Comparison between Giemsa, Harris Hematoxline & Eosin and Lieshman Stain in Peripheral Blood Picture

  

    
Hematoxline
    
2.5 g
  
  

    
Absolute alcohol
    
25 ml
  
  

    
Potassium alum
    
50 g
  
  

    
Distilled water
    
500 ml
  
  

    
Mercuric oxide
    
1.25 g or
  
  

    
Sodium iodide
    
0.5 g
  
  

    
Glacial acetic acid
    
20 ml
  

Table 1: Preparation of solution.

Preparation of solution: The alum which has previously been dissolved in the warm distilled water in a 2-liter ?ask. The mixture is rapidly brought to the boil and the mercuric oxide the hematoxline is dissolved in the absolute alcohol, and is then added to de or sodium iodide is then slowly and carefully added. Plunging the ?ask into cold water or into a sink containing chipped ice rapidly cools the stain. When the solution is cold, the acetic acid is added, and the stain is ready for immediate use. The glacial acetic acid is optional but its inclusion gives more precise and selective staining of nuclei. As with most of the chemically ripened alum hematoxline, the quality of the nuclear staining begins to deteriorate after a few months. This deterioration is marked by the formation of a precipitate in the stored stain at this stage the stain should be filtered before use, and the staining time may need to be increased. For the best results, it is wise to prepare a fresh batch of stain every month, although this may be uneconomical unless only small quantities are prepared each time [9].

Eosin stock solution

1. Eosin Y 1 g.
2. Distilled water, 100 ml.
3. Mix to dissolve.

Application: Harris hematoxline solution is widely used in the areas of histology and cytology as a component of hematoxline-eosin staining. Basically, it reveals the nuclear structure in both nucleoproteins and nucleic acids in samples of human tissue.

Principle: Hematoxline itself is a compound without staining properties. It is the product of its oxidation, haematein, which lends coloring properties to the solution. Oxidation can be produced either spontaneously, or by inducement. The first option is slow because it involves natural oxidation through air and light The second, rapid or instantaneous method, involves the use of an oxidizing agent such as mercury or iodine, however, this reduces the durability of the stain because the haematein may also be oxidized. Therefore, the hematoxline solutions where oxidization is induced are less durable, but they do not require a maturation process in order to achieve adequate results; they can be used practically an instant after their preparation. In order to reduce the problems caused by using an oxidizing agent, increase the concentration of ethanol and decrease the pH of the solution. Both strategies were adopted in the original formulation of Harris Hematoxline solution. In some cases these modifications are insufficient to preserve the coloring activity after dying many samples, probably because the oxidizing agent has worn out the haematein and because of the elevated number of samples which require dying.

Main advantages

Better Formulation

1. Better contrast and more color fixing power.
2. Increased capacity to process a higher number of samples.

Mercury free

1. Contributes to the preservation of the environment.
2. Safer for the user [10].

Peripheral smear procedure

Principle: When automated differentials do not meet specified criteria programmed into the automated hematology instrument, the technologist/technician must perform manual differential count from a prepared smear. There are two types of blood smears: the wedge smear and the spun smear. The wedge smear will be discussed in this procedure. Smears are prepared by placing a drop of blood on a clean glass slide and spreading the drop using another glass slide at an angle. The slide is then stained and observed microscopically. A well-stained peripheral smear will show the red cell background as red orange. White cells will appear with blue purple nuclei with red purple granules throughout the cytoplasm. A well made, well distributed peripheral smear will have a counting area at the thin portion of the wedge smear which is approximately 200 red cells not touching. A good counting area is an essential ingredient in a peripheral smear for evaluating the numbers of and types of white cells present and evaluating red cell and platelet morphology.

Reagents and equipment

1. Glass slides (frosted)
2. Wooden applicator sticks
3. DIFF-SAFE (an apparatus designed to avoid removing the tube top)

Specimen collection and storage

1. EDTA specimen or EDTA Microtainer.
2. Smears are made from EDTA.
3. Microtainers within 1 hour of collection.
4. EDTA blood within 2 to 3 hours.
5. Check all Microtainers for clots with applicator sticks.

Quality control: A random slide is picked after it has been stained and technologist/technician checks the quality of the stain for the WBCs and RBCs, platelets, and the distribution of cells [11].

Procedure

1. Insert the DIFF-SAFE dispenser through this topper of the tube held in an upright position.
2. Turn the tube upside down and apply pressure at the frosted end of the slide. When the drop of blood appears, discontinue pressure.
3. Using a second slide (spreader slide), place the edge of the second slide against the surface of the slide at an angle between 30 and 45 degrees.
4. Bring the spreader slide back into the blood drop until contact is made with the drop of blood
5. Move the spreader slide forward on the slide, so a smear is made approximately 3 to 4cm in length. The smear should be half the size of the slide, with no ridges, and a “feather edge “should be toward the end of the smear.
6. Label the frosted end of the slide with the patient’s last name and first initial, specimen number, and the date.
7. Allow the smear to air dry completely.
8. Proceed with staining. Manual Wright staining is not found often in the clinical laboratory setting. Most clinical laboratories have an automated.

Staining instrument attached to their automated CBC analyzer. If there is no automated stainer attached to the analyzer, there still is a separate staining instrument [11].

Staining the blood film: Prior to staining, cells must be fixed to the glass slide with acetone-free methanol, either alone or in solution with dye. Addition of a buffer solution to the dye changes the pH of the solution and ionizes the reactants to initiate the pH-dependent staining process. Acidic cellular elements such as nucleoproteins, nucleic acids and primitive cytoplasmic proteins, react with the basic dyes, Methylene blue and its oxidative products. These elements are basophilic and stain variations of blue. Basic cellular elements such as hemoglobin molecules and some cytoplasmic constituents in leukocytes, have an affinity for the acidic dye, eosin. These elements are acidophilic and stain orange-red. A neutrophil has neutral staining characteristics and stains blended shades of purple or pink, representing combinations of acidic and basic molecular groups. Azure dyes stain the primary or non-specific granules in most myeloid cells red-purple, hence, the term azurophilic granules [12].

Manual staining methods

Dip method (Rapid): The dip method is a quick staining method that uses a modified Wright-Giemsa stain buffered in methanol at pH 6.8. Slides are immersed in the stain in a coplin jar for a user-determined length of time. It is important that the Wright-Giemsa stain be kept tightly sealed in coplin jar when not in use and be replaced when water artifact appears in red cells (see: Causes and Corrections of Stain Deviations).

Note: Staining time may be increased for greater cellular detail as required.

Automated staining methods

Hema-Tek slide stainer - Miles scientific (platen-type stainer): The Hema-Tek slide stainer is a self-contained bench top slide stainer that uses a Hema-Tek Stain Pak. Three sensing switches are triggered sequentially to activate three solution pumps which deliver metered volumes of stain, buffer and rinse solution from the stain pack. A Wright-Giemsa Pak is most commonly used. Slides are advanced by two parallel conveyer spirals with the stain, buffer and rinse solutions pumped up between the blood film and platen.

Note:

1. Slides can be randomly added if required
2. the staining process takes approximately ten minutes
3. Time phases are constant
4. Pump volumes are user adjusted by control knobs
5. a stain/buffer ratio of 1:2 is desirable
6. Cleaning procedures and daily maintenance are vitally important to prevent stain deposit artifact

Hemastainer automatic slide stainer - Miles scientific (dip-type stainer): This is an automated staining instrument that stains up to fifty slides at one time. The slides are loaded into a slide basket and dipped into each of six stations in the staining process. Five individual, adjustable timers control the first five stations. The six stations consist of: methanol, stain, a second stain, buffered-water rinse, a phosphate buffer rinse and lastly, forced-air drying. The two stains commonly used in the stainer are May-Grunewald and diluted Giemsa.

Midas II automatic slides stainer- EM Diagnostic (dip-type stainer): This instrument is a completely automatic slide stainer that stains up to twenty slides at one time. The slides are loaded into a bucket and are cycled through six stations: methanol, stain, a second stain, water rinse, phosphate buffer rinse, followed by the drying station. The first five stations are on individual, adjustable timers. The Midas II stainer is similar in operation to the Hemastainer.

Note:

1. Slides cannot be added once the staining process is started.
2. The staining process takes approximately twenty minutes.
3. Produces good quality, reproducible staining [13].

Previous study

Giemsa stain: Clarke Dr. Gwendolyn - prephral Blood film: staining cell estimation and reviow, CPSAALQEP.MAY.2003 find that Red blood cells Salmon pink Nuclei of neutrophils Deep blue-purple Specific granules of neutrophils, granules of Light purple or violet lymphocytes, granules of platelets. Specific granules of Basophils Deep purple Specific granules of Eosinophils Orange Cytoplasm of lymphocytes Blue Cytoplasm of Monocytes Blue-grey (ground glass appearance).

Ramadas Nayak, Sharada Rai, Astha Gupta, Essentials in Hematology and clinical pathology first Edition 2012. Find that: The red blood cell was stained by red color; the white blood cytoplasmic granule was stained as following: Neutrophils are stained by fine purple cytoplasmic granule. Basophile was stained by dark stained granule. Monocyte was stained pale grayish blue cytoplasm. Lymphocyte was stained by pale blue cytoplasm and nuclear is course. The platelet was stained by purple color.

Dacia, Sir J.V., Lewis, S.M. Practical Haematology 7^th edition, pages 77 to 81, Churchill Livingstone, 1991 find that: Stain blood film by Romano sky stain the red blood cell was stained by dark pink color, the white blood cytoplasmic granule was stained as following: lymphocyte stained blue color, monocyte stained by gray –blue color, neutrophil stained by pink-orange and purple granule, basophile stained by blue color and purple black granule, eosinophil stained by red color and orange granule. platelet was stained by purple color.

Lieshman stain: In Version: MM2/130108 find that: Red blood cell was stained by: light pink to brown Cores. lymphocyte was stained by: deep, dark blue to blue-violet Cytoplasm of lymphocytes: light blue Nuclei of neutrophil, polymorph nuclear leukocytes: a deep blue to blue-violet Granules of neutrophilic polymorph nuclear leukocytes: red Cores of eosinophil leukocytes: blue violet Granules of eosinophilic leukocytes: deep red Cores of basophilic leukocytes: blue violet.

Hematoxline and eosin stain: No previous study for uses this stain in hematological lab. This stain use as routinely in histopathology lab. We modified it and use in hematological lab for staining peripheral blood picture that give reliable result.

Materials and Methods

Study design

Descriptive study

Study area

The study was conducted in kosti town -while Nile state, kosti is 315 km from Khartoum to south of Sudan state area in 39701km and population about 159500 [2008].

Study population

The target population of this study volunteer of Medical student in university of elemam almahdi.

Study duration

From September to November /2017.

Inclusion criteria

Healthy individual without any hematological disorder.

Exclusion criteria

Any individual suffer from hematological disorder.

Ethical approval

Collecting sample is done by agreement of individual.

Sampling

Amount of sample we take 2ml from each health individual in EDTA anticoagulant.

Sample size

Sample collected from 50 health’s individual.

Lab diagnosis

Giemsastain

Procedure

Giemsa May-Grunewald:

1. Dilute Giemsa Stain 1:20 with deionized water. For bluer coloration, water buffered at pH 7.2 may be used in place of deionized water.
2. Place slides in May-Grunewald Stain for 5 minutes.
3. Place slides in Working Phosphate Buffer or Trizma® Buffer (20-70 mol/L). PH 7.2, for 1.5 minutes.
4. Place slides in dilute Giemsa solution from step 1 for 15-20 minutes.
5. Rinse slides BRIEFLY in DEIONIZED water.
6. Air dry and evaluate.

Standard giemsa

1. Fix slides in methanol 5-7 minutes.
2. Air dry.
3. Dilute Giemsa Stain 1:20 with deionized water. Color can be varied by diluting in buffer.
4. Stain film for 15-60 minutes.
5. Rinse in deionized water.
6. Air dry and evaluate.

Quick stain giemsa

1. Place air dried blood film in undiluted Giemsa Stain for 1-2 minutes.
2. Place in deionized water for 2-4 minutes depending upon color preference.
3. Rinse in deionized water.
4. Air dry and evaluate.

Performance characteristics

Nuclei will be varying shades of purple. Cytoplasmic staining will be varying shades of blue to light pink. Fine reddish to lilac granules may be present in cytoplasm of some cell types. Basophiles will demonstrate dark blue black granules in the cytoplasm. Eosinophils will demonstrate bright orange granules in the cytoplasm. Red blood cells should be pink to orange. If observed results vary from expected results, please contact Sigma-Aldrich Technical Service for assistance [12].

Harris’ Hematoxline

Staining Procedure:

1. Deparaffinize sections, 2 changes of xylem, 10 minutes each.
2. Re-hydrate in 2 changes of absolute alcohol, 5 minutes each.
3. 95% alcohol for 2 minutes and 70% alcohol for 2 minutes.
4. Wash briefly in distilled water.
5. Stain in Harris hematoxylin solution for 8 minutes.
6. Wash in running tap water for 5 minutes.
7. Differentiate in 1% acid alcohol for 30 seconds.
8. Wash running tap water for 1 minute.
9. Bluing in 0.2% ammonia water or saturated lithium carbonate solution for 30 seconds to 1 minute.
10. Wash in running tap water for 5 minutes.
11. Rinse in 95% alcohol, 10 dips.
12. Counter stain in eosin-phloxine solution for 30 seconds to 1 minute.
13. Dehydrate through 95% alcohol, 2 changes of absolute alcohol, and 5 minutes each.
14. Clear in 2 changes of xylem, 5 minutes each.
15. Mount with xylem based mounting medium.

Results Nuclei should be blue, cytoplasm pink to red [14-16].

This histological method is causes red cells destruction

Method of staining thin blood film

1. Fixed thin blood film by absolute methanol.
2. Covered blood film by Harrishematoxline 5 min.
3. Washed by DW.
4. Add Eosin for 10 second.
5. Wash by DW.

Leishman’s Stain

Example Staining Protocol:

Blood staining according to Lieshman (covering technique):

1. Use smears that are as thin as possible and air-dried.
2. Fully cover the smears with Leishman’s Stain solution. Stain for 2 minutes.
3. Add twice the amount of distilled water and mix by swirling. Incubate for at least 10 min
4. Rinse thoroughly with distilled water.
5. Dry the slides using blotting paper and air-dry.
6. (Optional) for fixing, include the slides in balsam. (Canada balsam genuine AppliChem product No.A0569) or neutral Malinol [6].

Equipment

1. Glass slides 75.25.
2. Spreader slides.
3. baster pupate.
4. Anticoagulant EDTA.
7. ligh microscope.

Data analysis

Done by spass program

Data presentation

Result presented as tables.

Results

This was descriptive study was performed in EL-Imam El-mahdi University Faculty of medical laboratory science 50 blood sample was collected from healthy volunteer student in EDTA container. Thin blood film was made from this samples (three films) from each samples and stained with Giemsa, lieshman and H&E the comment of this films as the following. In all types of stain the morphology WBCs, RBCs, and platelet was normal. The different in color of RBCs and cytoplasmic granule of WBCs. In Lieshman stain RBCs was stained by [pale red color]. In Giemsa stain RBCs was stained by [pale gray color] and In hematoxline stain RBCs was stained by [strong red color]. In Lieshman stain RBCs was stained by [pale red color]. In Giemsa stain RBCs was stained by [pale gray color] and in hematoxline stains RBCs was stained by [strong red color].

In hematoxline and eosin stain the white blood cell cytoplasmic granules was stained as the following: neutrophils is stained by fine pink, with light purple or violate color of nucleus, lymphocyte: blue cytoplasm with light purple color nucleus. Eosinophils: course red granules. Monocyte blue: gray cytoplasm with clear vacuolated nucleus.

In Giemsa and lieshman stain white blood cell cytoplasmic granule was stained as the following: neutrophil is stained by fine purple with violate color of nucleus, lymphocyte: stain pale blue cytoplasm with condensed nuclear chromatin. Eosinophil: orange –red cytoplasmic granule. Monocyte: stained by blue–gray cytoplasm with clear nucleus.

Platelet in [giemsa stain is stained by purple color] in Lieshman stain platelet was stained by [purple –blue color] and in hematoxline & Eosin was stained by [coarse red color]. The type of stain is differing in WBCs cytoplasmic granule, deposit and background of stain.

In Giemsa stain the cytoplasmic granule from 50 blood film 35 is valid appear that represented about 70% while 15 is not appear that represented about 30% from total blood film, the background 40 blood film is valid clear that represented about 80%, while the 10 film is not clear that represented about 20%. And deposit of stain 40 blood film is absent from deposit that represented about 80%, while the 10 blood film present of deposit that represented about 20% from total blood film.

In Lieshman stain the cytoplasm granule from 50 blood film 33 is valid appear that represented about 66%, while the 17 blood film is not appear that represented about 33% from total blood film. The background 32 blood film is valid clear that represented 64% while the 18 is not clear that represented about 34% from total blood film. and deposit of stain 33 blood film is absent from deposit that represented about 66%, while the 17 blood film is present of deposited of stain that represented about 34% from total blood film.

 In hematoxline & Eosin stain the cytoplasm granule from 50 blood film 50 is valid clear that represented about 100%. The background 50 blood film is valid clear that represented about 100% from total blood film. The deposit of stain 50 blood film is absent of deposit of stain that represented about 100% from total blood film (Tables 2-5) (Appendixes).

Table 2: White blood cell cytoplasmic granules: Out of 50 films.

  

    
 
    
Giemsa
    
Lieshman
    
H&E
  
  

    
Clear
    
35 (70%)
    
33 (66%)
    
50 (100%)
  
  

    
Not clear
    
15 (30%)
    
17 (34%)
    
0 (0%)
  

Table 2: White blood cell cytoplasmic granules: Out of 50 films.

Close

Table 3: Deposit of Stain: Out of 50 films.

  

    
 
    
Giemsa
    
Lieshman
    
H&E
  
  

    
Absent
    
35 (70%)
    
33 (66%)
    
50 (100%)
  
  

    
Present
    
15 (30%)
    
17 (34%)
    
0 (0%)
  

Table 3: Deposit of Stain: Out of 50 films.

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Table 4: Background: Out of 50 films.

  

    
 
    
Giemsa
    
Lieshman
    
H&E
  
  

    
Clear
    
35 (70%)
    
32 (64%)
    
50 (100%)
  
  

    
Not clear
    
15 (30%)
    
18 (36%)
    
0 (0%)
  

Table 4: Background: Out of 50 films.

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Table 5: Time consumes & Cost: In about 30 ml of each stain.

  

    
 
    
Giemsa
    
Lieshman
    
H&E
  
  

    
Time consumes
    
10 minutes
    
10 minutes
    
6 minutes
  
  

    
Cost
    
3.5 SDG
    
19.5SDG
    
40SDG
  

Table 5: Time consumes & Cost: In about 30 ml of each stain.

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Discussion

In this descriptive comparative study 50 blood samples were collected from volunteer healthy student thin blood film was made from this sample (three films) from each samples and stained with Giemsa, lieshman and H&E the comment of this films as the following in all type of stain the morphology WBCs, RBCs, and platelet was normal. the different in color of RBCs and cytoplasmic granule of WBCs.

In Lieshman stain RBCs was stained by [pale red color]. In Giemsa stain RBCs was stained by [pale gray color] and in hematoxline stains RBCs was stained by [strong red color].

In hematoxline and eosin stain the white blood cell cytoplasmic granules was stained as the following: neutrophils is stained by fine pink, with light purple or violate color of nucleus, lymphocyte: blue cytoplasm with light purple color nucleus. Eosinophils: course red granules. Monocyte blue: gray cytoplasm with clear vacuolated nucleus.

In Giemsa and lieshman stain white blood cell cytoplasmic granule was stained as the following: neutrophil is stained by fine purple with violate color of nucleus, lymphocyte: stain pale blue cytoplasm with condensed nuclear chromatin. Eosinophil: orange –red cytoplasmic granule. Monocyte: stained by blue–gray cytoplasm with clear nucleus.

Platelet in [Giemsa stain is stained by purple color] in Lieshman stain platelet was stained by [purple –blue color]and in hematoxline & Eosin was stained by [coarse red color]. The type of stain is differing in WBCs cytoplasmic granule, deposit and background of stain.

In Giemsa stain the cytoplasmic granule from 50 blood film 35 is valid appear that represented about 70% while 15 is not appear that represented about 30% from total blood film, the background 40 blood film is valid clear that represented about 80%, while the 10 film is not clear that represented about 20%. And deposit of stain 40 blood film is absent from deposit that represented about 80%, while the 10 blood film present of deposit that represented about 20% from total blood film.

In Lieshman stain the cytoplasmic granule from 50 blood film 33 is valid appear that represented about 66%, while the 17 blood film is not appear that represented about 33%from total blood film.

The background 32 blood film is valid clear that represented 64% while the 18 is not clear that represented about 34%from total blood film. and deposit of stain33 blood film is absent from deposits that represented about 66%,while the 17 blood film is present of deposited of stain that represented about 34% from total blood film. In hematoxline & Eosin stain the cytoplasmic granule from 50 blood film 50 is valid clear that represented about 100%. The background 50 blood film is valid clear that represented about 100% from total blood film. The deposit of stain 50 blood film is absent of deposit of stain that represented about 100% from total blood film.

In most of this study agreement of Clarke, Dr. Gwendolyn, the Peripheral Blood Film: Staining, Cell Estimation and Review, CPSA ALQEP: May, 2003.

Clarke, Dr. Gwendolyn, the Peripheral Blood Film: Staining, Cell Estimation and Review, CPSA ALQEP: May, 2003. Red blood cells Salmon pink Nuclei of neutrophils Deep blue-purple Specific granules of neutrophils, granules of Light purple or violet lymphocytes, granules of platelets. Specific granules of Basophile Deep purple Specific granules of Eosinophils Orange Cytoplasm of lymphocytes Blue Cytoplasm of Monocyte Blue-grey (ground glass appearance)

Cytoplasm of neutrophils Light pink Cytoplasm of platelets Purple-blue to lilac. This was descriptive study was performed in EL-Imam El-Mahdi University Faculty of medical laboratory science 50 blood sample was collected from healthy volunteer student in EDTA container.

Ramadas Nayak, Sharada Rai, Astha Gupta first Edition 2012, in this study agree with me, the red blood cell was stained by red color; the white blood cytoplasmic granule was stained as following:

Neutrophils are stained by fine purple cytoplasmic granule. Basophile was stained by dark stained granule. Monocyte was stained pale grayish blue cytoplasm. Lymphocyte was stained by pale blue cytoplasm and nuclear is course. The platelet was stained by purple color.

Barbara j Bain, Imelda Bates, Michael A Laffan and S.Mitchell Lewis eleventh Edition 2011 agree with me that stain blood film by romanowisky stain the red blood cell was stained by dark pink color, the white blood cytoplasmic granule was stained as following: lymphocyte stained blue color, monocyte stained by gray –blue color, neutrophil stained by pink-orange and purple granule, basophile stained by blue color and purple black granule, eosinophil stained by red color and orange granule. platelet was stained by purple color.

Leishman's Eosin-Ethylene blue Product No. A4277 agree with me the red blood cell was stained by light pink to brown Cores color, lymphocytes was stained by: deep, dark blue to blue-violet Cytoplasm of lymphocytes was stained by: light blue Nuclei of neutrophil, polymorph nuclear leukocytes: a deep blue to blue-violet, Granules of neutrophilic polymorph nuclear leukocytes was stained by: red Cores of eosinophil leukocytes was stained by: blue violet Granules of eosinophilic leukocytes was stained by deep red Cores of basophilic leukocytes was stained by: blue violet color.

Conclusion

The study concluded that the hematoxline and eosin are found to obtain reliable result for staining peripheral blood picture that compared with giemsa and lieshman stain by modified method and NEW. And we improved that Harris hematoxline is best stain with all situation like general appearance and cell color and morphology and background and present of deposit. On the other hand the best stain used for deferential count and estimated cell morphology.

Recommendations

1. More studies should be done.
2. More modification for staining method of H&E.

Examine the possibility of addition of eosin to harris hematoxline to make one step staining method.

References

1. Clarke Dr. Gwendolyn-prephral Blood film: staining cell estimation and reviow, CPSAALQEP. MAY. 2003.
2. Romanowsky DL, St. Petersburg Med. Wshr. 1891; 16: 297-302.
3. Version: MM2/130108.
4. Carson Freida L, Christa Hladik Cappellano. Histotechnology: A Self-instructional Text. 4th ed. Chicago: ASCP Press. 2015; 113-114.
5. Sheehan Dezna C, Barbara B. Hrapchak. Theory and Practice of Histotechnology. St. Louis: Mosby. 1980. 142-144, 153-154.
6. Dacie JV, Lewis SM. Practical Haematology, Churchill Livingstone. 1991; 54-79.
7. Leishman WB. Note on a simple and rapid method of producing Romanowsky staining in malarial and other blood films. Br Med J. 1901; 2: 757-758.
8. Modifications developed by Newcomer Supply Laboratory.
9. MOHAMMED AE. Routine Histological Techniques. editions one. 76.
10. Bancroft John D, Marilyn Gamble. Theory and Practice of Histological Techniques. 6^th ed. Oxford: Churchill Livingstone Elsevier. 2008: 123-125.
11. PanReac Química SLU Garraf, 2Polígono Pla de la Bruguera E-08211 Castellar del Vallès Barcelona. Spain.
12. Hematology in Practice Betty Ciesla, MS, MT (ASCP) SH Faculty, Medical Technology Program Morgan State University Baltimore, Maryland Assistant Professor Villa Julie CollegeStevenson, Maryland.
13. Hematology: Principles and Procedures, Sixth Edition, Brown AB, Lea & Febiger, Philadelphia.
14. O’Connor, Barbara H, A Color Atlas and Instruction Manual of Peripheral Cell Morphology. 1984: 15-18.
15. Ramadas Nayak, Sharada Rai, Astha Gupta, Essentials in Hematology and clinical pathology first Edition. 2012.
16. Kiernan JA. Histological and Histochemical Methods: Theory and Practice. 4th ed. Bloxham, UK: Scion. 2008.

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Citation: Ali AS, Dafaalla AAEA and Eltyeb Babeker YM. Comparison between Giemsa, Harris Hematoxline & Eosin and Lieshman Stain in Peripheral Blood Picture. J Blood Disord. 2018; 5(1): 1051. ISSN 2379-8009

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