Topic 1: The Effect of Exercise on Blood Parameters

Article #1

“The comparison of blood parameters between morning and evening exercise”

Ibrahim Erdemir

School of Physical Education and Sports, Balikesir University, Balikesir, Turkey

Corresponding Author:
Ibrahim Erdemir
School of Physical Education and Sports
Balikesir University, Balikesir, Turkey

Abstract

The purpose of this study was to research to the differences of blood parameters between morning
exercise and evening exercises. 12 participants, in younger adults aged 20 years, were recruited and
their blood was taken four times, from 8:00 to 9:00 (pre and post) for morning exercise and from 20:00
to 21:00 (pre and post) for evening exercise. The results found that leukocytes (WBC, NE and LY),
erythrocytes (RBC, HGB, HCT, MCH and MCHC) and thrombocyte (PLT, MPV and PCT) show resulting
differences (p<0.05) between the morning and evening exercises. Additionally, no significant differences
were found in the other parameters in blood. In conclusion; Hematologic parameters display different
behaviors exhibit acute exercise at different times of day. Leukocytes, erythrocytes and thrombocyte
levels display different behaviors as exercises at morning and evening.

Introduction

Exercise is an important function of living systems. It affects many systems in our body. Human body
adapts to exercise by breathing and by cardiovascular systems; such as, cardiac output is 20-25 liters
during high intensity exercise [1] and also exercise may affect blood parameters. Physical and
physiological response also plays an important role, in hematology [2]. When hematology is analyzed,
the effect of acute exercise on hematological levels is seen different. It is stated that these differences
depend on the severity, duration, exercise at different times of day and frequency of exercise as well as
physical and physiological conditions of subjects [3].

Hematologic parameters, plasma, leukocytes and platelets, are very responsive to exercises which are
done different time of day and different exercise [4]. Some changes in erythrocyte's metabolism and in
affinity of hemoglobin to oxygen take place in organism of sportsmen-volleyball with high and low
qualification under training loading [5]. Research has been found contradictory concerning the effects of
training on red blood cells. The specific type and duration of exercise is of major importance in the
adaptations of the blood cell system [6]. Training at various altitudes above the sea level also seems to
increase hemoglobin more than training at sea level. When the number of red blood cells increases,
their hemoglobin content causes the blood to become thicker and more resistant to flow through the
body.

It is very common for national and international level of athletes to exercise several times a day during
their training regimes (i.e. perform multiple daily session). Most people do exercise at different times of
day. However, the impact of the different times of daily exercise and multiple daily exercise sessions
upon blood has not been researched thoroughly. Additionally, the effect of multiple daily exercise
sessions upon night-time blood level has not been evaluated. We therefore designed the present study
with the aims of determining to compare the hematologic parameters between the morning and
evening exercise in high school students.

Materials and Methods

Subjects

12 healthy, untrained male student subjects aged 20 years volunteered to participate in the study. All
subjects were non-smokers, used no drugs affecting the cardiovascular system and had no
contraindications to exercise. All of them submitted their written consents to participate after having
been informed about the study protocol and possible risks involved. The study was approved by the
local committee of ethics.

Physiological Parameters

Body weight, body height, body fat percentage, vertical jump and body-mass index (BMI) were
determined approximately a week prior to the first admission. The BMI was calculated with the
following formula: body weight (kg) /body height (m2) [7]. The 12-minute run/walk test was conducted
and aerobic exercise capacity estimated using Balke’s equation [2]. Average power was estimated using
the Lewis formula.

Exercise

Before the morning and evening exercise, the resting heart rate was taken with polar 610i and predicted
maximum heart rates estimated by using the formula 220 – Age [8]. The subjects did the same
(submaximal, %85 intensity) exercise at the same heart rate. The volume (including warm up and cool
down) of morning and evening exercise is about 70 minutes and intensity of exercises is about 85%.
After each drill, participants were rested passively between exercises. Passively means that they don’t
do any physical activity between the exercises. When the heart rate of participants fell down to between
115-125 beats per minute, participants continued the interval exercise again. All drills were performed
with the same intensity and to the same heart rate by all participants. They used a heart rate monitor
during exercise and recovery. The same procedure was used for evening exercise after a week.

Blood Parameters

Blood sample (for morning exercise), was taken at 08:00 and 09:00 (pre- and post- exercise). Blood
sample (for evening exercise) was taken at 20:00 and 21:00 (pre- and post- exercise). Four milliliters of
blood were collected from each participant in gel tubes. Hematological levels including Red Blood Cells
(RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean Red Cell Volume (MCV), Mean Cell Hemoglobin
(MCH), Mean Cell Hemoglobin Concentration (MCHC), Red Cell Distribution Width (RDW), White Blood
Cells (WBC), Lymphocyte (LYM), Neutrophil (NE), Monocyte (Mono), Eosinophil (Eos), Basophil (BASO),
Platelets (PLT), Platekri (PCT), Mean Platelet Volume (MPV) and Platelet Distribution Width (PDW) were
analyzed by means of an Archem H3000 Hematology Analyzer.

Statistical Analysis

All data were presented as mean ± standard deviation. Pre- and post-exercise values for the dependent
variables were analyzed to determine if the distributions were normal using Kolmogorov-Smirnov (K-S)
Normality test. Comparison, between pre- and post-exercise, was performed through the Paired-
Samples T test procedure in order to examine the differences between tests. It was also used for
comparing the differences for pre-test ME and EE and post-tests ME and EE. Significance level was set at
α = 0.05 and 0.01. The statistical package program was used to evaluate the results of the study [9].

Results

The subjects studied had a mean age of 20.0 ± 0.0yr, body weight of 69.8 ± 2.3kg, and body height of
174 ± 1.6cm. The subjects had a anaerobic power, 147.32 ± 12.10kg-m/s, VO2 max of 47.47 ±
1.97mL/Kg·min, vertical jump·61.10 ± 6.45cm, body mass index (BMI) 23.13 ± 1.62, and a percent body
fat of 10.63 ± 1.65%.

In leukocytes, there was significant increase on WBC count in morning pre- and post-exercise at the
significant level of p<0.01 and also that of evening pre-and post-exercise at the significant level of
p<0.05. The mean level of NE % and NE 10^3/μL increased at morning pre-and post-exercise at the
significant level of p<0.05. But NE percentage decreased at evening pre-and post-exercise at the
significant level of p<0.05. Meanwhile, the mean values of pre-test of LYM % and LYM 10^3/μL was
higher than that of the post-test at evening exercise.
In erythrocytes, the mean level of RBC at evening exercise was higher than the mean level of morning
exercise. At the same time, there were significant increase at the mean level of HGB and HCT of morning
pre-and post-exercise and also that of evening pre-and post-exercise at the significant level of p<0.05.
The mean level of MCH and MCHC at evening exercise increased more than the mean level of morning
exercise.

In thrombocyte, there were significant increase at morning pre-and post-exercise and also evening pre-
and post-exercise on PLT and on MPV at the significant level of p<0.05. Mean level of PLT at morning
pre-exercise increased more than morning post-exercise.

Discussion

As a result of the research, it was found that there were a significance increase in the value of
leukocytes; WBC and NE, and was a significance decrease LYM, between the morning and evening
exercise. When a comparison was made between these changes and the ones in other studies carried
out on hematological levels, both similarities and differences were observed.

The results are similar to the findings of Vogelaere et al. [10] who determined the effects of cold stress
on routine hematologic parameters when subjects were submitted on long-lasting exercise. They found
that WBC count was significantly increased during exercise in both 20°C and 0°C environmental
temperatures. Meanwhile, Vogelaere et al. [10] searched the effect of negative thermal stress on
hematological variables at rest, and during submaximal and maximal exercise were observed for young
males who volunteered in two experimental sessions, performed in cold (0°C) and in normal room
temperature (20°C). They found the same result that values for WBC also slightly increased during cold
stress exposure. However he explained that this increase can partly be related to hemoconcentration
but also to the cold induced hyperventilation activating the lung circulation. At the same time, Gimenez
et al. [11] researched the influence of work intensity and duration on the WBC and LYM count response
to exercise was studied. They found that WBC and LYM increased at 150W, cyclo-ergospirometric
protocols. However, during a 45min Square-Wave Endurance Exercise Test: WBC and LYM increased at
the 158th min, in the same research. Another similar research study, Temiz et al. [12] searched that RBC
mechanical alterations and oxidative damage were investigated after an acute exhausting exercise in
rats, together with the leukocyte activation. They found that the leukocyte phagocytic activity increased
significantly after the exhausting exercise and prolonged till 24 hours. RBC membrane lipid peroxidation
was gradually increased till 24 hours. Gonzalo-Calvo et al. [13] was to investigate the changes in a large
panel of emergent geriatric biomarkers in long-term trained elderly men to analyze the effects of long-
term exercise on an aged population. They showed that long-term training was associated with lower
levels of white blood cell counts and neutrophil counts.
We found significant differences in erythrocytes; RBC, HGB, HCT, MCH and MCHC, and in thrombocyte;
PLT, MPV, PCT between the morning and evening exercise. However, there were no significant
differences in the value of the remaining blood parameters. Similar results have been found in other
studies. For example, Vogelaere et al. [14] observed a slight but not statistically significant increase of
RBC, HGB and HCT and a plasma concentration during cold exposure. And they found that platelets
count significantly increased during exercise in both 20°C and 0°C environmental temperatures. Still
another study of Vogelaere et al. [10] found that at rest, hematological variables such as RBC and
derivate HGB and HCT significantly increased during cold stress exposure, while plasma volume
decreased. Remaining values for platelets also slightly increased during cold stress exposure. However
this increase can partly be related to hemoconcentration but also to the cold induced hyperventilation
activating the lung circulation. Maximal exhaustive exercise induced, in both experimental
temperatures, significant increments of RBC, HGB and HCT while plasma volume decreased. Green et al.
[15] researched to investigate the role of high-intensity intermittent exercise on adaptations in blood
volume and selected hematological measures. Total blood volume based on plasma volume and HCT
values increased by 4.5%, whereas red cell volume decreased by 6.4%. But in contrast to this study
Green et al. [15] reported that measurements of hematological indices indicated significant reductions
in whole-blood HCT, HGB concentration, HGB content, and RBC count. Their findings suggest that
exercise intensity is a major factor in promoting exercise-induced hypervolemia and that rapid
elevations in plasma volume can be induced early in training. Gimenez et al. [11] researched that the
influence of work intensity and duration on PLT count response to exercise was studied. They reported
that the increase was very small for HCT, [HGB], and RBC, in contrast with large changes for plasma,
total plasma volume, [H+] and lactate at VO2max cyclo-ergospirometric protocols. Temiz et al. [12]
report that the leukocyte phagocytic activity increased significantly after the exhausting exercise and
prolonged till 24 hours. RBC membrane lipid peroxidation gradually increased till 24 hours and there was
a significant correlation between leukocyte phagocytic activity and RBC lipid peroxidation. There was a
slight but significant decrease in MCV and increase in MCHC suggesting a cellular dehydration after 24
hours. Their results imply that activated leukocytes might play role in the RBC damage observed after
exhausting exercise encouraging oxidative stress [12].

Different from our research, some studies stated that neither exercise nor cold inducement significantly
modified the hematological indices (MCH, MCV and MCHC). Spiropoulos and Trakada [16] examined on
marathon runners, no significant differences were found in hematological parameters before and after
the marathon competitions in their study. Kara et al. [17] stated that there was no statistically significant
difference in HGB, HCT, RBC, WBC and PLT values in elite athletes in two different branches. Schumacher
et al. [6] showed that alterations of the red blood cell system and iron metabolism may influence
physical performance with no difference was found between athletes and control group in HGB and
HCT. They stated that reduced HGB, HCT and RBC levels were observed in endurance compared with
strength and mixed-trained. Physical training itself has no significant effect on selected hematological
variables in athletes compared with untrained control group. Cakmakçı [18] studied on taekwondoers
that there was not a significant difference in WBC, RBC, PLT and HCT parameters in blood samples taken
before and after the camp. Reitjens al. [5] compared the blood samples of athletes before and after the
season in their study on 11 Olympic athletes and determined that there was no significant change in
MCHC levels.

Conclusion

Hematologic parameters (leukocytes, erythrocytes and thrombocyte) are affected by exercise done in
morning and in evening exercise. When we compare the difference of hematological levels between
morning pre-exercise and evening pre-exercise, there was an increase in terms of leukocytes, NE % and
LYM % and in erythrocytes, MCH and MCHC. When we compare the difference of hematological levels
between morning post-exercise and evening post-exercise, there was an increase in terms of leukocytes,
NE % and LYM % and in erythrocytes, MCH and MCHC. There were differences between morning and
evening exercise in terms of increase or decrease in the hematological level of subjects. Some of them
were statistically insignificant and some were significant. These changes were within regular limits.
Hematologic parameters display different behaviors exhibit acute exercise at different times of day.

References

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Y. O. Schumacher, A. Schmid, D. Grathwohl, D. Bultermann, A. Berg, 2002, Med. Sci. Sports Exerc., 34(5):
869-875.
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2009, pp 18, 29, 30.
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J.H. Zar, Biostatistical analysis. Fourth edition.Prentice Hall. 1999
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34(1): 1-14.
M. Gimenez, T. Mohan-Kumar, J.C. Humbert, N. De Talance, J. Buisine, 1986, Eur. J. Appl. Physiol. Occup.
Physiol. 55(5):465-70.
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259.
D. Gonzalo-Calvo, B. Fernández-garcía, B. Luxán-Delgado, S. Rodríguez-gonzález, M. García-macia, F.M.
Suárez, J.J. Solano, M.J. Rodríguez-colunga, A. Coto-montes, 2012, Age, 34(3) : 761-771.
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56(1):145-9.
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E. Cakmakcı, 2009, Nigde Uni. J. Phys. Edu. Sport Sci., 3(1): 21-29.

Article #2:

“Exercise and interleukin-6”

Pedersen, BenteKlarlund MD, DMSc; Steensberg, Adam; Schjerling, Peter PhD

Strenuous exercise induces increased levels in a number of pro-and anti-inflammatory cytokines, natural
occurring cytokine inhibitors, and chemokines. Thus, increased plasma levels of tumor necrosis factor
(TNF)-α, interleukin (IL)-1 β, IL-1 receptor antagonist (IL-1ra), TNF-receptors (TNF-R), IL-10, IL-8, and
macrophage inflammatory protein (MIP)-1 are found after strenuous exercise. The concentration of IL-6
increases as much as 100-fold after a marathon race. It has recently been demonstrated that IL-6 is
produced locally in contracting skeletal muscles and that the net release from the muscle can account
for the exercise-induced increase in arterial concentration. Larger amounts of IL-6 are produced in
response to exercise than any other cytokine, IL-6 is produced locally in the skeletal muscle in response
to exercise, and IL-6 is known to induce hepatic glucose output and to induce lipolysis. These facts
indicate that IL-6 may represent an important link between contracting skeletal muscles and exercise-
related metabolic changes.

Article #3

“Effects of exercise on hematological parameters, circulating side population cells, and cytokines.”

Author information

Wardyn GG1, Rennard SI, Brusnahan SK, McGuire TR, Carlson ML, Smith LM, McGranaghan S, Sharp JG.

Department of Internal Medicine, Pulmonary Section, University of Nebraska Medical Center, Omaha,
NE 68198-2055, USA. gwardyn@unmc.edu

Abstract

OBJECTIVE

To determine the effects of exercise and/or training on hematologic indices, circulating side population
(SP) cells, and cytokines. Specifically hemoglobin (Hgb), hematocrit (Hct), white blood cell (WBC) and
platelet (Plt) numbers, SP cells and plasma vascular endothelial growth factor (VEGF) and interleukin-6
(IL-6) levels were analyzed before and following exercise to maximal fatigue.

MATERIALS AND METHODS

Thirty-seven nonsmoking subjects, aged 19 to 35 years, free of cardiopulmonary disease were enrolled
and characterized as "trained" or "untrained." Standard hematologic indices were measured. Blood cells
were stained with Hoechst 33342 vital dye and analyzed using flow cytometry for enumeration of SP
cells. The levels of IL-6 and VEGF were determined by enzyme-linked immunosorbent assay.

RESULTS

Trained individuals had higher oxygen utilization and significantly longer exercise times than untrained
individuals. Following exercise, significant increases were observed in Hgb, Hct, Plt, SP cell numbers, and
IL-6 levels. These changes occurred in both trained and untrained individuals of both genders. No
significant change in WBC numbers or VEGF levels was observed. Although circulating SP cell numbers
were significantly increased, the "quality" of SP cells, defined by the ratio of lower SP to upper SP cells,
was unchanged. Increases in SP cells did not correlate with cytokine levels.

CONCLUSION

Exercise increased Hgb, Hct, and Plt numbers, circulating SP cell numbers and IL-6 levels in young,
healthy individuals of both genders and all fitness levels. These changes in hematologic, hematopoietic,
and cytokine parameters, suggest that exercise can have a physiologic impact by potentially mobilizing
stem cells and thereby enhancing tissue repair mechanisms.

Article #4

“Exercise Is Medicine: Restoring Function in Older Adults with Hematologic Malignancy”

Ashley E. Rosko, MD , Sarah A Wall, MD , Carolyn Presley, MD , ReNea Owens , Desiree Jones , Ying
Huang, MA, MS , Michelle Naughton, PhD MPH

Abstract

Background: Exercise programs are proven to positively impact physical fitness, quality of life, and late
toxicities in cancer patients, and many recent reports document the benefit of exercise in patients with
diverse cancers.1-3 However, such programs are underutilized in patients with hematologic
malignancy.2,3 As anemia and thrombocytopenia associated with hematologic diseases are risk factors
for falls and bleeding complications, exercise has not been routinely recommended. Thus, exercise
programs have yet to gain traction in patients with hematologic malignancy and are rarely seen as a
preventative measure for functional decline. Of critical importance, functional decline is not an
inevitable part of illness or aging and is potentially modifiable.4,5 Here, we identified older adults with
functional decline and incorporated a preventative exercise program to attenuate complications
associated with disease- and therapy-related de-conditioning.

Methods

This is a single center, pilot prospective study of older adults (≥60 years) with hematologic malignancy
actively receiving chemotherapy. Patients enrolled had mild or moderate impairments in physical
function, as defined by a score ≤9 on the Short Physical Performance Battery (SPPB). The SPPB is an
objective, validated tool used to capture at risk patients and has been shown to be prognostic in
predicting decline in function, re-hospitalization, and mortality.6 The primary objective was to assess the
feasibility of implementing a structured exercise program; including recruitment and retention,
adherence, sustainability, adverse events and implementation challenges. Reasons why patients decline
exercise participation were also tracked. The Otago Exercise Program (OEP) has been found to be an
effective exercise regimen to improve functional balance, muscle strength, and prevent fall-related
injury and mortality.8 The OEP is a structured combination of physical therapist prescribed
individualized exercise plans with home-based exercise, demonstrated to improve balance and
functional decline.9 The OEP focuses on strengthening, balance retraining, and walking.

Results

Older adults actively receiving chemotherapy with a median age of 75.5 (62-83) with hematologic
malignancy (myeloma=18, NHL=6, leukemia=5) were enrolled. Chemotherapy regimens were variable
(e.g. R-EPOCH, venetoclax, IMiDS, proteasome inhibitors, bone marrow transplant). Patients were
approached (n=63) for participation of a structured exercise program and a target accrual of n=30 was
achieved over 17 months. Reasons for declining participation included travel (n=13), inconvenience
(n=12), not appropriate (n=5) or concern for side effects/cost/uninformed (n=3). There was no
relationship with distanced traveled and exercise completion, R=-0.01 (p=0.94). Adherence was
excellent with all 8 sessions complete (n=18) or 7 sessions complete (n=4), at time of analysis. Geriatric
assessment factors were analyzed at baseline (Visit 1) and following 4 months of exercise (Visit 2).
Physical health scores as measured by the MOS-PFS increased significantly [MOS-PFS: V1=55 (0-100),
V2=67.5 (30-100), p=0.005], where patient reported KPS were similar [KPS V1=80 (40-100), V2=85 (60-
100), p=0.065]. Importantly, objective measures of physical function improved to normal scores by visit
2 [SPPB V1=7(0-11), V2=11(2-12), p<0.001]. Moreover, quality of life scores by PROMIS demonstrated
improvement in physical health symptoms. No adverse events were attributable to exercise.
Conclusions

In this pilot study evaluating a structured exercise program for older adults undergoing chemotherapy,
physical deficits normalized for patients, resulting in improved subjective and objective measurements
of functional capacity. The program was feasible, sustainable and adherence was optimal. Here we
demonstrate that exercise programs can attenuate complications associated with disease- and therapy-
related de-conditioning and are feasible in older adults.

Article #5

“The Effects of Acute Aerobic and Anaerobic Exercise on Blood Parameters”

TulinAtan&HasanAlacam

Abstract

The study attempts to investigate the effect of acute aerobic and anaerobic exercise on blood
parameters and to determine whether blood parameters change between aerobic and anaerobic
exercise. To achieve the objectives of this study, 25 male athletes participated in the research. Aerobic
(Shuttle run test) and anaerobic (Running aerobic) Sprint test (RAST) exercise test were applied to the
test subjects with a one-week interval. Before the exercise (resting), 1 minute and 60 minutes after both
exercises’ protocol, subjects’ blood samples were taken to determine the blood hematologic values. A
rise in the values was observed in 1 minute after the exercise, but 60 minutes after the exercise it was
observed that most of the hematologic parameters returned to the resting levels. It was found out that
the effect of aerobic and anaerobic exercise on hematologic blood parameters was mostly similar.

Topic 2: Effects of Anticoagulants on Platelets

Article #1

“The Use of Anticoagulation in Patients With Thrombocytopenia”

AmarisBalitsky, MD, MSc

Adult Hematology Resident
Michael G. DeGroote School of Medicine; McMaster University, Hamilton, Ontario, Canada
Donald Arnold, MD, MSc
Director, McMaster Centre for Transfusion Research
Michael G. DeGroote School of Medicine; McMaster University, Hamilton, Ontario, Canada
Published on: April 24, 2018

The need for anticoagulation in patients with thrombocytopenia is a common, vexing problem and a
scenario in which clinicians have to commit to therapy without the benefit of solid trial data. Instead,
the decision to administer or withhold anticoagulation depends on the assessed risks of thrombosis and
bleeding. To make an informed decision, patients and clinicians must consider the clinical context, the
presence of additional risk factors, and the potential consequences of thrombosis and bleeding.
Management decisions result in a trade-off since administration of anticoagulation will increase the risk
of bleeding, and omission of anticoagulation will increase the risk of thrombosis. In addition to balancing
the absolute risks for individual patients, several principles should be considered when deciding on
anticoagulation in the setting of thrombocytopenia: 1) a low platelet count does not protect from
thrombosis; and 2) in general, thrombotic complications are more dangerous than bleeding
complications.

Patient 1

A 75-year-old woman with immune thrombocytopenia (ITP) develops atrial fibrillation with a CHADS2
score of 3. Her platelet count is 19 × 109/L. You contemplate administering anticoagulation.

As the population ages, the incidence of atrial fibrillation in patients with ITP will continue to increase.1
Additionally, as the management of stroke prevention becomes more aggressive,2 the need for
anticoagulation in patients with ITP will become a more common occurrence. In general, older ITP
patients have an increased annual risk of bleeding compared with younger patients: 10.4 percent in
those older than 60 years compared with 0.4 percent in those younger than 40 years.3 In a systematic
review of adults with ITP, the overall risk of severe bleeding was 9.6 percent (95% CI, 4.1-17.1%)4 and
the risk of intracranial hemorrhage was approximately 1 to 2 percent.4,5 Conversely, ITP patients may
have an increased risk of thrombosis compared with age-matched controls,6,7 and some ITP
treatments, including intravenous immune globulin8 and thrombopoietin receptor agonists,9,10 have
been associated with an increased thrombotic risk.

Data on the use of anticoagulation in ITP are lacking; thus, treatment decisions should be based on the
best available evidence, typically from studies in nonthrombocytopenic patients and patient values. For
this patient, stroke prevention would likely take priority at the expense of bleeding,11 especially since
the risk of thrombosis (including stroke) may be particularly high in patients with ITP,12 and the
bleeding risk can be mitigated by improving the thrombocytopenia. For patients with normal platelet
counts and a CHADS2 score of 3, the annual risk of stroke is 5.9 percent (95% CI, 4.6-7.3).13 In the
context of ITP and ITP treatments, that annual risk might be approximately twofold higher. With full-
dose anticoagulation (e.g., apixaban) in non-ITP patients, the annual risk of stroke is reduced to 1.3
percent14; the risk reduction is likely to be similar or greater in patients with ITP. The risk of major
bleeding with apixaban is approximately 2.1 percent for nonthrombocytopenic patients. Add to that the
risk of bleeding attributable to thrombocytopenia (approximately 10%), and the risk of major bleeding
for this patient might be closer to 15 percent4; however, raising the platelet count above 30 to 50 ×
109/L would partially mitigate her risk of bleeding.

Given the risks of bleeding and stroke in the patient, the treatment approach was to increase the
platelet count using ITP therapies (starting with low-dose corticosteroids) until it is above 50 × 109/L,
and then to administer anticoagulation.

Patient 2

A 34-year-old man who recently underwent autologous stem cell transplantation for relapsed diffuse
large B-cell lymphoma develops deep vein thrombosis in his right leg. His platelet count is 15 × 109/L.
You contemplate administering anticoagulation.

Cancer-associated thrombosis is common, occurring in up to 19 percent of patients depending on the

tumor type and cancer treatments.15,16 Anticoagulation is required to improve immediate and long-
term symptoms and to reduce the risk of recurrence. In patients without cancer, the risk of recurrence
after unprovoked venous thromboembolism (VTE) is 5 percent at one year.17 The risk of recurrence is
higher in patients with cancer — 20.7 percent (95% CI, 15-25.8%) at one year as reported in one
study.18 However, risk estimates are limited because anticoagulation is rarely stopped, and life
expectancy is reduced in this patient population. On the other hand, the risk of bleeding on
anticoagulants in patients with cancer is also increased — up to 12.4 percent (95% CI, 6.5%-18.2%) at
one year18 and even higher due to the severe thrombocytopenia.

In terms of management approach, in this patient, his one-year risk of VTE recurrence is likely higher
than his risk of bleeding, the thrombocytopenia is anticipated to be short-lived, the consequences of
thrombosis are more dangerous than bleeding in general, and his platelet counts and bleeding
symptoms can be closely monitored, the decision was made to administer a platelet transfusion and
start anticoagulation once the platelet count is greater than 50 × 109/L. Low-molecular weight heparin
(LMWH) is recommended for patients with cancer-associated thrombosis19 and the dose can be
adjusted for severe thrombocytopenia.20 Data from a recent randomized trial supported the use of the
direct oral anticoagulant edoxaban for patients with cancer-associated VTE21; however, patients with
thrombocytopenia were excluded from that trial and the generalizability of those data is limited.

Future Research: Clinical trials are needed to address pressing clinical questions related to the use of
anticoagulation in patients with thrombocytopenia. For example, in patients with ITP (platelets <50 ×
109/L) and atrial fibrillation, what is the risk of severe bleeding with full-dose anticoagulation? In
designing a trial to address this question, patients could be stratified by the severity of the
thrombocytopenia (bleeding risk) and CHADS2 score (thrombotic risk); the intervention could be to
administer or withhold anticoagulation; and the primary outcome could be bleeding (as assessed by an
ITP-specific bleeding tool22). While a randomized trial design could be used, the feasibility and buy-in
from clinicians and patients would be important limitations. An alternate approach could be a
prospective longitudinal registry, where the decision to administer or withhold anticoagulation is made
by the clinician and patient as per clinical practice, and bleeding and thrombosis outcomes can be
captured over time. Registries and other well-designed observational studies are needed therefore to
inform these difficult management scenarios.

References

Segal JB, Powe NR. Prevalence of immune thrombocytopenia: analyses of administrative data. J
ThrombHaemost. 2006;4:2377-2383.

McIntyre WF, Healey J. Stroke prevention for patients with atrial fibrillation: beyond the guidelines. J Atr
Fibrillation. 2017;9:1475.

Cortelazzo S, Finazzi G, Buelli M, et al. High risk of severe bleeding in aged patients with chronic
idiopathic thrombocytopenic purpura. Blood. 1991;77:31-33.

Neunert C, Noroozi N, Norman G, et al. Severe bleeding events in adults and children with primary
immune thrombocytopenia: a systematic review. J ThrombHaemost. 2015;13:457-464.

Arnold DM, Nazy I, Clare R, et al. Misdiagnosis of primary immune thrombocytopenia and frequency of
bleeding: lessons from the McMaster ITP Registry. Blood Adv. 2017;1:2414-2420.

Feudjo-Tepie MA, Le Roux G, Beach KJ, et al. Comorbidities of idiopathic thrombocytopenic purpura: a
population-based study . AdvHematol. 2009;2009:963506.

Doobaree IU, Nandigam R, Bennett D, et al. Thromboembolism in adults with primary immune
thrombocytopenia: a systematic literature review and meta-analysis. Eur J Haematol. 2016;97:321-330.

Paran D, Herishanu Y, Elkayam O, et al. Venous and arterial thrombosis following administration of
intravenous immunoglobulins. Blood Coagul Fibrinolysis. 2005;16:313-318.

Dultz G, Kronenberger B, Azizi A, et al. Portal vein thrombosis as complication of romiplostim treatment
in a cirrhotic patient with hepatitis C-associated immune thrombocytopenic purpura. J Hepatol.
2011;55:229-232.

Przespo E, Elefante A. Deep vein thrombosis associated wit a single dose of romiplostim in a high-risk
patient. Am J Health Syst Pharm. 2012;69:131-133.
Bed Freedman S, Gersh BJ, Lip GY. Misperceptions of aspirin efficacy and safety may perpetuate
anticoagulant underutilization in atrial fibrillation. Eur Heart J. 2015;36:653-656.

Balitsky AK, Kelton JG, Nazy I, et al. Use of anticoagulation in patients with immune thrombocytopenia.
Blood. 2017;130:2313.

Gage BF, Waterman AD, Shannon W, et al. Validation of clinical classification schemes for predicting
stroke: results from the National Registry of Atrial Fibrillation. JAMA. 2001;285:2864-2870.

Granger CB, Alexander JH, McMurray JJ, et al. Apixaban versus warfarin in patients with atrial fibrillation.
N Engl J Med. 2011;365:981-992.

Lyman GH, Khorana AA, Falanga A, et al. American Society of Clinical Oncology guideline:
recommendations for venous thromboembolism prophylaxis and treatment in patients with cancer. J
ClinOncol. 2007;25:5490-5505.

Streiff MB, Holmstrom B, Ashrani A, et al. Cancer-associated venous thromboembolic disease, version
1.2015. J NatlComprCancNetw. 2015;13:1079-1095.

Kearon C, Akl EA, Comerota AJ, et al. Antithrombotic therapy for VTE disease: Antithrombotic therapy
and prevention of thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical
Practice Guidelines. Chest. 2012;141:e419S-e496S.

Prandoni P, Lensing AW, Piccioli A, et al. Recurrent venous thromboembolism and bleeding
complications during anticoagulant treatment in patients with cancer and venous thrombosis. Blood.
2002;100:3484-3488.

Lee AY, Levine MN, Baker RI, et al. Low-molecular-weight heparin versus a coumarin for the prevention
of recurrent venous thromboembolism in patients with cancer. N Engl J Med. 2003;349:146-153.

Mantha S, Miao Y, Wills J, et al. Enoxaparin dose reduction for thrombocytopenia in patients with
cancer: a quality assessment study. J Thromb Thrombolysis. 2017;43:514-518.

Raskob GE, van Es N, Verhamme P, et al. Edoxaban for the treatment of cancer-associated venous
thromboembolism. N Engl J Med. 2018;378:615-624.

Page LK, Psaila B, Provan D, et al. The immune thrombocytopenic purpura (ITP) bleeding score:
assessment of bleeding in patients with ITP. Br J Haematol. 2007;138:245-248.
Article #2

“Influence of different anticoagulants on platelet aggregation in whole blood; a comparison between

citrate, low molecular mass heparin and hirudin.”

Author information

Wallén NH1, Ladjevardi M, Albert J, Bröijersén A.

Department of Laboratory Medicine, Karolinska Institute & Hospital, Stockholm, Sweden.

Abstract

Anticoagulants used for platelet function studies in vitro may affect platelet responsiveness. In the
present study we compared the influence of three different anticoagulants, sodium citrate, low
molecular mass heparin, and recombinant hirudin, on platelet aggregation in whole blood in vitro, using
impedance aggregometry. ADP and collagen induced aggregation was significantly lower in citrated
blood compared to hirudin treated blood, reflecting the importance of extracellular calcium for platelet
function. Inhibition of platelet aggregation by aspirin, was more pronounced in citrated blood compared
to hirudin treated blood, in agreement with the concept of artifactually enhanced thromboxane
generation in media containing low extracellular calcium levels. In blood anticoagulated with low
molecular mass heparin, platelet aggregation to collagen tended to be enhanced as compared to hirudin
treated blood, whereas platelet responses to ADP at a high concentration were slightly reduced. Of the
anticoagulants investigated, the selective thrombin inhibitor hirudin is the most suitable anticoagulant
for studies of platelet aggregation in vitro in whole blood.

Article #3

“Effects of different anticoagulants on human platelet size distribution and serotonin (5-HT) induced
shape change and uptake kinetics.”

Malmgren R, Beving H, Olsson P.

Abstract

The effect of collecting blood with disodium ethylene diaminetetraacetate (EDTA), citrate (NAC) or acid
sodium citrate-dextrose (ACD) as anticoagulants on platelet count and size distribution was investigated.
No difference between the three preparations regarding platelet count was found in whole blood.
Preparation of platelet-rich plasma (PRP) significantly reduced the platelet count in NAC-PRP (p less than
0.01) to a value of 288 X 10(9)/l compared to those of 365 X 10(9)/l and 368 X 10(9)/l in EDTA and ACD
blood respectively. A significant shift in the platelet size in EDTA-PRP towards larger cell volumes was
observed. There were no differences in the size distribution pattern between NAC-PRP and ACD-PRP in
spite of the differences in platelet count. Platelet 5-HT uptake kinetics in EDTA-PRP showed a 50 per
cent reduction in both Km and Vmax compared to that in ACD-PRP. The study shows that the receptor
mediating 5-HT induced shape change has a direct opposite pH dependence than that of the 5-HT
carrier. Interference of receptor-mediated responses in 5-HT uptake studies in human platelets is clearly
minimized at a lowered pH. The finding is probably of importance in disorders associated with platelet
hyperaggregability.

Article #4

“The effect of anticoagulants on the quality and biological efficacy of platelet-rich plasma”

Author links open overlay panelHuaLeiLaiGuiRanXiao

Show more

https://doi.org/10.1016/j.clinbiochem.2009.06.012Get rights and content

Abstract

Objectives

This study investigated the effect of anticoagulants on platelet-rich plasma (PRP) quality to determine
the appropriate anticoagulants for PRP production.

Design and methods

This study was carried out at the Plastic Surgery Hospital of Peking Union Medical College. The
microstructure of platelets collected with heparin, citrate, acid citrate dextrose (ACD) and citrate-
theophylline-adenosine-dipyridamole (CTAD) was observed. The extent of spontaneous activation of
platelets was detected by measuring sP-selectin in plasma. The amount of TGF-β1 released from PRP
and the effect of PRP on cell proliferation were also studied.

Results

ACD and CTAD were superior to heparin and citrate in maintaining the integrity of platelet structures
and preventing the platelet spontaneous activation. ACD-PRP and CTAD-PRP released more TGF-β1 and
significantly enhanced the proliferation of human marrow stromal cells compared to heparin-PRP and
citrate-PRP.
Conclusions

The PRP quality was closely related to the type of anticoagulants. ACD and CTAD are appropriate
anticoagulants for PRP production.

Article #5

“Differences between the effects of EDTA and citrate anticoagulants on platelet count and mean
platelet volume”

R.L. McSHINEFIMLS P.C. DAS MD, PhD, FRCPath C.TH. SMIT SIBINGA MD, PhD B. BROZOVIC MD, PhD,
FRCPath

First published: September 1990 https://doi.org/10.1111/j.1365-2257.1990.tb00038.x Cited by: 32

ePDFPDFTOOLS SHARE

Abstract

Summary Platelet counts on whole blood samples collected into tripotassium salt of EDTA, trisodium
citrate (Na3 citr), citrate phosphate dextrose adenine formula 1 (CPDA‐1) and acid citrate dextrose
formula A (ACD‐A), all showed a statistically significant drop (P < 0.01) after 1 h standing at room
temperature (RT) as compared with the immediate (within 30 min) counts. After 1 h the enumeration
became stable in the EDTA samples but the drop continued up to 4–6 h in those samples taken into
citrate. The decreases in citrate were significant (18–30%, P < 0.001). The addition of EDTA (1.5 mg/ml)
to the citrated samples after the sixth hour count created a significant rise (6–22%, P < 0.01) in the
counts between the sixth and the seventh hour. Our observations show that platelet counts in citrated
blood samples arc lower than those in EDTA and highlight the necessity to present citrated samples
mixed with dried EDTA when characterizationor quality control of blood and blood components is
required. Analysis of the mean platelet volume (MPV) showed significantly lower values (6–13%, P <
0.05) in the citrated samples as compared to the same samples in EDTA, and a significant increase (4–
6%, P < 0.01) on the addition of EDTA to the citrated samples after the sixth hour analysis.
Topic 3: Correlation between the climatic factors and the pathogenesis of
deep vein thrombosis

Article # 1

“Correlation between the climatic factors and the pathogenesis of deep vein thrombosis”

Abstract

Correlation between the climatic factors and the pathogenesis of deep vein thrombosis Damnjanović Z1,
Jovanović M1,2, Stojanović M2,3 1 Vascular Surgery Department, Clinical Centre of Niš 2 Medical Faculty
of Niš 3 General Surgery Department, Clinical Centre of Niš Niš, Serbia Abstract There are numerous
researches dealing with the correlation between the seasons and climatic factors and the pathogenesis
of deep vein thrombosis (DVT). The presented researches show an undoubted correlation between the
climatic factors and the pathogenesis of DVT. In the majority of researches, retrospection is noted as a
disadvantage. Further prospective researches could aim on testing the correlation between both
climatic and thrombotics factors and the pathogenesis of DVT. This may additionally clarify the
pathophysiological mechanism of the DVT incidence and contribute to the prevention and treatment of
risk groups of patients in certain periods of the year. Hippokratia 2013; 17 (3): 203-206

Introduction

There are numerous researches dealing with correlation between the seasons and climatic factors and
pathogenesis of deep vein thrombosis (DVT). The published results of the researches differ, which leads
to further studies. It is assumed that the obtained results are controversial due to different methodology
applied to conduct the researches, as well as to the fact that the influence of climatic factors is
geographically dependent.

Correlation between seasons and pathogenesis of DVT Only a few conducted researches show the
absence of correlation between the seasons and pathogenesis of DVT. In Geneva, Bounameaux et al1
conducted a retrospective study which included 7303 patients registered during the period from 1989 to
1994, with a suspected DVT. The presence of DVT was recorded in each seventh patient out of 300
patients with suspected DVT. The results of their research showed that there was no seasonal or
monthly pattern concerning the occurrence of DVT for patients both with suspected and confirmed DVT.
A retrospective research conducted by Galle et al, in Belgium2 during the period from 1982 to 1995,
included 512 patients with a diagnosed lower limb DVT, also showed no correlation between the
climatic factors and lower limb DVT. In their retrospective research in USA, which included data for a
twenty-one-year period, Stein et al3 showed the absence of the seasonal character of DVT incidence.
The absence of a correlation between the seasons and DVT incidence was also shown by Lee et al4 in a
research which included 2774 patients with a diagnosed DVT in 2002 in Taiwan. On the other hand,
studies showing a correlation between the seasons and pathogenesis of DVT are far more numerous. A
retrospective research by Boulay et al5 in France, which included 65081 patients with a diagnosed DVT,
showed that the number of patients is far larger in winter than in the summer. The incidence of DVT for
patients with protein C or protein S insufficiency was more frequent in the winter6 . In Austria, Fink et
al7 conducted a research which included 905 patients with a diagnosed lower limb DVT. The research
showed a seasonal pattern in the incidence of lower limb DVT, which was significantly more frequent
during the cold period of the calendar year (October – March). This study, which observed a correlation
between the seasons and the location of lower limb DVT, showed that DVT below the knees was more
frequent during the cold period of the year, while DVT above the knees was more frequent during the
warm period (April – September). Manfredini et al8 conducted a retrospective research which included
2119 patients with a diagnosed DVT, according to the data provided by 25 Italian hospitals, for the
period from 2002 to 2004. The results showed that the incidence of DVT followed a rhythmical pattern,
with its peak in September and October, and was the most frequent for men aged over 40, patients who
had previously suffered a DVT and in immobilized patients. Brown et al9 conducted a retrospective
research which included 37336 patients with DVT, based on data obtained from Scottish hospitals for a
twenty year period. The results proved a seasonal pattern of DVT incidence, with its peak in the winter.
Dentali et al10,11 conducted researches as well as a meta-analysis which confirmed the seasonal
character of DVT with its peak during the winter in January. In this meta-analysis, which included about
35,000 patients, 12 studies implied a research on the seasonal variations of DVT, while 10 studies
observed monthly variations of DVT. The study conducted by Jang et al12 in Korea, included 1495
patients with DVT during the period from 2001 to 2010 and confirmed the seasonal character of DVT
with its peak during the winter and in January. A retrospective research on the territory of South Serbia
showed a seasonal pattern in the incidence of the idiopathic lower limb DVT with the highest frequency
in the cold period of the year (October – March) and the peak in January13. There are different
explanations for the seasonal character of DVT. An acute, mainly respiratory infection, which is more
frequent in the winter, increases the risk for DVT incidence14. Infection increases the concentration of
fibrinogen, anticardiolipid antibodies and the C protein level, which leads to hypercoagulability that can
be the reason for the DVT incidence15-17. In this respect, Masoti et al18 conducted a research which
showed a statistically significantly higher level of C reactive protein, D–dimer and the platelets during
the winter than in the summer, whereas Keatinge et al19 showed that the level of fibrinogen and the VII
c coagulation factor were higher in the winter than during the summer period. During the cold periods,
peripheral vasoconstriction and decreased physical activity may also be one of the reasons for higher
frequency of DVT during the winter7 .

Correlation between atmospheric temperature and the pathogenesis of DVT

Cold conditions make changes in the erythrocytes quality and in the number of leucocytes, increase the
number of granulocytes and decrease the number of lymphocytes, which indicates the possibility of a
potential cause for the development of inflammation and hypercoagulability20-21. Exposure to low
temperature in the winter period can be a risk factor for DVT incidence22. Chung et al23 conducted a
study in 24 centers from 17 countries from Africa, Asia, Europe and South America (including the
Caribbean). The study showed that a change of temperature for 5o C was not related to DVT incidence.
A research conducted by Brown et al9 showed that the seasonal pattern in the incidence of DVT was
related to the minimum and maximum temperature during a calendar year. Explanations concerning the
effect of temperature on DVT can be found in the results obtained from experimental researches.
Marcer et al24 showed that a shortterm exposure to cold performed by healthy volunteers, leads to
haemoconcentration caused by the increase in the number of erythrocytes and granulocytes. Kawahara
et al25 reported that exposure to cold conditions performed by healthy volunteers resulted in the
increased activity of platelets, being thus a potential factor affecting the pathogenesis of DVT.

Correlation between atmospheric pressure and the pathogenesis of DVT

An experimental study conducted by LaCroix et al26 showed that the change of atmospheric pressure
did not lead to hemostasis disorder. Esquenet et al27 proved a correlation between atmospheric
pressure and DVT. Brown et al9 reported that each decrease of atmospheric pressure for 10 millibars,
nine days before DVT was diagnosed, was related to the increase of the DVT incidence by 2.1%. A
retrospective study which included patients from Niš, showed a correlation between the incidence of
lower limb DVT and the increase of atmospheric pressure. Each change of atmospheric pressure for 1
millibar on the day of the DVT diagnosis and 7 days before that, was related to the increase of the
incidence of lower limb DVT of 5.1%, as well as the increase of the incidence of the above-knee DVT of
5.9%28.

Correlation between air pollution and the pathogenesis of

DVT Researches dealing with the correlation between air pollution and the incidence of DVT reported
the existence of such correlation. Baccarelli et al29 were the first to study the relation between DVT and
air pollution. Compared to the control group, patients with DVT had higher exposure to particles with
the aerodynamic diameter of 10 micrometers. The second study conducted by Baccarelli et al30 showed
a correlation between air pollution and DVT in subjects residing near large traffic crossroads. A research
conducted in Santiago, Chile31, showed that the increased concentrations of ozone, sulphordioxide,
nitrodioxide and the increased number of particles smaller than or the same size as the size of the
aerodynamic diameter of 2.5 micrometers, were related to the increased incidence of DVT. There are
many hypotheses explaining this correlation. A direct effect would be explained by hypercoagulability of
vein circulation. An indirect effect of air pollution could be hypothetically explained by the increased
number of lung and heart diseases32,33 which increase the possibility for DVT incidence.

Correlation among rain fall and wind speed with pathogenesis of DVT

Brown et al9 showed that increased rain fall by 1 mm and increased wind speed by 1 nod, 10 days
before diagnosing DVT, caused an increase of the DVT incidence by 0.8% and 0.6% respectively. The
authors stated that the mechanisms of correlation between the climatic factors and DVT are so far
unknown and deserve further clarification. The main characteristics of the studies which connect the
influence of the climatic factors and the pathogenesis of DVT, are shown in Table 1.
Conclusion

The presented researches show a positive correlation between climatic factors and the pathogenesis of
DVT. In the majority of the researches, retrospection is a disadvantage. Therefore, further prospective
studies should aim on testing the correlation between both the climatic and the thrombotic factors and
the pathogenesis of DVT. This would additionally clarify the pathophysiological mechanism of the DVT
incidence and contribute to the prevention and treatment of risk groups of patients in various periods of
the year.

Conflict of Interest

The authors have no competing interests to declare.

References

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284-285.

2. Galle C, Wautrecht JC, Motte S, Le Minh T, Dehon P, Ferreira J, et al. [The role of season in the
incidence of deep vein thrombosis]. J Mal Vasc. 1998; 23: 99-101.

3. Stein PD, Kayali F, Olson RE. Analysis of occurrence of venous thromboembolic disease in the four
seasons. Am J Cardiol. 2004; 93: 511-513.
4. Lee CH, Cheng CL, Lin LJ, Tsai LM, Yang YH. Epidemiology and predictors of short-term mortality in
symptomatic venous thromboembolism. Circ J. 2011; 75: 1998-2004.

5. Boulay F, Berthier F, Schoukroun G, Raybaut C, Gendreike Y, Blaive B. Seasonal variations in hospital

admission for deep vein thrombosis and pulmonary embolism: analysis of discharge data. BMJ. 2001;
323: 601-602.

6. Bilora F, Boccioletti V, Manfredini E, Petrobelli F, Tormene D, Simioni P, et al. Seasonal variation in the
incidence of deep vein thrombosis in patients with deficiency of protein C or protein S. Clin Appl Thromb
Hemost. 2002; 8: 231-237.

7. Fink AM, Mayer W, Steiner A. Seasonal variations of deep vein thrombosis and its influence on the
location of the thrombus. Thromb Res. 2002; 106: 97-100.

8. Manfredini R, Imberti D, Gallerani M, Verso M, Pistelli R, Aqueno W, et al. Seasonal variation in the
occurrence of venous thromboembolism: data from the MASTER Registry. Clin Appl Thromb Hemost.
2009; 15: 309-315.

9. Brown HK, Simpson AJ, Murchison JT. The influence of meteorological variables on the development
of deep venous thrombosis. Thromb Haemost. 2009; 102: 676-682.

10.Dentali F, Manfredini R, Ageno W. Seasonal variability of venous thromboembolism. Curr Opin Pulm
Med. 2009; 15: 403- 407

11. Dentali F, Ageno W, Rancan E, Donati AV, Galli L, Squizzato A, et al. Seasonal and monthly variability
in the incidence of venous thromboembolism. A systematic review and a meta-analysis of the literature.
Thromb Haemost. 2011; 106: 439-447.

12.Jang MJ, Kim HJ, Bang SM, Lee JO, Yhim HY, Kim YK, et al. Seasonal variation in the occurrence of
venous thromboembolism: a report from the Korean Venous Thromboembolism Working Party. Thromb
Res. 2012; 13: e199-e202.

13.Damnjanovic Z, Jovanovic M, Ilic N, Bogdanovic D, Kudumovic M, Kamenov A, et al. Seasonal

variations in the incidence of idiopathic lower extremity deep vein thrombosis on the territory of South
Serbia. HealthMED. 2012; 6: 2477-2481.

14.Malone PC, Agutter PS. To what extent might deep venous thrombosis and chronic venous
insufficiency share a common etiology? Int Angiol. 2009; 28: 254-268.

15.Smeeth L, Cook C, Thomas S, Hall AJ, Hubbard R, Vallance P. Risk of deep vein thrombosis and
pulmonary embolism after acute infection in a community setting. Lancet. 2006. 367: 1075-1079.

16.Stout RW, Crawford V. Seasonal variations in fibrinogen concentrations among elderly people.
Lancet. 1991; 338: 9-13.
17.Macko RF, Ameriso SF, Gruber A, Griffin JH, Fernandez JA, Barndt R, et al. Impairments of the protein
C system and fibrinolysis in infection-associated stroke. Stroke. 1996; 27: 2005- 2011.

18.Masotti L, Ceccarelli E, Forconi S, Cappelli R. Seasonal variations of pulmonary embolism in

hospitalized patients. Respir Med. 2005; 99: 1469-1473.

19.Keatinge WR, Coleshaw SR, Cotter F, Mattock M, Murphy M, Chelliah R. Increases in platelets and red
cell counts, blood viscosity, and arterial pressure during mild surface cooling: factors in mortality from
coronary and cerebral thrombosis in winter. Br Med J (Clin Res Ed). 1984; 289: 1405-1408.

20.Neild J, Syndercombe-Court D, Keatinge WR, Donaldson GC, Mattock M, Caunce M. Cold-induced

increases in erythrocyte count, plasma cholesterol and plasma fibrinogen of elderly people without a
comparable rise in protein C or factor X. Clin Sci (Lond). 1994; 86: 43-48.

21.Hawes AS, Fischer E, Marano MA, Van Zee KJ, Rock CS, Lowry SF, et al. Comparison of peripheral
blood leukocyte kinetics after live Escherichia coli, endotoxin or interleukin-1 alpha administration.
Studies using a novel interleukin-1 receptor antagonist. Ann Surg. 1993; 218: 79-90.

22.Schuh A. [Not good for weak veins. Winter temperature promote thrombus formation]. MMW
Fortschr Med. 2003; 145: 31-32.

23.Chang CL, Shipley M, Marmot M, Poulter N. Lower ambient temperature was associated with an
increased risk of hospitalization for stroke and acute myocardial infarction in young women. J Clin
Epidemiol. 2004; 57: 749-757.

24.Mercer JB, Osterud B, Tveita T. The effect of short-term cold exposure on risk factors for
cardiovascular disease. Thromb Res. 1999; 95: 93-104.

25.Kawahara J, Sano H, Fukuzaki H, Saito K, Hirouchi H. Acute effects of exposure to cold on blood
pressure, platelet function and sympathetic nervous activity in humans. Am J Hypertens. 1989; 2: 724-
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26.LaCroix KA, Davis GL, Schneider DA, Lavoie P, Kintzing E, Waterfield DA. The effects of acute exercise
and increased atmospheric pressure on the hemostatic mechanism and plasma catecholamine levels.
Thromb Res. 1990; 57: 717-728.

27.Esquenet P, Boudet J, Sevestre-Pietri MA, Ganry O, Pietri J. [Effect of meteorological variations on the
emergence of deep venous thrombosis of the leg]. J Mal Vasc. 1997; 22: 244-248.

28.Damnjanović Z, Jovanović M, Bogdanović D, Smiljković I, Ilić N, Damnjanović I. Relationship between

the incidence of idiopathic lower extremity deep vein thrombosis and the location of the thrombus
depending on the changes of atmospheric pressure. Chirurgia (Bucur). 2012; 107: 483-487.

29.Baccarelli A, Martinelli I, Zanobetti A, Grillo P, Hou LF, Bertazzi PA, et al. Exposure to particulate air
pollution and risk of deep vein thrombosis. Arch Intern Med. 2008; 168: 920-927.
30.Baccarelli A, Martinelli I, Pegoraro V, Melly S, Grillo P, Zanobetti A, et al. Living near major traffic
roads and risk of deep vein thrombosis. Circulation. 2009; 119: 3118-3124.

31.Dales RE, Cakmak S, Vidal CB. Air pollution and hospitalization for venous thromboembolic disease in
Chile. J Thromb Haemost. 2010; 8: 669-674.

32.Martinelli N, Girelli D, Ciqolini D, Sandri M, Ricci G, Rocca G, et al. Access rate emergency department
for venous thromboembolism in relationship with coarse and fine particulate matter air pollution. PloS
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pollution on blood coagulation. J Thromb Haemost. 2007; 5: 252-260.

Article # 2

“Seasonal variation of venous thromboembolism in the subtropical climate of São Paulo,

Brazil”

Daniela KleinfelderI; Jó Luis AndradeII; Sascha Werner SchlaadIII; Francine Correa CarvalhoIV; Bonno van
BellenV
I
Graduate Program in Vascular Surgery and Angiology, Hospital Beneficência Portuguesa de São Paulo
(BPSP), São Paulo, SP, Brazil
II
Graduate Program in Integrated Vascular Surgery, BPSP, São Paulo, SP, Brazil. Professor, General
Surgery, Faculdade de Medicina, Universidade Estadual do Sudoeste da Bahia (UESB), Vitória da
Conquista, BA, Brazil
III
Graduate Program in Integrated Vascular Surgery, BPSP, São Paulo, SP, Brazil
IV
PhD in Cardiology, Instituto do Coração, Faculdade de Medicina da Universidade de São Paulo (InCOR-
FMUSP), São Paulo, SP, Brazil. Cardiologist, Vascular Surgery and Angiology Service, BPSP, São Paulo, SP,
Brazil
V
Professor, Peripheral Vascular Diseases, Universidade Estadual de Campinas (UNICAMP), Campinas, SP,
Brazil. Head, Vascular Surgery and Angiology Service, BPSP, São Paulo, SP, Brazil. Coordinator, Graduate
Program in Integrated Vascular Surgery, BPSP, São Paulo, SP, Brazil

ABSTRACT

Background: The understanding of the triggering factors of venous thromboembolic disease has
presented important improvements during the last years. External causes may influence its occurrence,
and some climatic factors have also been mentioned to interfere with its occurrence. There are not
studies about these specific interference in the subtropical climate.
Objectives: To determine whether there are seasonal variations in the incidence of venous
thromboembolism in a hospital-based population in São Paulo, Brazil, which has subtropical climate.
Methods: Medical records of patients admitted to Hospital da Beneficência Portuguesa de São Paulo
with the diagnosis of deep venous thrombosis or pulmonary thromboembolism were reviewed from
January 1996 to October 2003. Cases were grouped in trimesters (first trimester = January, February and
March; second trimester = April, May and June; third trimester = July, August and September; fourth
trimester = October, November and December). They were also grouped as to warm and cold months,
according to mean temperature (warm months = October through April; cold months = May through
September).
Results: A total of 955 cases of venous thromboembolism were found during the study period. The
ANOVA test was used for statistical analysis, showing no significant difference in the occurrence of
venous thromboembolism considering the four trimesters. Separate analysis of deep venous thrombosis
and pulmonary embolism incidence showed no differences either. Comparing warm and cold months,
there was an increased incidence of deep venous thrombosis during warm months (p < 0.05, Mann-
Whitney test).
Conclusion: Venous thromboembolism is not clearly related to climatic variations. The influence of
climate and temperature on blood coagulability is poorly understood and needs to be further studied.

Introduction

Venous thromboembolism (VTE) is a major cause of morbidity and mortality in Western countries.1 Only
in the USA more than 100 cases/100,000 people/year are reported.2 In Brazil and in other Latin
American countries, there are few studies on VTE epidemiology.3 Maffei et al. estimated an incidence of
0.6 cases of deep venous thrombosis (DVT) for each 1,000 inhabitants/year in the municipality of
Botucatu.4 Amari et al. found a 6% incidence of pulmonary thromboembolism (PTE) in a necropsy study
at Santa Casa de São Paulo.5

Mean population age has increased, as well as the absolute number of VTE cases.6 Some of these cases
will be manifested as PTE. In England, PTE is the cause of 200,000 deaths/year, accounting for 1/10 in-
hospital deaths7 and is the most common avoidable cause of death in hospitalized patients in the
USA.8 Of all PTE cases, 10% are fatal in less than 1 hour after event onset9 and approximately 5% of
patients with PTE develop pulmonary hypertension.9 Chronic venous stasis syndrome affects 11% of
patients with VTE, evolving to varicose ulcers in 3.7 ± 0.9% of patients in 20 years.9

Much has been done regarding the development of methods and drugs for VTE prophylaxis, and
consequently there has been significant progress regarding understanding of causes and epidemiological
phenomena of the disease.

Many studies have suggested variation of VTE incidence according to seasons of the year.2,710-
16
However, such studies were performed in countries with temperate climate. No study conducted in
subtropical climate was found.

This study aims at analyzing whether there is a difference in VTE incidence according to seasons of the
year at a hospital in São Paulo, located just on the Tropic of Capricorn, which is the limit between
tropical and temperate climate.
Population and methods

A retrospective survey of medical records of patients whose diagnosis of hospitalization or death was
VTE or PTE was performed from January 1996 to October 2003 at Hospital da Beneficência Portuguesa
de São Paulo.

For comparison and analysis, cases were grouped according to trimesters:

- First trimester: January, February and March;
- Second trimester: April, May and June;
- Third trimester: July, August and September;
- Fourth trimester: October, November and December.
The ANOVA test was used in the analysis.
The year was also divided in warm and cold months. Such division was performed considering the mean
monthly temperature ≥ 20 ºC (warm months) and < 20 ºC (cold months), respectively. Warm months are
between October and April, and cold months are between May and September. Data were obtained
from information on the website The Weather Channel17. The Mann-Whitney test was used for
statistical analysis.
Statistical significance was set in p < 0.05.

Results

There were 955 cases of VTE: 453 cases of DVT and 502 cases of PTE (mean of 10.2 cases of VTE per
month, standard deviation of 4.9).

In the first trimester, there were 253 cases of VTE (26.5%): 143 cases of DVT (31.6%) and 110 cases of
PTE (21.9%). In the second trimester, there were 232 cases of VTE (24.3%): 100 cases of DVT (22.1%) and
132 cases of PTE (26.3%). In the third trimester, there were 223 cases of VTE (23.3%): 94 cases of DVT
(20.7%) and 129 cases of PTE (25.7%). In the fourth trimester, there were 247 cases of VTE (25.9%): 116
cases of DVT (25.6%) and 131 cases of PTE (26.1%).

The ANOVA test did not show statistically significant difference in VTE incidence according to the
trimesters. When analyzed individually, there was no statistical significance in relation to PTE and DVT.

In warm months (October-April, mean temperature > 20 ºC) there were 578 cases of VTE (60.5%): 283
cases of PTE (56.4%) and 295 cases of DVT (65.1%). In cold months (May-September, mean temperature
< 20 ºC) there were 377 cases of VTE (39.5%): 219 cases of PTE (43.6%) and 158 cases of DVT (34.8%).
The Mann-Whitney test showed increase in DVT cases in warm months (p < 0.05) (Figure 1)
Discussion

Many studies have reported higher incidence of death and hospitalizations due to coronary disease and
stroke during the winter.10,18 Similarly, some studies have shown higher incidence of fatal PTE in the
winter than in the summer,2,7,11,18 well documented in a meta-analysis of 23 studies involving 11,000
cases.12,13 In an English necropsy study, there was a higher incidence of PTE in the fall and spring.14

Bounameaux et al.10 in 1996 found no seasonal variation in DVT incidence in a retrospective study of
9,208 patients from 1989 to 1994 in Geneva, a city with large temperature variations between the
summer and the winter, as well as the French study by Galle et al., performed in 1998,15 and the
American study by Stein et al.16 The latter evaluated seasonal variations of VTE over a 21-year period in
regions with wide temperature variations and in areas with little climatic difference.

However, Boulay et al.13 in 2001 published a study conducted in France that included 62,237 patients,
showing the existence of a seasonal variation in VTE occurrence, with number of hospitalizations 18 and
22% higher than the mean in cold months for DVT and PTE, respectively.

The likely reasons for seasonal variation were also discussed in many studies on this issue. White
considers that reduction in physical activity in the winter may be associated with development of
DVT.2 Boulay et al.13 speculate that cold-induced vasoconstriction and reduction in physical activity
produce a well documented reduction in blood flow in the lower limbs; in addition, respiratory tract
infections may induce hypercoagulability. Other authors believe that change in temperature is an
indirect factor and that reduction in day duration and, therefore, sunlight interferes with production of
melatonin and coagulability.12 Wilmshurst18 adds that plasmatic concentration of fibrinogen is inversely
related to room temperature, and part of such increase in fibrinogen in the winter may result from
seasonal respiratory infections. In cold conditions, some coagulation factors are increased in vitro such
as platelet count and platelet aggregation. Reduced plasma volume and increased blood viscosity during
exposure to cold tend to cause thrombosis. However, little is known about the seasonal fluctuation of
coagulability.13

In this study there were no differences in incidence for VTE cases according to trimesters, which
correspond to climatic seasons. However, there was in increased incidence in DVT in warm months.
Determination of warm and cold months was based on the mean temperature of all 12 months, which is
20 ºC.17 Such value was not used in other studies, as they were performed in European countries or in
the USA, where temperature variations are higher. They could not be matched with the Brazilian climate
to define the same cut-off point for temperature. There were no studies conducted in Brazil or in
countries with similar climate that could be used in the comparative analysis.

There was no theory in the literature that could explain increased DVT incidence in warmer months.
Absence of correlation between incidence of DVT and PTE is even harder to explain, as a parallelism
could be expected between both phenomena. As it is difficult to explain increased VTE in cold months in
the northern hemisphere, it might be speculated that the heat causes higher dehydration and maintains
people in a more sedentary condition, which in turn might cause increase in DVT.

Little is known about seasonal fluctuation of coagulability, and other variables should be analyzed to
discard the possibility of temperature being only a confounding factor in such studies.
Conclusion

VTE is a disease that has no well established relation with climatic variations. Influence of temperature
on coagulability still needs to be widely studied.

References

1. Arcelus JI, Caprini JA, Monreal M, Suárez C, González-Fajardo J. The management and outcome of
acute venous thromboembolism: a prospective registry including 4011 patients. J Vasc Surg.
2003;38:916-22.
2. White RH. The epidemiology of venous thromboembolism. Circulation. 2003;107(23 Suppl 1):I4-8.
3. Silva MC. Tromboembolismo venoso: epidemiologia e fatores de risco. In: Brito CJ, Duque A, Merlo I,
Murilo R, Fonseca VL, editores. Cirurgia vascular. Rio de Janeiro: Revinter; 2002. p. 1123-34.
4. Maffei FHA. Epidemiologia da trombose venosa profunda e de suas complicações no Brasil. Cir Vasc
Angiol 1998;14:5-8.
5. Amary J, Coli Jr DF, Pereira M, Bailone S. Embolismo pulmonar - levantamento em 13500 necrópsias.
Arq Hosp Santa Casa S Paulo. 1974;20:143-7.
6. Heit JA. Venous thromboembolism epidemiology: implications for prevention and management.
Semin Thromb Hemost. 2002;28 Suppl 2:3-13.
7. Cooke EA, McNally MA, Mollan RAB. Seasonal variations in fatal pulmonary embolism. Several
mechanisms contribute. BMJ. 1995;310:129.
8. Anderson FA, Spencer FA. Risk factors for venous thromboembolism. Circulation. 2003;107(23 Suppl
1):I9-16. 9. Kearon C. Natural history of venous thromboembolism. Circulation. 2003;107(23 Suppl
1):I22-30.
10. Bounameaux H, Hicklin L, Desmarais S. Seasonal variation in deep vein thrombosis. BMJ.
1996;312:284-5
11. Gallerani M, Manfredini R, Ricci L, et al. Sudden death from pulmonary thromboembolism:
chronobiological aspects. Eur Heart J. 1992;13:661-5
12. Allan TM, Douglas AS. Seasonal variation in deep vein thrombosis. Fatal pulmonary embolism is
increased in both autumn and winter. BMJ. 1996;312:1227.
13. Boulay F, Berthier F, Schoukroun G, Raybaut C, Gendreike Y, Blaive B. Seasonal variations in hospital
admission for deep vein thrombosis and pulmonary embolism: analysis of discharge data. BMJ.
2001;323:601-2
14. Green J, Edwards C. Seasonal variation in the necropsy incidence of massive pulmonary embolism. J
Clin Pathol. 1994;47:58-60.
15. Galle C, Wautrecht JC, Motte S, et al. Rôle de la saison dans l"incidence de la thrombose veineuse
profonde. J Mal Vasc. 1998;23:99-101.
16. Stein PD, Kayali F, Olson RE. Analysis of ocurrence of venous thromboembolic disease in the four
seasons. Am J Cardiol. 2004;93:511-3.
17. The Weather Channel [site na Internet]. The Weather Channel Interactive, Inc. Acess:
01/04. http://br.weather.com/weather/climatology/BRXX0232
18. Wilmshurst P. Temperature and cardiovascular mortality. BMJ. 1994;309:1029-30.
Article # 3

“Deep vein thrombosis related to environment”

Auhors:
Salvatore Santo Signorelli
Margherita Ferrante
Agostino Gaudio
Valerio Fiore

Published online on: March 24, 2017 https://doi.org/10.3892/mmr.2017.6395

Abstract

The first-time venous thromboembolism (VTE) is less frequent than other thrombotic events, however,
both the pulmonary embolism (PE) and the deep vein thrombosis (DVT) show a frequent morbidity.
Many factors play as risk situations in determining VTE, and the air exposure to the fine and ultrafine
particulate matter (PM) as PM10, PM2.5, PM0.1 is considered. Epidemiological studies have supported
this association although both the effective burden of the association and the mechanisms are to date
unclear. The PM concentrations and the exposure time are notable as emerging factors. Interestingly,
the seasonal climate variations resulted as effective risk factor for appearance of VTE or DVT. There is a
need to ameliorate the environment by reducing the air pollution at global scale.

Introduction

The incidence of first-time venous thromboembolism (VTE) affects a substantial number of subjects, in
fact it ranges between 62 and 143/100,000/year although it seems to differ ranging between 19 and
50/100,000 persons/years (1,2). Although the VTE incidence is lower than the arterial thromboses
(coronary and carotid arteries) both the pulmonary embolism (PE) and the deep vein thrombosis (DVT)
show morbidity cases. More situations are risk factors for the VTE in individuals. A number of factors are
able to promote the risk for VTE, and many epidemiological and interventional studies have focused on
several favourable situations (3–7). The conclusive statements of these have produced the scores to
know the possible risk for VTE and also to forward the early and useful approach to diagnose and to
manage VTE.

Pathophysiology

The low rate of shear and the raised activity of the coagulative cascade play a role for thrombotic
disease in the venous circle. The low rate of shear and the raised activity of the coagulative cascade play
a role for thrombotic disease in the venous circle. Such effects may comprise the occurrence of
immunologic reaction including the hypersensitivity that can be modulate with different
immunosuppressive treatments (8,9). The coagulative factor VII (FVII) and the tissue factor (TF)
represent the complex able to move other coagulative components inducing the thrombin generation
(T). The T seems to be the key enzyme leading to convert the monomers of fibrinogen (F) to polimers of
fibrin (Fib). In this way amplifying the coagulative cascade through the activation of other coagulative
factors such as V (FV), VIII (FVII) and X (FX) factors.
Air exposure and venous thrombotic disease

In 2004 the American Heart Association recognized the deleterious effect caused by exposure to air
components particularly by the fine particulate matter (PM) on cardiovascular system. The air pollutants
especially the particles sized <10 mm diameter (PM10) have been associated with an increased risk for
cardiovascular events (i.e., myocardial infarction (MI), stroke, arrhythmia and heart failure) (10). Most
recently, based on the negative effect played by the fine and the ultrafine air pollutants on coagulative
balance, several studies have postulated the possible association between exposure to the air pollutants
with the risk and/or appearance of VTE. Epidemiological studies have supported this association
although to date both the effective burden of the epidemiological association and the possible
mechanisms are unclear on the effect in promoting the pathologic link. The size of pollutants is closely
related to the pathogenic activities so far the particulate air pollutants are divided into several groups.
The two main are the coarse component with aerodynamic diameter between 2.5 and 10 mm (PM 10-2.5),
and the finest component with diameter <2.5 mm (PM2.5). These air components are different regarding
their sources and composition. Indeed, while PM2.5 particles result mainly from combustion of fossil
fuels from a variety of activities (e.g., traffic and industry), the PM10-2.5 particles are associated with non-
combustion surface or fugitive releases by a variety of human (e.g., agriculture) and natural (e.g.,
erosion) activities. The PM10-2.5 particles are found preferentially in the upper and larger airways of the
lung, while the PM2.5 particles are found in the smallest airways and in the pulmonary alveoli. The finest
particles (ultrafine particles sized 0.1 mm) can spread even into the systemic circulation throughout the
alveolar-capillary wall. All these particles have shown the capability of disarranging the coagulative
balance, in fact the PMs have been associated to changes in global hemostastic human capability and
also the exposure (short and prolonged) induces a dramatic hypercoagulative situation. Epidemiological
studies have demonstrated that the PM exposure shortened the prothrombin time (PT) and on the
other hand the PM increased the plasma level of Fib. These haemostatic disturbances were associated
to the DVT appearance. Unfortunately, studies have not supported the positive association between the
inhaled pollutants and the DVT (11–13). To explain the confounding results we can consider the role
played by the time of the PM exposure. It was found that prolonged time of exposure to PM over one
year is crucial in inducing risk of DVT. Differently, the short time of PM exposure (one week maximum)
did not result in positive correlation to the DVT. In this regard, experimental study demonstrated that
the direct short time intra-tracheal instillation of the pollutants raised the clot in the arterial bed but not
in the venous circulation (14). However, other experiments have found that high-doses (100 mg or
more) of PM induced in short time such hypercoagulative effects (15). It increased the Fib level and
conversely decreased the level of the C and S antithrombotic proteins. Interestingly, it has been
demonstrated that also a dose of 10 µg of PM10 may induce effects on coagulative balance. When a low-
dose of pollutant was directly instilled into the tracheal space in animals, it caused several
procoagulative effects. In fact a shortened PT was found and conversely an activated PT (aPTT) and the
platelet count, the V, VII and X coagulative factors and the Fib were found increased (11).

How do we explain the confounding results among the association?

It is notable that more experiences have demonstrated that the PM exposure affects the pro-coagulative
inflammatory pathway. Experimental findings found the absence of the PM prothrombotic pathway in
knockout IL-6 mouse, and this result recognized the association between PM exposure and
inflammation as possible major strength in inducing the relationship between the air pollutants and the
venous thrombotic diseases (15). This is a relevant issue to emphaticize the role of the inflammatory
markers in explaining the association between fine and ultrafine pollutants in VTE. In this context, we
must focus on the role played by such minor inflammatory markers as the Fib. The Fib is an acute phase
protein that is usually upregulated during inflammation. It is also a possible coagulative-inflammatory
marker of prothrombotic activation. However, it plays a minor role in clot and it cannot explain alone
the relationship between PM exposure and VTE. Indeed such studies have shown the negative effect of
the short time direct PM exposure in causing activation of coagulative cascade through release of
inflammatory markers (16–21).

Mechanisms to link inflammation with the PM exposure, and with VTE epidemiology

In contrast to the unclear knowledge concerning the mechanisms able to explain the association
between air pollutants and venous thrombosis, an interesting and potential role seems to exist in the
circulating number of the so-called microvescicles (or microparticles that are sized <1 µm derived from
stimulated or apoptotic cells. High number of these microvescicles has been found as circulating both in
subjects chronically exposed to high level of air pollutants and in patients affected by VTE. These
microvescicles negatively charged the phospholipids and TF, consequently, their surfaces attract more
procoagulative factors. Results from studies have highlighted the number and/or procaogaulative
capability of these microvesciscles found in patients with VTE (22,23). It was shown that long (acute,
subacute, chronic) duration of exposure to the pollutants caused different and increased number of the
circulating microvescicles. Current PM (short time) levels were associated with the lower numbers of
circulating microvesicles and with the decreased measurements of the inflammatory parameters. The
chronic PM exposure causes procoagulant tendency as shown by the thrombin generation and by
several markers of surface expression of negatively charged phospholipids (24–26). Upregulation of
procoagulant microvesicles could explain the pathophysiological mechanism underlying the found
association between chronic PM exposure and high number of microvesicles and their procoagulant
activity. Chronic PM exposure window seems to determine such procoagulant changes as the higher
microvesicle numbers have shown. High number of microvescicles both blood-platelet derived and red
blood cell-derived added to the increased microvesicular Annexin V binding reflect the surface
expression of negatively charged phospholipids (mainly phosphatidylserine) (26–29). The role of
inflammation and specific immunity is close debated among several situation also focusing on the
thrombotic process. Although, the link between environment variations with VTE is less established than
to arterial thrombotic events, however, the air pollutants negatively act on several targets (leukocyte,
platelets, coagulative factors, inflammatory markers and endothelial markers). These cells and factors
play roles in pathophysiology both of venous (i.e., DVT) and arterial thrombotic diseases (28).

Effect of climate
A large body of data postulated a seasonal effect on the frequency and/or incidence of VTE. In this
regard, such studies have highlighted on possible reasons, and firstly the haemostatic unbalance was
considered. The effects caused on packed cell volume, on platelet count and on their volume have been
demonstrated (30). Moreover, other factor to explain the seasonal variation of VTE frequency seems to
be a reduced physical activity during the winter. The limited physical activity acts particularly on subjects
(patients) suffering from chronic diseases (i.e., chronic pulmonary insufficiency, heart failure and
malignancies). It also particularly acts on older subjects. It is known that all the aforementioned
situations are usually considered as risk factors for VTE appearance. Furthermore, we must take into
account that the winter low temperature raises the urban motor traffic, and as consequence, it
increases the level of air pollutants (25,31–34). In turn, because the deleterious effect is known of
inhaled pollutants on haemostatic balance we can explain also the seasonal variations of the DVT during
the cold time compared to other seasons. Research performed in many countries and regions have
stated the association between epidemiology of the DVT with the cold climate. Interestingly, a positive
trend for the DVT appearance was found by Manfredini et al (35) in subset of hospitalized patients for
DVT that concomitantly were affected by pulmonary diseases. The chronobiological trend has been
demonstrated most recently by the multicenter study for a thromboembolism registry (MASTER) (36).
These results show that more VTE events appeared during the autumn compared to lower rate of VTE
events in the summer time. The seasonal variation affects the patients with coagulative deficiencies (i.e.,
deficiency of the C and S anticoagulant proteins). As we know the subjects are more prone to
thromboembolism. The frequency of the DVT (and/or VTE) in these subjects significantly increases in the
autumn (November) (37). Results from the Korean registry (38) has shown a raised frequency in DVT
diagnoses both in autumn and winter compared to the hot seasons. To date there is still an active
debate among the effective reasons to associate the climate variations and the environment changes
with the frequency of the DVT. However, we hypothesized that cold climate negatively acts both on the
coagulative balance and on the peripheral vasoconstriction, and it increases the vasoconstrictive tone of
artery-venous shunt (39). All these situations are risk factors for thromboembolic events in venous
circulation, and they may play a role in determining the frequency of VTE and DVT. On the contrary, the
positive effect of the sun exposure on the VTE risk has been demonstrated (40). Reduction of the VTE
risk up to 30% was found in subjects sunbathing both during winter and summer vacations. It is known
that the exposure to ultraviolet light improves the vitamin D status (41). Furthermore, the anticoagulant
capability of the 1,25 vitamin D is known as active metabolite of the vitamin D related to the
upregulation of the thrombomodulin generation and conversely effect on the downregulation of the
tissue factor (42,43). In addition, an inverse correlation was found between the levels of the 25-OH
vitamin D and the plasminogen activator inhibitor-1, and with the tissue-type plasminogen activator
antigen (44). These findings support the etiological effect of climate on the emerging risk and on the
clinical appearance of venous thrombotic disease of lower limbs as venous diseases potentially relate to
seasonal variations.
Conclusions

In conclusion, there is a large body of research focused on pathophysiologic and on epidemiologic

questions, and these have provided consistent evidence on the dangerous effects on the cardiovascular
system originated from current and prolonged inhalation of air pollutants. Particulate matters (PM10,
PM2.5, ultrafine PM) play a crucial role both in determining procoagulant disorders and in promoting an
inflammatory pathway. All these situations play a pathogenetic role on thrombotic diseases and
particularly on VTE. However, the link between environment variations with VTE is less established than
to arterial thrombotic events, however, the air pollutants negatively act on several targets (leukocyte,
platelets, coagulative factors, inflammatory markers and endothelial markers) which play crucial roles in
pathophysiology of the VTE and DVT. We agree with the conclusive remarks given by Emmerechts et
al (45) who considered the exposure to air pollutants as the highest risk factors for thrombotic events.
Therefore, there is a need to reduce such exposure both at individual and global level.

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27 Morel O, Toti F, Hugel B, Bakouboula B, Camoin-Jau L, Dignat-George F and Freyssinet JM:

Procoagulant microparticles: disrupting the vascular homeostasis equation? Arterioscler Thromb
Vasc Biol. 26:2594–2604. 2006.

28 Signorelli SS, Fatuzzo P, Rapisarda F, Neri S, Ferrante M, Conti Oliveri G, Fallico R, Di Pino L, Pennisi
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and oxidative parameters after treatment with propionyl L-carnitine in patients with peripheral
arterial disease requiring haemodialysis. Drugs Aging. 23:263–270. 2006.

29 Chirinos JA, Heresi GA, Velasquez H, Jy W, Jimenez JJ, Ahn E, Horstman LL, Soriano AO, Zambrano
JP and Ahn YS: Elevation of endothelial microparticles, platelets, and leukocyte activation in
patients with venous thromboembolism. J Am Coll Cardiol. 45:1467–1471. 2005.

30 Keatinge WR, Coleshaw SR, Cotter F, Mattock M, Murphy M and Chelliah R: Increases in platelet
and red cell counts, blood viscosity, and arterial pressure during mild surface cooling: factors in
mortality from coronary and cerebral thrombosis in winter. Br Med J (Clin Res Ed). 289:1405–1408.
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31 Ghio AJ, Hall A, Bassett MA, Cascio WE and Devlin RB: Exposure to concentrated ambient air
particles alters hematologic indices in humans. Inhal Toxicol. 15:1465–1478. 2003.

32 Baccarelli A, Martinelli I, Pegoraro V, Melly S, Grillo P, Zanobetti A, Hou L, Bertazzi PA, Mannucci
PM and Schwartz J: Living near major traffic roads and risk of deep vein thrombosis. Circulation.
119:3118–3124. 2009.

33 Dales RE, Cakmak S and Vidal CB: Air pollution and hospitalization for venous thromboembolic
disease in Chile. J Thromb Haemost. 8:669–674. 2010.

34 Martinelli N, Girelli D, Cigolini D, Sandri M, Ricci G, Rocca G and Olivieri O: Access rate to the
emergency department for venous thromboembolism in relationship with coarse and fine
particulate matter air pollution. PLoS One. 7:e348312012.

35 Manfredini R, Gallerani M, Boari B, Salmi R and Mehta RH: Seasonal variation in onset of
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36 Manfredini R, Imberti D, Gallerani M, Verso M, Pistelli R, Ageno W and Agnelli G: Seasonal

variation in the occurrence of venous thromboembolism: data from the MASTER registry. Clin Appl
Thromb Hemost. 15:309–315. 2009.

37 Bilora F, Boccioletti V, Manfredini E, Petrobelli F, Tormene D, Simioni P and Girolami A: Seasonal

variation in the incidence of deep vein thrombosis in patients with deficiency of protein C or
protein S. Clin Appl Thromb Hemost. 8:231–237. 2002.

38 Jang MJ, Kim HJ, Bang SM, Lee JO, Yhim HY, Kim YK, Kim YK, Choi WI, Lee EY, Kim IH, et al: Seasonal
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Thromboembolism Working Party. Thromb Res. 130:e199–e202. 2012

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40 Lindqvist PG, Epstein E and Olsson H: Does an active sun exposure habit lower the risk of venous
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41 Tangpricha V, Turner A, Spina C, Decastro S, Chen TC and Holick MF: Tanning is associated with
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density. Am J Clin Nutr. 80:1645–1649. 2004.

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1alpha,25-dihydroxyvitamin D3 on human myelogenous leukemia cells and monocytes. Blood.
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45 Emmerechts J, Jacobs L and Hoylaerts F: Air pollution and cardiovascular diseaseThe Impact of Air
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pp. 69–93. 2011

Article #4

“Raised Skin Temperature in the Early Diagnosis of Deep-vein Thrombosis of the Legs”

The reported incidence of deep-vein thrombosis in hospital patients varies from less than 1 % (Barker et
al., 1940 ; Felder, 1949) to over 28% (Sevitt and Gallagher, 1959). This wide variation reflects not only
differences in the populations at risk but also differences in the criteria of diagnosis and in the vigilance
with which they are sought. There is general agreement that the clinical diagnosis of deep-vein
thrombosis may be difficult or impossible, and the occurrence of pulmonary embolism without previous
recognition of its cause is common experience. The usually accepted signs of deep-vein thrombosis are
oedema, calf tenderness on pressure or on dorsiflex on of the ankle, and tenderness over the femoral
vein. Some authors add cyanosis of the limb and dilated superficial veins, and a few mention raised
temperature of the affected limb. Delayed cooling on exposure of the limb as an early sign of venous
thrombosis was first mentioned by Pilcher (1939), who stated, "I do not remember any patient showing
this sign who did not develop others " (of deep-vein thrombosis). This paper reports an attempt to
assess the value of this physical sign. The investigation was planned to answer three questions: (1) What
other factors than deep-vein thrombosis can cause delayed cooling on exposure ? (2) In the absence of
such factors is delayed cooling a reliable early sign of deep-vein thrombosis? (3) Is delayed cooling
always followed by other physical signs of deep-vein thrombosis?

Patients

During a period of six months 514 patients in an adult male and an adult female ward were examined in
the manner described below, 3,950 observations being made. The patients comprised a group of
general surgical cases, including emergencies and cold surgery, together with some thoracic and
vascular cases. Orthopaedic and genito-urinary cases were not usually resident in these wards and only
a few are included in the series. Eight of the 28 beds in the male ward were occupied by patients
receiving radiotherapy for malignant disease. Many of these were ill and confined to bed. Patients with
dressings or bandages on the legs were excluded.

Method

Each patient was examined at least every other day, th._ majority every day. The patient being supine,
both legs were extended with the feet wide apart and uncovered to above the knee. Particular care was
taken to ensure that the legs were not crossed and that the pat-ent was not lying on his side, and to see
that there was no constriction of the thighs by clothing. The legs were thus exposed for about 10
minutes, after which the observer, using one hand, relt the skin around both ankles and noted any
difference of temperature. The result was recorded simply as right warmer than left, or vice versa, or no
difference. The presence of associated local signs of deep venous thrombosis was also recorded,
together with the presence or absence of pedal pulses in those patients whose legs showed a
temperature difference. Finally, varicose ulcers, veins, or eczema and any other leg lesion, particularly
inflammatory ones, were looked for and noted. At the end of each round the age, diagnosis, length of
stay in hospital, and time post-operatively for each patient was written down. The observations were
made by two observers only, and the results of the previous day's observations were not known when
the patients were examined each morning at 8.30 a.m.

Discussion

This investigation was prompted by the generally accepted estimate that 5000 of pulmonary emboli
occur in patients without signs of venous thrombosis in the legs (Cosgriff, 1947). Sevitt and Gallagher
(1961) state that deep-vein thrombosis is symptomless in two-thirds of cases. In 1959 they
demonstrated that where thrombosis has been diagnosed in one leg necropsy may show that it is
bilateral. Whatever policy is adopted to deal with the hazard of pulmonary embolism, earlier and more
frequent recognition of deep-vein thrombosis is a worth-while aim.

The common site for the beginning of deep-vein thrombosis is the calf muscles (Gibbs, 1957), whence it
may or may not spread to the main deep veins of the limb. It is only when such spread occurs that the
full clinical picture with oedema and thigh tenderness will develop. The structure of pulmonary emboli
suggests that the propagation and separation of the clot is often rapid, and many believe that when the
signs of thrombosis are gross embolization is less likely. The stage at which diagnosis is needed is while
thrombosis is confined to the calf and before occlusion of main deep veins has occurred.

As an explanation of delayed cooling on exposure it is postulated that thrombosis in deep veins results
in shunting of the venous return into superficial veins and that increased flow in these raises skin
temperature. The presence of dilated veins below the knee has been noted by other observers
(Homans, 1934 ; Felder, 1949) in association with deep-vein thrombosis. Such dilated veins are most
easily recognized in thin subjects, but many patients with deep-vein thrombosis are fat. A rise of skin
temperature has also been noted by Homans (1934, 1947), Felder (1949), and Short (1952). Felder
found a raised leg temperature in 54 out of 105 legs, accompanied by tenderness and swelling in 42 of
them. He also states, however, that swelling often occurs without heat. This does not invalidate the
explanation of the sign, since with the progress of deep-vein thrombosis sufficient to cause oedema the
superficial venous return will also be slowed and the warming effect of increased flow will be lost. It is
suggested, therefore, that the sign of delayed cooling on exposure is most likely to be found early in the
process and may disappear as it either extends or resolves. This would explain the lack of a constant
association between a warm leg and other signs of deep-vein thrombosis.

The method of carrying out the test as described in the present paper is an attempt to exclude increase
in leg temperature due to crossing of the legs or other postural factors while the patient is in bed. The
period of 10 minutes was adequate for cooling to an equal temperature to occur in normal legs, but not
in those where a pathological condition of the leg was BRITISH MEDICAL JOURNAL present. The
simplicity of the test is its great advantage. It can be carried out quickly and easily, and a "leg round " on
48 patients takes about half an hour.

f a warm leg is indicative of venous thrombosis in that leg in the absence of the recognized physical
signs. Phlebography is notoriously unreliable as well as difficult and time-consuming to perform. Post-
mortem examinations have obvious limitations in a vital study such as this, and it was often found that
the sign of a warm leg, as well as the usual signs of thrombosis, disappeared after a few days. This may
be due to thrombolysis or to the development of deep venous collaterals, but if the former is true
necropsy evidence may have little value. The statistical evidence given does not prove that the relation
between a warm leg and deep venous thrombosis is causal, but in the absence of any other suggested
factor it seems very likely to be so. Additional support for the theory is given by the relation of the sign
of a warm leg to age, time of onset, and type of operation. This conforms closely to the findings of
Barker et al. (1940, 1941) at the Mayo Clinic for deep venous thrombosis.

The finding of warm legs from days 1 to 3 after admission is in keeping with the evidence of Browse
(1964) that hospitalization alone increases the period of a patient's inactivity from 33 % to 63 % per 24
hours. It may also increase his tendency to venous thrombosis at that time. The number of pulmonary
emboli in the series is too small for any conclusions to be drawn, but, using a warm leg with or without
the generally accepted signs as evidence of thrombosis, the diagnosed incidence of deep venous
thrombosis in this series (including those patients without a warm leg) is 10.500. If patients with a warm
leg only are excluded the incidence of deep venous thrombosis falls to 7.3 It remains to be seen how
often pulmonary embolism follows the finding of a warm leg alone.

Summary

leg) as an early physical sign of unilateral deep venous thrombosis of the lower extremities in 468
patients. Thirty-six of them had an otherwise inexplicable unilateral warm leg, and statistical analysis of
these patients shows that there is a highly significant correlation between the presence of a warm leg
and the signs of venous thrombosis in that leg. The same statistical correlation is present when these
patients are subdivided for age and type of operation, and the results are more significant in patients
undergoing major operations and in patients over the age of 59 years.

Fourteen of the 36 patients showing a warm leg subsequently developed other clinical signs of deep
venous thrombosis in that leg. The interval between the appearance of a warm leg and other signs of
thrombosis varied from one to seven days, but was usually three days or less. Fourteen patients who
developed a temporary warm leg did not produce other signs. Only two patients in the series had
pulmonary emboli; only one patient had leg signs first, but the numbers are too small to assess the
relation between a warm leg and pulmonary embolism. The use of this sign may draw early attention to
venous thrombosis, and, in this series, raises the diagnosis rate from 7.3 % to 10.5 %.

References

Barker, N. W., Nygaard, K. K., Walters, W., and Priestley, J. T. (1940). Proc. Mayo Clin., 15, 769. -~ ~~
(1941). Ibid., 16, 1. Browse, N. (1964). Brit. med. 7., 1, 669. Cosgriff, S. W. (1947). Amer. 7. Med., 3, 740.
Felder, D. A. (1949). Surg. Gynec. Obstet., 88, 337. Gibbs, N. M. (1957). Brit. 7. Surg., 45, 209. Homans, J.
(1934). New Engl. 7. Med., 211, 993. (1947). Ibid., 236, 196. Pilcher, R. (1939). Lancet, 2, 629. Sevitt, S.,
and Gallagher, N. G. (1959). Ibid., 2, 981. (1961). Brit. 7. Surg., 48, 475. Short, D. S. (1952). Brit. med. Y.,
1, 790.
Article #5

“Hyperhomocysteinemia as a Risk Factor for Deep-Vein Thrombosis”

Abstract

BACKGROUND
Previous studies have suggested that hyperhomocysteinemia may be a risk factor for venous
thrombosis. To assess the risk of venous thrombosis associated with hyperhomocysteinemia, we studied
plasma homocysteine levels in patients with a first episode of deep-vein thrombosis and in normal
control subjects.

METHODS
We measured plasma homocysteine levels in 269 patients with a first, objectively diagnosed episode of
deep-vein thrombosis and in 269 healthy controls matched to the patients according to age and sex.
Hyperhomocysteinemia was defined as a plasma homocysteine level above the 95th percentile in the
control group (18.5 μmol per liter).

RESULTS
Of the 269 patients, 28 (10 percent) had plasma homocysteine levels above the 95th percentile for the
controls, as compared with 13 of the controls (matched odds ratio, 2.5; 95 percent confidence interval,
1.2 to 5.2). The association between elevated homocysteine levels and venous thrombosis was stronger
among women than among men and increased with age. The exclusion of subjects with other
established risk factors for thrombosis (e.g., a deficiency of protein C, protein S, or antithrombin;
resistance to activated protein C; pregnancy or recent childbirth; or oral-contraceptive use) did not
materially affect the risk estimates.

CONCLUSIONS
High plasma homocysteine levels are a risk factor for deep-vein thrombosis in the general population.
Mild hyperhomocysteinemia is an established risk factor for atherosclerosis and vascular disease.1,2 In
classic homocystinuria, half the vascular complications are of venous origin,3 but until recently it has
been unclear whether mild hyperhomocysteinemia is also a risk factor for venous thrombosis.2,4,5 In a
case–control study, Falcon et al. found that hyperhomocysteinemia was a risk factor for thrombosis in
people younger than 40 years of age.6 They reported that the difference in homocysteine levels
between case patients and control subjects was particularly evident after methionine loading (since
methionine is a precursor of homocysteine). Recently, we found hyperhomocysteinemia to be a risk
factor for recurrent venous thrombosis in patients between 20 and 70 years of age, as compared with
controls from the general population.7 Although the results of these studies support the hypothesis that
mild hyperhomocysteinemia is a risk factor for venous thrombosis, the studies were not designed to
estimate the risk in the general population.
We measured homocysteine concentrations in patients and matched control subjects participating in
the Leiden Thrombophilia Study.8-11 This is a population-based case–control study designed to measure
the effect of several acquired and genetic risk factors for thrombosis in the general population. Because
of the data available on the study subjects, we were able to investigate whether the effect of
hyperhomocysteinemia was independent of other well-established risk factors for thrombosis, such as a
deficiency of protein C, protein S, or antithrombin; use of oral contraceptives; and pregnancy or recent
childbirth. Recently, resistance to activated protein C caused by a single point mutation in the factor V
gene (factor V Leiden) has been reported to be the most common hereditary cause of venous
thrombosis.12 Since hyperhomocysteinemia also appears to be common, we examined the risk of
thrombosis in persons with both abnormalities.

Methods

The methods by which blood samples were obtained and interview data were collected have been
described elsewhere.8-11 The study protocol was approved by the local ethics committee, and all
participants gave their informed consent. Briefly, consecutive patients younger than 70 years of age who
had a first episode of deep-vein thrombosis, objectively confirmed (by impedance plethysmography,
Doppler ultrasonography, compression ultrasonography, or contrast venography), between 1988 and
1993 and who had no known cancer were selected from the files of three anticoagulation clinics in the
Netherlands (in Leiden, Amsterdam, and Rotterdam). These clinics monitor the anticoagulant treatment
of virtually all patients in well-defined geographic areas. Each patient was asked to find his or her own
healthy control subject of the same sex and age (within five years) by asking neighbors or friends. We
restricted the present analysis to case patients and controls who were seen at the Leiden
Anticoagulation Clinic and whose blood samples were processed and frozen on site with minimal delay.
(Blood samples from participants in Amsterdam and Rotterdam were also processed in Leiden, which
caused delays of several hours, and homocysteine measurements were therefore less accurate than
those measured in samples from subjects in Leiden.13)
The total homocysteine concentration was measured in citrated plasma by automated high-
performance liquid chromatography with reverse-phase separation and fluorescent detection (with a
Gilson 232-401 sample processor, Spectra-Physics 8800 solvent-delivery system, and Spectra-Physics LC
304 fluorometer). We used the method described by Fiskerstrand et al.13 with some modifications.14 If
not otherwise stated, hyperhomocysteinemia was defined as a homocysteine level above the 95th
percentile in the control group (18.5 μmol per liter).
We calculated matched odds ratios as estimates of the relative risk
of thrombosis for homocysteine values above a given point, with
the matching factor taken into account. The univariate matched
odds ratio is the ratio of the number of pairs of case patients and
controls in which the homocysteine value for the case patient was
above the specified level and the value for the control was below
that level to the number of pairs in which the homocysteine value
for the control was above the specified level and the value for the
case patient was below that level. The 95 percent confidence
intervals were calculated from a conditional logistic-regression
algorithm by the maximum-likelihood method, with Egret
software. We also investigated a possible dose–response relation
by calculating odds ratios for several ranges of homocysteine
concentrations in a conditional logistic model. In addition, we
calculated odds ratios for men and women separately and for
several age groups in order to study possible differences in risk
among these subgroups.
We further explored the differences in risk between men and women by taking risk factors specific to
women into account — specifically, the use of oral contraceptives, pregnancy, and recent childbirth. We
analyzed the risk of thrombosis among women less than 50 years old, both with and without the
inclusion of women with these risk factors, by calculating unmatched odds ratios. The use of unmatched
odds ratios was necessary because in the restricted groups many matched pairs would not have been
complete. Since the matched and unmatched odds ratios did not differ substantially in any of our
analyses, we considered this approach justified.
We also assessed whether the increased risk associated with hyperhomocysteinemia in both sexes was
confounded by other risk factors, such as a deficiency of protein C, protein S, or antithrombin. We
repeated the analysis after excluding subjects with abnormally low levels of these proteins (measured,
as previously reported, with a single test8) and estimated the risk associated with
hyperhomocysteinemia in persons with normal protein C, protein S, and antithrombin levels.
Finally, we looked at the possibility of an interaction between hyperhomocysteinemia and
heterozygosity (carrier status) for factor V Leiden, a rather common defect that causes resistance to
activated protein C. We analyzed this interaction by calculating univariate odds ratios for thrombosis in
persons with both or either of these risk factors, as compared with persons with neither risk factor.

The ratio of male to female subjects among both the case patients and the controls was 1:1.3, and the
mean age was 44 years (range, 16 to 70 for the case patients and 16 to 71 for the controls); both these
variables were used in matching the case patients and the
controls.
Figure 1. Plasma Homocysteine Levels in 269 Patients with
Deep-Vein Thrombosis and 269 Controls.
The median plasma homocysteine level in the patients was
12.9 μmol per liter (range, 4.8 to 60.2), and that in the
controls was 12.3 μmol per liter (range, 6.4 to 37.5). The
homocysteine concentrations of individual case patients and
controls are shown in Figure 1.

Table 1. Pairwise Distribution of Plasma Homocysteine

Values in 269 Case Patients and Their Matched Controls,
According to Various Definitions of Hyperhomocysteinemia.
The 95th percentile of the homocysteine levels in the
control group was 18.5 μmol per liter. Of the 269 patients,
28 (10 percent) exceeded this cutoff, as compared with 13 (5
percent, by definition) in the control group. The matched
odds ratio for deep-vein thrombosis in subjects with a
homocysteine concentration above the 95th percentile, as
compared with those whose homocysteine levels were at or
below that value, was 2.5 (95 percent confidence interval,
1.2 to 5.2). When the cutoff was set at the 90th percentile,
the matched odds ratio was 1.9 (95 percent confidence interval, 1.1 to 3.3); it was 4.0 (95 percent
confidence interval, 1.4 to 12.0) when the cutoff was the 97.5th percentile (Table 1).
Figure 2. Odds Ratio for Thrombosis According to Plasma Homocysteine Level.
In order to evaluate the possibility of a dose–response relation, we stratified the patients and controls
according to their homocysteine concentrations and calculated odds ratios for thrombosis in the
patients at the higher levels as compared with those at the lowest level. As Figure 2 shows, the risk of
thrombosis did not increase among subjects with homocysteine levels up to 18 μmol per liter; the risk
was greatly increased above 22 μmol per liter, however, indicating a threshold effect rather than a
continuous dose–response relation.
Table 2. Odds Ratios for Thrombosis Associated with Hyperhomocysteinemia, According to Age and Sex.

Odds ratios for several age groups and for men and women separately are shown in Table 2. For both
sexes, there was a sharp increase in the risk of thrombosis associated with hyperhomocysteinemia at
increasing ages. The overall odds ratio for thrombosis associated with hyperhomocysteinemia in women
was 7.0 (95 percent confidence interval, 1.6 to 30.8), and in men it was 1.4 (95 percent confidence
interval, 0.6 to 3.4), with the cutoff set at the 95th percentile of the homocysteine levels in the control
group (P = 0.067 for the comparison between the sexes). When we calculated the 95th percentile of the
distribution of homocysteine levels for men and women separately, we found a 95th percentile of 17.1
μmol per liter among women and 20.0 μmol per liter among men in the control group. Using these
cutoffs for hyperhomocysteinemia, we found an odds ratio for thrombosis of 3.8 (95 percent confidence
interval, 1.4 to 10.2) for women and 1.8 (95 percent confidence interval, 0.6 to 5.4) for men.
The higher rate of hyperhomocysteinemia in women than in men was present at all ages, making it
unlikely that the difference was due to risk factors specific to women, such as the use of oral
contraceptives, recent childbirth, or pregnancy. Indeed, when we excluded women with these risk
factors, the unmatched odds ratio for thrombosis that was associated with hyperhomocysteinemia (with
the 95th percentile for both sexes — 18.5 μmol per liter — as the cutoff for hyperhomocysteinemia)
among women under the age of 50 was 11.3 (95 percent confidence interval, 2.7 to 46.0), whereas it
was 2.8 (95 percent confidence interval, 0.9 to 8.7) for all women, both those with and those without
these risk factors, under the age of 50.
Of the 269 patients, 15 had protein C deficiency, 7 had protein S deficiency, and 10 had antithrombin
deficiency. In the control group, four had protein C deficiency, eight had protein S deficiency, and eight
had antithrombin deficiency. After excluding these subjects, we found a matched odds ratio for deep-
vein thrombosis of 2.6 (95 percent confidence interval, 1.2 to 5.9), as compared with 2.5 (95 percent
confidence interval, 1.2 to 5.2) when those subjects were included; this result shows that the effect of
homocysteine is largely independent of these deficiencies in clotting-factor inhibitors.
With respect to the combination of factor V Leiden and hyperhomocysteinemia, we calculated odds
ratios for thrombosis in subjects with both risk factors or either one in relation to subjects with neither.
A total of 47 of the patients carried the factor V Leiden mutation, as compared with 7 of the controls.
The small number with both defects made the results statistically unstable and somewhat sensitive to
the cutoff chosen for elevated homocysteine levels. When the 90th percentile was used as the cutoff,
the odds ratio for thrombosis associated with the presence of both risk factors (factor V Leiden and
hyperhomocysteinemia) was 3.5 (95 percent confidence interval, 0.7 to 16.9); the odds ratios for
thrombosis associated with factor V Leiden alone and hyperhomocysteinemia alone, calculated
separately, were 9.5 and 2.2, respectively. With the 95th percentile used as the cutoff, the odds ratio for
the combination of risk factors was 2.0 (95 percent confidence interval, 0.4 to 10.9), whereas the odds
ratios for each risk factor separately remained virtually unchanged. The statistical uncertainty of results
based on these data is reflected in the wide confidence intervals, which do not exclude a relative risk as
high as 16.9.

Discussion

Our study shows that hyperhomocysteinemia is a risk factor for deep-vein thrombosis in the general
population. Moreover, our results suggest that the association between mild hyperhomocysteinemia
and venous thrombosis is similar in degree to that reported for hyperhomocysteinemia and arterial
vascular disease.15,16 An unexpected finding was the substantial increase in the risk of thrombosis at the
highest plasma homocysteine levels. Our data suggest that there may be a threshold level above which
homocysteine has a thrombogenic effect.
Falcon et al. reported that hyperhomocysteinemia was a risk factor for juvenile thrombosis.6 Our data
imply that hyperhomocysteinemia is a risk factor for thrombosis in adult subjects as well, since we found
an increasing odds ratio with increasing age.
When we analyzed men and women separately, we found a difference in the risk of thrombosis
associated with hyperhomocysteinemia. Even when we used different cutoff points for
hyperhomocysteinemia in men and women by calculating the 95th percentiles of their homocysteine
distributions in the control group separately, we found that the odds ratio was roughly twice as high for
women as for men. This suggests that women may be more susceptible to the pathologic effects of
elevated homocysteine levels, even though their homocysteine levels are in general lower than those of
men.1 This effect cannot be explained by risk factors specific to women (such as pregnancy, recent
childbirth, and oral-contraceptive use); an effect of these risk factors was unlikely in any case because
the difference between men and women who did not have such risk factors was even more
pronounced.
Hyperhomocysteinemia remained a risk factor for deep-vein thrombosis after we excluded subjects with
other well-established risk factors; that is, the association with thrombosis was not explained by the
presence of other hereditary risk factors for thrombosis, such as a deficiency of protein C, protein S, or
antithrombin. The same was true of the most common hereditary risk factor for deep venous
thrombosis, resistance to activated protein C, since hyperhomocysteinemia also increased the risk of
thrombosis in those without this abnormality. We investigated a possible interaction between resistance
to activated protein C (factor V Leiden) and hyperhomocysteinemia. Although we found that the risk of
thrombosis may be higher in carriers of the mutation who have hyperhomocysteinemia than in
noncarriers with hyperhomocysteinemia, the combined effect in our subjects seemed smaller than for
factor V Leiden alone. Because of the small numbers involved, the only reasonable conclusion is that the
two factors do not potentiate each other.
 Many hypotheses have been proposed to explain how hyperhomocysteinemia may lead to venous
thrombosis and atherosclerosis. One hypothesis is that homocysteine has a toxic effect on the vascular
endothelium and on the clotting cascade.1,2 Several in vitro studies seem to support this
view.17,18 However, virtually all these studies used amounts of homocysteine that produced higher-than-
physiologic concentrations. Alternatively, hyperhomocysteinemia may reflect abnormal methionine
metabolism that affects the methylation of DNA and cell membranes.19
Elevated homocysteine levels may result from low levels of folic acid, vitamin B6, or vitamin B12.
Moreover, several genetic alterations in enzymes involved in homocysteine metabolism have been
described.20-22 It remains unclear whether hyperhomocysteinemia of different causes entails the same
risk of thrombosis. Nevertheless, it is well known that vitamin supplementation lowers homocysteine
concentrations in almost all subjects with hyperhomocysteinemia, regardless of the underlying cause.
We conclude that mild hyperhomocysteinemia is a risk factor for deep-vein thrombosis in the general
population. The next question to be answered is whether homocysteine-lowering therapy — folic acid,
vitamin B6, or vitamin B12 — contributes to the prevention of recurrent venous thrombosis.23-25

References

1. Ueland PM, Refsum H, Brattström L. Plasma homocysteine and cardiovascular disease. In: Francis RB Jr,
ed. Atherosclerotic cardiovascular disease, hemostasis, and endothelial function. New York: Marcel
Dekker, 1993:183-236.
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