Optical Tweezers and Its use in Studying Red Blood Cells - Healthy and Infected
The experiment discussed in the next chapter was to conﬁrm the aforementioned bystander eﬀect. In the ﬁrst experiment we separated hosting and non-hosting mRBCs by the percol purification method and then measured the corner frequencies of them. The mean fc of the distribution is almost the same, and this conﬁrms the eﬀect of the parasite on the non-hosting mRBC. In the next experiment, we have incubated nRBCs in the spent media and measured the corner frequency at six-hours intervals to see how the fc changed with the incubation time. The results showed that within 24 hours, the fc of the incubated nRBCs increases to the level of the iRBCs. The fact that nRBCs are getting aﬀected by the spent media indicates that some substances must be released in the spent media which alter the physical properties of the nRBCs. This kind of eﬀect on non-host mRBCs was previously observed by some earlier works [Dondorp97, Sabolovic91a, Bambardekar08]. It has also been recently shown that the rosetting of the host mRBCs to the non-host mRBCs is also activated by the substances released in the medium [Handunnetti89, Wahlgren89], which are also somewhat similar to the bystander eﬀect observed by us. In addition to this, there are reports which suggest that sickle cell disease also shows binding properties [Roseﬀ08, Zhang12] which may be due to the substances released in the medium. So it was already observed that the released substances induced changes in the properties of RBCs, but our study gives a direct conﬁrmation of the same. The next study was to ﬁnd out the released substances which were responsible for the observed changes above. We incubated infected and uninfected RBCs in different drugs. Then, we measured them to see what kind of changes occur in the corner frequency of the incubated RBCs. The corner frequency of normal RBCs incubated in db-cAMP shows the maximum change. So the released substance that is responsible for the bystander eﬀect may be due to the db-cAMP. All the experiments above were done using samples cultured only in the lab. Since the environment of the blood taken directly from the patient may differ from the one that is cultured in the lab, it is natural to ﬁnd out if similar kinds of changes can be observed in the clinical sample or not. The study in chapter 6 was targeted to ﬁnd out the same. We took clinical samples from BMRI for patients having a conﬁrmed malaria infection by both P. falciparum and P. vivax. This also provided us the opportunity to work with the P. vivax infected sample as it is very difficult to culture them in the lab. The results shown in this chapter clearly indicate that similar kinds of changes occur in the clinical sample also. It is worth noting that even though P. vivax infects only immature RBCs (reticulocytes), changes were also observed in P. vivax samples. This gives us another strong conﬁrmation about the previously observed bystander eﬀect. This also indicates that this technique can be used as a tool to diagnose malaria. Although we cannot diﬀerentiate between P. falciparum and P. vivax, this technique combined with other well established techniques can give us more conﬁrmation. So, in all the experiment above we have shown an easy and novel technique which can be used to differentiate between normal and malaria-infected RBCs. We have also observed the bystander eﬀect and tried to ﬁnd out the released substances which are responsible for this eﬀect. We have shown that this technique can use the bystander eﬀect of malaria to identify malaria. It has also been shown that the RBCs taken from the patient sample also show the same changes as the cultured samples, which gives us the possibility that this technique can be used as a diagnostic tool combined with other technique. This technique can also be used in experiments like the eﬀects of drugs and to ﬁnd out drugs for diseases like malaria. Future outlook 1. We have observed the changes only for malaria. There may be other diseases like sickle cell anemia which can also alter the corner frequency of the distribution of RBCs. We have to ﬁnd out the specificity of the observed changes. 1 We can directly measure the elasticity of RBCs using dual traps in optical tweezers to ﬁnd out the eﬀect of different infections and drugs on the rigidity of RBCs and compare the with the data above. 2 We can also study other cells using the same method to see if we can ﬁnd out any difference between healthy and unhealthy cells.
- Physics (PHY) 
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