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ORIGINAL RESEARCH |
From the Lis Maternity Hospital and Internal Medicine "D, " Tel Aviv Sourasky Medical Center, Tel Aviv, affiliated to the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
Address reprint requests to: Michael J. Kupferminc, MD, Department of Obstetrics and Gynecology, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel; E-mail: ronn{at}post.tau.ac.il.
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ABSTRACT |
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METHODS: We recruited 25 women with pregnancy-inducedhypertension. Twenty-five age- and gestational age-matched normotensive volunteers took part in the study and served as controls. Blood smears were evaluated by an image analysis system (INFLAMET). Quantitative measures of erythrocyte aggregation were used to describe the state of erythrocyte adhesiveness/aggregation such as vacuum radius, which measures the spaces between the aggregated erythrocytes. The number of participants was established by power analysis (given 
of 0.05 and 80% power and considering a minimum difference to detect 4 µm in vacuum radius with a standard deviation of approximately 5).
RESULTS: A significant (P = .002) increment in the state of erythrocyte aggregation was noted in the study group compared with the controls, the vacuum radius values being 16.1 ± 1.3 and 10.3 ± 1.2, respectively. Erythrocyte sedimentation rate but not fibrinogen concentration was significantly elevated in the study group. The increased aggregation correlated significantly with fibrinogen concentration, systolic, and diastolic blood pressures.
CONCLUSION: We observed increased aggregability of red blood cells in hypertensive conditions of pregnancy. Our findings are significant in that they reveal blood pressure-related increment in red cell adhesiveness/aggregation despite there being no significant increment in clotable fibrinogen concentrations.
Red cell aggregation is one of the most important determinants of rheologic properties of blood1 and could affect blood flow in the microvascular bed. The adhesiveness/aggregation properties of the cells depend, in turn, on the shape and concentration of red blood cells (RBCs) as well as the presence of sticky proteins.2
The physiologic changes during normal pregnancy affect red cell aggregation. Physiologic hemodilution,3 microvascular vasodilatation,4 and an increase in concentration of plasma proteins such as fibrinogen5 are among the most dominant. Although the first two changes tend to reduce aggregation, the latter may lead to its increase.
The data regarding the summed effect of these changes in normal and pathologic pregnancies are scanty. In a cross-sectional study, Ozanne et al6 demonstrated that red cells aggregation increases during the course of normal pregnancy. Furthermore, Huisman et al7 reported in a longitudinal study that red cell aggregation considerably increases during normal pregnancy in spite of the physiologic hemodilution, mainly because of the increased fibrinogen concentrations.
Gestational hypertension is characterized by contracted plasma volume with hemoconcentration and abnormal vasospasm.8 In these pregnancies, the increased blood viscosity is the result of the concerted action of hematocrit, decreased RBC deformity, and increased RBC aggregation.9
Different methodologies have been adopted to detect the presence of aggregated RBCs in the peripheral blood10–12 including whole blood viscosity,2 erythrocyte sedimentation rate (ESR),13 zeta sedimentation ratio,14 and direct observation of RBCs in different conditions.11
We recently adopted a simple slide test and image analysis to reveal the state of erythrocyte adhesiveness/aggregation in the peripheral blood.15–19 This technology has the advantage of being inexpensive and simple as well as reporting results within a few minutes. The present study investigated women with pregnancy-induced hypertension (PIH) to see if previous observation in preeclampsia can also be seen in PIH using our slide test. In addition, we examined the question of whether increased erythrocyte adhesiveness/aggregation in women with PIH is associated with increased concentrations of clotable fibrinogen.
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MATERIALS AND METHODS |
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Control pregnant volunteers with normal BP were randomly selected by frequency matching of age, gestational age, and body mass index. The demographic and clinical parameters of study and control groups appear in Table 1
. Subjects gave an informed consent, and the procedure was approved by the local institutional review board according to the ethical standards for human experimentation. Overall, only two women (4%) refused to participate in the study.
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of 0.05 and 80% power and considering a minimum difference to detect 4 µm in vacuum radius with a standard deviation of approximately 5). The control group consisted of five women with elective induction of labor, seven with reduced fetal movement, five who were being observed after a fall, three observed because of single variable deceleration on routine nonstress test, two women with breach presentation before cesarean delivery, two women with passive cervical dilatation, and another with placenta previa (with no bleeding). The guidelines for the preparation of the peripheral blood slides were previously reported.15–19 In brief, blood was drawn into a syringe containing sodium citrate (one volume of 3.8% sodium citrate and three volumes of whole blood). Several large drops of blood were placed on a slide that was held for 2 to 3 seconds at an angle of 45° so that the blood could run down by gravity, leaving a fine film. This was done within 15 minutes. The slides were then dried within 10 minutes at room temperature while in a completely horizontal position. Automatic staining was performed by means of the HEMA TEK slide stainer (AMES, Elkhart, IN) and a HEMA TEK bloc colorant stain pack (Bayer Diagnostics, Puteaux, France). The time period between blood drawing and the end of the reading in the INFLAMET (INFLAMET, Tel Aviv, Israel) is less than 30 minutes.
For the analysis of the slides, we used an image analysis system (INFLAMET)15–19 consisting of a Pentium computer running Windows 95 (Microsoft CO, Redmond, WA), equipped with a Matrox Meteror color frame grabber, a color CCD camera (Videotronic, Bischke, Germany), and an Olympus BX 40 microscope (Olympus, Tokyo, Japan), which was operated at x200 magnification, resulting in an image resolution of 0.4 micron per pixel. Nine images were taken from each slide, three from the margins, three from the center, and three from the tail. The technician who chose the fields of view was masked to the source of the slides: PIH or controls. The fields of view were chosen systematically to sample different regions on the slide. Each image was processed separately, and the outputs were then averaged to yield the final slide outputs. The nine fields of view covered a total area of 0.6 mm2.
The state of erythrocyte adhesiveness/aggregation in the peripheral blood was determined by using the same image analysis system (INFLAMET). Three variables of erythrocyte aggregation were used to describe the state of erythrocyte adhesiveness/aggregation: the erythrocyte percentage, the aggregation radius, and the vacuum radius.15–19 For the definition of these variables, we used color characteristics to classify image pixels into two classes: class 1—aggregates of erythrocytes, and class 2—everything else (plasma, platelets, leukocytes).
A description of one-point and two-point statistics for this classification required very few parameters. The main reason for this is that the image statistics are homogenous (position-independent) and isotropic (direction-independent). The one-point statistics are completely described by the probability of a pixel belonging to class 1, which we named erythrocyte percentage. This is the proportion of image area covered by erythrocytes. The two-point statistics are described by the probability of a pixel belonging to class 1, given that it is a distance r from a pixel class 1, and, similarly, the probability of a pixel belonging to class 2, given that it is a distance r from a pixel of class 2. These probabilities are 1 for r = 0, and they decrease as functions of r.
The precise dependence of these probabilities on the interpixel distance r does not seem to convey biologically significant information. We found it sufficient to calculate the distances for which the probability fell below a threshold fixed at 0.7, and labeled them the aggregation radius for class 1 and the vacuum radius for class 2. These two parameters, measured in microns, provide an idea of what is the typical size of erythrocyte aggregates and plasma "spaces," respectively. Figure 1 depicts a typical picture obtained from a woman with PIH and significant erythrocyte aggregation, compared with a control.
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The blood cell count was determined by using the Coulter STKS electronic cell analyzer (Coulter Electronics, Miami Lake, FL), the ESR by the method of Westergren,21 the fibrinogen concentration by the method of Clauss,22 and a Sysmex 6000 automatic analyzer (TAO Medical Electronics, Kobe, Japan), whereas the quantitative C-reactive protein (CRP) was determined by laser nephelometry and specific antihuman CRP antibodies.
The t test and Pearson coefficient of correlation were performed by using the SPSS for Windows 9.0 statistical package (SPSS, Chicago, IL).
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RESULTS |
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presents the demographic and clinical data of the study and control groups. As expected, women in the study group had significantly higher systolic and diastolic BPs compared with controls.
There were no significant changes between study group and controls in the hematocrit, RBC count, hemoglobin, white blood cell count, platelet count, CRP, or fibrinogen concentration (Table 2). Significantly higher ESR values were noted in the study group compared with controls (P = .003) (Table 2). In addition, urate concentrations were also increased (P < .01) in the study group (4.8 ± 0.2 mg/dL, range 2.6–6.5) compared with controls (3.6 ± 0.2 mg/dL, range 1.7–6).
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).
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). In addition, significant correlations could be found between systolic and diastolic BPs and erythrocyte adhesiveness/aggregation test parameters (Table 4).
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DISCUSSION |
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The analysis of RBC aggregation in hypertensive conditions may have pathophysiologic relevance because of the possibility that the aggregated RBCs contribute to capillary slowed flow and increased peripheral resistance, as well as to relative tissue ischemia. Previously, Baskurt et al25 have proved that increased aggregation increases blood flow resistance. Furthermore, Cicco et al26 demonstrated reduced blood flow and tissue oxygenation that are related to increased red cell aggregation in hypertensive and vascular patients. The identification of increased RBC aggregation might have, therefore, therapeutic consequences in terms of drug therapy in PIH.23 We have recently adopted a simple slide test and image analysis to reveal the state of erythrocyte adhesiveness/aggregation in the peripheral blood during infection or inflammation.15–19 This test has the advantage of being simple, rapid, and inexpensive. Thus, it can be used as an almost bedside technique.
We have now extended our observations to women with PIH. We believe that real-time detection of RBC aggregation in women with PIH has a potential application. In effect, the erythrocyte adhesiveness/aggregation test is increased in these women and is related to the systolic and diastolic BP. Our findings are significant in that they show that erythrocyte adhesiveness/aggregation is enhanced in women with PIH and not only in patients with preeclampsia as previously described.9,23 Thus, further studies regarding RBC aggregation during pregnancy should include women with PIH as opposed to only women with the more severe condition of preeclampsia.
Multiple mechanisms are probably involved in the development of enhanced aggregation state in hypertensive conditions of pregnancy, among them blood cell hemoconcentration, vasospasm, and the appearance of increased amounts of sticky proteins in the circulation partly from placental origin8 as well as reduced deformability of the RBCs in preeclampsia.27
The pathophysiologic relevance of increased RBC aggregation is related to the known observation that it is one of the determinants of the whole blood viscosity.1,2 It has been suggested that this increased aggregation impairs the blood flow in the microvasculature and might contribute to the development of low placental perfusion and consequently villi ischemia and impaired fetal growth.9 Notwithstanding, Verkeste et al28 failed to demonstrate reduced uteroplacental blood flow in Guinea pigs with increased red cell aggregation induced by hemoconcentration. Our study is, however, too small to address this issue, and further studies are in progress to clarify the clinical significance of an increased erythrocyte adhesiveness/aggregation test in PIH.
Fibrinogen is an important determinant for the induction and maintenance of an increased RBC aggregability,29 although other proteins can contribute as well.30 Indeed, according to our findings, fibrinogen could explain only partly erythrocyte aggregation, and probably other factors including macromolecules and membranal changes have a role as well.
An important finding of the present study is that the erythrocyte adhesiveness/aggregation test was significantly enhanced in PIH patients compared with controls despite there being no significant difference in the concentration of fibrinogen (Table 2). In this regard, it should be noted that some fibrinogen molecules might be more adhesive regarding the RBCs, and this is not necessarily revealed by the clotable assay used to determine the concentration of this molecule.31 It has been previously shown that therapeutic interventions can reduce both hypertension and RBC aggregability in pregnant women.23
Our study is significant in that it suggests a simple way to identify these women who might have increased aggregation. Although the ESR correlates with the aggregability of RBC, the former is still an indirect measurement for RBC aggregation.32 In fact, we recently identified a group of vascular patients with relatively normal ESR who had significantly elevated erythrocyte adhesiveness/aggregation tests despite there being absolutely no difference in either ESR or fibrinogen concentration between them and controls.33 Therefore, we assume that the direct observation and measurement of RBC aggregation might be superior to indirect measurements such as ESR or clotable fibrinogen.
Partial but still statistically significant association between erythrocyte aggregation and BP is yet another important observation in the present study. Such association has been previously reported,25,26,34 although not in pregnancy. It is speculated that the increased aggregation may contribute to increased vascular resistance, thus leading to increase in systolic and diastolic BPs. Providing that increased blood aggregation appears early in the sequence of events leading to hypertension, it may have a prognostic quality as well.
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Footnotes |
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Received January 31, 2001. Received in revised form May 1, 2001. Accepted May 4, 2001.
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REFERENCES |
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