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Enhanced Erythrocyte Aggregation in Clinically Diagnosed Pelvic Inflammatory Disease

Almog, Benny MD*; Gamzu, Ronni MD, PhD*; Almog, Ronit MD*; Lessing, Joseph B. MD*; Shapira, Itzhak MD†; Berliner, Shlomo MD, PhD†; Pauzner, David MD*; Maslovitz, Sharon MD*; Levin, Ishai MD*

Sexually Transmitted Diseases: August 2005 - Volume 32 - Issue 8 - pp 484-486

Objective: Enhanced erythrocyte aggregation, revealed using a simple bedside test, has been found recently in several inflammatory conditions. The diagnosis of pelvic inflammatory disease (PID) is at times difficult because of the vague symptoms and signs, but is crucial because even mild PID can have future reproductive consequences. Our objective was to determine the degree of erythrocyte aggregation in PID. The demonstration of an increase in aggregation could be of additive value in cases in which the diagnosis is difficult.

Study design: A prospective case-control study was conducted. Fifteen consecutive women diagnosed clinically as having PID based on Centers for Disease Control and Prevention criteria, and 15 matched controls were enrolled. Blood samples were drawn for hematologic indices, C-reactive protein, fibrinogen levels, and red cell aggregation. We studied the degree of red cell aggregation using a simple slide test and image analysis. The variable measured was the erythrocyte percent (EP), equivalent to the slide area covered by erythrocytes.

Results: Erythrocyte percent was 59.6 ± 4.2 and 80.0 ± 3.6 for the study group and controls, respectively (P <0.001). A significant difference was noted also for the other hematologic and biochemical markers of inflammation between patients and the controls.

Conclusions: We conclude that the degree of erythrocyte aggregation is increased in PID. Its simplicity, rapidity, and low cost should be further evaluated as a diagnostic tool in the context of this frequent disease.

Enhanced erythrocyte aggregation, revealed using a simple bedside test, has been found recently in several inflammatory conditions. We found that the degree of erythrocyte aggregation is increased in pelvic inflammatory disease.

From the * Department of Gynecology, Lis Maternity Hospital, and † Internal Medicine D, Tel Aviv Sourasky Medical Center, affiliated with the Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel

Shlomo Berliner is a shareholder in the Inflamet Ltd. Co., Tel-Aviv, Israel.

Correspondence: Benny Almog, MD, Gynecology Department, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, 6 Weizman St., Tel-Aviv, 64239, Israel. E-mail:

Received for publication September 10, 2004, and accepted January 13, 2005.

A REAL-TIME, ONLINE, LOW-COST, and point-of-care method to identify the presence of a humoral acute-phase response might be of clinical relevance. This is true for most acute infections/inflammations and could help to single out individuals with a hyperinflammatory response to an invading microorganism.1 The prompt identification of a significant acute-phase response might have therapeutic implications in terms of early institution of appropriate antimicrobial and antiinflammatory treatments.2,3

We have recently adopted a simple slide test and image analysis to quantitate the degree of erythrocyte aggregation of peripheral blood slides.4,5 In this test, we use the patient's own erythrocytes as sensors for the presence of increased concentrations of sticky proteins that are synthesized during the acute-phase response. The erythrocytes react by enhanced intercellular interaction/aggregation.7 This enhanced aggregation correlates with the degree of the acute-phase response.8

We have presently focused on a model of pelvic inflammatory disease (PID) in women. PID is an inflammatory disease with clinical symptoms and signs that are at times vague, thereby making the diagnosis of this clinical entity a difficult one.9 Our results are encouraging in that they show that it is possible to differentiate effectively the affected women from matched controls. This is one of several studies that are currently conducted to reveal the usefulness of this new biomarker in daily practice.

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Materials and Methods

Patients and Controls

All women participating in the current study signed a written informed consent according to the instruction of the local ethics committee. There were 15 consecutive women admitted to the Department of Gynecology with PID according to the Centers for Disease Control and Prevention (CDC) criteria.9 The CDC minimal clinical criteria for diagnosis used were lower abdominal pain, adnexal tenderness, and cervical motion tenderness. Additional CDC criteria for diagnosis were elevated body temperature (above 38.3°C), enhanced erythrocyte sedimentation rate, and abnormal cervical or vaginal discharge. Fifteen healthy women, matched for age and body mass index (BMI), smoking status, and/or contraception use served as controls. None of the participants in the study had any chronic inflammatory underlying disease or was taking any steroidal or nonsteroidal antiinflammatory agent.

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Laboratory Tests

The blood count was performed using the Coulter STKS (Beckman Coulter, Nyon, Swiss) electronic cell analyzer, the erythrocyte sedimentation rate (ESR) by the method of Westergren,10 fibrinogen concentration by the method of Clauss,11 and a Sysmex 6,000 (Sysmex Corp., Hyaga, Japan) autoanalyzer, whereas the high-sensitivity C-reactive protein (hs-CRP) was performed by using the Boering BN II Nephelometer (DADE Boering, Marburg, Germany) and a method according to Rifai.12

The aggregation of red blood cells in the peripheral blood was performed by using a simple slide test.13 In brief, blood was drawn into a syringe containing sodium citrate (1 volume of 3.8% sodium citrate and 3 volumes of whole blood). One drop of the citrated whole blood was tricked onto a slide inclined at an angle of 30° and allowed to run down by gravity, leaving a fine film. The slides were left to dry in that position at room temperature. A technician who was masked to the clinical and laboratory results of the patients scanned the slides by using an image analysis system (INFLAMET; Inflamet Ltd., Tel Aviv, Israel). For the analysis of the slides, we used an image analysis system (INFLAMET)14 consisting of a Pentium computer running Windows 95 equipped with a Matrox Meteror (Matrox Ltd., Montreal, Canada) color frame-grabber, a color CCD camera, and a microscope, which was operated at ×200 magnification, resulting in an image resolution of 0.4 μm per pixel. Nine images were taken from each slide, 3 from the margins, 3 from the center, and 3 from the tail. The technician who chose the fields of view was not aware of any clinical or laboratory information regarding study participants. 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 9 fields of view covered a total area of 0.6 mm2. The variable that was used to describe the state of erythrocyte aggregation was the erythrocyte percentage (EP). This is the slide area covered by the erythrocytes. When there is no aggregation, 100% of the slide area is covered with erythrocytes, whereas during aggregation, this percentage is reduced as a result of the appearance of clear areas between the groups of aggregated cells. Variabilities of this methodology have been published elsewhere.15,16

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Statistical Analysis

All the variables were analyzed for the normality of their distribution by the one-sample Kolmogorov-Smirnov test procedure. Differences between parameters in the 2 patient groups were evaluated using Fisher exact test, 1-way analysis of variance, and Kruskal-Wallis when appropriate. The nonpaired t test or post hoc Bonferroni analysis were used to perform pairwise comparisons between group means. According to power analysis, 15 subjects were the minimum sample size required in each subgroup (α of 5% and power of 80%). A P value of below 0.05 was considered statistically significant. Calculations were performed using the SPSS software package (SPSS Inc., Chicago, IL).

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Mean age for the study and control groups was 31.5 ± 2.1 years. No difference was noted for the BMI in both groups (23 ± 1 and 27 ± 2 kg/m2, P = 0.14).

The various laboratory tests herewith performed are depicted in Table 1. It can be seen that although there was no significant difference between the 2 groups in the concentration of hemoglobin, a significant difference was noted for the variables of the acute-phase response, including the degree of erythrocyte aggregation. A typical picture obtained from a patient with PID versus a control is given in Figure 1.

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The prompt identification of a significant acute-phase response might have significant diagnostic and therapeutic implications in daily practice.1–3 This is especially true for clinicians who work in small clinics and group practices devoid of advanced laboratory facilities. A simple bedside low-cost and rapid evaluation of the acute-phase response might therefore be of special interest.

It is known that various proteins are synthesized by the liver after acute infections.6 Several of these proteins, and especially fibrinogen, immunoglobulins, haptoglobin, ceruloplasmin, α1 antitrypsin, orosomucoid, and even CRP itself, might be involved in the induction and or maintenance of erythrocyte aggregation.17–21 Although it was shown that fibrinogen has a dominant role in this aggregation,22 it is clear that other proteins are involved as well.23 We have taken advantage of this biologic phenomenon and used the erythrocytes as sensors for the presence of multiple adhesive proteins in their plasmatic milieu. By using accurate physical measurements, the degree of aggregation can turn into a diagnostic tool.24

An advantage of our slide test is that the images can be easily transmitted by telephone or Internet25 to an inflammation data center, where appropriate controls can be immediately matched.26 In fact, we have recently established such a data center where the inflammatory baseline profile of more than 2500 healthy individuals is available. These are individuals who are recruited during their routine annual checkup examination.

It can be argued that similar information can be obtained using the Westergren sedimentation rate. However, ESR is an indirect way of looking at the degree of erythrocyte aggregation. The advantage of looking directly at this aggregation has been shown by us in several clinical models in the past.27–30 In addition, results are obtained within a couple of minutes.

At first glance, it looks as if complicated systems of microscopy and image analysis are needed for our measurements. However, it is clear that by using simple electro-optical principles, our method could turn into a bedside test, similar to what is presently performed with glucometers. Such an approach might be relevant in women with PID, a frequent clinical dilemma for the gynecologist.

The CDC guidelines state that “Many episodes of PID go unrecognized.” Although some cases are asymptomatic, others are undiagnosed because the patient or the healthcare provider fails to recognize the implications of mild or nonspecific symptoms or signs (e.g., abnormal bleeding, dyspareunia, and vaginal discharge). Because of the difficulty of diagnosis and the potential for damage to the reproductive health of women even by apparently mild or atypical PID, healthcare providers should maintain a low threshold for the diagnosis of PID.9 We used the CDC clinical criteria, which are sensitive but not very specific. Laparoscopy and/or endometrial biopsy are more specific indicators of acute PID. Interestingly, in our study group, we noticed several women with increased aggregation results, whereas some of the other equivalent inflammation laboratory tests for PID were normal. There were 3 cases with normal ESR, 2 cases with a normal leukocyte count, and 2 cases with normal hs-CRP. These patients were clinically diagnosed as having PID despite some normal laboratory values. The difficult diagnosis of these patients was made easier because all had significantly elevated aggregation results. Increase in aggregation could be of additive value in these cases in which the diagnosis is difficult.

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We conclude that the degree of erythrocyte aggregation as determined by the erythrocyte percent is elevated in PID in a similar way that it has been shown for other clinical models. Its simplicity, rapidity, and low cost should be further evaluated in the context of this frequent disease.

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