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THE SUITABLE ANTICOAGULANT, TIME AND TEMPERATURE
FOR BLOOD SAMPLE COLLECTION
Adel M. Al zobidy*, Dhuha Adel Kareem**, Fawzi-Alasadi**
*Department of physiology and pharmacology, College of Veterinary Medicine,
University of Basrah, Basrah,Iraq
**Department of anatomy and histology, College of Veterinary Medicine, University of
Basrah, Basrah,Iraq
(Received 5 April 2020 ,Accepted 14 June 2020)
Keyword: Blood, Anticoagulant, Hematology.
* Corresponding Author: [email protected]
ABSTRACT
The blood is a connective tissue that composes of suspended cells in liquid
matrix. In this study ,we discuss the effect of three factors on blood sampling during
the collection of blood samples (anti-coagulants, temperature and duration of storage)
. The delay of storage processing led to hemolysis of blood sample , increase of
hemoglobin, decrease of red cells ,triglycerides, hematocrite, mean corpuscular
volume, mean corpuscular hemoglobin and its concentration and increase number of
white blood cells. The histological analysis of blood sample during long-term storage
showed a slight difference in the shape and size of RBC,WBC and platelets as well as
condensed nucleus bluish cytoplasm and basophilic segmented nucleus in neutrophils
in addition to decrease in sodium ,calcium and chloride. The effect of temperature on
blood sampling showed slight variation in shape and size of RBC , basophilic
neutrophils and platelets aggregation and decrease of leukocyte number and K levels .
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INTRODUCTION
The blood is a special type of connective tissue that consist of suspended cell in
liquid matrix, blood cells are characterized by continuous movement from one place
to other ( 1) . The total blood volume in most mammalian is about 7-8% of total body
weight .
it is composed of the cellular component and blood plasma. The cellular
components occupy 45%.The blood cell is divided into erythrocyte, leukocyte and
platelets or thrombocyte . The Red color of blood is due to hemoglobin staining in
RBC ( 2). The blood involved in 1- Body immunity.2-water balance 3-body thermo
regular 4- bleeding stop 5- Nutrition such as oxygen ,liquid, nutrient, hormones and
vitamins. Blood plasma is a transparent liquid material and represents about 55%
from total blood volume .It is composed of water 90% and protein7%. Plasma plays a
role in the transportation of nutrients which is necessary for cells and transportation of
the metabolic product in the body.
1-1 The effect of anticoagulants on hematology
There are few studies of the anticoagulant on blood sample in animals. In human,
they observed that heparin anticoagulant should not be used for blood smearing
because it stained the back ground blue and clotted the leuckocytes, whereas EDTA
may cause damage to blood cells(1). Researchers had noticed that EDTA
anticoagulant may increase hemolysis via effecting on cell membranes(3). Reece et al
compared three anticoagulant affecting the blood parameter in fish (Mugilcephalus)
(2)as shown in Table(1).
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Table (1): The effect of three anticoagulant on blood parameter in mugilcephalus in
day one.
Parameter EDTA Sodium citrate Heparin
RBC 2.55 ±0.41 2.26±0.76 2.34±0.91
HCT90 30.14±5.74 26.89±4.89 25.43±4.83
HB(g\dl) 7.34±1.18 6.13±1.83 6.54±1.91
WBC(x103\ML) 8.02±2.14 7.95±3.08 8.03±3.26
Lymphocyte% 96.8 ±2.60 90.10±2.62 91.40±3.80
Monocyte% 2.50±1.00 2.90±2.10 2.96±2.2.80
Neutrophile% 6.70±2.30 7.00±2.40 5.70±2.30
Esinophile 0.00±0.02 0.00±0.02 0.00±0.02
Basophile 0.00±0.02 0.00±0.02 0.00±0.02
TC (x103\M 22.36±3.87 24.59±3.65 24.99±3.14
MCV(FL) 119.84±22.66 130.67±44.23 117.93±32.27
MCH(pg) 28.89±1.88 27.88±5.16 28.99±3.74
MCHC(g\dL) 24.65±2.79 23.19±7.19 25.65±4.95
From Table (1) we can see that there are some significant effects of the three
anticoagulant on some blood parameters. There are significant effect of these
anticoagulants on Hb, HCT, TG, Lymphocytes, monocytes. From Table (1) above it is
also shown that Hb and HCT values in EDTA anticoagulant are more higher as
compared to sodium citrate and heparin .
Bas.J.Vet.Res.Vol.19, No.2, 2020.
1-2-The Effect of Duration
Sawant et al observed that t
avoid hemolysis(4) . Austic and Scott
during the first week of duration
effect of storage duration of blood on
as shown in human RBCs
Sawant et al observed that RBC begin
counts fall offered collection of blood
RBC disappear during two days of duration (6).
Michael et al observed that sodium citrate creatinin, alkaline phosphatase,
amylase decrease after one day of
dehydrogenase increase at
days, magnesium after seven day
Anita Tendulkar et al Noticed that hemoglobin (g
the Donors,14-30 at 48hours and 14
7-60 at 48hours and 7-58 at 72hours. (8). Fazio et al Noticed that there were
significant changes in RBCS,HCT,MCT and HB at 366 hours as in Table(2) as
compared to zero hour in blood samples of rainbow trout(oncorhynchus mykiss). (9)
Table (2): The effect of time (0 h and 366 h)on blood parameter of
rainbow trout (Oncorhynchus mykiss
119
on hematology
the storage duration of blood was ver
noticed that hemolysis of the RBC
ing duration(5) .They observed that there were differences in the
hematological parameters depending on species
deteriorate slowly during storage which rats RBCS
begins to swell while leukocyte and platelet
(4). Goossens et al also showed
uring se the collection, whereas Potassium and lactate
one day after the collection , inorganic phosphate after four
, days, uric acid after three days in a human.
g\dl)were 14-
14-32 at72hours, while WBC were 7
ffect ncorhynchus mykiss)
he very important to
increases
have .
that nucleated
(7) .
-25 at4 hours of
7-63 at 4hours,
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Ibrahim studied the effect of storage duration on rabbits blood cells. He noticed
that the Mean leukocyte count of 48hour storage was (7.055±0.09)as compared to the
mean leukocyte count of 24hour(7.075±0.08)(no significant differences)according to
histological changes(10).
He showed no any changes in RBC, WBC and platelets ,but there was a slight
difference in the size ,shape and rouleau appearance in normal RBC with central pallor.
There were also large condensed nucleus and bluish cytoplasm in lymphocytes, while
eosinophile had a bilobed nucleus and cytoplsm granules. There were also basophilic
segmented nucleus in neutrophil and also small nuclear fragments of purple staining
granules in the platelets Fig1,Fig(2).
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Fig (1):- Red cells with rouleau appearance. (R). Lymphocyte(L). Eosinophil (E) had
bilobed nucleus with many cytoplasmic granules. Neutrophil (N) had basophilic
segmented nucleus. The platelets (P). (Ibrahim K-I(2008)).
Fig(2):- red cells(R). Neutrophil (N). Platelets (P) aggregates. Early the central
biconcave area of RBCs appeared slit like. Normal platelets (P) . vesicles (V). White
cells (W) .( Ibrahim K-I(2008)).
In comparison of the complete blood count after taking blood immediately and at
incubation for 48hours by studying on human blood ,there was no significant difference
in leukocytes, hemoglobin and platelets befor and after incubation for 24 hours,
significant difference in RBC,HCT,MCV,MCH as well as MCHC as shown in table - 3
(11).
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Table (3): The effect of time (0 after incubation immediately and after 48 h) on blood
parameter.
1-3-The effect of Temperature on hematology.
Ibrahim observed that the heat blood sample at storage or processing is a risky
factor related to hemolysis via effecting the membrane deformity , .also he showed a
histological study on rabbits blood sample that at 4C˚few variation in the shape and size
of RBC is realized while at 24C˚,the RBC became as a spiky appearance but at 36 C˚
observed central biconcave region of RBC(10). Reece et al In the Leukocyte picture, at
4 C˚ , showed that there were no significant changes, while at 24 C˚ and at 36 C˚ the
WBC count were significantly decreased at 36 C˚.
It was found that at4 C˚ no morphological changes in lymphocytes,
eosinophil's and neutrophils , but at 24 C˚ the neutrophils became as homogenous
mass. At 36 C˚ the Leukocyte became weak basophilic and similar to the size of
platelets. Similar to the platelets morphology, there were no morphological changes at
4 C˚, but at 24 C˚ the platelets aggregate and avoid of granules (2).
Blood parameters Before incubation for 48 After incubation for 48
WBC 6.45±1.94 6.41±1.93
RBC 4.69±0.71 4.66±0.71
HB 14.08±2.15 14.07±2.16
HCT 40.54±5.56 41.81±5.60
MCV 87.04±8.22 90.37±8.55
MCH 30.29±3.86 30.44±3.84
MCHC 30.27±3.86 34.69±1.70
Platelets 226.41±7.70 226.75±7.45
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.Table (4):The Effect of Temperature on K+, Na++ and CL- ion of the blood sample.
Temperature K Na CL
9 3.83a 154.4a 117.6a
22 2.36b 156.3b 117.7a
30 1.82c 157.3b 117.6a
Table(4) above showed the effect of heat on the mean value of K,Na,CL shown
significant reduction( p<0.01)in k+. Plasma (Na+) increase ( p<0.05) when stored at
22C˚ as compared to9 C˚, the difference was not significant between the sample of 22
C˚ or 30 C. (Cl̄) no significant difference among samples stored at 9,22 and30.
.DISCUSSION
2-1- The Effect of anticoagulants on hematology
Walencik, and Witeska, noticed that the anticoagulant which applied in the
laboratory of human hematology are potassium and sodium salts of ethylene diamine
tetra acetic acid (EDTA), Sodium citrate and heparin (3). Witeska, and Wargocka
observed that the heparin is a common endogenous anticoagulant acting on vessels in
vivo and Exo vivo when added to blood samples. Heparin acts by inhibiting the
conversion of pro thrombin to active thrombin as well as it did not allow fibrinogen to
change into fibrin . EDTA and sodium citrate act as inhibitors of thrombocyte
aggregation via chelating factor to free ca++ ions(12).
Some researchers considered that heparin is the most suitable anticoagulants
(3),while other considered that EDTA is anticoagulant of choice .( 13).In human
hematology there is a disadvantage to use heparin in blood smear due to bluish of back
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ground staining and clotting W.B.C , while EDTA leads to damage of blood
cell(1).From our literature , the HCT and HB in anticoagulant (EDTA) increase
significantly as compared to heparin and sodium citrate .This may be attributed to
erythrocyte swelling and hemolysis(12)
The swelling of erythrocyte may be due to elevation in PCO2and acidification
which is caused by treatment with acidic EDTA(14) ,(15) . In fished in mammal blood
observed erythrocyte swelling with EDTA anticoagulant(12). While observed decrease
in hematocrite in human blood sample with EDTA as compared to heparin(16).The
present literature showed that a high number of RBC were found in the blood sample of
EDTA as compared to heparin and sodium citrate ,so it observed that EDTA is a better
preservation of cell.
2-2-Effect of time on the hematology.
Many literature noticed that the delay of blood sample processing led to hemolysis
of blood sample , increase of hemoglobin, decrease of RBC ,TC, hematocrit ,mean
corpuscular volum, mean corpuscular hemoglobin and its concentration and increase
number of WBC.The biochemical analysis of blood after delayed processing led to
decrease in sodium citrate ,creatine, alkaline phosphate and amylase while potassium
acetate and phosphate showed an increase as well as increase in magnesium.
The histological analysis of blood sample showed a slight difference in shape and
size of RBC,WBC and platelets as well as condensated nucleus bluish cytoplasm and
basophilic segmented nucleus in neutrophils.The decrease in Sodium ,Calcium and
Chloride may be contributed to the low molecular substance in the erythrocyte ,while
the increase in potassium and magnesium may be contributed to these ions moved to
the membrane of RBC but those slight elevations are due to low decrease of
erythrocyte.(17) . The increase in inorganic phosphatase is due to the hydrolysis of
phosphate ester (18). The slight elevation in urea may be attributed to cleavage of
ammonia from basic amino acid which cause slight urea interference and reaction of
glutamate dehydrogenase .
The amount of alkaline phosphatase amylase depends on the PH value ,so to the
determination of these enzymes is dependent on the amount of these substance .
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(19). The instability of dehydrogenase 3, dehydrogenase 4and dehydrogenase5 in
the cold state led to decrease in lactate dehydrogenase activity (20). The decrease of
RBC, triglycerides, hematocrit, hemoglobin, mean corpuscular volume and
concentration is due to the degeneration of erythrocyte due to long –duration . The
long periods storage of blood sample cause the dilatation of the pores of the
membrane of RBC which allow water to enter the cells . The decrease in HB value
caused change in MCH and MCHC value (9). The slight difference in shape and size
was due to decrease negative changes on the red blood cells membrane that caused
by high concentration of positively changed protein in the blood plasma such as
fibrinogen and immunoglobuline(21).Moreover, the spiky appearance was explained
by the changes in the distribution of phospholipids in the membrane and lipoprotein
of the blood plasma(22), (23) and enzymes which led to lysis of RBC.
2-3-Effect of temperature on hematology
The present review showed a slight variation in the shape and size of RBC.
These explained by inhibition of negative changes on the membrane of RBC that is
due to the elevation of the concentration of positively changed proteins in the plasma
(21) . The slight variation in shape and size of RBC that showed in this study may be
attributed to the changes in the distribution of phospholipids in the membrane of RBC
via the mature RBC cannot produce lipids (22),as well as the phospholipid that were
lost in storage (20)Our review noticed decreased leukocyte at storage which is due to
breaking that led to the release of chemicals and enzyme (H2O2 and proteases which
cause red blood cell lysis (23).
The decreased leukocyte in the present study was similar to the study of (24) in
wester. Many leukocyte derived lipids mediators, histamines and cytokines which
can lead to the destruction of leukocytes (25). The homogenous mass of neutrophils
during heat and characterization of basophilic was not consistent with(24) .The
platelet aggregation may contributed to the activation of the platelets by agonists
thrombin ,ADP and collagen ,that led to activate fibrinogen receptors which allow
platelets cross – linkeded(6)
The result of the present study noticed a decreased potassium in storage due to the
cyclic adenosine monophosphate –mediated potassium transport into the erythrocyte
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was activated by the production of catechol a – mines (26).Meyer et al Noticed that
catecholamine affects the distribution of Na, K and CL, that theirs function for the
volume –regulatory response of red cells in anisomotic conditions, as well as the Na+,
K and CL transport is activated by exposure to c – AMP (27).
مضاد التخثر و درجة الحرارة والوقت الملائمین لجمع عینات الدم
عادل موسى الزبیدی*، ضحى عادل کریم**، فوزی صدام الاسدی**
*- فرع الفسلجة والادویة کلیة الطب البیطری، جامعة البصرة.,البصره، العراق
**- فرع الانسجة والتشریح، کلیة الطب البیطری، جامعة البصرة،البصره ، العراق.
الخلاصة
یعد الدم من الانسجة الرابطة المتکونة من خلایا معلقة فی مادة سائلة . ھذه الدراسة بحثت تأثیر ثلاثة
عوامل على نماذج الدم وھی( مانعات التخثر ودرجات الحرارة ووقت الخزن).من خلال استعراض المراجع
بینت النتائج بأن بعض الباحثین سجلوا بان الھیبارین ھو مانع التخثر المفضل بینما اخرین لاحظوا بان ای دی تی
أی ھوالافضل.ان تأخیر فحص الدم یؤدی الى تحلل نموذج الدم،وزیادة فی الھیموغلوبین ونقص فی کریات الدم
الحمر،والدھون الثلاثیة، وزیادة فی عدد کریات الدم البیض .الفحص النسجی لنموذج الدم خلال الخزن الطویل
بین اختلاف بسیط فی شکل وحجم کریا الدم الحمر والبیض والصفائح الدمویة بالاضافة الى ظھور سایتوبلازم
ازرق ونواة کثیفة علاوة على نواة مقطعة قاعدیة للخلایا المتعادلة ،اضافة الى قلة فی کالسیوم وصودیوم
والکلور. ان تأثیر الحرارة على نموذج الدم اظھرت اختلاف بسیط فی شکل وحجم الکریات الدم الحمر ،وخلایا
متعادلة قاعدیة ،وتجمع للصفائح الدمویة وقلة فی عدد الخلایا البیض ومستوى البوتاسیوم. .
REFERENCES
1-Mariańska, B., Fabijańska-Mitek, J. and Windyga, J. 2003. Laboratory
hematological analyses. Manual for medicine students. Wyd. Lek. PZWL,
Warszawa.
127
Bas.J.Vet.Res.Vol.19, No.2, 2020.
2-Reece W. O., Trampe D. W., and Koehler K. J.. 2006. Effects of Storage Time
and Temperature After Blood Sampling from Turkeys on Plasma
Concentrations of Potassium, Sodium, and Chloride. Science 85:1095–1097.3-
Walencik, J. and Witeska, M. 2007. The effects of anticoagulants on
hematological indices and blood cell morphology of common carp (Cyprinus
carpio L.). Comparative Biochemistry and Physiology, 146: 331-335. DOI:
10.1016/j.cbpc.2007.04.004
4-Sawant RB, Jathar SK, Rajadhyaksha SB, Kadam PT: Red cell hemolysis
during processing and storage. (2007): Asian Journal of transfusion sience, 1
(2):47-51.
5-Austic, R. E., and M. L. Scott. 1997. Nutritional diseases. Pages47–73 in Diseases
of Poultry. 10th ed. V. W. Calnek, H. J. Barnes, C. W. Beard, L. R.
McDougal, and Y. M. Saif, ed. Iowa State Univ. Press, Ames.
6-Goossens, W., Van Duppen, V. and Verwilghen, R. H. (1991): K2- or K3-EDTA:
the anticoagulant of choice in routine haematology Clinical and Laboratory
Haematology, 13: 291.
7-Michael Heins, Wolfgang Heil and Wolfgang Withold.1995. Storage of Serum or
Whole Blood Samples? Effects of Time and Temperature on 22 Serum
Analytes.Eur J Clin Chem Clin Biochem. 1995:33:231-238.
8-Anita Tendulkar, Puneet Jain, Sumeet Gujral, Manisha Tambe, Ravindra
Kenjale and Bala Ganesh.2015. Stability of Selected Hematological
Parameters in Stored Blood Samples. Journal of Cell Science & Therapy ,
DOI: 10.4172/2157-7013.1000220ISSN: 2157-7013 JCEST, an open access
journal.
9-Fazio .F. , Ferrantelli .V., Saoca .C., Giangrosso. G., Piccione .G. 2017. Stability
of haematological parameters in stored blood samples of rainbow trout
Oncorhynchus mykiss (Walbaum, 1792) 62,(07): 401–405. DOI:
10.17221/51/2017-VETMED.
10-Ibrahim K. I. Ragab. 2008 . Histological study of effects of storage duration and
temperature on the rabbits blood cells. Vol., 30: 14– 24.
128
Bas.J.Vet.Res.Vol.19, No.2, 2020.
11-Narges Obeidi , Eisa Safavi and Habib Emami. 2012. Evaluation of the effect
of temperature and time of incubation on complete blood count (CBC) tests.
African Journal of Biotechnology Vol. 11(7), pp. 1761-1763.
12-Witeska, M. and Wargocka, W. 2011. Disodium EDTA used as anticoagulant
causes hemolysis in common carp blood. Turkish Journal of Veterinary and
Animal Sciences, 35: 99-104. DOI:10.3906/vet-0908-51.
13-Blaxhall, P.C. and Daisley, K.W. 1973. Routine haematological methods for use
with fish blood. Journal of Fish Biology, 5: 771-781. DOI: 10.1111/j.1095-
8649.1973.tb04510.x.
14-Smit, G.L., Hattingh, J. and Schoonbee, H.B. 1977. Observations on some
effects of disodium ethylen ediamine tetra-acetate and heparin on fish blood.
Comparative Biochemistry and Physiology, 57: 35-38. doi: 10.1016/0306-
4492(77)90074-0.
15-Korcock, D.E., Houston, A.H. and Gray, J.D. 1988. Effects of sampling
conditions on selected blood variables of rainbow trout, Salmo gairdneri
Richardson. Journal of Fish Biology, 33: 319-330. DOI: 10.1111/j.1095-
8649.1988.tb05474.x.
16-Morris, J.D., Fernandez, J.M., Chapa, A.M., Gentry, L.R., Thorn, K.E. and
Weick, T.M. 2002. Effects of sample handling, processing, storage, and
hemolysis on measurements of key energy metabolites in ovine blood. Small
Ruminant Research, 43: 157-166. DOI: S0921-4488001)00266-8.
17-Henry RJ, Cannon DC, Winkelmann JW. Phosphate. In: Henry RJ, Cannon
DC, Winkelmann JW, editors. Clinical chemistry, principles and technics. 2nd
edition New York: Harper and Row, Publishers; 1974:720-8.
18- Richterich R, Colombo JP, editors. Klinische Chemie - Theorie, Praxis,
Interpretation, 4. Auflage Basel: Karger Verlag, 1978:403-11.
19- Sawant RB, Jathar SK, Rajadhyaksha SB, Kadam PT: Red cell hemolysis during
processing and storage. (2007): Asian Journal of transfusion sience, 1 (2):47-51.
129
Bas.J.Vet.Res.Vol.19, No.2, 2020.
20- Jacobs E, Hissin PJ, Propper W, Mayer L, Sarkozi L. Stability of lactate
dehydrogenase at different storage temperatures. Clin Biochem 1986; 19:183-8.
21- Zipursky, A., Brown, E., Palko, J., and Brown, E.J. (1983): The erythrocyte
differential count in newborn infants. American Journal of Pediatric Hematology and
Oncology, 5: 45.
22- Mary M, Christopher, DVM, PhD, DACVP, DECVCP USA (2004): University of
California, Davis, Department of Pathology, Microbiology, and Immunology School
of Veterinary Medicine, Davis, CA,. Interpretation of Red Blood Cell Morphology
(Advanced Level). Red Blood Cell Morphology, 29th World Congress of the World
Small Animal Veterinary Association,
23- Heaton WA, Holmes S, Smith K, Brecher ME, Pineda A, AuBuchon JP, et al (1994):
Effects of 3-5 log10 pre-storage leucocyte depletion on red cell storage and
metabolism. Br J Hematol ., 87:363-8.
24-Hume-Moir, P. Clark, PBS Spencer.(1999): Effects of temperature and duration of
sample storage on the haematological characteristics of western grey Kangaroos
(Macropus fuliginosus). Australian Veterinary Journal 84 (4), 143-147.
25- Kristiansson M, Soop M, Saraste L, Sundqvist KG.(1996): Cytokines in stored red
blood cell concentrates: promoters of systemic inflammation and simulators of acute
transfusion reactions? Acta Anaesthesiol Scand.., 40:496-501.
26-Lumeij, J. T. 1985. The influence of blood sample treatment on plasma potassium
concentrations in avian blood. Avian Pathol. 14:257–260.
27-Meyer T, Monge PK, Sakshaug J. Storage of blood samples containing alcohol. Acta
Pharmacol Toxicol (Copenh) 1979; 45:282-6.
