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The Evaluation of Postmortem Ocular Fluid Analysis

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The Evaluation of Postmortem Ocular Fluid Analysis as a Diagnostic Aid in Sows
Article in Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc · February 1990
DOI: 10.1177/104063879000200103 · Source: PubMed
3 authors, including:
Sylvie D'Allaire
Madeleine Chagnon
Université de Montréal
Université de Montréal
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Journal of Veterinary Diagnostic
The Evaluation of Postmortem Ocular Fluid Analysis as a Diagnostic Aid in Sows
Richard Drolet, Sylvie D'Allaire and Madeleine Chagnon
J VET Diagn Invest 1990 2: 9
DOI: 10.1177/104063879000200103
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J Vet Diagn Invest 2:9-l3 (1990)
The evaluation of postmortem ocular fluid analysis
as a diagnostic aid in sows
Richard Drolet, Sylvie D’Allaire, Madeleine Chagnon
Abstract. This study was undertaken to evaluate the diagnostic usefulness of postmortem ocular fluid analysis
in estimating the antemortem status of various serochemical constituents. Chemical values of serum and aqueous
and vitreous humors were compared following different procedures. A blood sample and the 2 eyes were collected
from each of 100 sows at a nearby abattoir. The results obtained from immediate centrifugation of ocular fluids
after sampling were compared with those samples in which centrifugation was delayed by 2 hours. Two different
postmortem intervals were used for sampling ocular fluids, 2 and 24 hours. Concentrations of urea, calcium,
phosphorus, potassium, sodium, and chloride were determined from serum and humors. Delayed centrifugation
did not affect chemical values of ocular fluids nor the relationships between serum and humors. Phosphorus
and potassium values increased significantly with the postmortem interval in both aqueous and vitreous humors.
The relationships between chemical values of ocular fluids and serum were determined using simple linear
regression. There was a poor correlation between ocular fluid and serum values for all electrolytes; a significant
correlation was found only for urea concentrations in both humors.
Determination of biochemical abnormalities could
be a useful diagnostic aid to ascertain the cause of death
or to rationally evaluate the significance of many anatomic lesions found during necropsy. Because of the
chemical and physical alterations occurring in blood
after death, this body fluid is usually considered unsuitable for biochemical analysis. However, intraocular humors appear to deteriorate more slowly after
death and are an easily accessible source of fluid.3,7,21
In human forensic pathology, most studies have dwelt
on the determination of potassium content of the vitreous humor as an aid in estimating the time of
death 1-3,6,9,14,21,22 The information published in the veterinary literature in the last decade pertains mostly to
the relationships of ocular fluids with serum chemical
values, and to changes that occur with postmortem
interval and incubation temperature. There are very
few recently published data concerning the concentrations and variations of eye fluid contents in swine13
compared with other animal species.4,8,10-l3,18-20,23
The aims of the present study Were to evaluate in
sows, for different biochemical constituents, the effect
of delayed versus immediate centrifugation of ocular
fluids after sampling, the changes occurring up to 24
hours after death, and, more importantly, the diagnostic value of postmortem ocular fluid analysis to
predict antemortem serum electrolytes and urea concentrations.
Materials and methods
A total of 100 sows were used from a nearby abattoir. A
blood sample was collected from each sow at exsanguination
time at slaughter and was kept on ice in a cooler for transportation to the laboratory. The serum was removed and
kept at -20 C until analyzed.
At the abattoir, the eyes of each sow were enucleated with
a pair of curved scissors and placed in a plastic bag on ice
in a cooler for transportation to the laboratory. The 100 sows
were divided into 4 groups of 25 sows each, corresponding
to 4 different treatments in which either the postmortem
interval (PMI) or the time between sampling and centrifugation (CEN) varied. For each eye, aqueous and vitreous
humors were collected and analyzed separately. Aqueous humor was gently aspirated with a syringe using a 16-gauge
needle inserted under the cornea. Vitreous humor was withdrawn with a syringe using a 16-gauge needle inserted through
the sclera caudal to the limbus. Samples containing cellular
debris were discarded.
Centrifugation was ‘either done immediately (CEN 0) or
delayed by 2 hr (CEN 2) after sampling of eye fluids. For
CEN 2, ocular humors were kept at room temperature (22
C) for 2 hr. Samples were centrifugated at 11,400 x g for 15
min. The supernatants were collected and stored immediately at -20 C until analyzed. The 2 postmortem intervals
selected were 2 (PMI 2) and 24 hr (PMI 24). The eyes were
kept at 4 C until sample extraction when the incubation time
was 24 hr. We compared the effect of CEN or PMI on ocular
fluid values in the same animals (Table 1). We also compared
serum, aqueous humor, and vitreous humor values in the
same animals for different PMI’s or CEN’s.
Biochemical analyses were all done on the same day to
From the Departement de pathologie et microbiologic (Drolet,
Chagnon), and the Groupe de recherche sur les maladies infectieuses
du port (GREMIP) (D’Allaire), Faculte de Medecine veterinaire,
Universite de Montreal, C.P. 5000 Saint-Hyacinthe, Quebec, Canada J2S 7C6.
Received for publication August 17, 1989.
Drolet, D’Allaire, Chagnon
Figure 1. Means and SD of chemical constituent concentrations in serum and aqueous and vitreous humors. Serum was obtained at
time of death, and humors were obtained either 2 (PMI 2) or 24 hours (PMI 24) after death. Differences were significant at P < 0.05: a,
between serum and ocular fluids for PMI 2 groups; b, between serum and ocular fluids for PMI 24 groups; c, between aqueous and vitreous
humors within each PMI group. Numbers within bars represent means of ratios of ocular fluid values to serum values.
minimize technical errors. Chemical analyses on ocular fluids
and serum included urea, calcium, phosphorus, potassium,
sodium, and chloride. Albumin concentrations were determined in serum to adjust total serum calcium. Urea, calcium,
phosphorus, and albumin concentrations were measured with
an automated batch analyzer.a The calcium, phosphorus, and
albumin assay was based on a calorimetric method, and the
urea assay was based on a UV kinetic method. Determination
of potassium, sodium, and chloride concentrations was done
on an automated analyzerb using an ion-selective method.
Student’s paired t-test was used to compare values of serum, aqueous humor, and vitreous humor and to determine
whether the time between centrifugation and sampling or the
postmortem interval influenced the chemical concentrations
Table 1. Experimental design. PMI = postmortem interval (hours)
and CEN = interval between eye fluid sampling and centrifugation
Aqueous and vitreous humors
Left eye
PMI 2, CEN 0
PMI 24, CEN 0
PMI 2, CEN 0
PMI 2, CEN 2
Right eye
PMI 2, CEN 2
PMI 24, CEN 2
PMI 24, CEN 0
PMI 24, CEN 2
CEN at PMI 2
CEN at PMI 24
PMI at CEN 0
PMI at CEN 2
of ocular fluids. Ratios of eye fluid to serum concentrations
for each constituent were calculated. Correlation coefficients
were used to measure the association between ocular fluid
and serum chemical values. Simple linear regression was used
to obtain a regression equation for estimation of the antemortem serum concentration from the postmortem eye fluid
concentration. A P value <0.05 was chosen for statistical
significance. The results were computed and analyzed with
the Statistical Analysis System. l7
Table 2. Correlation coefficients between ocular fluid values and
serum values for various chemical constituents at 2 different postmortem intervals (PMI-in hours).
Aqueous humor
vs. serum
Vitreous humor
vs. serum
PMI 24
PMI 24
*P < 0.05.
Ocular humors in sows
Figure 2. Regression line revealing the lack of correlation between postmortem aqueous humor calcium concentration and serum
calcium concentration (r = 0.09, P = 0.43). Aqueous humor calcium
concentrations were obtained 2 hours after death.
Figure 3. Scatter diagram and fitted regression line of serum urea
concentration on aqueous humor urea concentration. Aqueous humor urea concentrations were obtained 2 hours after death.
There was a high ly significan t correlation on1y between
ocular fluid and serum urea concentrations (Fig. 3).
A difference in chemical values of ocular fluids was
not observed between CEN 0 and CEN 2 subgroups
whether the aqueous or vitreous humors were collected
It has been suggested that immediate centrifugation
at PMI 2 or PMI 24. Therefore, results of samples taken or filtration of ocular fluids after sampling is desirable
at CEN 0 and CEN 2 were pooled for subsequent anal- for improving the repeatability of the results.23 In our
study, values of chemical constituents of both humors
Potassium and phosphorus levels increased signifi- were not significantly different when centrifugation was
cantly with the postmortem interval in both aqueous done immediately or delayed by 2 hours after collecand vitreous humors. The increases in potassium and tion at either postmortem interval. These results might
phosphorus were greater in vitreous humor, 2-fold and have been different if ocular fluids had been sampled
2.5-fold, respectively, compared to 1.7-fold and 1.8- immediately after death (PMI 0). Centrifugation or
fold, respectively, for aqueous humor. None of the filtration of vitreous humor, however, has the obvious
other chemical constituents varied with the postmor- beneficial effect of removing the gel structure of the
vitreous body that may interfere with the handling of
tem interval.
Concentrations (mean ± SD) of the various chem- the sample.
To verify the stability of the different chemical conical constituents in serum and aqueous and vitreous
humors for PMI 2 and PMI 24 groups are reported in stituents after death, 2 postmortem intervals were seFig. 1. Means ± SD of adjusted serum calcium levels lected to reflect, as closely as possible, the situation
for sows in the PMI 2 and PMI 24 groups were 2.61 that prevails in a diagnostic laboratory. At our insti± 0.16 and 2.66 ± 0.17 mmol/liter, respectively. Means tution, most dead sows are submitted for necropsy
of ratios of aqueous and vitreous humors to serum within 24 hours of death. A postmortem interval of 2
values are also presented for both PMI groups (Fig. 1). hours probably represents the minimum interval of
For all electrolytes, a significant difference was ob- time between the death of an animal on a farm and
served between values in aqueous and vitreous humors the beginning of a necropsy at a nearby laboratory.
at either PMI. Urea concentrations were similar in Results of this study showed that with increasing postaqueous and vitreous humors until 24 hours after death. mortem interval there was, in both humors, a signifCorrelation coefficients between ocular fluid and se- icant rise in the concentrations of potassium and phosrum chemical values are reported in Table 2. All chem- phorus, whereas the values of calcium, sodium,
ical constituents except urea showed a poor correlation chloride, and urea stayed relatively stable up to 24
between ocular fluid and serum values. Similar results hours after death. Similar results were obtained in a
were found using adjusted serum calcium instead of recent study using porcine vitreous humor. l3 This intotal calcium. Figure 2 represents the regression line crease in potassium concentration after death has been
of serum calcium on aqueous humor calcium concen- widely used in forensic medicine as an indicator of
trations at PMI 2 as an example of poor correlation. postmortem interval. 1-3,69,14,2l,22 The potassium con-
Drolet, D’Allaire, Chagnon
centrations in ocular fluids also rise with an increased
environmental temperature as demonstrated in several
This study was supported in part by the Fonds du centeanimal species. 12,13,18 In our study, the temperature did naire, Faculté de Médecine vétérinaire, Université de Monnot vary; eyes were kept at 4 C from the time of enucle- tréal. We are grateful to L. Héroux, G. Dumas, R. Vigeant,
ation until collection of ocular fluids.
and M. Sicotte for their assistance.
Because concentrations of most chemical constituSources and manufacturers
ents are different in ocular fluid versus serum, some
investigators have used ratios of ocular fluid values to
a. Hitachi Automatic Analyzer 705, Boehringer Mannheim Canada, Dorval, Quebec, Canada.
serum values to express this relationship for a given
b. Beckman System E4A Analyzer, Beckman Instruments, Inc.,
In our study, ratios of vitreous huconstituent.
Brea, CA.
mor values to serum values for constituents that remained stable after death were similar to those published for swine. l3 However, ratios calculated from
aqueous humor could not be compared to other reports
1. Adelson L, Sunshine I, Rushforth NB, Mankoff M: 1963, Vitbecause data available for ocular fluids in animals usureous potassium concentration as an indicator of the postmorally pertain to the vitreous humor. Moreover, values
tem interval. J Forensic Sci 8:503-514.
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death by potassium levels in the vitreous humor. Forensic Sci
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from published reports have been commonly used to 3. Coe JI: 1969, Postmortem chemistries on human vitreous humor. Am J Clin Pathol 51:741-750.
estimate the antemortem serum values. This has been 4. Crowell WA, Duncan JR: 1974, Potassium concentration in
considered to be of some practical application for
the vitreous humor as an indicator of the postmortem interval
in dogs. Am J Vet Res 35:301-302.
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humor calcium concentration with antemortem serum calcium
correlation and the regression equation would be more
concentration. J Forensic Sci 27:889-893.
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a given postmortem ocular fluid value.
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There was a poor correlation between ocular fluid
of death. J Forensic Sci 11:390-394.
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mined, even those not varying with the postmortem
8. Lane VM, Lincoln SD: 1985, Changes in urea nitrogen and
interval. Because these results applied to serum values
creatinine concentrations in the vitreous humor of cattle after
that were within normal limits, it is possible that these
death. Am J Vet Res 46:1550-l552.
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However, in human medicine, a recent study also
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