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52 Chapter 5/Role of Physiologic Testing in Venous Disorders
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popliteal fossa, and the posterior tibial vein just behind the
medial malleolus.
With the pencil-like probe positioned toward the venous
fl ow and at 60 degrees to the fl ow streamline, the target
velocity is optimized. The fact that a velocity is identifi ed
means the vein is patent at the target level; this is the fi rst
of three major diagnostic criteria. The second diagnostic
criterion is associated with the spontaneous and phasic
nature of the signal. When veins are not obstructed proximal
to the target vein, the local pressure is low and local velocity changes as a function of respiration. Low-pressure
veins collapse and local velocity often is reduced to zero
shortly after inspiration. This is due to the fact that when the
diaphragm moves down on inspiration, pressure in the closed
abdominal cavity increases and collapses veins at low pressure. With proximal obstruction this phasic velocity is disturbed in the sense that velocity is no longer phasic with
respiration and in fact may be continuous. The third criterion
is associated with velocity response secondary to distal compression. When veins are unobstructed proximal to the target
and compression is performed distally, the local velocity
will increase in response to compression. In a high resistance
proximal venous system, distal compression will not evoke
increased velocity.
If a subject demonstrates at the femoral, popliteal, and
posterior tibial veins good velocity signals that are phasic
with respiration and augment with distal compression, the
chance of DVT involving the iliac, common femoral,
femoral, or popliteal veins is very low. DVT limited to the
calf veins is more problematic due to vein duplication at this
level. As mentioned earlier, when CW Doppler is combined
with venous plethysmography (SVC and MVO) the sensitivity and specifi city of the combined package is 85%,
respectively.
11
Venous Insuffi ciency
The main use of CW Doppler in venous insuffi ciency is
in assessing refl ux in the major deep veins of the lower
extremity (common femoral, femoral, and popliteal veins).
This procedure is most effectively performed with the
subject standing. To the extent possible, weight should be
shifted to the contralateral leg. A bidirectional CW Doppler
with a stereo audio signal and printout is recommended. For
venous work an ultrasound frequency range of 5 to 7 MHz
is suggested. As a quick review, the pencil-like probe of the
CW Doppler should be aligned toward the fl ow and at an
angle of approximately 60 degrees to the anticipated fl ow
streamline. Unlike Duplex Ultrasound, with CW Doppler
the exact path of the target vein is not well defi ned. Therefore, in practice the operator will have to adjust the probe
angle manually to obtain the maximum signal (audio level
and velocity level). The concept is quite simple; target veins
are assessed for reversal of fl ow velocity after rapid manual
limb compression and release. The more reversal, the more
refl ux. In terms of diagnostic criteria, a normal vein demonstrates no evidence of refl ux using this technique. Flow
reversal can be assessed both by audio signal and by examination of velocity versus time printouts.
17
ASSESSMENT OF THE DEEP AND
SUPERFICIAL VENOUS SYSTEMS
USING DUPLEX ULTRASOUND
The two sections preceding this text described pure physiologic measures. This section will focus on the combination
of physiologic and imaging measures. Further, Duplex
Ultrasound has become the “gold standard” in the diagnosis
of both deep venous thrombosis and venous insuffi ciency.
The method is so pervasive that it has replaced in most
venous centers the use of venous plethysmographs and CW
Dopplers. It also should be stated that the accuracy, speed,
and cost of this procedure to diagnose deep venous thrombosis have been so attractive that venography is rarely indicated or necessary.
Power Color Pulsed-Wave Doppler and High-Resolution
B-mode Imaging characterize state-of-the-art Duplex Ultrasound. Descriptions of these devices are found elsewhere in
this book and are commonplace in medical literature. The
remaining sections describe our approach to the assessment
of the deep and superfi cial systems using duplex ultrasound.
This essentially follows the worksheet and procedures
our technologists use at the Miami Vein Center (see
Figure 5.3).
Risk Factors, Vascular History,
Presenting Signs and Symptoms,
and CEAP Classifi cation
In addition to demographic data we suggest risk factors
and associated history be recorded. This includes parameters
like obesity, pregnancy, hormone use, and hypercoagulability. Also recorded for each leg are presenting signs and
symptoms like edema, pain/tenderness, skin changes, varicose veins, and previous DVT. We have found the CEAP
Classifi cation to be helpful in describing degree of disease
and in developing management plans.
20
Deep Venous System Assessment
We recommend that the subject be studied on a fl at examining table in which the lower extremities may be placed in
the dependent position at approximately 15 degrees. This
slight angle dilates the deep system, which makes the identifi cation of veins easier and improves the velocity signals.
We recommend deep vein interrogation from the level of the
Assessment of the Deep and Superfi cial Venous Systems Using Duplex Ultrasound 53
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inguinal ligament to the distal calf. This includes the common
femoral, popliteal, and tibial veins. In special cases the deep
femoral vein may be added to this list. Effective imaging
requires the technologist have a comprehensive understanding of venous and arterial anatomy.
The evaluation begins by obtaining a B-mode image of
the structures at the level of the inguinal ligament. In a single
FIGURE 5.3 Worksheets used by vascular technologists at the Miami Vein Center in the assessment of the lower
extremity deep and superfi cial venous systems.
transverse view it is generally possible to see the common
femoral vein, common femoral artery, and great saphenous
vein. In Florida, we refer to this image as the “Mickey
Mouse Image” because of the similarity to the Disney character. We have found keeping the lateral (arterial) structures
on the left side of the screen for both the right and left leg
to be helpful. This requires that the technologist rotate the
54 Chapter 5/Role of Physiologic Testing in Venous Disorders
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linear array probe 180 degrees when moving from the right
to the left leg. The marker on the probe should be oriented
to the lateral aspect of the leg. With this orientation, Mickey’s face is the common femoral vein and is the larger and
lower of the three structures. The common femoral artery is
Mickey’s right ear and the great saphenous vein (GSV) is
Mickey’s left ear. As the probe is moved distally, the GSV
will disappear, and the common femoral artery will divide
into the superfi cial femoral artery and the deep femoral
artery. As the probe continues distally the technologist
should focus on keeping the superfi cial femoral artery and
the femoral vein in clear view. The popliteal artery and the
popliteal vein are diffi cult to visualize in the adductor canal,
therefore, these structures are identifi ed by placing the probe
in the popliteal crease. Below the knee, the duplicated posterior tibial and peroneal veins with their associated single
arteries can be viewed with the probe at a medial location.
In general, the anterior tibial veins can be ignored because
they are rarely pathologic.
During this examination a number of maneuvers are necessary. First, the technologist may change from transverse
to longitudinal views. When longitudinal views are used, the
vein walls (proximal and distal) should be seen across the
entire screen, left to right. The technologist may use the
Doppler portion of the Duplex system to verify artery versus
vein and determine fl ow direction.
With the probe, the technologist can compress the vein.
The ability to fully compress the vein walls confi rms vein
patency and absence of thrombus formation. The technologist also looks for visible thrombus formation in the vein
structures. Acute thrombi are characterized by vein dilatation and noncompressible echo lucent intraluminal material.
Chronic thrombi take on a speckled ultrasonic appearance.
If the evaluated system from the common femoral vein
through the tibial veins is compressible and no evidence of
thrombus formation is seen, the study is considered negative
for DVT. Color Doppler, Power Doppler, compression
maneuvers, and respiratory maneuvers can be used to supplement this procedure, if necessary.
Superfi cial Venous System Assessment
Most investigators agree assessment of the superfi cial
venous system is more challenging for the technologist and
interpreting physician than the deep system.
with this contention. In our facility we always perform deep
system assessment in advance of superfi cial venous system
assessment.
In contrast to the deep system, for superfi cial assessment
we always evaluate subjects in the erect position. We have
our subjects stand on a standard medical step, which is
approximately 8 inches high. The patient is asked to rotate
the leg of interest to expose the medial surface of the lower
extremity from the groin to the ankle. To the extent possible,
21,22
We agree
weight should be shifted from the leg of interest in order to
relax the musculature. A degree of arm support may be
needed.
With the subject properly positioned, the technician
moves the probe to the inguinal ligament and produces the
Mickey Mouse Image described earlier. The Mickey Mouse
Image is the most important landmark of the venous examination. At his point the focus is on the Great Saphenous
Vein. Starting from the three-vessel image in the transverse
view the probe is moved slowly down the leg following the
course of the GSV. The GSV is kept near mid-screen. The
normal GSV extends from the saphenofemoral junction to
the distal calf and is surrounded by superfi cial fascia above
and muscular fascia below. As a minimum we record
diameter measurements in mm and the presence of
refl ux (positive or negative) at three locations in the GSV
(saphenofemoral junction, mid-thigh, and below knee).
Refl ux is determined at locations of interest using the
following technique. The technologist adjusts the color box
of the Duplex system in the measurement location. The
velocity scale is adjusted (maximum 25 cm/sec). While a
signal is being obtained the technologist compresses the calf
(below the probe) in a brisk manner. The vein highlighted
in the color box should demonstrate an increase in velocity
toward the heart with compression. On release the vein
should demonstrate no velocity or minimal velocity away
from the heart. We have found that refl ux (venous fl ow
away from the heart after release) lasting between 0.5
to 2.0 seconds is mild. Refl ux is severe if present >2.0
seconds.
The same evaluation is repeated for the Small Saphenous
Vein (SSV). This vein originates in the distal calf and can
terminate in the upper thigh. We access this vessel with
ultrasound by rotating the subject to expose the back of the
legs. We identify the SSV at the distal calf, and advance
over the course of the SSV. Multiple levels may be assessed;
however, we generally record a characteristic SSV diameter
(mm) and assess refl ux in the most diseased location.
At this point it is important to note that there are variations in superfi cial venous anatomy. For example, the GSV
may be quite small and complemented by an Anterior Accessory Saphenous Vein (AASV), which may be competent or
incompetent. Further, the GSV may be duplicated in portions of its course. It is worth repeating that these variations
are common and must be known and anticipated by the
technologist, if a comprehensive report is to be generated.
The lower extremity has some common perforators that
play signifi cant roles in venous insuffi ciency. Hunterian perforating veins are located in the mid-thigh. Dodd perforating
veins are located at the distal thigh. The Boyd perforating
vein is located below the level of the popliteal fossa. Finally,
we have Cockett #1, #2, and #3 perforating veins located,
respectively, between the ankle and the lower calf. This
assessment must also be part of this work-up. If present,
References 55
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perforators should be assessed regarding diameter, degree of
refl ux, and extension to other superfi cial structures.
Finally, there are tributaries of the GSV and SSV that
deserve attention. We look for seven tributaries in the GSV
and three in the SSV. If present, we record diameter, degree
of refl ux, and connection to other superfi cial structures.
In closing, we emphasize that Duplex Ultrasound is not
only diagnostic, but also plays crucial roles in endovenous
ablation, ultrasound-guided sclerotherapy, and monitoring
the success of vein closure procedures.
References
1. Barron HC, Ross BA. Varicose veins: A guide to prevention and treat-
ment. Facts on File, Inc., New York, NY (An Infobase Holdings
Company). 1995. p. vii.
2. Marston WA. PPG, APG, or Duplex: Which noninvasive tests are most
appropriate for the management of patients with chronic venous insuffi ciency? Semin Vasc Surg. 2002. 15(1): 13–20.
3. Prandoni P, Villalta S, Bagatelle P, et al. The clinical course of deep
vein thrombosis: Prospective long-term follow-up of 528 symptomatic
patients, Haematologica. 1997. 82(4): 423–428.
4. Heit JA, Silverstein MD, Mohr DN, et al. Predictors of survival after
deep vein thrombosis and pulmonary embolism: A population-based,
cohort study, Arch Intern Med. 1999. 159(5): 445–453.
5. Wheeler HB. Diagnostic tests for deep vein thrombosis: Clinical use-
fulness depends on probability of disease, Arch Intern Med. 1994. 154:
1921–1928.
6. Huisman MV, Buller HR, ten Cate JW, Vreeken J. Serial impedance
plethysmography for suspected deep venous thrombosis in outpatients.
The Amsterdam General Practitioner Study, NEJM. 1986. 314: 823–
828.
7. Croal S, Birkmyre J, McNally M, Hamilton C, Mollan R. Straingauge
plethysmography for the detection of deep venous thrombosis, J
Biomed Eng. 1993. 15: 135–139.
8. Maskell NA, Cooke S, Meecham Jones DJ, Prior JG, Butland RJA.
The use of automated strain gauge plethsmography in the diagnosis
of deep vein thrombosis, British J of Radiology. 2002. 75: 648–
651.
9. Fronek A. Photoplethysmography in the diagnosis of venous disease,
Dermatol Surg. 1995. 21(1): 64–66.
10. Schultz-Ehrenburg U, Blazek V. Value of quantitative photoplethysmography for functional vascular diagnostics: Current status and prospects, Skin Pharmacol Appl Skin Physiol. 2001. 14(5): 316–323.
11. Raines J, Traad E. Noninvasive evaluation of peripheral vascular
disease, Medical Clinics of North America. 1980. 64: 283–304.
12. Owens LV, Farber MA, Young ML, et al. The value of air plethysmography in predicting clinical outcome after surgical treatment of
chronic venous insuffi ciency, J Vasc Surg. 2000. 32(5): 961–968.
13. Asbeutah AM, Riha AZ, Cameron JD, McGrath BP. Reproducibility
of duplex ultrasonography and air plethysmography used for the evaluation of chronic venous insuffi ciency, J Ultrasound Med. 2005. 24(4):
475–482.
14. Fukuoka M, Sugimoto T, Okita Y. Prospective evaluation of chronic
venous insuffi ciency based on foot venous pressure measurements and
air plethysmography fi ndings, J Vasc Surg. 2003. 38(4): 891–895.
15. Nicolaides AN, Christopoulos D, Vasdekis S. Progress in the evaluation of chronic venous insuffi ciency, Ann Vasc Surg. 1989. 3: 278–
292.
16. Abramowitz HB, Queral LA, Flinn WR, et al. The use of photoplethysmography in the assessment of venous insuffi ciency: A
comparison to venous pressure measurements, Surgery. 1979. 86:
434–441.
17. Needham T. Assessment of lower extremity venous valvular insuffi ciency examinations, J Vasc Ultrasound. 2005. 29(3): 123–129.
18. Feied C, Weiss R, Hashemiyoon RB. Varicose veins and spider veins,
emedicine. 2004. www.emedicine.com/derm/topic475.htm.
19. Strandness DE. History of ultrasonic duplex scanning, Cardiovasc
Surg. 1996. 4(3): 273–280.
20. Porter JM, Moneta GL. Reporting standards in venous disease: An
update. International Consensus Committee on Chronic Venous
Disease, J Vasc Surg. 1995. 21(4): 635–645.
21. Labropoulos N, Touloupakis E, Giannoukas AD, et al. Recurrent varicose veins: Investigation of the pattern and extent of refl ux with color
fl ow duplex imaging, Surgery. 1996. 119: 406–409.
22. Valentin LI, Valentin WH, Mercado S, et al. Venous refl ux localization: Comparative study of venography and DU, Phlebology. 1993. 8:
124–127.
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CHAPTER
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6
Inappropriate Leukocyte
Activation in Venous Disease
PHILIP COLERIDGE SMITH
INTRODUCTION
Venous ulceration remains a common problem in medical
practice. A great deal has been learned about the causes of
this problem, but a simple solution for all patients remains
elusive. Although the presence of a leg ulcer is easy to
establish, it may be the result of a number of diseases, not
just venous disease. In a recent study patients with leg ulcers
had venous disease, arterial disease, diabetes, lymphedema,
and rheumatoid disease.1 In this study patients had combined
pathologies in 35% of cases. The overall prevalence of open
venous ulceration in published epidemiological studies in
adults over the age of 18 years is about 0.3%.
patient with an open ulcer there are probably three or four
with healed venous ulcers. This means that approximately
1% of the adult population are affected by ulceration, either
open or healed. Since these ulcers require regular management by health care services the cost of this disease remains
high.
Venous ulceration occurs when valves fail in the deep,
superfi cial, or perforating veins. This results in impairment of the venous muscle pumps in the lower limb.6 Superfi cial venous refl ux accounts for 20 to 50% of venous leg
7,8
ulcers,
in many. The consequence of incompetent lower limb vein
valves is that the pumping mechanism no longer reduces
the pressure in the superfi cial veins to low levels during
walking. This is refl ected in the microcirculation of the skin
leading, in some patients, to lipodermatosclerosis and leg
ulceration.
with deep vein and perforating vein refl ux involved
2–5
For every
MECHANISMS OF ULCERATION
Fibrin Cuffs
In 1982 Browse and Burnand proposed that oxygen diffusion into the tissues of the skin was restricted by a pericapillary fi brin cuff that they had observed histologically.9
They suggested that increased capillary pressure as a consequence of venous hypertension results in an increased loss
of plasma proteins through the capillary wall. This includes
fi brinogen, which polymerizes to provide the fi brin cuff that
may be seen around capillaries in the skin, using both histochemical and immunohistochemical methods. Subsequent
measurements of fi brinolysis have shown that patients with
venous disease have reduced fi brinolytic activity in the
blood and veins, which might explain why the fi brin cuff
persists.
of the effi ciency of the microcirculation in handling a molecule of similar size to oxygen had been measured. This gas
has a molecular weight four times that of oxygen, so its diffusion rate would be half that of oxygen, assuming similar
solubility for oxygen and xenon in body fl uids (water). Measurements were made in the liposclerotic skin of patients
with venous disease, and compared to control subjects under
conditions of reactive hyperemia after fi ve minutes of cuff
occlusion of the arterial supply to the leg. No difference in
xenon clearance was found between patients with venous
disease and control subjects.
conclusion that in patients with chronic venous insuffi ciency,
10
The clearance of
133
xenon from the skin as an assessment
11
These fi ndings lead to the
The Vein Book
57
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Copyright © 2006, Elsevier Inc.
58 Chapter 6/Inappropriate Leukocyte Activation in Venous Disease
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skin changes are not principally attributable to failure of skin
oxygenation.
The White Cell Trapping Hypothesis
The search for alternative mechanisms of skin damage in
venous disease has resulted in investigation of the blood
itself. Thomas investigated a series of patients and control
subjects who were subjected to experimental venous hypertension by sitting with the legs dependent for a period of 60
minutes.12 Blood samples were taken from the great saphenous vein at the ankle. After 60 minutes patients with venous
disease were trapping 30% of the white cells and control
subjects were trapping 7%.
White cell margination is a normal event in the arterioles,
capillaries, and venules. This phenomenon is thought to be
important in the mechanism that results in tissue injury
following ischemia. White blood cells are substantially
larger than red cells and are responsible for many of the
rheological properties of blood. White cells take 1000 times
longer than red cells to deform on entering a capillary bed,
and are responsible for about half the peripheral vascular
resistance despite their small numbers in the circulation
compared to red cells.13 In myocardial infarction they
cause capillary occlusion, which can be prevented in experimental animals by fi rst rendering the animal leukopenic.
White blood cells have been implicated as the mediators of
ischemia in many tissues including myocardium, brain,
lung, and kidneys.
16–19
Polymorphonuclear leukocytes, particularly those attached to capillary endothelium, may
become activated, in which cytoplasmic granules containing
proteolytic enzymes are released.20 In addition, a nonmitochondrial respiratory burst permits these cells to release free
radicals, including the superoxide radical, which have nonspecifi c destructive effects on lipid membranes, proteins,
and many connective tissue compounds.21 Leukotactic
factors also are released, attracting more polymorphonuclear
cells.
In conjunction with other authors I published a hypothesis suggesting that white cell trapping resulted in
neu trophil activation, causing damage to the tissues (see
Figure 6.1).
22
Based on the literature on myocardial ischemia, we proposed that white cells might cause occlusion of capillaries.
If some of the capillaries were occluded this might result in
heterogeneous perfusion and therefore tissue hypoxia and
ischemia. This seemed a reasonable suggestion at the time,
since it predated our attempts to measure the severity of the
diffusion block, and we included this to explain the hypoxia
observed by transcutaneous oximetry. I subsequently have
concluded that this part of the original hypothesis is not of
major importance in producing skin damage in patients with
venous disease.
14,15
White cell trapping hypothesis
Reduced blood flow
on standing
Reduced shear rate in microcirculation
favors white cell margination
Capillary plugging and
heterogeneity of perfusion
result in hypoxia
Tissue damage
FIGURE 6.1 White cell trapping hypothesis as originally published in
Reference 22.
Leukocyte Activation
White cell activation
Release of free radicals,
proteolytic enzymes, cytokines,
and chemotactic substances
The effect of venous hypertension on leukocyte activation subsequently has been studied in human volunteers
using a series of plasma and cellular markers. Control subjects exposed to lower limb venous hypertension produced
by standing were studied by taking blood samples from the
hand and the leg veins. Degranulation of neutrophils was
studied by measuring plasma levels of neutrophil elastase
(a primary neutrophil granule enzyme) and lactoferrin (a
secondary neutrophil granule enzyme). After a 30-minute
period of experimental venous hypertension, a rise in plasma
lactoferrin concentration was observed in the blood taken
from both the foot and the arm.
23
When venous hypertension
was produced by infl ation of a cuff around one lower limb,
a rise in lactoferrin was observed only in that limb. Subsequently expression of the surface neutrophil ligand, CD11b,
has been investigated as a marker of neutrophil activation.
The experiment was repeated as before on control subjects.
Blood was taken from a dorsal foot vein. CD11b expression
was assessed by fl uorescent labelled monoclonal antibody
used to label neutrophils in whole blood, which were counted
using fl ow cytometry. During the period of venous hypertension in control subjects no rise in CD11b expression was
seen in the lower limb blood.24 Following return to the
supine position, when neutrophils might be expected to
leave the lower limb, according to the studies of Thomas,12
increased levels of CD11b were observed. This indicates
Mechanisms of Ulceration 59
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that neutrophils were upregulated by their period of adhesion to normal endothelium. An increased white cell : red
cell ratio also was observed during this phase, confi rming
white cell egress from the lower limb.
A similar study also has been conducted in patients with
venous disease, including only subjects with unulcerated
skin to avoid the possibility that the infl ammatory processes
involved in the ulcer may result in up-regulation of infl ammatory mediators in a way unrelated to the development of
the ulcer. Two groups of patients were studied: one group
with uncomplicated varicose veins and one with skin changes
(lipodermatosclerosis) attributable to venous disease. The
adhesion of neutrophils and monocytes to endothelium was
investigated. This is a two-stage process. Initially these cells
roll along the endothelium, binding in a loose manner using
a ligand on the leukocytes known as CD62L or L-selectin.
When binding occurs a fragment of L-selectin is released
into the plasma (soluble L-selectin) and can be detected by
an ELISA. It was found that the concentration of soluble
L-selectin rose during venous hypertension, confi rming that
endothelial : leukocyte binding had occurred. There was no
major difference in magnitude between the two groups of
patients.
25
Subsequently, fi rm binding of neutrophils and monocytes
occurs using CD11b/CD18 ligands, which link to endothelial ICAM. This is refl ected in the peripheral blood by a fall
in the cells expressing most CD11b. Just such a fall was seen
in the blood taken from the leg in both groups of patients.
On return to the supine position I had expected to see an
egress of leukocytes expressing more CD11b in these
patients, but this was not observed, in contrast to the studies
on control subjects. In the time-scale of this experiment (up
to 10 minutes following venous hypertension), the more
activated neutrophils and monocytes remained bound to the
endothelium of the lower limb.
26
Plasma lactoferrin and elastase have been assessed in
groups of patients with active venous disease. Blood was
taken from the arm veins (not the lower limb veins) of
patients with varicose veins, liposclerotic skin change, and
active venous ulceration.
27,28
In all samples, the levels
of lactoferrin and elastase were higher in the patients than
the age and sex-matched control groups (see Figures 6.2
and 6.3).
However, it was found that the highest levels of plasma
lactoferrin were present in patients with active varicose
veins. Subsequently blood was taken from the arms of
patients for measurement of neutrophil CD11b expression.
This was elevated in patients with varicose veins, but
depressed in patients with lipodermatosclerosis.29 The explanation may be that the more active leukocytes are attracted
to the region of the infl ammatory process and do not circulate in the peripheral blood. Alternatively, such patients may
have high circulating levels of neutrophil inhibitors.
p=.0054
60
50
40
30
20
10
60
50
40
30
20
10
0
Controls
p<0.04 p<0.01
(94.5)
0
ControlVVs ControlLDS ControlUlcer
(94, 162)
Patients
p<0.03
(162)
Plasma
elastase
(ng/ml)
Plasma
elastase
(ng/ml)
FIGURE 6.2 Results of plasma neutrophil elastase measurements in
patients and control subjects. Error bars show the median and interquartile
range of data. Statistical signifi cance was tested by the Mann-Whitney U
test.
Histology
Histological studies have been used to investigate the
biological processes at work in the skin in chronic venous
disease. A quantitative histological study has been reported
in which three groups of patients were studied.
was a group of patients with no evidence of skin changes
as a consequence of their venous disease. The next group
exhibited lipodermatosclerosis without a history of ulceration. The third group had healed ulcers with residual lipodermatosclerosis. Patients with normal skin had a low
number of white blood cells visible (4 per mm2) in the upper
0.5 mm of the skin. There were eight times as many in
patients with liposclerotic skin, and 40 times as many in
patients with healed venous ulcers. Subsequently an immunohistological study was undertaken to determine the types
of white cell present in this infi ltrate.31 The majority of cells
30
The fi rst
60 Chapter 6/Inappropriate Leukocyte Activation in Venous Disease
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(1050,
1101,
800
Plasma Lf
(ng/ml)
600
400
200
0
Controls Patients
1424)
A
1200
Plasma Lf
(ng/ml)
900
600
300
0
Control
(1424)
Control Control
VV
LDS
Active
ulcer
Control
Healed
ulcer
FIGURE 6.3 Results of plasma neutrophil lactoferrin measurements in
patients and control subjects. Error bars show the median and interquartile
range of data.
are macrophages with a T-lymphocyte component, but no
excess of neutrophils compared with control sections taken
from normal limbs. So this infi ltrate is a refl ection of a
chronic infl ammatory process.
The Endothelium
The microcirculation of the skin has been investigated
by histology32 and by capillary microscopy.33 Both methods
demonstrate capillary proliferation in patients with CVI—
vastly more capillaries are visible by both techniques (see
Figure 6.4). However, capillary microscopy shows that these
probably arise from a single capillary loop and appear like
a glomerulus, rather than an increase in the numbers of
capillaries. Quantitative measurement of the capillary convolution in patients from each of the CEAP clinical classes
has been published (see Figure 6.5).
investigations have shown that the pericapillary cuff con-
34
Immunohistochemical
B
FIGURE 6.4 Images from the capillary microscope. Normal capillary
loops in the skin of the lower limb show one reversal of direction as the
vessel rises to the top of the papillary dermis and then descends (a). In a
patient with lipodermatosclerosis, numerous convolutions are seen in each
capillary (b).
tains far more than fi brin. The capillary endothelium is perturbed, expressing increased amounts of factor VIII–related
31,35
antigen
and adhesion molecules, especially ICAM-1.
ELAM-1 may be slightly upregulated but VCAM appears
to be normal in patients without venous ulceration. Perturbed endothelium is more likely to attract the adhesion of
leukocytes. The presence of the peri-capillary fi brin cuff has
been confi rmed, but it also contains collagen IV, laminin,
fi bronectin, and tenascin.36 A strong leukocyte infi ltration
has been measured in patients with venous disease.37 These
cells are macrophages and T-lymphocytes. The cytokines
involved include IL-1α and IL-1β. TNFα has not been
detected in these histological sections. The presence of the
perivascular fi brin cuff (with other components) is a refl ection of the infl ammatory process and is seen in other chronic
infl ammatory conditions. In patients with venous disease,
increased plasma D-dimer levels have been observed, sug-
Mechanisms of Ulceration 61
https://t.me/med1917
Convolutions per capillary loop
20
15
10
5
No. convolutions
0
FIGURE 6.5 This graph shows the results of analysis of the number of
convolutions per capillary loop in images such as those shown in Figure
6.4. The vertical axis shows the number of convolutions per capillary in
CEAP stages C0–C5. Capillary abnormalities are mainly present in C4a,
C4b, and C5 limbs.
C0 C1 C2 C3 C4a C4b C5
gesting enhanced deposition of fi brin.38 The perturbed state
of the endothelium allows the passage of large molecules
though the endothelium permitting their perivascular accumulation, and explains the presence of the fi brin cuff.
A search for systemic markers of endothelial activation
has been performed by undertaking measurements of plasma
levels of soluble endothelial adhesion molecules and von
Willebrand factor.
39
Patients with chronic venous disease (a
group with uncomplicated varicose veins and a group with
skin changes) again were studied and compared to normal
controls. The concentration of soluble VCAM (vascular
endothelial adhesion molecule) was elevated in both patient
groups compared to control subjects, and was highest in the
group with skin changes (see Figure 6.6).
Histological Search for Angiogenic Factors
The vascular proliferation seen in the skin of patients
with venous disease has been known for many years, but has
not been explained. In recent years many angiogenic factors
that stimulate the growth of blood vessels have been recognized. Immunohistochemistry was used to evaluate the presence of a number of such factors in the skin of patients with
venous disease.
surgery for varicose veins from the legs of patients with and
without skin changes as well as of breast skin in patients
without clinical evidence of venous disease, for use as a
control. There was an increase in platelet derived growth
factor, subtype BB (PDGF-BB) in patients with venous
disease. This was found in the capillary wall in vessels of
the dermal papillae. There was also considerable upregulation of the production of vascular endothelial growth factor
(VEGF) in the epidermis of patients with venous disease,
most marked in those with skin changes. It seems likely that
VEGF may account for at least some of the vascular prolif-
40
Skin biopsies were taken at the time of
sVCAM-1 (ng/ml)
1400
p=0.001, Wilcoxon
1200
p=0.001, Mann-Whitney
♣
p<0.001, Wilcoxon
1000
800
600
400
♣
♣
200
Controls Patients
0
L S L S
FIGURE 6.6 Plasma VCAM-1 levels in normal controls and patients
with chronic venous disease (with and without skin changes), before and
after venous hypertension produced by sitting with the lower limbs dependent for 30 mins. Descriptors: medians and inter-quartile ranges; statistics;
Wilcoxon and Mann-Whitney U tests for unpaired data. L = lying, S =
standing.
eration seen in the skin of patients with venous disease. This
growth factor is also responsible for increased vascular permeability to large molecules, a feature of the skin microangiopathy that has been reported from capillary microscopy
studies.
33
The mechanism of stimulation of epidermal VEGF
production is unclear at present.
Skin Fibrosis in Venous Disease
The role of TGF–β1 in the skin damage of CVI has
been studied in considerable detail by Pappas et al. using
im munohistochemical examination, electron microscopy,
and examination of TGF–β1 gene expression.
gation indicated that activated leukocytes traverse perivascular cuffs and release active TGF–β1. Positive TGF–β
staining of dermal fi broblasts was observed and suggests
that fi broblasts are the targets of activated interstitial leukocytes. A potential mechanism for quick access and release
is storage of TGF–β1 in the extracellular matrix. TGF–β1
was elevated exclusively in areas of clinically active disease,
indicating a localized response to injury. These data suggest
that alterations in tissue remodelling occurs in patients with
CVI and that dermal tissue fi brosis in CVI is regulated by
TGF–β1.
The fi brosis seen in the skin of patients with lipodermatosclerosis also has been investigated by other authors.42
This study shows that enhanced cell proliferation and an
increase in the number of procollagen mRNA-expressing
fi broblasts contribute to the development of LDS (see Figure
6.7). The fi brotic changes that result may not only be
41
This investi-
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