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Evaluation of Two New Fixatives for Peripheral Malaria Smear

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Abstract

Introduction: Malaria is still a leading killer disease affecting millions of people worldwide, particularly in the tropical and subtropical areas. Till now, the cost-effective, gold standard test for diagnosing malaria with best sensitivity and specificity is stained peripheral smear for different stages of Plasmodium spp., even after invention of newer tests like Quantitative buffy coat and PCR. Staining involves use of absolute methanol as fixative. Using methanol is often very dangerous because its systemic absorption can lead to serious toxicities in humans like ocular damage and metabolic acidosis. Also it can be costly to use. Hence we evaluated three new fixatives, namely 70% ethanol, absolute (95%) ethanol and mixture of acetic acid (20%) and formalin (10%) as fixative for staining peripheral blood smear for malaria. Material and methods: We prepared four thin and four thick smears from each patient’s blood after obtaining written informed consent from them, fixed them with absolute methanol, 70% ethanol, 95% ethanol and acetic acidformaldehyde mixture, dried the smears, stained them with dilute Giemsa stain and observed the dried, stained smears microscopically under oil immersion. Results: The alcoholic fixatives, 70% ethanol and 95% ethanol were found to be good substitutes for methanol, and 70% ethanol even gave better results than methanol after staining as seen by different observers. Acetic acid-formaldehyde mixture was not found to be a good fixative for this purpose. Conclusion: Ethanol at both concentrations(70% and 95%) was a good fixative and can substitute methanol for the purpose of observing stained peripheral blood smear for malaria.
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International Journal of Contemporary Medical Research
ISSN (Online): 2393-915X; (Print): 2454-7379 | ICV: 77.83 | Volume 5 | Issue 1 | January 2018 1
Section: Microbiology
Evaluation of Two New Fixatives for Peripheral Malaria Smear
Ankur Kumar1, Sayan Bhattacharyya2, Asim Sarfraz3
ORIGINAL RESEARCH
ABSTRACT
Introduction: Malaria is still a leading killer disease affecting
millions of people worldwide, particularly in the tropical and
subtropical areas. Till now, the cost-effective, gold standard
test for diagnosing malaria with best sensitivity and specicity
is stained peripheral smear for different stages of Plasmodium
spp., even after invention of newer tests like Quantitative
buffy coat and PCR. Staining involves use of absolute
methanol as xative. Using methanol is often very dangerous
because its systemic absorption can lead to serious toxicities
in humans like ocular damage and metabolic acidosis. Also it
can be costly to use. Hence we evaluated three new xatives,
namely 70% ethanol, absolute (95%) ethanol and mixture of
acetic acid (20%) and formalin (10%) as xative for staining
peripheral blood smear for malaria.
Material and methods: We prepared four thin and four thick
smears from each patient’s blood after obtaining written
informed consent from them, xed them with absolute
methanol, 70% ethanol, 95% ethanol and acetic acid-
formaldehyde mixture, dried the smears, stained them with
dilute Giemsa stain and observed the dried, stained smears
microscopically under oil immersion.
Results: The alcoholic xatives, 70% ethanol and 95% ethanol
were found to be good substitutes for methanol, and 70%
ethanol even gave better results than methanol after staining
as seen by different observers. Acetic acid-formaldehyde
mixture was not found to be a good xative for this purpose.
Conclusion: Ethanol at both concentrations(70% and 95%)
was a good xative and can substitute methanol for the purpose
of observing stained peripheral blood smear for malaria.
Keywords: Malaria, Fixative, Methanol, Smear
INTRODUCTION
Malaria is a leading killer infectious disease with about 200
million new cases and 0.4 million deaths worldwide per
year recorded in the year 2015 as per records of the World
Health Organisation1. It is caused by Plasmodium spp.,
namely P. vivax, P. falciparum, P. malariae and P. ovale and
a fth new species, P. knowlesi2. Cases are mostly reported
from tropics and subtropical areas of the world but are also
found in other areas2. Diagnosis is achieved best by staining
stained peripheral smear, the stain used being Giemsa stain
and methanol is used as xative for this purpose3. However,
methanol is toxic to man, and can produce effects like
metabolic acidosis and ocular damage following period of
latency4. Moreover methanol is expensive and not suitable for
routine use in busy labs in developing countries5. So keeping
these things in mind, we tried to assess the efcacy of 3 new
xatives (70% ethanol, 95% ethanol, and mixture of 10%
Formalin and 20% acetic acid in water, for malaria smear
staining using Giemsa stain, and comparing it individually
with methanol.
MATERIAL AND METHODS
This was a lab based observational study, carried out in the
department of Microbiology of the institute from July 2017
to December 2017. Ethical clearance was obtained before
the study from institute, and informed written consent was
taken from patients. Routinely venous blood samples (1
ml) were received in the lab in EDTA vials. Tongue-shaped
peripheral blood smear was prepared in glass slides that
were made grease-free by dipping overnight in absolute
methanol. Smear was dried, xed with (a) methanol, (b) 70%
ethanol, (c) 95% ethanol and (d) 10% Formalin and 20%
acetic acd in water, for 5 minutes. So there were 8 slides
prepared from blood of one patient (4 thick smears and 4
thin smears). After this, thin smears were stained using a 1
in 10 dilution of Giemsa stain in Phosphate buffered saline,
pH 7.2, prepared in-house by mixing 3.8 Grams of Giemsa
powder (Thomas Baker Chemicals private limited, Mumbai,
India), 500 ml glycerol (Khona IINC, Thane, Maharashtra,
India) and 500 ml methanol (Himedia, Mumbai, India)
and ripened in dark for 1 week. Thick smears were dried
and 2 ml of distilled water was added over the slides for
dehemoglobinisation. After 10 minutes, water was decanted
and slide were again xed with the xatives mentioned
above and stained likewise. After drying sides, they were
observed microscopically under 100X objective by cedar
wood oil immersion for malaria parasites. Methanol xed
smear was considered as Gold standard and other xative-
treated smearswere compared with it.
RESULTS
Fifty four (54) blood samples were tested in this period.
Out of these, only 2 came out to be positive for malaria
parasite (One for P. vivax and one for P. falciparum). They
were positive using the rst three xatives (methanol, 70%
ethanol, 95% ethanol) but not the fourth (10% Formalin and
20% acetic acid in water). Alcohol (70%) and 95% ethanol
were found to be equally good as compared to methanol for
this. Morphology of parasite was well appreciable using both
these two new xatives. In all positive cases using the rst
two new xatives, parasite trophozoite nucleus appeared
1Tutor, 2Assistant Professor, 3Senior Resident, Department of
Microbiology, AIIMS Patna, India
Corresponding author: Dr Sayan Bhattacharyya, Assistant
Professor, Department of Microbiology, AIIMS, Patna, India
How to cite this article: Ankur Kumar, Sayan Bhattacharyya, Asim
Sarfraz. Evaluation of two new xatives for peripheral malaria
smear. International Journal of Contemporary Medical Research
2018;5(1):1-2.
Kumar, et al. Two New Fixatives for Peripheral Malaria Smear
International Journal of Contemporary Medical Research
Volume 5 | Issue 1 | January 2018 | ICV: 77.83 | ISSN (Online): 2393-915X; (Print): 2454-7379
2
Section: Microbiology
red and cytoplasm blue. In fact, the clarity of stained smear
was qualitatively best viewable using 70% ethanol and
equally good as compared to methanol using 95% ethanol,
as tested by 3 different observers for each stained smear
where staining was done using the methanol and the rst two
new xatives. Even for smears negative for malaria parasite,
staining of RBC and WBC was best with 70% ethanol
followed by methanol and 95% ethanol. The mixture of 10%
formalin and 20% acetic acid was not a good xative as per
our study, since all RBCs were damaged on using it a xative
and nothing was visible post staining. Even positive smears
tested using other xatives, gave negative results when xed
using the mixture (fourth xative).
DISCUSSION
A large number of cases of malaria are diagnosed each
year in our country, and rapid and early diagnosis is
essential to reduce mortality and also disease transmission
in the community6. Staining of blood lms and subsequent
microscopy remains the gold standard test for detecting
malaria in the laboratory accurately, even after 100 years of
its discovery7. Our study found out, for the rst time, the
utility of three new xatives for malaria smear staining. This
is important since methanol is toxic and costly as mentioned
above. Also, methanol has doubtful toxic effect on HIV
virus, and thus testing for malaria in known HIV virus
positive patient can be very dangerous8. Humans normally
ingest or inhale small amounts of methanol, and about 0.5
gram of Methanol per day is the upper safe limit for humans
for consumption9. There are reports that methanol can cause
blindness and even death if swallowed accidentally in
any quantitity10. Though methanol is needed for preparing
Giemsa stain and making it grease-free, still by substituting
it with other chemicals for xing smear, its toxicity can
somewhat be mitigated. This nding can be applied for other
tests also where blood or even bone marrow smear staining
is required, like for LD-bodies in visceral leishmaniasis or in
babesiosis or lariasis. One drawback of our study was the
small sample size and the lesser number of positive smears.
Nevertheless, these things have not been tested earlier and
further such studies are needed. As far as we know, this is the
rst type of such a study at least in this part of the country.
These ndings should show the way for further research in
this area of medical microbiology.
CONCLUSION
Ethanol in 70% concentration and also in 95% concentration
can be suitable substitutes of methanol for xing peripheral
blood smear before staining for malaria parasite.
REFERENCES
1. World Health Organisation. Malaria. http://www.who.
int/features/factles/malaria/en/.
2. Plasmodium species. Transfusion Volume 49, August
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tm/eid/Documents/224s.pdf.
3. Staining for malaria parasites. Available at: https://
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malaria_staining_benchaid.pdf.
4. Tephly TR. The toxicity of methanol. Life Sci.
1991;48:1031-41.
5. Methanol (Laboratory), Fisher Chemical. https://www.
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6. Bhattacharyya PK. Diagnosis and treatment of malaria.
British Med J 2007;334:375.
7. Tangpukdee N, Duangdee C, Wilairatana P, Krudsood S.
Malaria Diagnosis: A Brief Review. Korean J Parasitol
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8. Ghannoum M, Haddad HK, Lavergne V, Heinegg J,
Jobin J, Halperin ML. Lack of toxic effects of methanol
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61.
9. Jahan K, Mahmood D, Fahim M. Effects of methanol
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Bioallied Sci. 2015; 7: 60–64.
10. World Health Organisation. Giemsa staining of malaria
blood lms. http://www.wpro.who.int/mvp/lab_
quality/2096_oms_gmp_sop_07a_rev.pdf.
Source of Support: Nil; Conict of Interest: None
Submitted: 20-12-2017; Accepted: 18-01-2018; Published: 29-01-2018
... A small drop of blood was taken on a clean dry slide and a uniform film was smeared. Slides were dried in air, fixed in methanol 58,59 and stained in Giemsa and Leishman stain 60,61 . ...
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