to establish the effectiveness of therapeutics, an accurate, reproducible and convenient assay of parasitaemia is necessary Towards this end, we modified a flow cytometric (FC) method based on thiazole orange fluorescent intercalating dye to detect parasite DNA, by using a lower fluorochrome concentration (0.8 mu M) and micro-cultures of parasites subsequently fixed with a standard formaldehyde-based solution. A linear relationship was observed between classical microscopically determined parasitemias and those from FC (r = 0.98), as well as between experimental FC values and parasitaemias calculated from the serial dilution of either unfixed or fixed stock cultures (r > 0.98). The applicability of the FC method was confirmed during quantitation of the extent of inhibition of parasitaemia by chloroquine treatment of Plasmodium falciparum-infected human erythrocytes. Results obtained with flow cytometric analysis in this instance correlated with those from both classical Giemsa-stained blood films and [H-3]hypoxanthine incorporation (IC50 = 70-76 nM). The modified flow cytometric procedure is therefore suitable for the rapid cost-effective in vitro estimation of parasitaemias in micro-cultures and the evaluation of agents with anti-malarial potential.
--------------------------------------------------------------------------------
----- Original Message -----
From: Gabriel Alespeiti
To: Cytometry Mailing List
Sent: Tuesday, July 30, 2002 9:45 AM
Subject: RE: anti-malarial antibodies
Barren,
I appreciate your help. Thank you.
I did not run the Giemsa stained RBC's through a flow cytometer. Therefore I don't know the answer.
About your other question. Richard Haugland (Molecular Probes) responded that "several of our SYTO dyes have been used this way too but I believe not published. They are excitable at 488 nm."
He also mentioned the paper:
"Cytometry 1993;14(3):276-80. Flow cytometric screening of blood samples for malaria parasites. Philip H. van Vianen PH, van Engen A, Thaithong S, van der Keur M, Tanke HJ, van der Kaay HJ, Mons B, Janse CJ.Laboratory of Parasitology, Medical Faculty, University of Leiden, The Netherlands."
They lysed the erythrocytes and are able to detect parasitemias as low as 0.001%. They use Hoechst 33258, which cannot be detected in a FACScalibur. Samples contain mainly white blood cells and free parasites. When lysing erythrocytes only 5000 events are needed to detect parasitemia levels under 0.01%, and advantage compared to working with whole blood, where ~1000X more events are needed to detect the same level of parasitemia.
I would like to know if someone has modified this method and tried it with 488nm excitable dyes?
I may not be of much help., but I am curious if you ever ran the Giemsa stained RBC's through a flow cytometer. I wonder if the Giemsa positive staining RBCs ( with parasites) would not show up as an increase in SSC?
Another thought, Human RBC's have no nucleus, could you stain with a DNA dye (one that permeates live cells , Syto dye series from Molecular Probes?) and then look for RBC's with DNA (i.e. parasite)?
Pb
-----Original Message-----
From: Gabriel Alespeiti [mailto:gabriel_alespeiti@nybc.org]
Sent: Thursday, July 25, 2002 3:13 PM
To: Cytometry Mailing List
Subject: Re: anti-malarial antibodies
Dear colleagues,
I have read the replies on anti-malarial antibodies and I hope to get some help from the experts in this field. A researcher in the Institute is conducting parasitaemia studies of Plasmodium falciparum infected erythrocytes using Giemsa stained microscopy, a tedious, time consuming and technically imprecise method. We want to replace the microscopic counts with a flow cytometry method. Peripheral blood will be collected in Brazil and will need to be fixed before shipping. A FACScalibur is available for this work. We would like to analyze 25 samples a day, the expected parasitemia level is between 0.05% and 8%.
I tried formaldehyde fixation followed by propidium iodide staining and even though there is a direct correlation between cytometric and microscopic determination, the manual counts are significantly higher than the flow counts.
What I'm looking for is a simple, proven protocol for daily use that gives directly the % of parasitaemia by flow. In our case, determination of erythrocyte or parasite antigens; even though useful, is not necessary. What is important for us is to be able to detect low infection levels.
Does anybody know, what is the lowest level of infection that can be detected by flow?
Please post the reply on this site, so other people can benefit from it. Thank you very much.
--
**************************************
Gabriel E. Alespeiti
Supervisor, Flow Cytometry
Lindsley F. Kimball Research Institute
New York Blood Center
310 E.67th St. Room 2-16E
New York, NY 10021
Phone: (212) 570-3346
Fax: (212) 570-3307
E-mail:gabriel_alespeiti@nybc.org
**************************************
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:26:17 EST