Transcription
Foreign [Music] presents to you simply pathology. And today, we are back with a very important lecture. Today, we are going to read about the peripheral blood smear. So, this topic is very, very important, not only for the undergraduates but it is also very important for the postgraduate MD, DNB pathology students. Because knowing about the peripheral blood smear is the most basic thing that you have to know, and it is a must for you to pass the MD or DNB practical exams. And one question on the peripheral blood smear is 100% there in your practical exams, both in MD, DNB, as well as in the undergraduate practical exams in MBBS. Okay, so let us see what are the objectives. What are we going to learn in today's topic of discussion?
So, first of all, we have to understand that what is a peripheral blood smear and what are the uses. Why are we doing the peripheral blood smear? Secondly, how do you make a peripheral blood smear? Okay, what is the principle? What is the method of staining of the PBS? Okay, you have to know in details about the staining process and what are the stains used. Normally, we are using the Romanowsky stains. Okay, so we are going to read what are the Romanowsky stains, why are they used for the peripheral blood smear? Okay, what are the features of an ideal peripheral blood smear? How to identify the different blood cells that you see in the peripheral blood smear? How to have a systematic approach to a PBS? Okay, so it is very important for the first-year residents as well as the third-year residents. Okay, it is important for both of you. For the first-year residents, you have to develop a plan, a systematic approach, how to study a particular PBS and how to report. That is very important. It is a bread and butter for a pathologist. And for the third year, because you are the, you know, exam-going batch, so lot of questions will be asked from the peripheral blood smear, and you have to answer all of them. Okay, if you fail in the PBS, then you will fail in your MD, DNB exam as well. So, this topic, though it is very easy and it is a basic topic, but it is a must-know topic. Okay, you should know that what are the things that you are going to study under the 10x, under the 40x, under the 100x? Okay, and in the end, I'm going to show you the normal peripheral blood smear reporting. So, in this particular session, we are only going to understand what are the basics of the peripheral blood smear, details about the morphological evaluation of the RBCs, morphological evaluation of the WBCs, platelets, as well as the parasites. It will be discussed in details in the subsequent lectures. Okay, so in the first part, we will understand the basics of the peripheral blood smear. So, what is a blood smear and what are its uses?
So, basically, if you see a blood smear, it is a specimen for microscopic examination which is prepared by spreading a drop of blood across a glass slide, followed by staining with one of the Romanowsky stains. Okay, so this is a basic definition of a blood smear. So, what is the use? When is it that you require a blood smear? So, the first very important use when you want to diagnose any kind of anemia or thrombocytopenia. So, for example, there are clinical signs, symptoms of anemia, then the treating physician is going to write down a complete CBC gram or the complete blood count. Okay, so there they are suspecting anemia. So, for the diagnosis of anemia or for the diagnosis of thrombocytopenia. For example, there is a recurrent bleeding or there is signs, symptoms of bleeding like petechiae, ecchymosis, like that. So, in that situation, okay, you have to get a CBC done. Then for identifying and suggesting the type of leukemia. So, for example, if there is a, there are signs, symptoms of leukemia like fever is there, anemia is there, infections are there. Okay, but you know, bone pain is there. So, to confirm, you are going to write down a peripheral blood smear. Okay, in addition to the CBC, you will write down the PBS. Okay, so wherein the pathologist has to examine the slide and comment on the presence or absence of abnormal cells. In the detection of hemoparasitic infection. So, sometimes they are going to write down MPD. Okay, along with that, they might also write down MP. So, when they are writing down just MP, that means a slide has to be made and you have to see these, the you have to, you know, search for the malarial parasites in the peripheral blood smear. If only the dual antigen test is required, then in that case, you don't have to examine the slide personally. But if MP is written or if a PBS is written and they're suspecting malaria, then you have to examine the slides for any malarial parasite or any other parasite. Okay, then to monitor the effect of chemotherapy and radiotherapy on the bone marrow. Okay, then to detect pre-analytical or analytical error in the output of automated analyzer. So, what is very important over here? That for example, suppose if your machine is working nicely or no, whether your machine, for example, you are having a three-part or a five-part analyzer, so they are going to carry out quality control on that. They are going to run certain controls like low control, high control or whatever they are going to run. Okay, so what happens that whatever finding you are getting in the automatic analyzer, whether that is matching completely with what you are examining under the microscope in the slide? Okay, so that is very important. So, if there is any kind of, if for example, the machine findings are not correlating with the findings that you are finding under the microscope, that that means something is wrong with the machine or something is wrong with the analyzer. So, that means that there is some kind of problem in the machine. So, therefore, this diagnosis can be made only in that particular way because whatever you are looking under the PBS, that is completely correct because what you are seeing is what is true. So, to find out any discrepancies between the machine finding and between your finding, this is very important. So, for quality control, to find any pre-analytical or analytical error, we are doing the PBS as well. Okay, then to provide direction for further investigation that will help in arriving at the correct diagnosis. Okay, now, for example, sometimes you have to do further investigation. So, for example, if you are suspecting some kind of infections in a, in a particular person, or you are suspecting some kind of sepsis or something like that. Okay, or you are suspecting some drug effect, then in that situation also, you are, you know, to order, you know, for further investigation and for further confirmation, you are carrying out a PBS. Okay, to correlate the instrument flag. Instrument flag means what? That's for example, your machine has given a platelet count of, for example, 15,000. Okay, so in that case, it becomes mandatory for you because when the platelet count is less than 1 lakh. Okay, so routinely in our lab, when the platelet count is less than 1 lakh, then a peripheral blood smear is prepared so that we can confirm whether really the platelet counts are that much less. Similarly, for example, this is about the platelet count. Similarly, for example, if the WBC count is coming around 40,000. Okay, it is very high, or for example, it is coming around even 20,000. So, the normal range is around 4,000 to 11,000. So, if it is so high, then we have to confirm, okay, why it is so much high, whether it is neutrophilia or whether it is any kind of myeloproliferative or lymphoproliferative disorder. So, to rule out any abnormalities, okay, to find out the cause of this increased count, we have to examine the peripheral blood smear slide. So, similarly, any kind of instrumental flag. Sometimes the instrument cannot give you differential count, they will just flag it. Okay, so as a result, you have to, or for example, sometimes the mixed percentage is very high. So, in that case, you have to examine the particular peripheral blood. We have to confirm, you know, those cells and to confirm the nature of those cells to carry out complete differential count. Okay, that is why sometimes to correlate with the instrument flag, sometimes machine is giving some flag, machine is saying that this, you have to check this parameter. In that case, you have to examine that particular parameter under the microscope. And also, before examining a bone marrow aspiration slide, you have to do a PBS. So, what is very important? Before you are carrying out a bone marrow study, okay, you always have to see the blood picture, that is the peripheral blood picture. You have to see that is seen with the help of PBS. So, these are some of the very practical uses why you are going to make use of a PBS. So, you have to make sure in your mind that when a PBS is coming to you, what are the requirements? What is the treating clinician want you want from you? Okay, so blood smear examination is therefore indicated in clinically suspected cases of anemia, thrombocytopenia, or hematological malignancies like leukemia, lymphoma, multiple myeloma, disseminated intravascular coagulation. In conditions where you suspect parasitic infection, for example, fever with chills is there, okay, with diurnal variation of the chills and fever, so you will suspect malaria. So, if there's a season of malaria, so absolutely you are going to ask for an MP. That can be confirmed by examining the peripheral blood smear. Similarly, if you suspecting any kind of viral infection, so the lymphocyte count will be very high. So, in that situation, it is very important to understand. Okay, and for further investigations, you are giving the PBS. Okay, and for various other inflammatory and malignant diseases, you are requiring a PBS examination. So, these are the basic uses or indications of carrying out a peripheral blood smear. So, you should be very swift with your answer. So, if your question in your viva is, what are the indications of doing a peripheral blood smear? So, these are the things that you have to say in your viva as well. Okay.
Then, how do you make a peripheral blood smear? So, let us try and understand this point. So, to prepare a peripheral blood smear, you take a small drop of blood, around two to three millimeter in diameter, is placed in the center line about one centimeter away from one end of the glass slide. So, you can see you have taken one drop over here. You have taken a drop of blood over here. You have taken exactly around one centimeter from one of the ends. Okay, the typical size of the glass slide that we are using is 75 into 25 millimeter with a thickness of about one millimeter. With a wooden stick or a glass capillary, you are placing the drop of blood in this particular slide. Now, the slide that you use, it should be clean, dry, and grease-free. Now, the blood sample may be venous blood, okay, that is anticoagulated with EDTA, or it can be capillary blood from the fingerprint. Okay, remember, for the best blood cell morphology, if you want to study the morphology of a particular cells, it is best to obtain this sample from the finger prick, directly from the skin puncture. So, better blood cell morphology is obtained if the smear is made directly from the skin puncture. Okay, if EDTA anticoagulated venous blood is used, the smear should be prepared and stained within two hours of blood collection. If venous blood collected in a syringe is used, the last drop of blood in the needle after withdrawing, or the first drop while dispensing, should be used for making the peripheral blood smear. So, what you are doing? You are taking one drop with the help of a wooden stick or a glass capillary and you are placing it mainly on the center, just one centimeter from one end of a particular glass slide. The standard size of the glass slide is 75 into 25 millimeter and the thickness of approximately one millimeter. Now, now then you can see this is the glass slide where you are going to have the peripheral blood smear. Okay, and you have taken approximately one or two drops of blood over here. Okay, now you want to spread this blood uniformly across this slide. Okay, so you have to take a spreader slide. So, this slide over here, this slide that you can see, this is the spreader. This is the spreader slide. Okay, so it is a glass slide which should have absolutely smooth edges. Okay, and one or both corners at one end of the slide should be broken off. So, the corners, if you see, they are saying that this corner, if I say you the corner, this corner and this corner should be broken off. Okay, ideally, usually they do not, but if possible, you should break off the corners. Okay, the spreader slide should be narrower. Okay, they should have a width of 15 millimeter, whereas the breadth of the particular PBS slide is 25. Okay, that of the spreader should be around 15 millimeter, but it is not always possible. Okay, it is not always possible in day-to-day activity. Okay, so why is this required? Because finally, whatever you are getting the smear, so that the edges of the smear can be examined microscopically. Okay, so what they are trying to say, let me just tell you, if this is a particular glass slide, okay, and if for example, we want a smear to be somewhat like this, so that these edges, okay, these edges can be examined nicely or can be examined carefully, that is why they are saying that we should use a spreader, the thickness of which is lesser as compared to that of the peripheral blood slide, okay, where we are going to make the smear. Okay, this is very, very important, but usually this is not always possible. This is not always possible in day-to-day activity. This is the ideal situation. Now, a spreader slide, if you see, it is placed at an angle of 30 degrees in front of the drop. Now, you have to place this spreader space. So, for example, this drop is there, and the spreader has to be placed in front. It has to be placed in front of the drop. Okay, and it is placed at a particular angle over here. It can range between 30 to 45. Usually, they are keeping it at an angle of 30 degrees in front of the drop, and then it is little bit drawn backward. So, as you are drawing it backward, what is happening? This drop is actually spreading along the line of the spreader. Okay, so it is spreading along the line. So, a spreader slide is placed at an angle of 30 degrees in front of the drop and then drawn back to touch the drop of the blood. The blood spreads across the line of contact of the two slides. So, the spread is now spreading across the line of contact. So, this is how, so you can see now the blood sample was over here. I have kept the spreader in front. Just I have taken little bit the spreader back. So, once you take back this, the entire blood is basically spreading along the line of contact between the two slides. This is the spreader slide, and this is the slide in which the PBS is made. So, it is spreading like this. Okay, and once it happens, the smear is made by smooth forward movement of the spreader along the slide. So, the smear is now made by smooth forward movement of the spreader along the slide. The whole drop should be used up up to one centimeter before the end of the slide. The length of the smear should be about three centimeters. The spreader should not be raised above the slide surface till the whole drop of blood is spread out. Okay, so you have to remember these points very importantly. They are saying over here that when you are spreading it like this, okay, you should spread till the entire blood has been used up. Okay, so you should take a very less amount, not a very high amount. Usually, one or two drops, a drop of blood is enough. Less than 10 microliter of blood can be taken. Okay, and you have to make, okay, just remember, if this is a particular slide, the smear should come, okay, at least a distance of one centimeter has to be kept from the end, okay, till which the smear is drawn. Okay, so this was where you have started. Okay, and from here the smear has been made. Okay, so at least one centimeter has to be, you know, some distance has to be there from the end of this slide, okay, till the smear is basically ended. Okay, so as you can appreciate over here, once the smear is made, so you have to rapidly air dry it. Okay, so smear is rapidly dried by waving it in the air or keeping it under an electric fan. Slow drying will cause shrinking artifact of the red cells. So, you have to write very rapidly. Okay, so again, you can see over here that they are again making. See, already the blood has spread over here, and now they are going to make. Now, what we have, what we are trying to say that you can appreciate over here in this particular smear that the smear length, if you see, okay, it is not going till the end. Okay, it is ending over here. So, there is some distance between the end as well as between this particular peripheral blood smear that is being created over here. Okay, why it is very important? Because when you are examining under the microscope, okay, so the microscope, the stage cannot move after further length. So, if for example, a smear is reaching till here, you will not be able to examine this area. That is why your smear should end at least one centimeter from the end. Okay.
Not only that, now the patient's name or the laboratory number and data are written. So, you can see over here, a bar coding has been done for individual slides. A bar coding has been done. In our country, you can just give a particular number with a lead pencil or with a permanent marker pen or a diamond pencil on the thicker end of the smear, and always the marking is done at this particular end. Okay, why it is done at this particular end? So, it is very important to understand because it is at this end that we have to examine. So, we should not write anything over here. It should always be written. The marking should be done at this particular end, that is the head end of the particular PBS slide. So, I hope you have understood in details that how to make a particular, you know, peripheral blood smear. So, how do you make a peripheral blood smear? You have to understand in details about that. And very, very importantly, for the postgraduate students, I am telling again, in the exams, you are required to make your own slide. So, you will have to take the sample, you will have to make the smear by yourself. So, it is very important that you start practicing making the peripheral blood smear slide by your own. Okay, and this is the way you should make it ideally because you will be marked on these particular parameters. Okay.
Now, after the smear is particularly, you know, after it is air dried, after the smear has been air dried, it is fixed immediately with absolute methyl alcohol, which should be moisture and acetone-free, and the fixation is done for two to three minutes in a covered jar. Okay, so absolute ethyl alcohol can also be used, but not methylated spirit, as methylated spirit is containing water. So, first you have to fix your particular smear. So, ideally, this is the ideal situation. Okay, but usually in day-to-day practice and routine practice, that is not being done. Why? Because the stain that we use, that is the Leishman stain, already it is containing methanol, which can act as a fixative. But apart from that, if you are asked about the ideal situation, so ideally, after you air dry the smear, you should fix the smear immediately with absolute methyl alcohol for two to three minutes. Okay, now, why do, why are we fixating or why is the fixation done? The aim of fixation, it is to prevent washing off of the smear from the slide. So, whatever smear you have created over here, okay, so in the particular slide, you have created a smear, so if you do not fix it, then what is going to happen? During the process of staining or something, that is a high chance that your smear will, you know, will will will come off. Okay, it is going to come off the particular slide. So, to put, the aim of fixation is to prevent washing off of the smear from the slide. Following this, the color of the smear becomes light brown in color. So, after you do the fixation, the color becomes light brown. Now, this fixation is desirable even when Leishman stain is used, which contains methyl alcohol. As I told you, this fixation is required or is desirable. The term is desirable, it is not a must, but it is desirable. So, this fixation that we are doing with methyl alcohol is desirable even if the Leishman stain is used, which is also containing methyl alcohol. Okay, so but ideally, this is not followed everywhere routinely. If you go, they are just using Leishman stain with a mixture of methyl alcohol. Why? Now, now why is this, you know, done? This is because Leishman stain may have absorbed. So, you must be thinking that if Leishman stain is containing the fixative methyl alcohol, then why are we doing fixation separately? Why is it desirable? Because over a period of time, the Leishman stain that you are using may have absorbed moisture, that will lead to poor fixation. If methanol is contaminated with water, it will, the sharpness of the cell morphology is lost, and there is vacuolation of the red cells. So, that is why it is desirable to carry out fixation separately. Okay, methanol should be, the methanol that is used for fixation should be acetone-free. Since acetone washes out the nuclear stain. So, why should it be water-free? The absolute methyl alcohol should be moisture or water-free because if it is not water-free, then the fixation will not be proper, and the cell morphology is not very crisp or sharp. And why it should not have any acetone? Because if the acetone is there, it is going to wash out nuclear stain. It should be acetone-free and moisture-free. Now, in many laboratories, the slide is stained immediately after air drying without prior fixation, and the results are satisfactory. This is what being, this is what is being done in majority of the laboratories. So, they are not doing any proper fixation first. So, directly after air drying, they are using the Leishman stain, which is containing methyl alcohol. However, if you are suspecting a delay of more than four hours is anticipated between the air drying and staining, the slide should be fixed. Okay, so very importantly, separately fixation is not required if you are, if you are going to stain it immediately after air drying. But if you think that some amount of time will be required, and in that situation, you should fix the slide immediately. If not, background gray blue staining of the plasma will occur. So, they will take a background stain of grayish blue. Okay, gray blue staining of the plasma will occur in the background. So, the first step after air drying, so rapidly you should air dry, then you should carry out fixation. Okay, ideally, fixation should be done before carrying out Leishman stain. But if you are, no, but if after air drying, you are immediately going to stain that particular slide within, then you might skip the step of fixation because the Leishman stain already is containing methyl alcohol. But for ideal situations, absolute methyl alcohol should be used for fixation before going for Leishman staining. Okay.
So, a well-spread blood smear. Okay, if you see, what are the ideal characteristics? What are the ideal characteristics of a, you know, well-prepared smear? So, it is tongue-shaped and having a very smooth tail. It does not cover the entire area of the slide. It has both thick as well as thin areas with gradual transition. It does not contain any lines or any kind of holes. Okay, so these are very important points. Okay, so this is what an ideal smear should look like. So, you can appreciate over here, this is an unstained, this is unstained peripheral blood smear. Okay, you can see there is a lot of distance between the end of this slide and this particular peripheral blood smear. You can also see over here how this particular smear is tongue-shaped. Okay, it is tongue-shaped at one end. So, how it is tongue-shaped? We can appreciate this was the drop where we had taken, we had taken initially. Okay, then the spreader was taken little bit a bit back. So, as a result, this drop of blood has spread like this, and then after that, very gently we have taken, smoothly we have drawn a particular smear, as we can appreciate over here in this particular slide. Now, you can see very, very importantly, there is no hole, and there is a gradual transition. You can see from this thicker area, okay, this thicker area, there's a gradual transition to this thinner area, as you can appreciate over here. Okay, also, if you see it, this particular peripheral blood smear, it is not covering the entire area. So, the entire area should not be covered. Okay, as I told you, if a smear is going till this length, then because the stage of the microscope is limited, it will not be able to move the lens till this portion, and you will not be able to examine this area. That is why your smear should end at least one centimeter from the end. Okay.
Very importantly, what is very important? The area of interest is always this area, the end of this smear. Okay, this is the area which is ideal for examination. Ideally, this area is suitable for examination. From here, you go inwards for your examination. This is the area where the morphology is best observed. Okay, so these are the basic important characteristics of a smear. It is tongue-shaped, it with a smooth tail, as we can appreciate. It is tongue-shaped with a smooth tail over here, as you can see. It does not cover the entire area of the slide. So, the entire area of the slide is not covered, as you can see, only this area of the slide is covered. Okay, it is having both thick and thin area with a gradual transition. So, this is the thick area, and this is the thin area. Okay, and there is a gradual transition from the thick to the thin area. Okay, and they do not contain any lines or holes. So, there are no lines or holes that you can appreciate over here. Okay.
Now, what are the parts? What are the parts of the peripheral blood smear that we are seeing? So, if you see over here on the left-hand side, that you can see on the side from where the point, from this point we have made the smear, so this area from where the smear was made, this is called as the head. This is the head area. Okay, after the head comes the second area, that is the body, and the third area is your tail area. This is the tail. This is the area. This is the tail area. So, there is the head area, body area, and the tail area. Okay, this is the area. This the tail area is the area which is most suitable for morphological evaluation of the blood cells or for examination. So, the first-year residents, usually you people make a lot of mistakes. You directly go and you focus in these areas. So, this is completely wrong. You should focus completely at the tail end. Okay, so you should be very aware what is the head, what is the body, what is the tail area. Okay, so this is very, very important for your exam purpose. Okay.
Now, this is, these are all the examples of poorly made slides. So, if you see over here, what are the problems? So, in this slide, if you see, it is covering the entire portion. Also, there are some holes over here in this area. Such a big hole. See over here, what has happened basically? Proper fixation was not there, and as a result, the particular slide completely has come off. You can see over here. See, this is not a very smooth slide. Okay, and you can see this area is dark, this area is light. It shouldn't be like this. See, this is not a tongue-shaped smear. This is some other shape. Okay, this is not a tongue-shaped smear. Again, over here, you can see, you can see multiple holes over here and lines. Again, multiple holes and lines can be appreciated. Again, if you see these particular slides, they are ending abruptly like this. They should end very nicely. They should end up. They should not end abruptly. Again, you can see lines over here. Okay, so these are all the types in which you should not make. Again, if you see this particular smear, it is completely starting from here and is going towards the end. So, you will not be able to examine this area over here. Okay, and also they are basically stopping abruptly. They are not completely, see this area, they are stopping abruptly over here. Okay, so they are stopping abruptly over there. So, you cannot examine nicely. Okay, again, if you see, there is no gradual transition. Okay, over here, the tail area is darker as compared to the head area, as we can appreciate. So, there are holes over here. Okay, so these are exactly the ways in which your, you know, slides should not be made. Okay, so it should ideally be something like this. This is how you should make a particular peripheral blood smear slide. I hope this part is clear to everyone. Okay.
Now, as you can appreciate, you can tell a lot, okay, about a patient, okay, looking at the gross image of particular slides, looking at the gross image of a slide, you can say clearly what is wrong with a particular patient. So, if you see all of these are very properly stained or not stained, sorry, all of them are very properly drawn PBS smear, but what is very important? If you see on the right-hand side, this particular PBS, this is very, very light. Okay, so even looking at the PBS grossly, you can say whether the hemoglobin is less and the patient might be anemic. Okay, so this kind of slide is reflecting anemia. Okay, whereas on the other hand, sometimes you might get a very, very dark smear like this. Okay, so for example, this is very, very dark. Okay, so grossly, this is pointing towards polycythemia. Okay, so even just looking at the slide, even before microscopic examination, you can get a rough idea about what is wrong with the patient. This is normal. This is a normal slide. Somewhat looks like this. If it is much lighter, it is pointing towards anemia. If it is very dark, it can point towards polycythemia. Okay.
Now, again, we are having different kinds of smears also, like the thin. Now, the one that I have shown you over here, the one that I have shown you over here, this particular smear, okay, that I have shown you, that is done routinely. So, this particular peripheral blood smear, which is done routinely, okay, this is called as a thin smear, as we can appreciate over here. This particular smear is a thin smear, as we can appreciate over here. Okay, whereas there is another kind of soft smear that is made, which is very thick. This is called as a thick smear. This is called as a thick smear. Okay, this is another slide where we have demonstrated the thin and thick. This is the thin smear. Okay, and that you see over here, this is the thick smear. So, you must be thinking, what is the use? So, one of the uses of thin smear or the routine smear is to carry out routine morphological evaluation that we do in the lab, that you do on the day-to-day basis. Not only that, thin smear is also good for the species identification of malaria. So, it is very important for these species, okay, so to identify the species, okay, of malaria. Whereas thick smear is only required when you want to detect the presence of malaria, detection of malaria. So, why the thick smear is very good for detection? So, over here, you cannot say what kind of malarial species is there, but you will be able to say whether malaria or parasite is present or no. Why? Because in the thick smear, you are having more amount of blood. So, if more amount of blood is there, the chances of of getting or the chances of identifying a malarial parasite will be very high. So, thick smear is used for malarial detection or parasite detection, whereas thin smear is basically used for routine examination and for species identification of malaria. Okay, so we can say that the thin smear is more specific for malarial identification, and the thick smear is more sensitive for malarial parasite identification. Okay.
Now, there is something called as a wet preparation always. So, I am just telling you different kinds of preparation. Okay, so we know about the thin smear, thick smear. We also have what is called as a wet preparation. So, wet preparation is classically used for detection of microfilaria and trypanosomes. So, what you can appreciate over here? Okay, what is this? This is your classical microfilaria that you can appreciate. So, what is that, uh, wet preparation? In a wet preparation, a drop of anticoagulated whole blood is placed on a glass slide and it is covered with a coverslip and immediately examined under the microscope. So, what is happening over here? We are not doing the Leishman staining over here. We are not doing any kind of staining. Okay, we are just adding, you can see the RBCs, they are not stained. Okay, we are directly, we are, you know, in the, you know, we are directly taking the blood in the glass slide, we are covering it with a coverslip and we are examining under the microscope. So, nothing is done over here. So, what is the use of wet preparation? They are used for detection of motile organisms like your microfilaria or trypanosomes. Okay.
Now, we are going to see. So, till now, what all we have read about? We have read about what are the indications. So, when are you going to, you know, examine a peripheral blood smear slide? Number one. Number two, we have seen that, very importantly, we have seen that how do you prepare a particular smear? What is the correct way of preparing a smear? What are the characteristics of a particular smear grossly? What kind of information you can get for from a particular smear? Okay, we have seen all these things over here. And what are the different kinds of preparation that we can see like thick smear, thin smear, wet preparation? Now, we are going to read about, and we have also read about the fixation till now. Okay, so till this step, we have read. Now, we want to stain a particular peripheral blood smear. So, now we are going to see the routine staining of the peripheral blood smear. Okay, so first, we are going to see the principle of staining. Okay, what is the principle of staining? So, first of all, the first exam question, viva question, that is when that will be asked to you, that blood smears are routinely stained by one of the Romanowsky stains. So, Leishman stain is a type of a Romanowsky stain. So, what is a Romanowsky stain? Romanowsky stain is a type of a stain which is having two main components. It is present in all Romanowsky stains. They are having one acidic dye or eosin Y, as well as a basic dye. Okay, basic dye, for example, oxidized methylene blue or for example, Azure B. These are all basic dyes. So, they have a mixture of acidic dye and a basic dye. The staining properties of Romanowsky stains are dependent on the two synthetic dyes, one acidic, one basic. Example of acidic is your eosin, example of basic dye is methylene blue. So, the basic or the cationic dye that we see, it is positively charged, and it binds to the anionic sites and imparts a bluish gray color to the nucleic acid, nuclear protein, and the granules of the basophil. So, the example is methyl blue, methylene blue, or Azure B. These are examples of basic dye, and the principle is that it is positively charged, so it will bind with the negatively or ionic sites, like, for example, they will give bluish color to the, bluish gray color to the nucleic acid, nuclear proteins, granules of the basophils. Okay, so they are stained mainly by the basic dyes. Acidic or anionic dyes, they are negatively charged, and they will bind to cationic sites, and they will impart an orange red color to hemoglobin and to eosinophilic granules. Example of the acidic dye that we see is your eosin Y, as we can appreciate over here. So, the first question will be your first important question will be that what is Leishman? It is the type of a Romanowsky stain. What is the Romanowsky stain? It is a combination of acidic and basic. The principle being one basic which is positive will bind to anionic, and one acidic dye will bind to the cationic side. So, what are the examples of Romanowsky stain? So, you have MGG, Giemsa, Leishman, Wright's stain. So, these are all examples of Romanowsky stain. Okay, remember, Romanowsky stains, they are insoluble in water, but they are soluble in methyl alcohol. Methyl alcohol, therefore, acts as a solvent as well as a fixative. So, as I told you, if you are carrying out staining with a Leishman stain, that it is containing both methyl alcohol as well as this methyl alcohol is serving both as a solvent for the Leishman as well as as a fixative. So, usually if you are staining with Leishman stain routinely, you do not require, you know, as a fixative. Routinely it is not carried out in any labs. But in higher labs, in an ideal situation, in first-world countries, they are first carrying out fixation, after that they are followed by Leishman staining. But the Leishman staining is also having a fixation part, okay, which is because of the methyl alcohol that is present. Now, Romanowsky stains imparts more colors than just blue as well as red orange. Okay, so the usefulness or the principle of Romanowsky stains lies in their ability to differentially stain the different parts. So, they will stain the cytoplasm with a different color, the cytoplasmic granules with a different color, and the nucleus with a different color. So, this ability to to give impart different colors to different components of a cell, okay, is the is the basis or the principle of Romanowsky stain, and which is allowing for differentiation between the different blood components, that is the usefulness of the Romanowsky stain. Okay, so the usefulness lies in their ability to differentially stain different components of a cell. So, you have to use this term in the principle. The staining reaction is pH dependent, and these stains, they have a tendency towards precipitation, and therefore, the Leishman stain should always be filtered before use, else a lot of, you know, stain precipitates is going to deposit on the slide, and it is going to create a problem for you. Okay, so the Leishman stain should always be filtered. At least they should be filtered on a weekly basis in very good labs and very high-quality labs. If you go in in first-world countries, they are filtered every day. Okay, this is very important. Okay.
Now, we are going to see. Sometimes you will have excess bluish coloration in a slide. So, excess blue coloration can occur if your smear is very thick, or there is a low concentration of eosin and more amount of methylene blue. If the dye used is impure, if you are staining for a very long period of time, then that that has been prescribed in the protocol, if you are carrying out inadequate washing, if excessive alkaline pH is there for the stain buffer or water, then your particular peripheral blood smear might appear more bluish. Similarly, your slide can become more reddish because of an impure dye or incorrect proportion of dye with excessive amount of, for example, eosin or and less amount of methylene blue, or there is an excessive acidic pH of the stain buffer or water. Okay, or there is too short staining time or excessive washing is there. If there are granules of stain precipitate, that is masses of small black dots on smear, the stain needs to be filtered. Okay, sometimes in while you are examining, you will see multiple stain deposits, that means that you have to filter the particular Leishman stain. So, a well-stained smear, as we can see, it is pink in color in the thinner portion. So, you can see this is the stain, this is your stained thin blood smear, as we can see. So, at the tail end, this is the tail end where the slide is thinner, where the PBS is thinner, so at the tail end, where the particular smear is thinner, if you see, it is pinkish in color, whereas at the head end, that is this is the the thicker portion, or you can say this is the head end, you can see they are purple blue, they are purple blue in a thicker portion. So, after staining, this is how grossly you can say whether your particular smear has been made properly or no. And mind it, these questions are asked in your exam. So, just by looking, the particular examiner will give you a slide and he will tell you whether this staining is proper or no. So, how you can say that? So, over there, you are not replying anything in the exam. So, in that case, you have to say these points very importantly. I'm repeating again. Okay, as I already told you that there are that the Romanowsky stain that we see, okay, they are a mixture of two dyes, one is your basic dye, that is methylene blue, another is your acidic dye, that is your eosin Y. Okay, so the methylene blue. So, first I will be speaking about the methylene blue. Methylene blue and Azure B, they are the basic cationic dyes, and they have affinity for the acidic components of the cells like the nucleic acids or the basic granules, and also they are imparting a purple violet color to the nuclear chromatin. Okay, they are giving a purple violet color to the nuclear chromatin, dark blue violet color to the basophilic granules, and a deep blue color to the cytoplasm of the lymphocytes. So, there are three important components and the ways in which they are imparting color, a purple violet color to the nuclear chromatin, dark blue violet color to the basophilic granules, and a deep blue color to the cytoplasm of lymphocytes. Okay, this is about the methylene blue. Okay, now the second important dye is your eosin, which is an which is basically an acidic or an anionic dye. It has affinity for the basic components of the cells like the hemoglobin, which is stained pink red by eosin, and the granules in the eosinophil, which are stained orange red or brick red. Neutrophil granules are also slightly basic and stained violet pink or lilac. Actually, it is very light pink in color. Okay, or violet pink or lilac in color. Okay, so this is how the staining is done, and this is called as differential staining. So, the basic principle of any Romanowsky stain is differential staining, and they are having a mixture of acidic and basic dye. So, you have to, you know, say about these points in the exam when you're asked about the principle. Okay.
Now, we see what is the method of Leishman staining. Okay, so we will try to understand the method. So, Leishman stain into the Romanowsky stains, it is containing two dyes, one is basic, that is methylene blue, and one is acidic, that is your eosin, which is dissolved in absolute methyl alcohol. Now, this methyl alcohol, it is acting as a major solvent in which the Leishman stain is dissolved, and the absolute methyl alcohol is also acting as a fixative. Okay, commercially, the Leishman stain powder is available. So, 0.6 gram of Leishman stain powder, you take, and you have to mix it with water-free as well as acetone-free absolute methyl alcohol, approximately 400 ml. Now, the prepared stain should be kept tightly stoppered in a brown colored bottle. So, whatever stain you are preparing, you should keep in an amber or a brown colored bottle, and they are stored in a cool, dark place at room temperature. Okay, they are stored at room temperature. Ideally, if you ask me, whatever Leishman stain is is made, it should be filtered twice. Okay, and after you filter it twice, you should keep it in the incubator for seven days before you make the first usage of that. Because once you keep in the incubator at 37 degrees, the Leishman stain is properly, you know, ripened. There is the ripening of the Leishman stain which is taking place in the incubator. So, this is a very, very important point. Okay, you should be able to make, if you have made the Leishman stain, then you will remember and recall. Okay, nowadays, ready-made Leishman stain is also available, or you can make Leishman.
Stain yourself as well, okay? I have done both of them, so I'm just sharing my experience with you. So, in whatever way you do, you should always keep it in a dark-colored bottle, away from sunlight. And you should ideally keep them in the incubator at 37 degrees. And you should keep, now the longer you keep it, the better it is. There is better staining, there is better ripening of the Leishman's stain. Not only that, uh, not only that, on a weekly basis, you have to filter as well. So, the first time you prepare the Leishman's stain, you filter it twice, okay? And then you keep it in the incubator for seven days, okay? Now, exposure to direct sunlight will cause deterioration of the stain. After preparation, the stain should be kept, uh, for three to five days before using it, since it will improve the quality of the stain. So, as I told you, that once you prepare the stain, it is not used in that time. At least you give one week time and you keep it in the incubator so that a process called ripening occurs.
So, the first reagent required is the Leishman's stain. The second reagent which is required is the buffered water with a pH of around 6.8. Now, what is the method? How do you carry out Leishman staining? So, you have made, so for example, in your exam, you have been given a particular, you know, blood sample. You take the blood sample, take a drop of blood, you make the proper smear. After that, you air dry it. And after you air dry it, you are basically fixing it with methanol for two to three minutes. Now, usually, I am sure in your colleges, if it is done, then you follow the step. If it is not, then you can omit this step, okay? Basically, we directly cover the entire smear with the Leishman stain for two minutes, okay? For two minutes, you cover the smear with Leishman's stain. After two minutes, you, after two minutes, you add twice the volume of buffered water, which we are having a pH of 6.8, that we have already seen. And you leave it for five to seven minutes. Now, once you add the buffer, you will see a scum of metallic sheen forms on the surface. Now, once it is formed, also in the process, maybe after an interval of 30 seconds or 15 seconds, you do air agitation with the help of a dropper. You do air agitation, okay, of the mixture with the help of a dropper.
Now, wash the stain away in a stream of buffered water, okay? Tap water can also be used for washing if it is not highly alkaline or highly acidic. Usually, we are using tap water for washing, okay? You wipe the back of the slide clean. Now, once you have the staining is there, okay, the back of the slide should be completely clean, okay? It should be made clean with particular, you know, you can use cotton, you know, wet cotton can be used to basically clean. Else, your back of the slide will be completely bluish and it will not give a very good color. And after that, you set it upright in the draining rack to dry. After that, you mount the slide in a suitable mounting medium, that is DPX, with a clean and dry cover slip of approximately 25 by 25 millimeters.
Now, I, now on a day-to-day, on a routine basis, we are not using the cover slip, but for routine steps, we are using these steps, okay? So, you should be able to do and stain a particular slide very nicely in your exams, okay? And this will be very, very important for you because you are expected to know everything about the Leishman staining. Now, one important way from where you can learn these methods of staining is to, you know, see the medical technologists in your laboratory. So, you go to them and you see the way they are staining. In that way, you will be able to identify how to carry out the process of staining.
Now, the very important step. Now, the other important competency over here is to identify the blood cells because once you will be going to the central lab duties, you need to identify the blood cells, okay? So, let me just tell you. So, when you are using a Leishman stain, okay, then the red cells, they are pink red or they are deep pink in color. The polychromatic cells, that is the reticulocytes, they are grayish blue in color. The neutrophils, they have a pale pink cytoplasm with pinkish purple granules or light pink granules or lilac granules. The eosinophils, they are also pale pink cytoplasm with orange red or they have a brick red granules. Basophils, if you see, they have a blue cytoplasm, but the important feature is the presence of the dark blue violet granules. Monocytes, they are having a grayish blue cytoplasm with cytoplasmic vacuolation and they have very fine reddish granules and vacuoles in the cytoplasm. Small lymphocytes, they have, they are recognized by the presence of a dark blue cytoplasm, whereas large lymphocytes, if you see, they are basically having a little bit pale blue cytoplasm. So, I'm just writing it over here. The large lymphocyte, if you see, they are having a pale blue cytoplasm and they have more amount of cytoplasm compared to the small lymphocyte, and sometimes they contain one or two cytoplasmic granules are there, okay? The platelets, they are purple in color, and the nuclei of all the cells are stained purple violet in color. So, this is how the normal staining occurs with the help of Leishman's stain, and this is a differential staining, okay?
So, now, one by one, we are going to see how the individual components look like. So, as you can see this particular slide, okay? So, as you see over here, can you see the individual RBCs? So, these are the individual RBCs. So, if you look at the normal RBCs, they are seven to eight micrometers in size. They are round, as we can see, and they have a very smooth contour, and they stain deep pink at the periphery. So, at the periphery, they are very deep pink in color, and they become pale at the center, okay? There's a central pallor, okay? The area of the central pallor for a normal, for a normal RBC, it is one-third the diameter of the red cell. If this pallor becomes more, we call it as hypochromia. If it becomes less, it will become hyperchromia. Now, the size of the normal red cell corresponds roughly with the size of the nucleus of the small lymphocyte. So, the normal RBC size, so for example, if I have drawn a small lymphocyte with a nucleus, so the size of this nucleus actually should correspond to the RBC size. So, the size of the nucleus of the small lymphocyte is same as the, is reflecting the size of the normal RBC. So, you can compare the size of the RBC comparing it with the size of the small lymphocyte. The normal red cells, they are described as normocytic of normal size, normochromic with normal staining intensity, that is the hemoglobin content, okay?
Now, the segmented neutrophils. So, how do you identify the segmented neutrophils over here? As you can see, the polymorphonuclear neutrophils, they are characterized by the presence of two to five lobes over here. One, two, three lobes are there over here. One, two, three lobes are there again, okay? And these are joined by very thin, you can see over here, very thin chromatin strands. The nuclear chromatin scores. The cytoplasm, if you see, it is pale pink. It is pale pink in color, and they are containing numerous small, small dot dots like granules, okay? What are these? These, these purple colored stuffs? These are the platelets, as you can appreciate over here. So, platelets are purple color, okay? Very, very important, okay?
Now, again over here, in this slide, what you can appreciate, what are these cells that you can see? Yes, these are basically the eosinophils. The mature eosinophil, they are 15 to 16 microns. They are slightly larger as compared to the neutrophil. Usually, they are bi-lobed. So, you can see they are having two lobes over here, okay? Not only that, they contain numerous bright orange red granules. So, this presence of these kinds of granules helps in identification of the eosinophils. The eosinophilic granules they contain major basic protein that is toxic to many parasites, and what is the function? It includes anti-helmetic activity and allergic response, okay?
Then we have the basophils. So, the way that you will identify the basophils, you can see is the presence of these dark violet color, okay? Dark purple black granules which is obscuring the nucleus. This is helping in the identification of basophils. So, basophils are 9 to 12 microns in size, and the cytoplasm is filled with numerous deep purple black granules that obscures the nucleus. Nuclei is segmented into two to three lobes. The basophil granules contain histamine and heparin. They may have, they play a role in allergic and anaphylactic reaction, that is immediate variety of hypersensitivity reaction, okay?
Now, if you look at the monocytes, okay? Ideally, if I show you, they are the largest white blood cells in the peripheral blood. So, whenever you see the largest cell in the peripheral blood, there's a high chance that is a monocyte. Now, always remember one thing in your book, okay? In your book, the monocytes are always shown somewhat like this, having a bean-shaped nucleus, okay? But ideally, in the ideal situation, it is not that the shape of the nucleus of the monocyte can have several kinds of variation. So, if you see these are two monocytes. The most important distinguishing feature is the presence of the gray grayish blue cytoplasm. Sometimes they will show vacuolation, okay? There will be small vacuolations over here, as we can appreciate over here. So, you will find vacuolations over here. They have a ground glass appearance if you look at the cytoplasm. Cytoplasm, they are giving a ground glass appearance, okay? And they have very fine, very small dotted azure of like reddish granules and vacuoles in the cytoplasm, okay? They are the largest cell. Now, if you see over here, the nucleus shape, you can see they are kidney-shaped, but not always. Sometimes you might have some abnormal shapes like this. So, the nucleus of the monocytes can have a wide variety of shapes. This is my practical experience, okay? So, monocytes, they are the largest white blood cells in the peripheral blood, around 15 to 20 microns.