The Progression of Glucose Consumption by Red Blood Cells Once Extracted From
Domestic Canines, Canis familiaris
Jeanette Coffin and Nadine Muallem
Department of Biological Science
Saddleback College
Mission Viejo, CA 92692
The integrity of uncentrifuged specimens for diagnostic evaluation is a
continued problem amongst clinical laboratories. Prolonged contact of red blood
cells with other analytes may be detrimental to the stability of the sample being
analyzed. It is pertinent that a patient receives the utmost ideal medical treatment
for their condition. If blood results are compromised due to ongoing metabolism by
erythrocytes, improper medical treatment may ensue. Medical conditions such as
diabetes and hypoglycemia rely directly on diagnostic evaluation. To determine if
RBCs continued to consume glucose, uncentrifuged, canine blood specimen’s
glucose levels were tested at intervals of 0, 1, 3, 6, 9 and 12-hours post venipuncture.
Samples remained in BD microtainer tubes containing lithium heparin to prevent
coagulation. The results yielded were significant in the depletion of glucose from the
initial blood draw beyond the third hour (p0.05 ANOVA). However, further
statistical analysis (Bonferroni post-Hoc) revealed no remarkable decrease in
glucose from the initial venipuncture to the first hour (p0.05).
Introduction
Blood is comprised of a multitude of analytes (such as sugars, clotting factors,
hormones, and other nutrients), platelets, red blood cells, and white blood cells. During
the lifespan of the red blood cell, it’s job is not only to carry oxygen rich hemoglobin to
the capillaries, but also includes, production of various chemical compounds from the
absorption and consumption of other chemicals in the surrounding environment (Deaton
et al. 1984).
Red blood cells (also called erythrocytes) continue consumption of
surrounding analytes even after blood specimens have been extracted from their host
environment (Zehnder et. al. 2008). Therefore, accuracy is key when evaluating a
patient’s blood for any type of diagnostic testing. Blood samples from an individual, must
be properly stored and maintained in order to obtain an accurate evaluation of chemical
analyses (Hrubec et al. 2002). The cells must be separated from the plasma or serum to
prevent continued metabolism (Blick and Boyanton 2002).
Diagnostic results of a patient’s blood play a vital role in the management of their
medical treatment (Herbert et al. 2005). For instance, diabetes and hypoglycemia are
serious medical conditions that directly rely on accurate analysis of glucose levels within
the bloodstream. Differentials in blood glucose levels are also known to accompany
critical conditions, such as, stroke, shock and sepsis. Even glucose concentrations within
the blood directly coincide with glucose concentrations in cerebrospinal fluid and can be
used to diagnose bacterial meningitis (Dujmovic and Deisenhammer 2010). In a study by
Christopher and O’Neill (2000), human blood was sampled and glucose depletion was
significantly noted in vitro when not properly stored. The rate of consumption of various
analytes by RBCs is dependent on the makeup of the blood (Han et. al. 2003). Using
blood from ten, randomly selected, healthy canines, Canis familiaris, the study tested the
hypothesis that over a period of twelve hours glucose concentrations would notably
decrease when blood samples were left uncentrifuged.
Materials and Methods
Ten privately owned domestic canines, Canis familiaris, were used in this study
with signed authorization from their owners. The name, mass, species, age, and sex was
recorded for each dog. All testing took place at Animal Urgent Care of South Orange
County, (Mission Viejo, CA), between 2 November 2011 to 7 November 2011. Three
milliliters of blood was obtained from the jugular vein of each subject using a sterile 6.0mL syringe with an attached 22-gauge needle. Immediately post draw, an initial packed
cell volume (PCV) was measured by placing a small portion of the blood into two
heparinized hematocrit tubes then centrifuged for three minutes. The PCV measurement
was obtained by then lining a single tube up to a standardized hematocrit chart. The
second hematocrit tube was to insure for accuracy. PCV values were recorded to the
nearest whole percent. Along with PCV values an immediate initial blood glucose
reading was performed using the Abaxis, VetScan i-STAT analyzer.
The remaining 2.5mLs of blood was then placed into five sterile BD microtainers
containing lithium heparin, 0.5-mL per tube. Each microtainer was labeled with the
subjects name and hour of testing, and then placed at room temperature (25C) onto a
blood rocker to prevent natural separation of the plasma. The blood glucose levels for
each sample were tested with the VetScan i-STAT analyzer at one, three, six, nine, and
twelve hours post draw. All glucose data were then entered into Microsoft Excel (2008)
where statistical analyses were performed. An analysis of variance (ANOVA) and
Tukey HSD post hoc analysis were run to determine if there were any statistical
differences between the groups.
Results
The mass for each canine was recorded in kilograms, and a mean mass for the
group was calculated out to be 15.1-kg. Glucose values for all draws were compiled by
the hour of measurement (1, 3, 6, 9, and 12) and the mean mass for each was calculated.
Glucose levels are as follows: Initial draw= 86.0mg/dL (Mean ± SEM), one hour-post
draw= 82.9mg/dL (Mean ± SEM), three-hours post draw= 65.6mg/dL (Mean ± SEM),
six-hours post draw = 40.1mg/dL (Mean ± SEM), and nine-hours post draw = 26.1mg/dL
(Mean ± SEM). All samples for the twelve-hour post draw registered on the analyzer as
 20mg/dL; therefore, with no exact numerical value, the twelve-hour recordings were
disregarded for statistical processing. This indicated that there was an overall decrease in
the amount of glucose from the initial sample. Figure 1 displays the depletion of glucose
values (mg/dL) over time (hour).
An ANOVA confirmed that the decrease in glucose between the groups was
significant (p0.05). A Tukey HSD post hoc analysis was run comparing each group to
the other. There were significant differences between each group (p 0.05), except for
the initial to one-hour post mean (p 0.05).
Initial
1
3
6
9
Time Post Draw (hour)
Figure 1. There was no significant difference in the time between the mean initial blood
glucose value (mg/dL) of the Canis familiaris and the 1-hour post samples (p0.05). An
increase in time significantly decreased the amount of blood glucose for all other
variants (p0.05, ANOVA with Bonferroni Correction, Error bars indicate mean ±
SEM).
Discussion
Breed, gender and age were obtained for records, but did were not factored into
any diagnostic results of this study. Initial PCV values were obtained to ensure that each
subject that participated was within the normal documented levels of 37-55%. It was
assumed that a participant with a normal PCV would less likely have constituents that
could potentially interfere with the project. There is documentation that describes the
hemolytic process of which RBCs undergo when in contact with glucose due to
glycolysis and the production of lactate (Sakota et. al. 2009). Therefore, supporting the
tested hypothesis that glucose is consumed by erythrocytes when blood samples remain
uncentrifuged.
By running an ANOVA with Bonferroni correction it was noted that from the
initial glucose value to 1-hour post draw, there wasn’t a significant difference (p 0.05).
For all other time variants, there was a significant drop in the number of glucose
molecules within the specimen (p0.05). After a 12-hour period of testing the glucose
levels of all ten canines have decreased below 20mg/dL. The chemical analyzer used
does not read numerical values below that of 20mg/dL. Just for comparison, a One
Touch Ultra 2 Glucometer was used to see if a numerical value, below 20mg/dL, could
be obtained. This instrument’s reading was “lo”, therefore, not successful.
Taken into consideration, although not accounted for, the rate at which glucose is
depleted is directly dependent on the chemical makeup of the blood itself. Many studies
test different variants and their influence on consumption and/or absorption by
erythrocytes, one factor being pH sensitivity (Mulquiney and Kuchel 1997). For the
conclusion of this study, it is warranted that to obtain the most accurate measurement of
blood glucose within a specimen, plasma or serum must be separated immediately from
erythrocytes. In a similar study by Blick and Boyanton (2002), there was a pronounced
decrease in glucose levels over a 24-hour period due to RBCs undergoing glycolysis.
They too concluded that for optimal analyte stability, separation by centrifugation must
be immediate.
Acknowledgments
We would like to sincerely thank Professor Steve Teh for his guidance and
assistance; the staff at Animal Urgent Care of South Orange County for their willingness
to allow their personal pet dogs to take part in scientific research, without which this
project would not have been possible; and lastly Our four-legged furry friends: Seamus,
Chicken, Noodle, Keira, Shredder, Motto, Paisley, Delilah, Quincy and Jedi for their
gracious “donations”.
Literature Cited
Blick, K.E. and Boyanton, B.L. 2002. Stability studies of twenty-four analytes in human
plasma and serum. Clinical Chemistry 48(12): 2242-2247
Christopher, M.M. and O’Neill, S. 2000. Effect of specimen collection and storage on
blood glucose and lactate concentrations in healthy, hyperthyroid and diabetic
cats. Veterinary Clinical Pathology 29(1): 22-28
Deaton, M.L. Higgins, J.J., and Smith, J.E. 1984. A model for the synthesis of chemical
compounds in red blood cells, with an application to diabetes. Biometrics
40(1): 77-84
Dujmovic, I. and Deisenhammer, F. 2010. Stability of cerebrospinal fluid/serum glucose
ratio and cerebrospinal fluid lactate concentrations over 24 h: analysis of repeated
measurements. Clinical Chemistry Laboratory Med 48(2): 209-212
Han, Tan H., Qamirani, E., Nelson, A.G., Hyduke, D.R., Chaudhuri, G., Kuo, L., and
Liao, J.C. 2003. Regulation of nitric oxide consumption by hypoxic red blood
cells. Proceedings of the National Academy of Sciences of the United States of
America 100(21): 12404-12509
Hebert, Paul C. et al. 2005. A pilot trial evaluating the clinical effects of prolonged
storage of red cells. Anesthesia and Analgesia 100(5): 1433-1458
Hrubec, T.C., Whichard, J.M., Larsen, C.T., and Pierson, W. 2002. Plasma versus serum:
specific differences in biochemical analyte values. Journal of Avian Medicine
and Surgery 16(2): 101-105
Mulquiney, P.J., and Kuchel, P.W. 1997. Model of the pH-dependence of the
concentrations of complexes involving metabolites, haemoglobin and magnesium
ions in the human erythrocyte. European Journal of Biochemistry 245: 71-83
Sakota, D., Sakamoto, R., Yokoyama, N., Kobayashi, M., and Takatani, S. 2009. Glucose
depletion enhances sensitivity to shear stress-induced mechanical damage in red
blood cells by rotary blood pumps. Artificial Organs 33(9): 733-739
Zehnder, L., Schulzki, T., Goede, J.S., Hayes, J., and Reinhart, W.H. 2008. Erythrocyte
storage in hypertonic (SAGM) or isotonic (PAGGSM) conservation medium:
influence on cell properties. Vox Sanguinis 95: 280-287
http://www.saddleback.edu/faculty/steh/bio3bfolder/peerreviewpage.htm
Review Form
Department of Biological Sciences
Saddleback College, Mission Viejo, CA 92692
Author (s):_ Jeanette Coffin and Nadine Muallem
Title:
The Progression of Glucose Consumption by Red Blood Cells Once Extracted From
Domestic Canines, Canis familiaris
Summary
Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the
paper.
The paper was about testing the drop of glucose levels in uncentrifuged
RBCs, as time elapses. The glucose concentrations were tested via blood samples
obtained from the jugular vein in domestic canines. The samples where taken in a time
interval of 0, 1, 3, 6, 9 and 12-hours post venipuncture.
The glucose concentration were tested via blood samples obtained from the
jugular vein in domestic canines. The samples where taken in a time interval of 0, 1, 3,
6, 9 and 12-hours post venipuncture.
After the first hour hours post venipuncture, there was no significant drop in
the glucose level in the blood. After 3, 6 and 9 hours post venipuncture, each had a
significant drop in glucose level. The concentration after 12 hours was too low for
device to measure.
ANOVA was run that determined a significant difference. Bonferroni post-Hoc
was then run which showed a signinficant difference between all variants except
change from 0 to 1 hour post venipuncture.
General Comments
Generally explain the paper’s strengths and weaknesses and whether they are serious, or important
to our current state of knowledge.
Many sentences are worded that does not come of to present the paper particularly
well. Some sentences have more hints of colloquialism of academic writing content.
The structure of the sentence can prevent the paper from receiving the value the
authors should receive based on their significant discovery.
Sentences tend to be too passive. When writing a scientific paper, one needs to be
passive and just present fact. However, one is also trying to convey a potential
significance and meaning with the research made. This however, does not mean that
the information and/or data should be presented in a misleading way in order to
enhance the view of the results. For example shifting the graph to make the bar
graphs look bigger or smaller than they should due to scaling. This is an important
aspect of writing a scientific paper.
There are inconsistencies with the way data is showed. For example in the text
numerical data is shown is all three ways: 2.5mLs, 6.0-mL 3.0 mL. Stay consistent
with the spacing between ## and unit. DO NOT “use” the one with no spacing in
between. Very important with consistencies in paper (I did not label all of them).
All the information is there to produce a great scientific paper. Just need to revise
some parts of the paper and make it scientifically presentable.
Abstract was well written (short and sweet) and covered key the aspects of paper.
Technical Criticism
Review technical issues, organization and clarity. Provide a table of typographical errors,
grammatical errors, and minor textual problems. It's not the reviewer's job to copy Edit the paper,
mark the manuscript.
This paper was a final version
This paper was a rough draft
Although not wrong, try to avoid making sentences that include many commas.
Some words beginning of a sentence are spaced more than one space away from the
period in the previous sentence.
On the materials used, would be good if the name of the manufacturer was included.
As mentinon before, the spacing between values and units should be consistent.
Most error are made as comments at the location revision is recommended. Some
has also been make that has been tracked in order for undoing the changes.
Recommendation
 This paper should be published as is
 This paper should be published with revision
 This paper should not be published