European Journal of Cardiovascular Medicine

Peripheral blood film examination plays a vital role in the diagnosis of all haematologic diseases. A well stained peripheral blood film with good morphological details is very essential for an accurate diagnosis from blood films. Hence ensuring the supply of best quality stain is one of the prime concerns of hematology laboratories. Romanowsky stains are compound neutral dyes using a partly polychrome methylene blue in combination with eosin Y or eosin B. The polychroming process gives differentiation ability to the Romanowsky dyes creating a wide range of hues in different types of leukocytes and help in their easy identification in smears.^[1,2,3]

Affinity of a cell or its constituents for a particular stain depends on its chemical nature and the pH of the medium. In appropriate pH 6.8, the acidic structures take up basis dyes (which are therefore, called basophilic) and the basic structures stain with acidic dyes (Therefore called acidophilic).^[4] The exceptional property of the Romanowsky stains of making subtle distinctions in shades of staining granules differentially depends on two components, namely azure B (trimethionine) and eosin Y (tetrabromofluorescein).^[5] Leishman stain is the stain of choice in the staining of blood cells for differential leucocyte count analysis.^[6]

Though many modifications of different Romanowsky stains have been done, only few modifications of Leishman stain have been introduced to reduce the duration of staining of peripheral blood smear. Leishman stain is commonly used in hematology labs to stain peripheral blood films. Conventional Leishman staining method wont be helpful in rapid diagnosis. Modified Leishman stain seeks to optimize the staining of cellular elements of blood by a completely different approach. This method uses the accentuating property of phenol to optimize staining. Phenol was first used as one of the component dyes in Ziehl-Neelsen stain, where it acted as an accentuating agent.

We hypothesized that if phenol optimized staining in Ziehl- Neelsen procedure, it should do the same in Leishman stain. Phenol act by changing the pH of the solution thereby increasing the rate of stain uptake by the tissue.^[7] Phenol also helps in ripening of the Leishman staining solution and thus shortens fixing and staining time. This will help in overall reduction in turnaround time (TAT) of peripheral smear reporting.This study was undertaken to compare the quality of modified Leishman stain. To assess the efficiency of modified Leishman stain by comparing with conventional Leishman stain.

To evaluate and compare the quality of peripheral smears stained by modified Leishman stain and with conventional Leishman stain. To find the appropriate concentration of phenol to Leishman powder ratio in modified leishman stain.

Study was a cross-sectional observational study conducted in clinical pathology laboratory, Department of Pathology for a period of one month (May 2025).

Study Size

N = d (zα+zβ) PQx2/d²

P =P1+P2/2

P1-Acceptable nuclear pattern in conventional Leishman stain is 71.8%^[8]

P2-Acceptable nuclear pattern in modified Leishman stain is 93%

P=82.4

D=21.2

Q=17.6

N=50.3

50 nos in each group

Methodology

A total of 60 EDTA samples from healthy individuals with normal complete blood count were collected from the clinical lab after getting their concent. And from each sample 2 smears was made, one smear was stained by conventional Leishman method and the other one stained by modified Leishman method.

Procedure

For Conventional Method

• Took an air dried smear prepared by manual wedge method and covered the smear with Leishman staining solution, kept for 1-2 minutes.
• Added twice the volume of buffered water (pH 6.8) to dilute the stain.
• Mix the water with stain by gently blowing with a plastic bulb pipette or with a straw. Allowed to stain for 12-15 minutes.
• Washed off the stain with tap water and wiped the back of the slide and kept for drying.

Components of Modified Leishman Stain

Ingredients of modified Leishman stain are,

• Leishman powder
• Absolute methanol
• Phenol crystals or liquefied phenol

Preparation of Modified Leishman Stains with Varying Phenol Concentrations

Phenol crystals were weighed in five different quantities: 30 mg, 37.5 mg, 50 mg, 75 mg, and 150 mg. Each was combined with 150 mg of commercially prepared Leishman powder in separate brown screw-capped bottles, yielding phenol-to-Leishman powder ratios of 1:5, 1:4, 1:3, 1:2, and 1:1, respectively. Each mixture was dissolved in acetone-free methanol and the volume was made up to 100 ml to prepare the modified Leishman staining solutions.

All preparations were carried out at room temperature (18–25°C), and the bottles were protected from direct sunlight throughout the process^[8] . The modified Leishman stains were assessed for quality and staining efficacy three days after preparation.

The blood films were assessed after staining with the modified stain prepared in a 1:3 ratio, which was established as the reference phenol-to-Leishman powder proportion yielding optimal staining performance. Inadequate fixation and improper staining durations led to under-staining or over-staining of the blood films, ultimately resulting in inaccurate differential count results. The optimal fixation and staining times were determined through experimental adjustments of the procedure, which led to the development of the new staining protocol.

Modified Leishman Staining Procedure

1. Initial Staining: The air-dried blood smear was covered with the prepared modified Leishman stain for 1 minute to allow fixation.
2. Dilution: An amount of buffered water twice the volume of the stain was added directly to the slide to dilute the stain.
3. Staining Duration: The mixture was allowed to act on the smear for 1½ minutes to facilitate staining.
4. Washing: The slide was gently rinsed with clean water to remove excess stain.
5. Drying: The back of the slide was wiped clean, and the slide was then placed in a near-horizontal position to air dry completely.

Examination

We examined 60 smears for both conventional and modified Leishman stains was be examined by two pathologists under low power objective(10X) first, then under high power objective (40X) and finally under oil immersion objective(100X) for RBC pattern, nuclear pattern, neutrophil granules, eosinophil granules, platelets and background clarity.

Total Score System

Scoring system assigned based on the morphology of RBC, WBC, granules, platelets, staining quality, shape of blood smear, background clarity, presence of stain deposit. ^[9,10]

Statistical Analysis

Microsoft version 10, EXCEL was used for data entry and SPSS software version 26β for analysis

Statistical Analysis of Staining Quality

The observed p-value for the red blood cell (RBC) staining pattern was 1.00, while that for the white blood cell (WBC) nuclear staining pattern was 0.625. These results indicate no statistically significant difference between the modified and conventional Leishman staining methods in terms of RBC morphology and WBC nuclear staining quality (p > 0.05).

The observed p value is 1.00 in neutrophil granules and 0.791 in Eosinophil granules, both showed no significant difference between modified and conventional method in the aspect of neutrophil and Eosinophil granules.

The observed p value is 1.00 in Platelets and 0.727 in background clarity, there is no significance difference between modified and conventional method in the aspect of platelets in staining and background clarity.

Thus, from these values we can interpret that the observed p value is greater than 0.05 and the modified method is as acceptable as that of the conventional method.

McNemar test and total score system values suggest that Modified Leishman method is acceptable and can be used for peripheral smear staining similar to conventional method.

Leishman stain, belonging to the group of Romanowsky stains, consists of a combination of acidic and basic dyes. The basic dye, which carries a positive charge, stains nucleic acids and basophilic granules, imparting a gray coloration. On the other hand, the acidic dye, being negatively charged, stains hemoglobin and eosinophilic granules with a red-orange hue. In the conventional Leishman staining procedure, both dyes are dissolved in acetone-free methyl alcohol, and the staining process typically requires about 15–20 minutes to complete peripheral blood smear staining. Therefore, the present study was conducted to develop a modified staining technique aimed at reducing the staining time of peripheral blood smears, while maintaining optimal staining quality.

In this pilot study, a total of 60 EDTA samples of healthy individuals were collected which were received in a clinical lab for CBC. And from each sample 2 smears were made, one smear is stained by conventional method and the other one is stained by modified method. A great percentage of smears stained with modified Leishman stain in the ratio 1:3 had shown appropriate nuclear and cytoplasmic staining of neutrophils, eosinophils, and red blood cells. Moreover, we observed that unlike conventional Leishman staining, modified Leishman staining time takes only 2.5 minutes to complete staining, which is an advantage over conventional Leishman stain in case of emergency situations. When compared to conventional which take 2 minutes for fix, here we take only 1 minute due to the accentuating agent present in the Modified Leishman stain and staining take 10-12 minutes in conventional method reduced to 1.5 minutes due to the acceleration activity of accentuators in staining.

In the study done by Fasakin et al^[11] he concluded that the 1:5 (overall staining time : 75 seconds) and 1:3 (4 minutes) ratio of modified Leishman stain gave better morphology than the conventional Leishman stain.(table 6) In our study the 1:5 ratio the leukocyte granules were not well appreciable and the staining procedure also different.

Our study was in comparison to done by Premkumar et al^[12] in which he compared Modified Leishman stain similar to our that 1:3 ratio which gave similar result in leukocytes, erythrocytes, and platelet staining(table 4, 5 & 6) Our study also gave similar results.

Rizwana Abdul Hye et al^[8] use a scoring system and quality index to assess the efficacy of Modified Leishman stain which was similar to our study. The parameters evaluated included RBC morphology, nuclear pattern, neutrophil granules (Fig. 2), eosinophil granules, platelets, and background staining quality. Eosinophil granules were clearly visualized and demonstrated better differentiation from neutrophil granules compared to the conventional method. The nuclear pattern was also more distinctly appreciated with the modified staining technique. Additionally, RBCs exhibited superior staining with well-defined central pallor. Platelets were more distinctly visible (Fig. 1), and the background staining quality was optimal, further supporting the effectiveness of the modified method.

Atchyuta Mathi, IV Renuka and Santhi Imandi in 2022,^[13] which was conducted in the department of clinical Hematology laboratory of a tertiary care Hospital, South India. They statistically concluded that modified Leishman stain prepared by adding phenol to conventional Leishman stain can giving better staining results in 4 minutes unlike the conventional Leishman stain which takes 15-20 minutes of time for staining peripheral blood smears. They concluded that phenol to conventional Leishman stain preferably at a concentration of 1:5 and can be use for staining the peripheral blood smears thereby reducing the turnaround time. They took 4 minutes to complete staining. In our study 1:3 at 2.5 minutes give optimal results.

Our study on the topic “A comparative study between conventional and modified Leishman stain” shows that there is no statistically significant difference between modified Leishman staining and conventional Leishman staining. It was observed that most of the smears stained with modified Leishman stain with the ratio of 1:3 have given appropriate stain of RBCs,platelets, nuclear and cytoplasmic staining of neutrophils and eosinophils. And the overall staining quality of the Modified Leishman stain was satisfactory. More over modified Leishman stain have an advantage over conventional Leishman stain that is, because phenol in the modified Leishman stain is an accentuating agent. It takes only a few minutes to complete staining. Hence this method is applicable in emergency hematological cases where rapid diagnosis with peripheral smear examination is highly essential.

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