BLOOD SMEAR

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BLOOD SMEAR

Introduction

The testing of any abnormal thing that is observed in the blood of an individual is done by utilizing the technique of blood smear. The testing of the blood sample is done on a special slide that is treated with chemicals. By observing the sample under the microscope the professional determines whether the condition is appropriate. If any difference is observed, then it is considered that some abnormality has been undergone in the cells of the blood. The cells that are examined are the red blood cells which are responsible for carrying oxygen from the lungs to the whole body. The white blood cells are also analyzed and the function of the white blood cell is to fight against the infection. Examination of the platelets is also done because these platelets are the ones that are responsible for the clotting of blood in the human body. The requirement for smearing of blood comes if it is observed that the “complete blood count” (CBC) is not normal as per the requirement. Lesihman’s stain was used while doing this experiment, but the other stains mentioned have their own role in blood smear.

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Objectives

The main objective of the experiment is:

  • To prepare a blood film and then identify the cells of the blood by using the Leishman stain, the cells are found at the periphery of the blood smear.
  • To calculate the count of reticulocytes by utilizing the methylene blue stain along with their identification.

Methods

The blood film was prepared on a slide where a drop of a sample of the blood was placed and it was done very carefully in such a way that it did not touch the corner of the slide. Then the blood is spread over the slide by using the spreading slide and the slide will spread at an angle of 45 degrees to the slide and then move back the slide till it comes in contact with the blood. Then the spreading slide is moved forward very quickly without any break in the movement process. The film is kept for air dry after fixing it with methanol. Leishman’s stain was used to do the staining of the blood film which was left for 2 minutes to complete the staining process. After this buffer is added it is then allowed to wait for 5-7 minutes preventing the slide from getting dried. The smear was then washed using distilled water and after washing it was again kept for drying. After drying the stain is studied under a light microscope.

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To count the reticulocyte, the EDTA blood is mixed with new methylene blue in a test tube and then the tube is allowed to stand at room temperature. Instead of this, the incubator can also be utilized at 37 degrees Celsius for 15-20 minutes. Then again the mixing of the mixture is done and then the mixture is placed on the slide to make 2 thin wedges of the film which was previously left for drying. The slides were then studied under the microscope by using the objective of 10X so that the area of uniform distribution of red blood cells could be seen. For a further detailed study that is for locating the area that has 100-200 red blood cells 100X objective is used. At last, the counting of the red blood cells is done averaging between the two slides.

Results

Part 1: Staining of Blood smear

BLOOD SMEAR

Figure 1: Blood smearing utilizing the Leishman’s stain

(Source: Nakasi et al. 2020)

The result that is obtained in the lab shows the red blood cells that are matured, neutrophils, platelets, and monocytes. The red blood cells that are matured are seen to be in pink color.  The purple color which is the round structure represents the platelets. The faint pink colors are the neutrophils which are stained by their granules (Pattanaik et al. 2020). The identification of the monocytes can be done by observing the nucleus and also by observing their shapes. The shape of the monocytes is either horseshoe-shaped or kidney-shaped. The monocytes also have a transparent cytoplasm.

 

Type of cell Description Number Percentage
Monocyte Presence of Nucleus, horseshoe-shaped or kidney-shaped, purple. Translucent

Cytoplasm can also be bi-lobed

2 0.20%
Neutrophil stain light pink, have granules. Dark purple is the nuclei 3 0.45%
Erythrocytes Pink in color. 615 93.20%
Platelets Purple in color, round in shape 72 12.10%

Table 1: Different types of cells with description and quantity

Part 2: Identification of types of blood

BLOOD SMEAR

Figure 2: Reticulocyte count

(Source: Zaninetti & Greinacher, 2020)

The identification of the RNA can be done by observing the color. The deep blue color is the RNA. The Medium green color represents the red blood cells.  The total number of reticulocytes is 12 and it is calculated by using the “2 Heinz bodies”. The number of red blood cells that have matured is 512.

Reticulocyte count = (12 X 100)/1000 Reticulocyte count = 1.2%

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Discussion

Question 1

Red Blood cells are the most common type of cells which are mainly found in the blood. It is also known as erythrocytes (Umer et al. 2020). The main specification of this cell is that each cubic millimeter of blood can contain 4-6 million cells in it.

Question 2

As per the scientific records, it is observed that the monocytes are much larger as compared to other blood cells. Its actual size is four times the actual size of the red blood cells only. The monocytes are around 7% of the leukocytes which is itself a larger size. This is also capable of being folded rather than the multi-lobed.

Question 3

Mainly there are two considerable types of leukocytes which include lymphocytes as well as monocytes.

Question 4

The proposed granulocytes are white blood cells that help in understanding the immune system that fights off infection. Some characteristic morphologies incorporate large cytoplasmic granules that can be strained with the help of the basic dyes. This also takes into account the bi-lobed nucleus.

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Question 5

There are some different types of anemia which mainly include pernicious anemia, iron-deficiency anemia, and hemolytic anemia. The common reason for this disorder is respiratory disorders as well as hypo- or hypertension (Rehman et al.2018). The basic difference between the types of anemia is its diagnosis with the present red blood cells.

Question 6

Several blood disorders are caused due to microorganisms. But among them, the most common disorders are malaria as well as syphilis.

Question 7

The cancer which mainly originates from the blood-forming tissues is called blood cancer. Blood cancers are also categorized in different aspects including leukemia, lymphoma, and myeloma. Leukemia is a cancer that occurs in the bone marrow. Secondly, lymphoma is a cancer that infects the body’s germ-fighting network. Thirdly, myeloma is a cancer that mainly occurs in the white blood cells.

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Question 8

It is observed that two major blood group antigens are considered medically relevant in the field of cancer. This includes ABO (with types as A, B, AB, and O) and the second is Rh (with Rh D-positive and the second one is Rh D-negative)

Question 9

Mainly there are antibodies as well as proteins found in plasma. These are acting as a body’s natural defense. These are useful in understanding the foreign substances, including the germs. This also helps in alerting the immune system of the body (Ahnach et al. 2020).

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Question 10

Rh typing implements the same methods as the ABO typing system. The Rh typing is the one that has given a positive reaction as well as produced a positive result. The Rh factors when being used get surfaced on the red blood cells and protect the cell proteins. This is a type that contains type-A antigens with the presence of proteins.

Question 11

The type of blood that is a universal donor is “O type blood” because this blood type can be utilized in transfusion for any type of blood.

Question 12

The type of blood that is a universal acceptor is “AB positive” because this blood has no antibodies so it can receive the blood from every group without having the risk of getting any adverse effect.

Question 13

Types of antibodies present in the plasma of an individual with a blood group A is Anti-B.

Question 14

It is acceptable to give O+ blood to a patient who has B+ blood because the O+ blood is considered to be the universal donor (Poostchi et al. 2018). It is so because A, B, or Rh antigens are absent in the red blood cells of these blood groups.

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Question 15

The most common blood type that is found frequently is O+.

Case Study 1

  1. Organs mainly responsible for removing old worn red blood cells from circulation are the Liver and spleen.
  2. The hemoglobin is at first degraded into globin, then the components of the protein are broken down, the iron is preserved for future usage, and at last, the disintegration of the red blood cells occurs. At the initial stage, the splitting of heme occurs into biliverdin.
  3. The treatment of neonatal jaundice can be done by using phototherapy (Safuan et al. 2018). This treatment is done by using a special kind of light and not using sunlight.

Case Study 2

BLOOD SMEAR

Figure 3: A peripheral blood smear was done on this patient

(Source: Salamah et al. 2019)

Finding of the peripheral blood smear

The cell types that are identified from the blood smear are of 3 types, they are platelets, lymphocytes, and red blood cells. The above figures explain that the red blood cells have undergone hypochromia 2+ and this can be understood because of the color deficiency that appears to be greater than half of the cell diameter (approx. ⅔).

Diagnose for the patient

Thalassemia, iron deficiency, and iron deficiency are the possible diagnoses, and all these are connected to hypothermia.

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Other relevant investigations and their expected findings

To confirm the diagnosis Mean Corpuscle/Cell hemoglobin investigation is done. The average weight of hemoglobin in a single red blood cell is calculated by this test. The result is <27 pg/cell (Grochowski et al. 2019). Mean Cell Hemoglobin Concentration is another test that can be done and is used to calculate the average concentration of hemoglobin in the red blood cell. The expected result is <32g/dl.

Conclusion

It can be concluded that 4 types of cells present are neutrophils, platelets, erythrocytes, and monocytes. The highest percentage of cells are the erythrocytes with 93.20%. The percentage of platelets observed in the experiment is 12.10%    and the value of neutrophil is 0.45%. The lowest percentage value obtained is 0.20% for the monocytes. All these are obtained in Figure 1. Figure 2 represents the reticulocyte count at the various stages of development and the value calculated is 12 which is 1.2%.

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References

Journal

Ahnach, M., Ousti, F., Nejjari, S., Houssaini, M. S., & Dini, N. (2020). Peripheral blood smear findings in COVID-19. Turkish Journal of Hematology37(4), 301. Retrieved From: https://www.scienceopen.com/document_file/fc66406e-54eb-4cd0-86a7-3991a41fe00/PubMedCentral/fc66406e-54eb-4cd0-86a7-c3991a41fe00.pdf [Retrieved On: 03.06.2022]

Aris, T. A., Nasir, A. S. A., Mohamed, Z., Jaafar, H., Mustafa, W. A., Khairunizam, W., … & Shahriman, A. B. (2019, June). Color component analysis approach for malaria parasites detection based on thick blood smear images. In IOP conference series: materials science and engineering (Vol. 557, No. 1, p. 012007). IOP Publishing. Retrieved From: https://iopscience.iop.org/article/10.1088/1757-899X/557/1/012007/pdf [Retrieved On: 03.06.2022]

Grochowski, M., Wąsowicz, M., Mikołajczyk, A., Ficek, M., Kulka, M., Wróbel, M. S., & Jędrzejewska-Szczerska, M. (2019). Machine learning system for automated blood smear analysis. Metrology and Measurement Systems26(1). Retrieved From: https://yadda.icm.edu.pl/baztech/element/bwmeta1.element.baztech-e5dc574b-7ef5-466b-b00b-60b9141c61e0/c/Grochowski_Machine_MMS_1_2019.pdf [Retrieved On: 03.06.2022]

Nakasi, R., Tusubira, J. F., Zawedde, A., Mansourian, A., & Mwebaze, E. (2020). A web-based intelligence platform for diagnosis of malaria in thick blood smear images: A case for a developing country. In Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition Workshops (pp. 984-985). Retrieved From: http://openaccess.thecvf.com/content_CVPRW_2020/papers/w57/Nakasi_A_Web-Based_Intelligence_Platform_for_Diagnosis_of_Malaria_in_Thick_CVPRW_2020_paper.pdf [Retrieved On: 03.06.2022]

Pattanaik, P. A., Mittal, M., & Khan, M. Z. (2020). Unsupervised deep learning CAD scheme for the detection of malaria in blood smear microscopic images. IEEE Access8, 94936-94946. Retrieved From: https://ieeexplore.ieee.org/iel7/6287639/8948470/09097238.pdf [Retrieved On: 03.06.2022]

Poostchi, M., Ersoy, I., McMenamin, K., Gordon, E., Palaniappan, N., Pierce, S., … & Jaeger, S. (2018). Malaria parasite detection and cell counting for humans and mice using thin blood smear microscopy. Journal of Medical Imaging5(4), 044506. Retrieved From: https://www.spiedigitallibrary.org/journals/Journal-of-Medical-Imaging/volume-5/issue-4/044506/Malaria-parasite-detection-and-cell-counting-for-human-and-mouse/10.1117/1.JMI.5.4.044506.pdf [Retrieved On: 03.06.2022]

Rehman, A., Abbas, N., Saba, T., Mahmood, T., & Kolivand, H. (2018). Rouleaux red blood cells splitting in microscopic thin blood smear images via local maxima, circles drawing, and mapping with original RBCs. Microscopy research and technique81(7), 737-744. Retrieved From: http://researchonline.ljmu.ac.uk/id/eprint/8510/3/Rouleaux%20Red%20Blood%20Cells%20Splitting%20in%20Microscopic%20Thin%20Blood%20Smear%20Images.pdf [Retrieved On: 03.06.2022]

Safuan, S. N. M., Tomari, M. R. M., & Zakaria, W. N. W. (2018). White blood cell (WBC) counting analysis in blood smear images using various color segmentation methods. Measurement116, 543-555. Retrieved From: https://www.researchgate.net/profile/Razali-Tomari/publication/319859950_White_blood_cell_counting_analysis_of_blood_smear_images_using_various_segmentation_strategies/links/5ae2ef7ea6fdcc9139a106e4/White-blood-cell-counting-analysis-of-blood-smear-images-using-various-segmentation-strategies.pdf [Retrieved On: 03.06.2022]

Salamah, U., Sarno, R., Arifin, A. Z., Nugroho, A. S., Rozi, I. E., & Asih, P. B. S. (2019). A robust segmentation for malaria parasite detection of thick blood smear microscopic images. Int. J. Adv. Sci. Eng. Inf. Technol.9(4), 1450-1459. Retrieved From: https://www.researchgate.net/profile/Ismail-Rozi/publication/336585613_A_Robust_Segmentation_for_Malaria_Parasite_Detection_of_Thick_Blood_Smear_Microscopic_Images/links/603ee86692851c077f129a84/A-Robust-Segmentation-for-Malaria-Parasite-Detection-of-Thick-Blood-Smear-Microscopic-Images.pdf [Retrieved On: 03.06.2022]

Sunarko, B., Bottema, M., Iksan, N., Hudaya, K. A., & Hanif, M. S. (2020). Red blood cell classification on thin blood smear images for malaria diagnosis. In Journal of Physics: Conference Series (Vol. 1444, No. 1, p. 012036). IOP Publishing. Retrieved From: https://iopscience.iop.org/article/10.1088/1742-6596/1444/1/012036/pdf [Retrieved On: 03.06.2022]

Umer, M., Sadiq, S., Ahmad, M., Ullah, S., Choi, G. S., & Mehmood, A. (2020). A novel stacked CNN for malarial parasite detection in thin blood smear images. IEEE Access8, 93782-93792. Retrieved From: https://ieeexplore.ieee.org/iel7/6287639/8948470/09093853.pdf [Retrieved On: 03.06.2022]

Zaninetti, C., & Greinacher, A. (2020). Diagnosis of inherited platelet disorders on a blood smear. Journal of Clinical Medicine9(2), 539. Retrieved From: https://www.mdpi.com/2077-0383/9/2/539/pdf [Retrieved On: 03.06.2022]

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