What Does Pathology Review in a Giemsa Stain
Islamic republic of iran J Vet Res. 2019 Spring; 20(2): 147–150.
Introducing a combined Leishman-Giemsa stain as a new staining technique for avian blood smears
A. Akhlaghi
1BSc Student in Veterinary Laboratory Science, Kinesthesia of Veterinary Medicine, Semnan University, Semnan, Iran;
Grand. Ahmadi-Hamedani
2Department of Clinical Sciences, Faculty of Veterinary Medicine, Semnan University, Semnan, Iran
Received 2018 Feb 12; Revised 2018 Sep 5; Accepted 2018 Nov xiv.
Abstruse
Background:
The use of conventional stains including Giemsa, Wright and Leishman have become an essential tool for differential diagnosis of cells in peripheral claret.
Aims:
The aim of the study was to develop a new combination of Leishman-Giemsa (L&G) stain for avian blood smears and to compare its efficacy with conventional staining methods.
Methods:
3 sets of peripheral blood smears, ane smear for L&Thousand stain and two other smears for Leishman and Giemsa stains, created from 50 broiler chickens blood samples. All the three sets of slides were blind screened by two adept clinical pathologists and scored based on the staining characteristics (4 parameters) such as nuclear features of blood-red blood jail cell (RBC) and white blood cell (WBC), cytoplasmic features and cytoplasmic granularity of WBC. The boilerplate grading score assigned by ii experts for each staining method were compared.
Results:
The average grading score of two conventional Leishman and Giemsa staining methods were significantly lower (P<0.001) than new L&One thousand staining method in avian nuclear features of the RBC and WBC. The L&G stain gave a ameliorate clarity of nuclear features of avian RBC and WBC. The new L&G staining technique created significant differences (P<0.001) in cytoplasmic features of avian WBC compared to the other two methods.
Conclusions:
For the first time, the results of the present study showed that the avian claret cells are more desirable stained with a new combination of L&G stain. In improver, it gives a meliorate nuclear and cytoplasmic differential staining than the conventional Giemsa and Leishman stains when used lonely.
Key Words: Avian, Blood smear, Leishman and Giemsa stain
Introduction
The peripheral blood and bone marrow smear exam for differential diagnosis of cells are associated with the staining characteristic and correct noesis of the different blood cells morphology. Study of stained blood smear is considered as ane of the most important methods in hematology analysis. A well-stained claret smear helps in the diagnosis of the avian leukocytes. Precise assessment of the morphological characteristics of the granulocytes depends on an adequately prepared blood motion picture (Robertson and Maxwell, 1990 ▶). Most Romanowsky stains include Wright stain, Giemsa stain, Wright-Giemsa stain, Leishman stain, Wright-Leishman stain, May-Grunwald stain and May-Grunwald-Giemsa stain commonly used for staining avian blood films as well equally human and mammalian blood pic staining (Garbyal et al., 2006 ▶). In the literature it has been proven that Romanovsky stains give a ameliorate clarity of nucleus and cytoplasm characteristics. In general, Romanovsky stains contain two components that include bluish azure and eosin Y. Blue azure tends to nucleic acids and the nuclear and cytoplasmic proteins, although eosin Y connects to the basic groups of hemoglobin (Kass et al., 2002 ▶). Leishman-Giemsa (L&Grand) cocktail as a relatively new technique has been previously used for cytology smear so far only one study has shown the benefits of this method for staining of human peripheral claret/os marrow smears (Belgaumi et al., 2013 ▶; Gajendra et al., 201 ▶v; Suryalakshmi et al., 2016 ▶; Doddagowda et al., 2017 ▶). It has been shown that a new modification of Wright-Giemsa stain gives results similar to the standard Wright-Giemsa stain and can reduce the staining time (Teerasaksilp et al., 2005 ▶; Kondo et al., 2011 ▶, 2012; Nakada et al., 2014). We developed for the outset fourth dimension a new combination of L&G stain for avian blood smears and compared the efficacy of this staining technique with conventional staining methods.
Materials and Methods
The nowadays written report was carried out in the Veterinarian Hospital of Semnan University from September 2017 to November 2017. Fifty 30-24-hour interval-onetime healthy Ross chickens without hematological abnormalities were included in the study. Iii sets of peripheral claret smear, one smear for L&G stain and the other two smears for Leishman and Giemsa stains, were created from 50 broiler chicken blood samples. The slides were bullheaded reviewed by two practiced haematopathologists who evaluated and scored all 3 sets of slides, based on the staining characteristics such every bit the nuclear features, cytoplasmic features, caste of granularity of the cytoplasm and other morphological ruddy claret prison cell (RBC) and white blood cell (WBC) characteristics every bit follows:
1- Staining criteria of mature bird RBC include: yellowish orangish ovalocyte with a tinge of blue
2- Staining criteria of nucleus of mature WBC include: purplish blueish, appropriate nucleus morphology according to the different WBC types
3- Staining criteria of cytoplasm of granulocyte, lymphocyte and monocyte include: pale pink, sky blue and gray blue, respectively
iv- Staining criteria of granules of heterophiles and eosinophils include: rod or speculated dark orange to brown-ruby-red shaped and circular or oval orange to carmine shaped, respectively
The slides were compared by giving scores for staining criteria of four parameters of each staining methods equally, score 0 (worst) to score 4 (best). The derived data were collected and compiled for further statistical analysis to compare the staining efficacy of each method. The average grading score for each staining method from the two experts was compared to test the interpersonal variability, and analysis of variance (ANOVA) was used to assess the difference among the average grading scores by each expert (Table ane).
Table 1
Comparing of average grading scores of four parameters for three staining methods
| Parameters | Leishman stain (A) | Giemsa stain (B) | Leishman-Giemsa stain (C) |
|---|---|---|---|
| Grading (mean±SD) | |||
| Nuclear features of the RBC | 3.27 ± 0.25 | 3.17 ± 0.24 | 3.seventy ± 0.25 **, ## |
| Nuclear features of the WBC | three.32 ± 0.24 | 3.15 ± 0.23 | 3.77 ± 0.25 **, ## |
| Cytoplasm features of the WBC | 2.88 ± 0.36 | 3.28 ± 0.37 | 3.74 ± 0.29 **, ## |
| Granules of the WBCs | three.08 ± 0.24 | 2.96 ± 0.24 | 3.80 ± 0.24 **, ## |
1- Staining procedures of slides with Leishman stain:
The smear was covered with Leishman stain for 2 min. Double volume of Sorensen buffer (pH=half-dozen.8) was added to the slide and mixed gently for fifteen min. Washed off with running water and dried (5 min) (Gajendra et al., 2015 ▶).
ii- Staining procedures of slides with Giemsa stain:
The smear was covered with absolute methyl alcohol for 5 min (fixation). The alcohol was drained off and the slide was covered with freshly diluted Giemsa stain (1:10 with Sorensen buffer, pH=half dozen.8) for 20 min. Done off with running water and dried (5 min) (Gajendra et al., 2015 ▶).
three- Staining procedures of slides with Leishman and Giemsa stain:
The slide was covered with Leishman stain for 2 min. The slide was washed in running water. Fresh diluted Giemsa stain (1:x with Sorensen buffer, pH=half-dozen.viii) was poured for fifteen min. Washed off with running water and dried for five min (Gajendra et al., 2015 ▶).
Results
The comparison of average grading scores of four parameters such equally nuclear features of RBC, nuclear and cytoplasmic features of WBC and degree of granularity of the cytoplasm among three staining methods was presented in Table 1. The boilerplate grading score of two conventional Leishman and Giemsa staining methods were significantly lower (P<0.001) than new 50&K staining method in avian nuclear features of the RBC and WBC (Table ane). The L&G stain gave a ameliorate clarity of nuclear features of avian RBC and WBC (Figs. 1A-C). The new Fifty&G staining technique created significant differences (P<0.001) in cytoplasmic features and cytoplasmic granulations of avian WBC (Figs. 1B and C) compared to the other two methods (Figs. 2B and C and 3B and C).
The avian RBC and WBC. The nuclear (blue pointer) and cytoplasmic (red arrow) features of avian RBC (A), eosinophil (B), and heterophil (C) in Leishman-Giemsa stain (×chiliad)
The avian RBC and WBC. The nuclear (blueish pointer) and cytoplasmic (cerise arrow) features of avian RBC (A), eosinophil (B), and heterophil (C) in Leishman stain (×yard)
The avian RBC and WBC. The nuclear (blueish arrow) and cytoplasmic (crimson arrow) features of avian RBC (A), eosinophil (B), and heterophil (C) in Giemsa stain (×thou)
Discussion
In this pilot written report, the efficacy of 50&Chiliad stain, as a new staining technique, on the features of avian RBC and WBC included in the study was evaluated and compared to Leishman and Giemsa conventional stains. We selected an skilful clinical pathologist to subtract Pearson's variability to smears evaluation and to optimize the results of reports. The evaluated parameters from each staining method were compared with the boilerplate grading score given by two experts. We found that the grading scores calculated past both experts were almost the same in the morphological evaluation of avian blood cells. The coefficient of variation (CV%) calculated for nuclear features of the RBC in Leishman, Giemsa and L&G stain were 0.076, 0.075 and 0.067, respectively. The CV% calculated for nuclear features of the WBC in Leishman, Giemsa and L&G stain were 0.072, 0.073 and 0.066, respectively. The CV% calculated for cytoplasm features of the WBC in Leishman, Giemsa and Fifty&Yard stain were 0.125, 0.113 and 0.077, respectively. The CV% calculated for granules of the WBCs in Leishman, Giemsa and Fifty&1000 stain were 0.078, 0.081 and 0.063, respectively.
Statistical assay of average grading score from each staining method showed that the nucleus and cytoplasm of avian claret cells in L&M staining was much better staining than Leishman and Giemsa staining as presented in Table 1 (P<0.001).
Leishman stain is an excellent nuclear stain and using this stain lonely leads to an intense staining nucleus and understained cytoplasmic granulations. Giemsa stain is a good cytoplasmic stain and when used alone gives fainter staining of nucleus and its combination with Leishman stain provides a proper staining of nucleus, cytoplasm with cytoplasmic granulations (Gajendra et al., 2015 ▶; Suryalakshmi et al., 2016 ▶). The results of the staining efficacy of the modified L&Grand stain on the man peripheral claret/bone marrow smears showed that this staining method was superior to the other two conventional stains when used lone (Gajendra et al., 2015 ▶).
We found that the new L&G stain is useful for morphological assessment of nucleus and cytoplasm in both the avian RBC and WBC (Figs. 1A-C), whereas Leishman and Giemsa stain alone created a poor staining of the nucleus and the cytoplasmic granules with fainter contrast between nuclear and cytoplasm (Figs. 2A-C and 3A-C). The granules of heterophils and eosinophils were improve stained by the new L&G stain than Leishman and Giemsa stains as given in Table 1. Eosinophilic granules of heterophils and eosinophils are amend visualized in L&G stain (Figs. 1B and C) than the ii conventional stains (Figs. 2B and C and 3B and C). On the other hand, the new Fifty&Chiliad stain made a significant divergence in heterophil detection (Table 1). As mentioned in the previous section Leishman stain unlike Giemsa stain, has a methanolic base and the new 50&G stain does not need any further fixation (Gajendra et al., 2015 ▶). As is evident in sure figures, heterophiles in the figures of this manuscript are non degranulated, and these figures are themselves evidence of this merits.
During the study of avian blood it was constitute that this stain improves blood cell detection better than other formerly used stains such equally Leishman and Giemsa. Finally, we establish that the new L&G stain has nearly all the features of a good stain for morphological cess of the avian claret cells.
For the showtime fourth dimension, the results of the present study showed that the avian blood cells are more desirable stained with a new combination of L&Thou stain. In addition, it gives a better nuclear and cytoplasmic differential staining than the conventional Giemsa and Leishman stains when used lonely. Further studies should be undertaken to evaluate the staining technique abilities for identification of boom cells in avian bone marrow smears.
The authors have indicated that they have no affiliations or financial involvement with any arrangement or entity with a fiscal interest in, or in financial competition with, the subject field matter or materials discussed in this article.
Acknowledgment
We are thankful to Yard. Khodadi for the excellent technical help.
Conflict of interest
We declare that there is no disharmonize of involvement.
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Articles from Iranian Journal of Veterinary Research are provided here courtesy of Shiraz University
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716277/
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