STAINING TECHNIQUES OF BODY FLUIDS

BY GIEMSA STAIN

Submitted by – Kartikey
Chauhan
Batch 2020
Roll No- 206053061

Supervised by – Dr Alok Mohan

(Assistant Professor)
Department Of
Pathology
Muzaffarnagar Medical College & Hospital
 INTRODUCTION

Giemsa stain is a nucleic acid stain used in hematology, histology, cytology and bacteriology
for in vitro diagnostic (IVD) use.Giemsa stain is also a differential stain, such as when it is
combined with Wright stain to form Wright-Giemsa stain. It can be used to study the
adherence of pathogenic bacteria to human cells. It differentially stains human and bacterial
cells purple and pink respectively.[1]

Giemsa stain is also used to visualize chromosomes. This is particularly relevant for
detection of Cytomegalovirus infection, where the classical finding would be an "owl-eye"
viral inclusion.It is a prototypical stain used to detect malaria and Trypanosoma-infected
blood.[2]

It belongs to a group of stains known as Romanowsky stains. These are neutral stains made
up of a mixture of oxidized methylene blue, azure, and Eosin Y and they performed on an
air-dried slide that is post-fixed with methanol. Romanowsky stains are applied in the
differentiation of cells, pathological examinations of samples like blood and bone marrow
films and demonstration of parasites e.g., malaria.[3]

Wright giemsa stained cytocentrifuged slides are useful adjuncts in body fluid cytology and
may improve the detection of leukemia and lymphoma.Giemsa stain is one of the best
histological stain colouring the nuclei dark and cytoplasm blue to pink according to the
acidity of the cytoplasmic contents.Erythrocytes stain pink, platelets show a light pale pink,
lymphocyte cytoplasm stains sky blue, monocyte cytoplasm stains pale blue, and leukocyte
nuclear chromatin stains magenta.[4]

Its utility is well established in hematology for blood and bone marrow specimens,
bacteriology, clinical cytology specimens, histological biopsies, and tumor
samples.Giemsa’s
solution is registered as an “in vitro diagnostic medical device” (IVD) and bears the CE
mark. It is produced to the highest quality standards to enhance the visibility of cell
structures and ensure clear results. Other advantages include excellent batch consistency,
long shelf life, and detailed certificates of analysis (CoA).[5]

Giemsa stain is performed on paraffin sections. It is used to stain the blood cells of
hematopoietic tissues. It can also be applied to all tissue sections in which the presence of
microorganisms is suspected. Gram + and Gram Bacteria are not differentiated with this
staining.
 Aim and Objectives
Aim
To study different staining techniques of body fluids using GIEMSA stain.

Objectives
● To detect and identify the parasites in the given body fluid.
● To accurately prepare a Giemsa stock solution.
● To stain and identify blood cells.
● To differentiate blood cells nuclei from the cytoplasm
 Material and Methods

1. Study design - Hospital based observational study.

2. Study place - Department of Pathology, Muzaffarnagar Medical College and Hospital.
3. Study population - Fluids sent for routine examination in department of Pathology, Muzaffarnagar
Medical College
4. Duration of study- 15 days
5. Sample size – 20 patients will be selected from the pathology department.
6. Sampling technique – Purposive sampling.
7. Inclusion criteria - All body fluids sent for examination.
8. Exclusion criteria – Haemorrhagic fluids.
9. Procedure :
a. Fix air dried smear in methanol for 7 minutes.
b. May-Grunwald solution for 5 minutes.
c. Running water for 1 minute.
d. Giemsa solution for 15 minutes.
e. Running water for 1-2 minutes.
f. Air dry mount with DPX

May-Grunwald Reagent-Working solution:

May-Grunwald stock solution - 40 ml

Absolute methanol. - 20 ml

GiemsaStain-Working solution:

Giemsa stock solution. - 10 ml

Distilled water. - 90 ml

10. Statistical Analysis - Appropriate statistical tests will be used .

11. Ethical clearance - Will be taken up by institutional committee.

12. Conflict Of Interest - Nil

Review Of Literature

According to Stuart T. Fraser , Margaret H.Baron. (1998.)the peritoneal, pleural , cerebrospinal

and pericardial fluids comprise the major chunk of body fluids. It provides information about the
inflammatory or malignant nature of effusion, cause of effusion and detection of unknown primary
lesion.[6]

By the efforts of Neal P Barney , Frank M Graziano (2008) the cytological examination of
exfoliated cells in various effusion fluids is very challenging and is the paramount importance for
early diagnosis and management of various pathological processes . It is of at most significance in
identification of malignant cells and hence throws light on the cause, staging and prognosis of
various cancers. [7]

According to Elizabeth Mary Rush , Hillar Klandorf (2014) psychological

examination of body fluids is commonly done by conventional smear method.
However the cellblock method is one of the oldest but less commonly used
techniques in the evaluation of body fluids even today Therefore CB findings are
compatible to the histopathological diagnosis of primary tumors.[8]

In the review by Mariana C.C Silva, Jan-Michael Peters (2020) chromosome

spreading combined with Giemsa staining is well suited for visualizing overall
chromosome morphology. The harsh fixative, containing acetic acid, denatures
proteins and is therefore often incompatible with antibody staining of proteins. [9]

According to Joseph Caperna , Alfredo Tiu . D.O (2021)The stock solution for
Giemsa stain is obtained from commercial sources. The working stain solution is
prepared fresh by diluting the stock solution (1:9; v/v) using PBS buffer, pH 7.4.[10]
 References

1. Westerhoff, Verma Methods in enzymology.Research methods in biochemistry.

Vol 68 aug 1977 312.
2. Barcia J, Barcia. The Giemsa stain: its history. Origin of Giemsa
stain.2020;30:1505
3. Shapiro HM, Mandy F “Cytometry in malaria: moving beyond Giemsa”.
Analysis of cells at high speed -2002;46;1658.
4. VPVKumavat MP Kulkarni KR sulhayan cytological study of effusions Indian
Med Gazette 2013.
5. Wadha B Mohan A agarwal AK, varshney A kumar R garg B Sharm P. A study
of malignant serous effusion in a tertiary teaching hospital in western UP.
UPO 2016:3(2): 276-280
6. Stuart T. Fraser , Margaret H.Baron."Methods of enzymology" 32(6):128-133.
1998.
7. Neal P Barney , Frank M Graziano "ocular disease". 12(4): 556-560. 2008
8. Elizabeth Mary Rush , Hillar Klandorf "Therapy in surgery".23(4):110-118
2014
9. Mariana C.C Silva, Jan-Michael Peters "Methods in cell biology"42:1206; 2020
10. Joseph Caperna , Alfredo Tiu . D.O "Medical secrets"14(2): 878-882: 2021.