Alexandra Păvăloiu PhD Thesis Summary · Alexandra Păvăloiu PhD Thesis Summary iv Keywords: systemic inflammatory response syndrome, adult horses, myeloperoxidase, peritoneal

Alexandra Păvăloiu PhD Thesis Summary

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UNIVERSITY OF AGRICULTURAL SCIENCES AND

VETERINARY MEDICINE CLUJ NAPOCA

DOCTORAL SCHOOL

FACULTY OF VETERINARY MEDICINE

PĂVĂLOIU ALEXANDRA NICOLETA

PhD THESIS SUMMARY

SCIENTIFIC ADVISOR

Prof. univ. dr. IONEL PAPUC

CLUJ-NAPOCA

2012


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UNIVERSITY OF AGRICULTURAL SCIENCES AND

VETERINARY MEDICINE CLUJ NAPOCA

DOCTORAL SCHOOL

FACULTY OF VETERINARY MEDICINE

PĂVĂLOIU ALEXANDRA NICOLETA

PhD THESIS SUMMARY

SYSTEMIC INFLAMMATORY

RESPONSE SYNDROME IN THE HORSE

SCIENTIFIC ADVISOR

Prof. univ. dr. IONEL PAPUC

CLUJ-NAPOCA

2012


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TABLE OF CONTENTS

Keywords ................................................................................................................ iv

Structure of the thesis ............................................................................................. iv

Thesis originality ..................................................................................................... v

Personal contributions- Results ............................................................................... v

Personal contributions- Conclusions and recommendations ................................. xii


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Keywords: systemic inflammatory response syndrome, adult horses, myeloperoxidase,

peritoneal fluid, emergency, neutrophilic degranulation

Structure of the thesis

This study is divided into 12 chapters, organized into two main parts. Part I

consists of the first seven chapters and is followed by Part II with the following 5

chapters. The thesis contains 141 pages, with 23 figures, 11 charts, 11 tables and 38

histograms, structured and drafted in accordance with the requirements and standards set

by the Doctoral School of the Veterinary Medicine Faculty of Cluj-Napoca

The first part refers to the current state of knowledge in the field of Systemic

Inflammatory Response Syndrome; the first chapter provides definitions for various

syndromes related to SIRS. The second chapter deals with inflammation and

pathophysiology of SIRS, describing intimate cellular and humoral responses in this

syndrome, as well as inflammatory markers and the coagulopathy met in SIRS. Chapter

three deals with endotoxaemia and endotoxins that can lead to the apparition of SIRS.

Chapter four is a synthesis of the acute phase proteins and the role they play in the

syndrome treated. The fifth chapter describes neutrophilic activation and oxidative stress

mechanisms involved in the syndrome and the sixth chapter continues the idea of

neutrophilic degranulation and myeloperoxidase importance. The seventh chapter deals

with the problem of peritoneal fluid and its importance in the diagnosis of equine

abdominal pathology.

The second part of the thesis is intended to convey the results of this research. It

begins with an overview of the purpose, in chapter eight, and objectives, in chapter nine,

and presents the experimental part in chapters ten and eleven, followed by the general

conclusions in chapter twelve and then references. The bibliography includes 115 titles.


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Thesis originality

The thesis originality lies in the use of the myeloperoxidase index of neutrophils

found in peritoneal fluid as diagnostic and prognostic indicator in systemic inflammatory

response syndrome in adult horses, correlated to the corresponding index in blood.

Although the results negate the possibility of using myeloperoxidase index as a

diagnostic or prognostic method, due to the early degradation of peritoneal neutrophils,

this study helps the clinician by the correlations made with traditional markers of

inflammation: the total number of leukocytes, fibrinogen, serum iron and

myeloperoxidase index in the blood, and by providing an overview of systemic

inflammatory response syndrome in horses, in terms of a paraclinical view.

Personal contributions- Results

The aim of the thesis is to identify, as accurately as possible, the paraclinical

diagnostic markers relevant to the systemic inflammatory response syndrome in adult

horses, so as to facilitate a swift and effective intervention. Also, this thesis aims to

identify and classify horses with systemic inflammatory response syndrome by age, race,

sex, primary symptoms, etiology and make a complete assessment of disease status of

patients with generalized inflammatory states and correlate these events with the blood

picture and peritoneal fluid characteristics. We hope that this work will become a guide

for clinicians working with horses, to help in the diagnosis of systemic inflammatory

response syndrome in order to maintain the health and welfare of horses.

The main objectives have framed the systemic inflammatory response syndrome

were in terms of hematological and peritoneal fluid characteristics by developing a more

complete picture, with the focus point in using peritoneal myeloperoxidase index as an

indicator of neutrophilic degranulation and thus indirectly of systemic inflammation.

The study initiated by us has both a retrospective character and a prospective one.

The retrospective part was done by interpreting laboratory results acquired from the


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peritoneal fluid and blood collected from 155 horses. Correlations between markers of

systemic inflammatory response syndrome in peritoneal fluid and blood of horses were

performed. The diagnosis of systemic inflammatory response syndrome was made based

on clinical symptoms and biochemical markers: serum iron, fibrinogen and

myeloperoxidase index. Diagnosis of SIRS followed the protocol described by Schwarz

et al. (2011), in which patients were classified as healthy, with local inflammation, with

SIRS or sepsis (the last two may overlap). Samples were processed with ADVIA 120,

manufactured by Siemens, in the Clinical Laboratory of the University of Veterinary

Medicine, Vienna.

Our results were reported in the 155 patients taken into study, and interpretation

and discussion of results took into account both the parameters of peritoneal fluid and

blood.

Patients were classified according to race, age, sex, primary symptoms and

diagnostic categories.

Following parameters were determined in the peritoneal fluid: time lapsed since

sampling, color, turbidity level, composition, cell number, total protein, lactate

dehydrogenase, total leukocytes, erythrocytes in absolute numbers, neutrophils, relative

lymphocytes and monocytes, myeloperoxidase index and quality of transudate / exudate.

The values of these parameters were subjected to statistical calculation using descriptive

statistics, calculating the mean and standard deviation.

In blood samples we determined WBC, plasma iron, fibrinogen values which were

correlated with the myeloperoxidase index, making a comparison of these values in

horses diagnosed with SIRS and non-SIRS ones. Finally we tried to see if there is a

correlation between myeloperoxidase index in blood and peritoneal fluid in horses

diagnosed with SIRS.

The descriptive statistics for the 155 samples is presented in chapter 11, section

11.1. The breeds were mostly warmbloods (Shagya, Austrian Warmblood, Andalusian,

etc.), followed by ponies (Icelandic, Fjord, Falabella etc.) mixed breeds and a very small

percentage of heavy breeds (Noriker).


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In terms of sex, patients were distributed almost equally, with 46.5% geldings,

65% mares and 17% stallions. In terms of age, distribution was more or less

homogeneous, most being adult horses with fewer foals and seniors, the average stood at

12.28 years, with standard deviation = 6.742 , N = 152.

All samples studied were accompanied by a brief history that has been so designed

as to characterize briefly the reason for presentation and may be considered in

establishing a diagnosis. Based on history, patients were classified into nine categories:

colic, fever, anorexia / cachexia, suspected malignancy, castration complications, rectal

prolapse / crash mount, diarrhea (without other symptoms), rigid walking, or study. Most

problems for which patients were brought to consult were classic symptoms of colic,

followed by fever (usually of unknown origin, chronic) and anorexia / cachexia.

Based on clinical and laboratory diagnosis, the final diagnosis was established and

based on this evidence, patients were divided into 12 categories, of which most horses

(30.52%) suffered from peritonitis, followed by displacement of the colon / tympanism /

gastritis (12.34%) and gastroenteritis / colitis / ileus (11.04%), cancer (7.79%), colon

dysregulation (7.14%), parasites (6.49%) viral fever (5 19%), problems other than

gastrointestinal (3.90%), complications after castration and rectal prolapse (3.25%),

strangles (2.60%), inflammatory bowel disease/ MEED / EGS (1.95%). 7.79% of the

animals were classified as clinically healthy.

Parameters in the peritoneal fluid that were determined were: time lapsed since

sampling, centesis liquid color, degree of turbidity, consistency, number of cells, total

protein (TP g/dl), lactate dehydrogenase (LDH U/l ), total leukocytes in absolute numbers

(WBC/ml), erythrocytes in absolute numbers (RBC/ml), relative neutrophils (Neutro%)

relative lymphocytes (lymph%) relative monocytes (mono%), myeloperoxidase index

(MPXI), quality of transsudate / exudate. Studied parameter values are represented as

histograms.

Total protein expressed as g/dl peritoneal fluid had an average of 16.24, with a

standard deviation of 12.109. Total leukocytes expressed in absolute numbers stood at an

average of 23948.29, with standard deviation76690.167.


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Mean MPXI and percentiles are much lower than the ones in blood. Thus,

neutrophils that come through blood capillaries seem to have a more advanced states of

degranulation as soon as they get in the peritoneal cavity compared with those who

remain in the blood.

In approximately 75% of horses diagnosed with systemic inflammatory response

syndrome MPXI values are negative, but although this percentage is clearly lower for

healthy horses, the difference is not statistically significant.

Peritoneal fluid type (such as high or low protein: transsudate / exudate) had no

effect on MPXI values. It seems that the abdominal fluid character does not play an

important role in low levels of MPXI in the abdominal space of healthy or diseased

animals.

Effect of storage on MPXI-To monitor the effect of elapsed time from sampling to

processing peritoneal fluid, the data obtained were processed statistically, pursuing the

same variables as in the case of myeloperoxidase index, the addition calculation

correction of rank, U test (Mann Whitney), and Wilcoxon test for nonparametric methods

to assess the size of differences. It seems that a storage period of more than 4 hours had

no significant effects on peritoneal fluid samples, but it should be noted that MPXI was

already quite low in some cases.

MPXI prognostic utility within the systemic inflammatory response in adult

horses- MPXI used as prognostic indicators of survival was studied by comparing the

charts, which showed an easy recognizable model. 132 surviving horses were compared

with 21 who did not survive (either died or were euthanized). Thus, MPXI abdominal

fluid is not a useful parameter for prognostication of survival. To complete this analysis a

ROC curve was built in a later chapter.

Also to discover possible prognostic indicators, we studied whether peritoneal

fluid was an exudate or transsudate, and we statistically processed other data, such as

survival. 65.8% of samples were transsudate or modified transsudate and 29.7% were

exudates. 86.3 of patients were alive, and 13.5% did not survive (either died or were

euthanized). Other factors studied in order to find a relationship with the finality of the


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case were tested using Mann-Whitney U test for calculation of rank correlation

(myeloperoxidase index, total number of cells, total protein, blood leukocyte counts and

fibrinogen in the blood). These parameters do not differ statistically significant within

groups of survivors and non-survivors.

We also performed a combination of some of these parameters between them,

studying relationships through correlation calculations with non-parametric Kendall Tau

and Spearman Rho test (non-normally distributed data) between cell count., total protein,

total leukocytes, neutrophils, immature white blood cells and myeloperoxidase index,

evaluating the correlation coefficient.

Finally, a calculation and parametric correlation was performed by determining

Pearson correlation index after as a measure of association between two continuous

variables, and which is the best estimate of the parameters. The overall level of peritoneal

myeloperoxidase index correlates with some parameters of inflammation, but it is a rather

weak correlation in terms of statistical significance (neutrophils, total protein, total

leukocyte count, etc).

Digitization of the database as transsudate versus exudate gave somewhat better

results, using thell Pearson method, to assess the strength and direction of association

between several variables (depending on the nature of peritoneal fluid as transudate,

modified transsudate and exudate). The explanation for this is the fact that neutrophils do

not degranulate to release cytotoxic products in a random or gradual manner, but in an

explosive way and more or less the same time the invasion takes place into the peritoneal

cavity, issues that are facilitated by their rapid migration. Rapid degranulation will take

place due to the extreme mobility and short life of these cells. Neutrophilic mobilization

in the abdominal cavity takes place easily with any sign of peritoneal irritation, within 24

hours of its occurrence. This will lead to non-normally distributed peritoneal MPXI in the

granulocytes of sick and healthy horses.

ROC curve (Receiver Operating Characteristic): Any possibility that MPXI

can be used as an aid in diagnosis as well as its cut-off levels was analyzed using ROC

curves. These are graphic distributions illustrating the performance of a binary system


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performance by distributing true positives (TPR, true positive rates) versus false positives

(FPR, false positive rates). TPR (y-axis) is called sensitivity while FPR (x-axis) is called

specificity. The groups determined were horses with infection and healthy horses or with

other pathology than infection.

Area under the curve (AUC) is equal to the probability that a classifier will

classify in rank one positive instance higher than the negative one, it is similar to Mann

Whitney U test (which tests if positive values lie higher than the negative ones) and

Wilcoxon test and is useful for comparing a model. In our case, we calculated the AUC

for MPXI. Thus, standing at 0.463 (with a standard error of 0.48), the MPXI in peritoneal

neutrophils as diagnostic criteria for general or even local inflammatory status is not

valid.

Log modeling is one of multivariate analysis methods that try to find a good

explanatory model which describes relationships between variables. This model considers

that all variables used for classification are independent and the dependent variable is the

number of cases in the contingency table data. If the MPXI data is log normalized (elog)

and are looked at in terms of non-inflammatory states, it appears as quite a weak

diagnostic test (area under curve, AUC = 0.64). Cut off value for sensitivity of 0.80 is

2.9364 (MPXI = 18.8) while the maximum sensitivity is 0.62. This will lead to too many

false positives with this test. Also, many negative values of MPXI were taken out of the

question because we were not able to calculate logarithms. Area under the curve (AUC)

for the log myeloperoxidase index is also not strongly statistically significant, 0.639 with

standard deviation 0.101.

We also tried to adapt by adding the value of 75 for all data, but even this change

did not help very much. The curve was not so weak even before normalization, but still

looks random. Not even the use of cosine and arctangens normalization brought results.

Some features of MPXI of peritoneal fluid: transudates and modified transudate,

effusion: Regarding MPXI value in the transsudate or modified transudation, the average

value was -9.54, with a standard deviation of 32,006 (N = 102). Peritoneal fluid classified

as exudate had a MPXI with an average value of -21.47, 18.684 standard deviation (N =


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46). For animals clearly diagnosed with peritonitis, MPXI had an average value of -

17.79, with a standard deviation of 28.093 (N = 47). In patients with conditions other

than peritonitis, the average was -11.80, 28.804 standard deviation (N = 107). Patients

with peritonitis (N = 47) showed a mean negative myeloperoxidase index (-17.787,

standard error 4.09), but those without peritonitis (N = 107) also had a negative value (-

11,803, standard error 2.78). Levene test was used limited the sample to test (independent

samples), it had a weak statistical significance (p = 0.365), so we tried and tested random

samples by using the t test (basically as for a single small sample size), but had

statistically insignificant results (t = 1.196 for equal variances and for unequal variances t

= 1.208). We tried to find a statistically significant difference by comparing MPXI of

peritoneal neutrophils according to diagnosis (healthy versus diseased), depending on the

presence or absence of inflammation as well as rank data usage and utilization of

coefficients correlation (Mann Whitney U, Wilcoxon and Z), but that did not reveal any

statistically significant difference.

Blood samples were statistically evaluated both in terms of descriptive and a few

parameters correlating with each other, with surprising results. 41.37% percent of the

patients in whom inflammatory markers were tested, were suffering from SIRS with low

MPXI values. Patients with leukocytosis / leukopenia represented 45% of total blood

samples, whereas patients with white blood cells within the reference interval were 56%.

It is interesting that statistical analysis showed no statistically significant correlation in

the cases taken by our study, unlike research conducted by other research teams. The

correlation between myeloperoxidase index in blood and plasma iron showed a

regression line with a positive trend, but statistically insignificant, R2 being 0.0295. The

correlation between blood myeloperoxidase index with blood fibrinogen, showed a

regression line with a negative trend but statistically insignificant, R2 being 0.0552. The

correlation of blood myeloperoxidase index with the absolute number of leukocytes

showed a regression line with a negative trend but statistically insignificant, R2 = 0.008.

Comparison between blood samples and samples of peritoneal fluid-Another

element of statistical processing was comparing the blood samples MPXI with peritoneal


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fluid samples MPXI to show regression curves. MPXI of the average blood neutrophils

was 6.4, significantly higher than the value of -5.1 in abdominal neutrophils, but there

was a weak association (R2 = - 0.031) between MPXI blood and peritoneal fluid. Thus, it

appears that degranulation is more severe in the peritoneal level than in the blood. T test

was used for paired random samples. Pairs were made between blood myeloperoxidase

index and the MPXI of peritoneal fluid, but no test either parametric or non-parametric,

dedicated or random samples revealed any correlation statistically significant (MPXI

mean was 11.4945 with standard error of 5.246, T = 2.191, the Pearson correlation result

was -0.177). As we expected from the weak association and of both variables MPXI,

there was no linear relationship between abdominal neutrophi degranulation and

degranulation of neutrophils in the blood. Blood and abdominal MPXI regression was

statistically insignificant.

We have also tried and taken into account only the first sample / case analyzing

data of 51 horses. Thus, the mean peritoneal fluid MPXI value was – 3.657 with standard

deviation 37.2476 and the blood MPXI value was 6.686, with standard deviation 8.8251

(in both cases N = 51). In these cases, a quite weak correlation was found but almost

significant (R = -0.23, p = 0.054) between MPXI of blood neutrophils and abdominal

neutrophils. However, this correlation could not be confirmed by a significant linear

regression between both MPXI parameters.

Personal contributions- Conclusions and recommendations

Our research results in systemic inflammatory response syndrome in adult horses,

as the proposed experimental design, allowed us to extract the following conclusions:

• Systemic Inflammatory Response Syndrome is a challenge for clinicians and a

swift diagnosis is a medical priority, ensuring successful therapy.


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• The diagnosis of certainty in systemic inflammatory response syndrome in

horses is determined by biochemical constituents represented by myeloperoxidase index,

serum iron, fibrinogen, leukocytes, whose values are modified both in blood and

peritoneal fluid.

• Only half of the patients in the study and diagnosed with systemic inflammatory

response syndrome showed leukopenia / leukocytosis, with white blood cell counts

remaining within the normal range.

• Diagnostic criteria of SIRS in human medicine correspond only partially to

equine medicine, leukopenia / leukocytosis which are constantly encountered in the

diagnosis of systemic inflammatory response syndrome in humans are inconsistent in

horses, with no statistical significance.

• Increase of serum fibrinogen occurs within 48 hours so that it cannot be

considered a good indicator for monitoring the occurrence of systemic inflammatory

response syndrome.

• Diagnosing this syndrome requires a comprehensive evaluation of suspected

equine blood picture correlated with markers of systemic inflammation.

• Peritoneal fluid may be a good indicator of acute abdominal pathology.

• Myeloperoxidase index determined in peritoneal fluid does not accurately reflect

the systemic inflammatory state.

• The peritoneal myeloperoxidase index does not correlate with the blood

myeloperoxidase to aid in the diagnosis of systemic inflammatory response syndrome in

adult horses.


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• Peritoneal myeloperoxidase index has no predictive value in systemic

inflammatory response syndrome in adult horses.

• Corroboration of the blood results with the peritoneal fluid results from horses

suspected with systemic inflammatory response syndrome gives the best results in the

diagnosis of this pathology, especially due to light clinical symptoms or ones that hide

the systemic problem.

• Light and equivocal clinical symptoms in the systemic inflammatory response

syndrome in horses require the clinician to use laboratory tests as soon as possible to

narrow the search field to establish the diagnosis; the disease should be considered a

medical emergency.

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Alexandra Păvăloiu PhD Thesis Summary · Alexandra Păvăloiu PhD Thesis Summary iv Keywords: systemic inflammatory response syndrome, adult horses, myeloperoxidase, peritoneal