Differential Leukocyte Count Practical

I am Adi Prabhu. Today I will be performing the practical on differential leukocyte count. In this experiment we will be calculating the percentage composition of different types of WBCs. For this we will be needing slides, a rack, droppers, a tube to blow, lancets spirit for sterilization, some cotton, distilled water containing buffer and a stain. Now there are three types of stains which are commonly used in this experiment. The first one is Lishman stain, the second is Gimsa stain and third is the Wright stain. In this experiment we will be using the Lishman stain. The three major component of this stain are methylene blue which stains the nucleus and the basophilic granules. The another component is eosin which stains the cytoplasm and eosinophilic granules. Another very important component of this stain is acetone free alcohol, acetone free methyl alcohol. The function of this alcohol is it preserves the cells and helps in fixation of the smear on the slide. So first of all we will be proceeding with the sterilization of finger and removal of blood. So I am taking the spirit. The ring finger is usually preferred for pricking. Well, sterilization, I will rub the cotton downwards. Now we have to let it dry. So now I am going to prick the finger. Now take a clean slide, it should be clean. and place a drop of blood on the slide. So I have collected blood on three slides. We prepare multiple slides so that we can later compare the slides and choose the better one. So now I am going to take another slide in order to spread this blood in order to form a tongue shaped smear. So what we have to do is, first of all keep it at an angle of 45 degree, pull it a little backward, allow the blood to form a red color line as you can see here and then gently push it forward. Now I am going to spread this blood into a tongue shaped smear. For this I will place a slide at an angle of 45 degree, pull it a little backward, allow the blood to form a red colored line, then gently push it forward. Now we have prepared four slides out of which you can see these two are not that perfect. There are gaps in the smear so we are going to discard these slides. Now these are the two we will be using for carrying out the further experiment. An ideal smear should be a tongue shape. It should cover the entire width of the slide. It should cover at least two-thirds of the slide. There should be no striations or vacuoles or like patches of air in it. The thickness of the layer should be uniform. Now we have prepared the smear and now we will be proceeding towards the staining and fixation of the smear. So I will be using the Lishman stain. First of all I will be adding the stain to cover the entire slide. I will keep it for one or two minutes and then dilute it. Now what is the reason for diluting a concentrate? concentrated stain. The reason is first of all we add a concentrated stain because we want to fix this smear to the slide. After the fixation which takes about two minutes we will be adding water to dilute it in order to stain the slide. So now I will be proceeding with the addition of stain. Make sure you count the number of drops because you will have to add equal amount of water containing buffer. Now we will have to wait for time of 2 minutes after which we will be adding buffer. So now we have added the stain. You have to take few precautions. The stain should not dry. Like if the weather around you is hot. So you have to make sure that the stain does not dry and stick to the smear. In such a case if you see that the stain is getting dried, you should add more stain to it. So 2 minutes have been completed. Now we will be adding the distilled water. Now we will keep this for about 7 to 8 minutes for staining. Now we have completed waiting for 8 minutes. Now the slide has been stained. Now we have to wash the excess stain. So what I will do is, you have to avoid direct application of water on the slide because it will disturb the smear. So what I will do is, I'll wash it indirectly. So you can see we have washed the stain and this is our smear which we will be observing under the microscope to count the leukocytes. I am repeating this procedure once again. Now we have to dry these two slides. So I have completed washing these slides. Now I am going to keep it in inclined position in order to drain excess water and allow it to dry for some time. I am going to add the cedar wood oil. Now we are going to observe the slide under the microscope under oil immersion. Now I have placed the slide on microscope. Now sometimes people find it difficult to focus so what you have to do is you have to touch the oil to the lens. Now start observing and slightly just raise the lens and then shift to fine adjustment. In this way you can easily focus on the slide. Now I have placed the slide on the microscope. Now we will proceed to the observation. For focusing the slide what you have to do is raise the slide in such a way that the oil touches the lens. Okay, now start observing. Now slightly move the stage downwards and then proceed to fine adjustment. In this way you will find it easier to focus the slide. Now under the microscope you have to count the number of different types of WBCs. I have already counted them. And you have to write the number in this grid. This is a grid of 10 by 10. You have to write it here. In the form in which L-abrevates, lymphocytes, N for neutrophils, E for eosinophils, B for... ...basophils and M4 monocytes. Now while counting the leukocytes under the microscope you have to take a precaution that you don't count the same number of leukocytes again and again. For this what you are going to do is under the microscope start moving in any particular direction then move shift a little little bit downwards and then move in the opposite direction. In this way you are going to travel the entire course of slide. Now in the slide I was able to observe 34 neutrophils, 4 monocytes, 52 lymphocytes, 10 eosinophils and 0 basophils. How do you identify different types of leukocytes? The different types of leukocytes are classified basically on the presence of granules and absence of granules. There are granulocytes and agranulocytes. Granulocytes include neutrophils, eosinophils and basophils. Agranulocytes include lymphocyte and monocyte. In case of neutrophil, the observable characteristic is presence of a lobed nucleus. There are generally 3 to 5 lobes. The 3 lobed neutrophils are most abundant. In case of basophil, you will observe a shaped nucleus. In case of eosinophil, you will observe a shaped nucleus. cells, you will be observing a spectacle or a goggle shaped nucleus. In lymphocytes, the nucleus is round and large and covers almost the entire volume of cell. And in monocytes, the nucleus is kidney shaped or horseshoe shaped. double the size of RBC. What is the composition of Lishman stains and what are the functions of which you constitute? Lishman stain contains methylene blue which stains the nucleus and the basophilic granules. It also contains eosin which is going to stain the cytoplasm and eosinophilic granules and it has got acetone free methyl alcohol which help in fixation of the stain fixation of the smear on the slide. Now Adi tell me what is the necessity of adding concentrated stain and then diluting it. The concentrated stain is first added after which it is diluted the reason is that the concentrated stain helps in fixation of the smear to the slide and dilution is done in order to stain the slide. Tell me what are the functions of different types of WBCs? Neutrophils and macrophages help in the phagocytosis of bacteria and dead cells. The lymphocytes are involved in the cell mediated and humoral immune response. The eosinophils help in detoxification of histamines and engulfing of antigen antibody complexes. Therefore, their number is increased during allergies. It also secretes larvae cells. Therefore in case of parasitism, infections their number increases. Basophil release histamine and heparin which is again involved in allergic response. Okay tell me what is the useful information that we get from RNET count? RNET count is distribution of neutrophils based on the number of lobes in the nucleus. If the nucleus is unlobed it means that the neutrophil is immature. and if the number of lobe is 5 it means it has achieved its mature state and it is going to undergo lysis very soon. So the important function of this Arnath count is we get to know what is the status of bone marrow activity. Say for example if the concentration of unlobed neutrophils is higher, it means that the bone marrow is functioning very rapidly, like in case of leukemias. That's the reason immature cells are being sent into the blood flow. If the neutrophil count, neutrophil having five lobes is very high, it means that there is a defect or there is a depressed bone marrow activity. What is the significance of doing a differential WVC count? Differential WVC count helps us in determining the composition of each type of leukocyte in the blood. Now, in case there is the neutrophil count, it means that there is a back... bacterial or viral infection. If there is increased monocyte count, it implies there is any chronic infection. Increased basophil count generally occurs during myeloid leukemias and chronic nasal infections. And lymphocyte count also increases during chronic infections. This is absolute WBC count. Absolute WBC count is the actual number of WBC present in the blood. It is calculated by the method of we take the percentage value determined by counting which we did just now and we are going to multiply it with the total leukocyte count. Okay, tell me what is the significance of absolute WBC count? Differential leukocyte count gives us a relative idea about the different leukocytes. However, absolute count gives us the absolute or the real value of the WBCs, different type of WBCs. ok tell me what are the stains can be used instead of leishman stain gimza stain or right stain can be used instead of leishman stain tell me what are the other things that could be seen from the blood The structure of RBC can be observed from this slide and any defect might be attributed to the presence of anemia. Then parasites like filarial worms, trichomoniasis and malarial parasite can also be observed. Then depending upon the Rnet count we can get to know about the bone marrow activity.

I am Adi Prabhu. Today I will be performing the practical on differential leukocyte count. In this experiment we will be calculating the percentage composition of different types of WBCs.

For this we will be needing slides, a rack, droppers, a tube to blow, lancets spirit for sterilization, some cotton, distilled water containing buffer and a stain. Now there are three types of stains which are commonly used in this experiment. The first one is Lishman stain, the second is Gimsa stain and third is the Wright stain. In this experiment we will be using the Lishman stain.

The three major component of this stain are methylene blue which stains the nucleus and the basophilic granules. The another component is eosin which stains the cytoplasm and eosinophilic granules. Another very important component of this stain is acetone free alcohol, acetone free methyl alcohol.

The function of this alcohol is it preserves the cells and helps in fixation of the smear on the slide. So first of all we will be proceeding with the sterilization of finger and removal of blood. So I am taking the spirit.

The ring finger is usually preferred for pricking. Well, sterilization, I will rub the cotton downwards. Now we have to let it dry. So now I am going to prick the finger. Now take a clean slide, it should be clean.

and place a drop of blood on the slide. So I have collected blood on three slides. We prepare multiple slides so that we can later compare the slides and choose the better one.

So now I am going to take another slide in order to spread this blood in order to form a tongue shaped smear. So what we have to do is, first of all keep it at an angle of 45 degree, pull it a little backward, allow the blood to form a red color line as you can see here and then gently push it forward. Now I am going to spread this blood into a tongue shaped smear.

For this I will place a slide at an angle of 45 degree, pull it a little backward, allow the blood to form a red colored line, then gently push it forward. Now we have prepared four slides out of which you can see these two are not that perfect. There are gaps in the smear so we are going to discard these slides.

Now these are the two we will be using for carrying out the further experiment. An ideal smear should be a tongue shape. It should cover the entire width of the slide.

It should cover at least two-thirds of the slide. There should be no striations or vacuoles or like patches of air in it. The thickness of the layer should be uniform. Now we have prepared the smear and now we will be proceeding towards the staining and fixation of the smear.

So I will be using the Lishman stain. First of all I will be adding the stain to cover the entire slide. I will keep it for one or two minutes and then dilute it. Now what is the reason for diluting a concentrate? concentrated stain.

The reason is first of all we add a concentrated stain because we want to fix this smear to the slide. After the fixation which takes about two minutes we will be adding water to dilute it in order to stain the slide. So now I will be proceeding with the addition of stain. Make sure you count the number of drops because you will have to add equal amount of water containing buffer.

Now we will have to wait for time of 2 minutes after which we will be adding buffer. So now we have added the stain. You have to take few precautions.

The stain should not dry. Like if the weather around you is hot. So you have to make sure that the stain does not dry and stick to the smear. In such a case if you see that the stain is getting dried, you should add more stain to it.

So 2 minutes have been completed. Now we will be adding the distilled water. Now we will keep this for about 7 to 8 minutes for staining.

Now we have completed waiting for 8 minutes. Now the slide has been stained. Now we have to wash the excess stain. So what I will do is, you have to avoid direct application of water on the slide because it will disturb the smear.

So what I will do is, I'll wash it indirectly. So you can see we have washed the stain and this is our smear which we will be observing under the microscope to count the leukocytes. I am repeating this procedure once again. Now we have to dry these two slides. So I have completed washing these slides.

Now I am going to keep it in inclined position in order to drain excess water and allow it to dry for some time. I am going to add the cedar wood oil. Now we are going to observe the slide under the microscope under oil immersion.

Now I have placed the slide on microscope. Now sometimes people find it difficult to focus so what you have to do is you have to touch the oil to the lens. Now start observing and slightly just raise the lens and then shift to fine adjustment.

In this way you can easily focus on the slide. Now I have placed the slide on the microscope. Now we will proceed to the observation.

For focusing the slide what you have to do is raise the slide in such a way that the oil touches the lens. Okay, now start observing. Now slightly move the stage downwards and then proceed to fine adjustment. In this way you will find it easier to focus the slide.

Now under the microscope you have to count the number of different types of WBCs. I have already counted them. And you have to write the number in this grid. This is a grid of 10 by 10. You have to write it here.

In the form in which L-abrevates, lymphocytes, N for neutrophils, E for eosinophils, B for... ...basophils and M4 monocytes. Now while counting the leukocytes under the microscope you have to take a precaution that you don't count the same number of leukocytes again and again.

For this what you are going to do is under the microscope start moving in any particular direction then move shift a little little bit downwards and then move in the opposite direction. In this way you are going to travel the entire course of slide. Now in the slide I was able to observe 34 neutrophils, 4 monocytes, 52 lymphocytes, 10 eosinophils and 0 basophils.

How do you identify different types of leukocytes? The different types of leukocytes are classified basically on the presence of granules and absence of granules. There are granulocytes and agranulocytes. Granulocytes include neutrophils, eosinophils and basophils. Agranulocytes include lymphocyte and monocyte.

In case of neutrophil, the observable characteristic is presence of a lobed nucleus. There are generally 3 to 5 lobes. The 3 lobed neutrophils are most abundant.

In case of basophil, you will observe a shaped nucleus. In case of eosinophil, you will observe a shaped nucleus. cells, you will be observing a spectacle or a goggle shaped nucleus.

In lymphocytes, the nucleus is round and large and covers almost the entire volume of cell. And in monocytes, the nucleus is kidney shaped or horseshoe shaped. double the size of RBC.

What is the composition of Lishman stains and what are the functions of which you constitute? Lishman stain contains methylene blue which stains the nucleus and the basophilic granules. It also contains eosin which is going to stain the cytoplasm and eosinophilic granules and it has got acetone free methyl alcohol which help in fixation of the stain fixation of the smear on the slide.

Now Adi tell me what is the necessity of adding concentrated stain and then diluting it. The concentrated stain is first added after which it is diluted the reason is that the concentrated stain helps in fixation of the smear to the slide and dilution is done in order to stain the slide. Tell me what are the functions of different types of WBCs?

Neutrophils and macrophages help in the phagocytosis of bacteria and dead cells. The lymphocytes are involved in the cell mediated and humoral immune response. The eosinophils help in detoxification of histamines and engulfing of antigen antibody complexes.

Therefore, their number is increased during allergies. It also secretes larvae cells. Therefore in case of parasitism, infections their number increases. Basophil release histamine and heparin which is again involved in allergic response. Okay tell me what is the useful information that we get from RNET count?

RNET count is distribution of neutrophils based on the number of lobes in the nucleus. If the nucleus is unlobed it means that the neutrophil is immature. and if the number of lobe is 5 it means it has achieved its mature state and it is going to undergo lysis very soon. So the important function of this Arnath count is we get to know what is the status of bone marrow activity. Say for example if the concentration of unlobed neutrophils is higher, it means that the bone marrow is functioning very rapidly, like in case of leukemias.

That's the reason immature cells are being sent into the blood flow. If the neutrophil count, neutrophil having five lobes is very high, it means that there is a defect or there is a depressed bone marrow activity. What is the significance of doing a differential WVC count?

Differential WVC count helps us in determining the composition of each type of leukocyte in the blood. Now, in case there is the neutrophil count, it means that there is a back... bacterial or viral infection. If there is increased monocyte count, it implies there is any chronic infection. Increased basophil count generally occurs during myeloid leukemias and chronic nasal infections.

And lymphocyte count also increases during chronic infections. This is absolute WBC count. Absolute WBC count is the actual number of WBC present in the blood. It is calculated by the method of we take the percentage value determined by counting which we did just now and we are going to multiply it with the total leukocyte count. Okay, tell me what is the significance of absolute WBC count?

Differential leukocyte count gives us a relative idea about the different leukocytes. However, absolute count gives us the absolute or the real value of the WBCs, different type of WBCs. ok tell me what are the stains can be used instead of leishman stain gimza stain or right stain can be used instead of leishman stain tell me what are the other things that could be seen from the blood The structure of RBC can be observed from this slide and any defect might be attributed to the presence of anemia.

Then parasites like filarial worms, trichomoniasis and malarial parasite can also be observed. Then depending upon the Rnet count we can get to know about the bone marrow activity.

Transcript for:Differential Leukocyte Count Practical

Transcript for:
Differential Leukocyte Count Practical