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OJHAS: Vol. 5, Issue
3: (2006 Jul-Sep) |
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Study
on the effect of green
pit viper venom (Trimeresurus albolabris)
on platelet morphology by flow cytometry |
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Suphan Soogarun
Department
of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn
Univesity, Bangkok, Thailand 10330
Montri Choewbamrungkiat
Snake farm,
Thai Red Cross, Thailand
Viroj Wiwanitkit
Department
of Clinical Laboratory Medicine , Faculty of Medicine , Chulalongkorn
University, Bangkok, Thailand 10330
Jamsai Suwansaksri
Department
of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn
University, Bangkok, Thailand 10330
Waykin Nopanitaya
Professor,
System Development Expert
Paweena
Pradniwat
Department
of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn
Univesity, Bangkok, Thailand 10330
Attakorn Palasuwan
Department
of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn
Univesity, Bangkok, Thailand 10330
Supantitra Chanprasert
Department
of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn
Univesity, Bangkok, Thailand 10330 |
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Address For Correspondence |
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Suphan Soogarun
Department
of Clinical Microscopy,
Faculty of Allied Health Sciences,
Chulalongkorn
Univesity,
Bangkok, Thailand 10330.
E-mail:
supunsug@yahoo.com |
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Soogarun S, Choewbamrungkiat M, Wiwanitkit V, Suwansaksri J, Nopanitaya W, PradniwatP, Palasuwan A, Chanprasert S. Study
on the effect of green
pit viper venom (Trimeresurus albolabris)
on platelet morphology by flow cytometry.
Online J Health Allied Scs. 2006;3:5 |
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Submitted: Sep 10,
2006; Revised: Nov 7, 2006;
Accepted: Nov 15, 2006; Published Dec 7, 2006 |
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| Abstract: |
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The
incidence of venomous snake bites, especially by green pit viper, has been increasing every year in Thailand. The bite
of green pit viper causes bleeding because of thrombin–like property of the venom
and it has been reported that the mean
platelet volume decreases in those bitten by this snake. The objective of this
study was to study the effect of green pit viper venom
(Trimeresurus albolabris) on platelet volume (MPV), number
of platelets and platelet morphology in vitro.
The test was carried out by washing platelet in phosphate buffer pH
7.2 so as to get rid of fibrinogen, then the washed platelets were mixed
with green pit viper venom. The mean platelet volume and number of platelets
were determined by flow cytometry. The results showed that there was a decrease
in the number
of platelets (216±101 x 109/l and 78.1±43.4
x 109/l; P< 0.05) and also the MPV
(8.9±1.2fl and 4.8±1.3fl, P< 0.05). The platelet size was smaller than
normal, ranging from 1.1-1.2 micrometers. In conclusion, the green
pit viper venom had a direct effect on platelet morphology, especially
by decreasing platelet volume and numbers.
Key Words:
Green pit viper,
Venom, Platelet |
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The
green pit viper (Trimeresurus albolabris and Trimeresurus
macrops) is a common venomous snake in Thailand and incidence of its bites
has increased dramatically up to 73.58%.[1] Its venom has thrombin-like effect
in vitro and causes a defibrination syndrome in vivo[2] and the clinical features of this venomous
snake bite vary from asymptomatic to fatal bleeding. The venom of
Trimeresurus albolabris can increase fibrinolytic activity by shortening
euglobulin time.[3] A recent study of a group of patients who had
been bitten by green pit viper (Trimeresurus albolabris
and Trimeresurus macrops) found that fibrinolytic
system activation was very common as indicated by low plasminogen,
low antiplasmin and elevated fibrin-fibrinogen degradation products (FDPs).[4] Significantly decreased total
platelet count and mean platelet volume (MPV) were demonstrated in envenomous blood. The changes might be partly
due to the effect of green pit viper toxin on platelet morphology.[5]
In this study we performed the in vitro study by mixing green pit viper
venom and platelet rich solution. Then the changes on MPV and platelet
number were measured by flow cytometry, while the platelet morphological
changes were observed by SEM.
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Lyophilized crude
venom (Trimeresurus albolabris) was obtained from snake farm
of Thai Red Cross. One milligram of crude venom was dissolved in normal
saline solution (NSS) as described in an earlier study.[6]
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Fibrinogen-free
platelet was prepared by using 10ml of EDTA blood mixed with 150ml
of 0.1 M phosphate buffer pH 7.2 (40.5 ml of 0.2M dibasic sodium phosphate
and 9.5 ml of monobasic sodium phosphate then added equal volume of
distilled water).[7] The solution was then centrifuged
in refrigerated centrifuge at 3,000 x g for 15 minutes. The supernatant
was discarded and added another 145 ml. Gentle agitation was performed
so as to disperse clumping platelets. The solution was re-centrifuged
at 50 x g for 10 minutes. The platelets in supernatant were used to
measure the MPV and platelet count by flow cytometry (Technicon H*3). The number of platelets subjected to this experiment
was not less
than 100x109/L. Small amount of red cells could be found
in the supernatant.
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Morphological changes after exposure to green pit viper venom
were determined. Two hundred microlitres of fibrinogen-free platelet concentrate were mixed
with 100μl
of venom, then incubated at 37°C at different periods of time (1-30 minutes),
after which the MPV and platelet counts were determined
by the Technicon H*3. This process was repeated twenty times.
Before
treatment, the platelets were within normal limits at 216±101x109/L while the MPV was 8.9±1.2fl. By electron micrograph, the
platelets appeared regular shape with smooth surface, ranging from 1.4-2.0
micrometers. The red cells that still existed in the supernatant had smooth surface,
round, disc-like sphere, measuring ranged from 5-6 micrometers
in diameter. After addition of green pit viper venom to the platelet
solution at one minute, the red blood cells were irregular with multiple cytoplasmic projections. Most red cells showed shrinkage
with the diameter ranging from 3-4 micrometers. The number of platelets
decreased dramatically from 216±101x109/L to 78.1±43.4x109/L
(P< 0.05). The MPV also decreased
from 8.9 ±1.2fl to 4.8±1.3fl, difference being significant (P<0.05), as shown
in Table 1. The decrease in the number of platelets and that of MPV occurred at the same time as seen in Fig.
1 and Fig. 2.
Table 1. Changes occurred after
treatment of the platelets with green pit viper venom
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Parameters |
Before treatment |
Post treatment |
P value |
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Platelets |
216 ± 101 x 10 9/l |
78 ± 43.4 x 109/l |
<0.05 |
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MPV |
8.9 ± 1.2 (fl)
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4.8 ± 1.3 (fl) |
<0.05
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Fig. 1: Platelet number
after addition of green pit viper venom (1mg/ml in normal saline solution)
to platelet rich solution for 30 minutes |
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Fig.2: The Mean Platelet
Volume (MPV) after addition of green pit viper venom (1mg/ml in normal
saline) for 30 minutes |
This
study found that the numbers of platelet decreased after the
exposure to green pit viper venom. The decrease in platelets in vivo
might be in part the result of direct reaction of venom and partly due to consumption by clot formation. This study supports the
view that the decreased MPV in vivo might be due to
the direct effects of the snake venom.[5] The same may be the cause for decreased MCV
also, as suspected in a previous report.[8] This study found that
the red cell morphology treated with green pit viper venom have morphologically
changed very much like those treated with Russell’s viper venom.[9]
However, Russel’s viper causes a significant increase in hematocrit
value. Such altered morphology was observed immediately at 1 minute
and reached maximum at 30 minutes.[6,9] The green pit viper venom might have
some properties different from that of the Russell’s viper, even though
both could cause sphero-echinocytes. The decrease of platelets
at the first minute might be due to cell lysis, but some of them could
tolerate and persist in toxic environment, thus beyond that time, both graphs
were constant. However, further research is necessary on this issue.
- Dumavibhat B. A
study of epimiology, risk factors and preventive measures against snake
bites. J Med Assoc Thai 1997;80(9):547-556
- Visudhiphan S, Dumavibhat
B, Trishnananda M. Prolonged defibrination syndrome after green pit
viper bite with persisting venom activity in patient’s blood. Am J Clin Pathol
1981;75(1):65-69
- Kamnerdnond C, Jitprommeta
J. Using green pit viper venom for coagulation study. A thesis , Faculty
of Allied Health Sciences, Chulalongkorn University, 2004
- Rojnuckarin P, Intragumtornchai T, Sattapiboon R, Muanpasitporn C, Pakmanee N, Khow O,
Swasdikul D.
The effects of green pit viper (Trimeresurus albolabris and
Trimeresurus macrops) venom on the fibrinolytic system in human. Toxicon.
1999;37(5):743-755
- Soogarun S, Wiwanitkit V, Suwansaksri J. A
trend of platelet indices in patients with green pit viper toxin.
Clin Appl Thromb Hemost. 2003;9(4):337-339
- Soogarun S, Chiowbamrungkiat,
Lertlum T, Pradniwat P, Jarujaron S, Palasuwan A, Jitprommetta, Kamnerdnond
C. Does green pit viper (Trimeresurus albolabris) venom act against
antithrombin III? Hema 2005;8(3):532-533
- Common buffer (Online,
Cited 2005 Sep 30). Available at:
http:/www.ou.edu/research/electron/bmz5364/buffers.html
- Wiwanitkit V, Suwansaksri
J. Effect of green pit viper toxin on red blood cell index (an interim
analysis). Toxicology 2001;164 (suppl):178
- Nopathorn S, Tejachokviwat
M, Maneesri S, Kasantikul V. Effects of Russell’s viper venom on human
erythrocytes in vitro. J Nat Toxins. 1998;7(1):73-85
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