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Comparative analysis of different water samples as Buffer in Leishman stain.
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African Journal of Laboratory Haematology
and Transfusion Science Volume 1, Issue 2&3: page 120 – 134 2022. ARTICLE ID: 2022–AJLHTS–0002
www.hbtssn.org/ajlhts ISSN (Print): 2814–0591; (Online): 2814–0605
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ORIGINAL ARTICLE
Comparative analysis of different water samples as Buffer in Leishman stain.
Okafor, Ifeyinwa Maryann, ‘Anagha Grace Olachi, Okoroiwu Henshaw Uchechi and ‘Etura, Joyce Ezekiel
Department of Haematology and Blood Transfusion Science, Faculty of Medical Laboratory Science, College of Medical Sciences, University of Calabar, Nigeria. Department of Medical Laboratory Science, Faculty of Basic Medical Sciences, Arthur Javis University, Akpabuyo, Cross River State.
Abstract Introduction Staining of blood films remains one of the most essential part of haematology. The different blood cells have different intracellular structure that take up stains according to chemical nature. Leishman stain is one of the Romanowsky stains used in the haematology laboratory for the staining of blood films. It contains methylene blue and eosin dye prepared in an alcohol medium and is diluted with a buffer. Phosphate buffer is used during Leishman stain as it acts as a mordant enhancing staining reactions and it is the recommended and best buffer for staining.
Aim This comparative study was aimed at determining if different water samples used as buffer show significant effects on the morphology of blood cells during Leishman staining.
Corresponding author: Dr. Okafor, Ifeyinwa Maryann Department of Haematology and Blood Transfusion Science, Faculty of Medical Laboratory Science, University of Calabar, PMB 1115 Calabar, Cross River State, Nigeria.
Methods pH analyses of different water sources such as water from borehole, waterboard, rainwater, fridgewater, distilled water and air conditioned water were carried out and used as buffers to carry out Leishmann staining on blood films. The stained slides were examined macroscopically and microscopically using 100x objective.
E–mail: okaforify maryann@yahoo.c om Tel: +2348080680620
Received: March 03, 2022, Accepted: July 8, 2022, Published: September 30, 2022
Results The study has shown that deionized and tap water used as buffer could produce moderately good film with well stained red cells, white cells and platelets. Rainwater, distilled, fridge and air conditioned water showed a significant change in the morphology of blood cells both in
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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.
an apparently healthy film and a diseased film
Conclusion This study demonstrates that deionized water and tap water devoid of chlorine can be used as substitutes in the right pH of 6.8 in a situation where phosphate buffer is not available. It is therefore recommended that quality control should be carried out to check the quality of stains and buffer used for staining films in order to produce quality results. Keywords: Leishman stain, Buffer, Water
Introduction Different blood cells in our body have different shapes and sizes, which take up stains according to their structures. Some of the components of the blood cells are basophilic, that is they have great affinity to acidic dyes while some components of blood cells are acidophilic meaning they have affinity to basic dyes and also some components neutral and have high affinity to neutral dyes [1].
Romanowsky stains are types of stains employed for staining of blood cells. Almost all Romanowsky stains have two essential components; acidic and basic dye. Methylene blue is a basic dye with high affinity to the acidic component of the cell which is the nucleus, Eosin is the acidic dye that has high affinity to the basic component of the cell which is the cytoplasm and some granules of the cell [2,3]. Many Romaowsky stains are prepared with methanol which acts as a fixative and also as a cellular stain. These are four types of romanowsky stains commonly used in haematology laboratory for staining blood cells, they include: leishman stain, giemsa stain, field stain and wright stains[4,5).
Leishman stain is a type of Romanowsky stain containing a mixture of methylene blue and eosin dye prepared in an alcohol medium and diluted with buffer of pH6.8. The buffer acts as a mordant, it enhances staining reactions
and give better morphology of the blood cells under the microscope[6,7].
Leishman stain is used to stain various components of blood cells and also used to study bacterial cells. It is the basic routine stain for staining and examination of peripheral blood films under the microscope. It is also good for the examination of haemoparasite such as plasmodium species. Phosphate buffer is highly water soluble and has high buffering capacity that inhibits enzymatic activity and will precipitate in ethanol. It is one of the most used buffer employed in the haematology laboratory during Leishman staining [2,8]. Occasionally because of unavailability of phosphate buffer especially in resource–limited areas, available water samples are used as buffer.
These are different water sources that were analysed in this study: Tap water, fridge water, Distilled water, rain water and air conditioned water. Tap water is water supplied through a tap (valve) and distributed through pipes (plumbing). Fridge water is gotten from refrigerator after it has defrosted. Distilled water is steam from boiling water that has been cooled and returned to its liquid state. Rain water is collected from rainfall directly or from roof like surfaces. [3,5]. Air conditioned water is water formed from the cold coils of the air conditioner through squeezing and
in is
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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.
The different water sources were blindly labelled 1,2,3,4,5, and 6 for identification.
condensation of hot air in the room [4,6].
The different water types have different pH and different chemical elements in them, which may exert significant effect on the morphology of the blood cells when used in leishman staining. However, the effects of these water samples in leishman stain have not been extensively studied, hence, the reason for the study.
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Materials and Methods Study Design This was a comparative and analytical study that was carried out for four months, between March to July, 2021 at University of Calabar
Teaching Hospital Calabar.
Making of blood film 1. A microscope slide was cleaned with a
cotton wool to remove debris and make it
grease free. 2. A small drop of well mixed blood sample
was placed at the centre 2cm of the length of the slide. A clean smooth edged spreader was used; the spreader is held at angle of about 45° and is drawn back to touch the drop of blood; blood is allowed to spread to the edge of the spreader. The spreader was pushed gently and quickly forward, making a film in the process; the edge of the spreader is wiped
clean. 5. The blood film was allowed to dry
completely; and slide labelled appropriately.
a
was
Study Area The study was carried out in Calabar Metropolis, Cross River State, Nigeria specifically at Haematology Laboratory, University of Calabar Teaching Hospital Calabar.
Sample collection
Water samples from the respective sources were collected and stored in 4 litres Jerry can and labeled accordingly as Tap water (bore hole), Tap water (water Board), Fridge water, Rain water, Air conditioner
water and Distilled water. 2. About 2ml of blood was collected from
patients with different pathological conditions (Sickle cell anaemia) and apparently healthy Individual which was used to prepare blood films.
Leishman staining 1. The air dried thin film was placed on a
staining rack with smear surface upwards. 2. The entire area of the smear was flooded
with Leishman‘s stain. 3. The flooded slide was allowed to stand for
2 minutes. To ensure cells are fixed
properly by methanol 4. The stain was double diluted with
standard buffer of pH 6.8 and also water
from other sources as buffer differently. 5. The slide was blown gently to enhance
mixture of stain with buffer; a metallic sheen is formed over the surface of the
fluid and allowed to stand for 10 minutes. 6. The stain was rinsed with tap water. 7. The reverse side of the slide was carefully
wiped with a soft absorbent tissue. 8. Slide was kept on a slide rack and allowed
to air dry.
Water Analysis Water from different sources; Tap water(bore hole), Tap water(water Board), Rain water, Fridge water, Air conditioned water and Distilled water was collected and put inside a clean container and sent to CROSS RIVER STATE WATERBOARD LTD. for analysis.
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Constituents of phosphate buffer: 28.39g of morphology of the cells. disodium hydrogen phosphate dissolved in one PLATE 4 shows well–stained erythrocyte liter of distilled water. The pH range is checked € with faint staining of monocyte (M) and using a pH meter.
platelet (P) stained with Leishman stain using
tap water from waterboard as buffer and viewed Microscopy
with x 400 magnification. In this plate, central The stained film was air dried and viewed under pallor is not well defined and the faintly stained the microscope using x10 and x100 oil monocyte could be as a result of the amount of immersion lens. The film was chlorine present in the water used as buffer. photomicrographed using photomicrograph P LATE 5 shows well–stained erythrocyte microscope and the films were compared. €, lymphocyte (L) and platelets (P) stained using
Leishman stain using rainwater as buffer and Results
viewed with x 400 magnification. Appearance of stained blood films
PLATE 6 shows well–stained erythrocyte A well stained thin blood film appeared salmon €, neutrophil (N), and platelet (P) stained using pink.
Leishman stain using fridge water as buffer and Nucleus of the cell appeared purple–violet. viewed with x400 magnification. The Eosinophil granules appeared orange–red. erythrocytes appear crenated and not clearly Neutrophil granules appeared mauve purple. defined. Basophil granules appeared dark blue.
PLATE 7 shows erythrocyte € with Red blood cells appeared Salmon pink.
fluorescence at the center, well–stained Platelets appeared small with violet granules. neutrophil (N), and faintly–stained platelets (P) Lymphocytes and monocytes appeared pale stained using Leishman stain using distilled blue
water as buffer and viewed with x 400
magnification. The erythrocytes are not clearly PLATE 1 shows the film of an apparently defined and they appear crenated. healthy individual stained with leishman stain PLATE 8 shows moderately–stained using phosphate buffer and viewed with x 400 erythrocyte €, lymphocyte (L), and platelet (P) magnification. It shows well stained stained using Leishman stain using air erythrocytes €, eosinophil (EO), neutrophils conditioned water as buffer viewed with x 400 (N), basophils (B), monocytes (M) and magnification. The erythrocytes show crenated lymphocytes (L).
cells and are not well defined. PLATE 2 shows well–stained erythrocyte PLATE 9 shows a sickle cell film stained €, eosinophil (EO), neutrophil (N), and with leishman stain using phosphate buffer and platelets(P) stained with Leishman stain using viewed with x400 magnification. It shows well deionized water as buffer and viewed with x400 stained erythrocytes and sickle red cells. magnification. The morphology of the cells are PLATE 10 shows poorly–stained well defined without any distortion and erythrocyte €, well–stained lymphocyte (L), presence of crenated red cell.
and moderately–stained platelet (P) stained PLATE 3 shows well–stained erythrocyte using Leishman stain using deionized water as €, Lymphocyte (L), and platelets (P) stained buffer viewed with x400 magnification. The red with Leishman stain using tap water from cell morphology is distorted and appear like borehole as buffer and viewed with x400 crystals. magnification. There is no distortion in the
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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.
PLATE 11 shows poorly–stained erythrocyte €, moderately–stained neutrophil (N), and poorly–stained platelet (P) stained using Leishman stain using tap water from borehole as buffer and viewed with x400 magnification. The morphology of the red cells are not well defined.
PLATE 12 shows poorly–stained erythrocyte €, lymphocyte (L), and platelet (P) with numerous artefacts (A) in the background stained using Leishman stain using tap water from waterboard as buffer and viewed with x400 magnification. The red cells are not exactly well defined which could be as a result of the amount of chlorine present in the water.
PLATE 13 shows poorly–stained erythrocyte €, neutrophil (N), and platelet (P) with numerous artefacts (A) in the background stained using Leishman stain using rainwater as buffer and viewed with x400 magnification.
PLATE 14 shows poorly–stained erythrocyte € and neutrophils (N) with numerous artefacts (A) in the background stained using Leishman stain using fridge water as buffer and viewed with x400 magnification.
PLATE 15 shows poorly–stained erythrocyte €, lymphocytes (L) and neutrophil (N) with numerous artefacts (A) in the background stained using Leishman stain using distilled water as buffer and viewed with x400 magnification.
PLATE 16 shows poorly–stained erythrocyte € and lymphocytes (L) with numerous artefacts (A) in the background stained using Leishman stain using air conditioned water and viewed with x400 magnification.
Table 1 and 2 shows the result of water analysis. The table shows that different water sources has different Ph and different chemical constituents.
leishman staining by enhancing staining reaction [1]. It intensifies the stain and helps to attach the dyes methylene blue and eosin to the blood cells.
This study has shown that other water samples as buffer indeed interferes with staining results. Deionized water used as buffer was able to stain the red cells, white cells and platelets very well in a normal film as shown in plate 2, it could not stain the red cells and platelets properly but could barely stain the white cells well in sickle cell film as shown in plate 10.
Tap water from borehole used as buffer was able to stain the red cells, white cells and platelets in a normal film as shown in plate 3, it could not stain the red cells and platelets properly but moderately stained the white blood cells in a sickle cell film as shown in plate 11 whereas Tap water from waterboard used as buffer was able to stain the red cells and platelets well but was not able to stain the white blood cells properly in normal film as shown in plate 4, in sickle cell film, the red cells, white cell and platelets appeared poorly stained as shown in plate 12. The difference in staining properties that were observed in tap, deionized and waterboard water may be attributed to chlorine which is present in waterboard water but absent in both tap and deionized water.
However, Rainwater used as buffer as shown in plate 5 was able to stain the white cells, platelets and red cells very well in a normal film but in plate 13, it poorly stained the red cells, white cells and platelets in sickle cell film. Whereas Fridge water used as buffer stained the white cells and platelets very well, red cells appearing crenated as shown in plate 6 in normal film but in sickle cell film, it stained the red cells, white cells and platelets very poorly as shown in plate 14. The crenation of red cells observed in fridge water could be as a result of the slight acidic pH of the water.
Furthermore, distilled water used as buffer stained white cells well, platelets
Discussion Phosphate buffer acts as mordant during
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appeared faintly stained and it stained the red cells poorly with the cells appearing crenated and with fluorescence at the center as shown in plate 7 in normal film while in sickle cell film, it stained very poorly the red cells, white cells and platelets as shown in plate 15.
On the other hand, air conditioned water used as buffer, moderately stained the red cells, white cells and platelets as shown in plate 8 in normal film but in sickle cell film, the red cells, white cells and platelets are poorly stained as shown in plate 16. The water sources used as buffer could not stain the cells properly because
of its inability to intensify and fix the dyes to the blood cells. Another reason the water sources were not able to stain the blood cells well could be because of their varying pH. In normal blood film, the study shows that some of the water samples such as deionized water, tap water without chlorine could be used as substitutes for phosphate buffer in a situation where it is not available or in areas where it is not easily accessible. In diseased film, the study has shown that it is best to use the recommended phosphate buffer during leishman staining for the production of better results.
TABLE1: COMPONENTS OF DIFFERENT WATER SAMPLES ANALYSED
S/N
PARAMETERS
Units
NIS
2 (Urban
10 Guideline Undergrou
nd
Board Water)
(Rain Water)
(Fridge Drain Water)
(Distilled Water)
(AC Water)
(Deionized Water)
Borehole)
Aluminum
0.00
0.11
0.00
0.12
0.00
0.01
0.00
Ammonia NH3-N
Mg/L
0.05
0.12
0.12
0.16
0.22
1.16
0.21
0.22
Appearance
Calcium Ca
24.0
55.0
10.1
9.80
8.10
10.0
0.00
Mg/L Mg/L
50 100
0.251
2.162
0.112
0.241
0.112
0.121
0.00
Chlorine C1 Coliforms (Feacal) counts/100ml
Cfu
105
300
113
320
74
Coliforms (Total) counts/100ml
Colour
Hu
1.62
1.68
0.92
4.21
0.81
0.85
0.00
Copper Cu
0.12
0.11
0.11
0.15
0.16
0.15
0.00
Mg/L
Mg/L
2.44
2.31
3.20
3.11
3.20
3..20
0.02
Dissolved Oxygen
Electrical coductivity
148.0
504.0
88.0
24.0
24.0
0.00
Fluoride F
0.54
0.44
0.46
0.56
0.54
0.55
0.00
Mg/L Mg/L
1.0 100
10.54
34.1
0.44 68.4
13
17.1
17.1
12.1
Hardness (Total) as Caco,
17.1
Temperature
C
28.0
28.0
28.0
28.0
28.0
28.0
28.0
15
6.5–8.5
3.6
3.9
5.6
5.7
6.2
6.1
6.1
6.7
Ambient 6.5–8.5 200 250
– Mg/L Mg/L
16
28.0 3.6 6.25 8.69
2.61
6.55
2.18
1.82
1.92
pH Phosphate Total Alkalinity as CaCO, Turbidity
1.92
0.00
2.61 8.16
17
250
8.16
9.60
9.62
120
12.21
0.00
18
NTU
5
104
4.16
2.61
17.3
1.88
1.47
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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.
0,80 °•.
Plate 1– showing well–stained erythrocyte €, eosinophil (EO), neutrophil (N), monocytes (M), basophil (B), Lymphocytes (L) and platelets (P) using Phosphate
buffer. Leishman x400 magnification
Plate 2– showing well–stained erythrocyte €, eosinophil (EO), neutrophil (N), and
platelets(P) using deionized water as buffer. Leishman x400 magnification
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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.
Plate 3– showing well–stained erythrocyte €, Lymphocyte (L), and platelets (P) using tapwater gotten from underground borehole as buffer. Leishman x400
magnification
Plate 4– showing well–stained erythrocytet with faint staining of monocyte (M) and platelet (P) using tapwater gotten from waterboard as buffer.
Leishman x400magnification
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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.
Plate 5– showing well–stained erythrocyte €, lymphocyte (L) and platelets
(P) using rainwater as buffer. Leishman x400 magnification
Plate 6–showing well–stained erythrocyte €, neutrophil (N), and platelet
(P) using fridge drain water as buffer. Leishman x400 magnification
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‘Okafor et al/ Comparative analysis of different water samples as buffer in Leishman Staining.
Plate 7–showing erythrocyte € with fluorescence at the center, well–stained neutrophil (N), and faintly–stained platelets (P) using distilled water as
buffer. Leishman x400 magnification
00
Plate 8–showing moderately–stainederythrocyte €, lymphocyte (L), and platelet (P)
using air conditioned water as buffer Leishman x400 magnification
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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.
Plate 9– showing well stained erythrocytes including a sickled red cell and
platelets (P) using phosphate buffer
Plate 10–showing poorly–stained erythrocyte €, well–stained lymphocyte (L), and moderately–stained platelet (P) using deionized water as buffer.
Leishman x400 magnification
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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.
Plate 11–showing poorly–stained erythrocyte €, moderately–stained neutrophil (N), and poorly–stained platelet (P) using tap water from borehole as buffer. Leishman x400 magnification
LV
erous
Plate 12– showing poorly–stained erythrocyte €, lymphocyte (L), and platelet (P) with numerous artefacts (A) in the background using tap water from water board as buffer. Leishman x400 magnification.
Afr J Lab Haem Trans Sci 2022, 1(2): 120 – 134
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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.
Plate 13–showing poorly–stained erythrocyte €, neutrophil (N), and platelet (P) with numerous artefacts (A) in the background using
rainwater as buffer. Leishman x400 magnification.
Plate 14–showing poorly–stained erythrocyte € and neutrophils (N) with numerous artefacts (A)
in the background using fridge water as buffer. Leishman x400 magnification.
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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.
Plate 15–showing poorly–stained erythrocyte €, lymphocytes (L) and neutrophil (N) with numerous artefacts (A) in the background using
distilled water as buffer. Leishman x400 magnification.
Plate 16–showing poorly–stained erythrocyte € and lymphocytes (L) with numerous artefacts (A) in the background using air conditioned water as
buffer. Leishman x400 magnification
WIG
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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.
Acknowledgements We wish to acknowledge the Department of Haematology and Blood Transfusion Science, University of Calabar Teaching Hospital, Calabar for use of their laboratory facility.
Conclusion This study has emphasized that the type of buffer used during staining interferes with the staining procedure and affects the quality of slides produced. The study further shows that deionized and tap water from borehole could be used as substitute buffer for leishman staining in cases where the recommended buffer is not available because they are devoid of minerals such as chlorine in them.
conflict of interest: The authors declare that they have no competing interests.
Funding: There was no external source of funding.
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