Equations generated using SMA regression can be used to exchange values bidirectionally, while those from OLS regression should only be used to predict a dependent variable from values of an independent variable unidirectionally because of caveats surrounding error structure and slope estimates [ 30 — 32 ]. Whole blood and non-hemolytic and hemolytic plasma samples were collected from 23 nesting leatherback turtles for a total of 26 samples 3 turtles were sampled twice at two different time points during the nesting season.
Three electrophoretograms without and with hemolysis are presented in Fig 4. The line represents the median, the boxes represent the first and third quartiles, while the whiskers represent the range. Data is presented in SI units. Electrophoretograms of three individual nesting leatherback sea turtles Dermochelys coriacea without left column and with right column hemolysis showing a prominent alpha 2 -globulin fractions as highlighted in orange in hemolyzed samples. The orange boxes serve to highlight the alpha 2 -globulin fraction.
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Reference intervals are indicated for non-hemolyzed plasma. Whole blood hemoglobin results also shown. Equations for the regression lines are provided, in addition to the confidence intervals CI of the slopes of the regression lines and the y-intercept. We used these model results to create conversion equations and report how well the models fit the data using r 2 values Table 3. Values can be converted birectionally. R 2 values indicate how well models fit the data; values close to zero indicate low fit, while those close to one indicate higher fit.
Analytes not included in this Table showed non-significant equations and are not reported here. Blood analyte data from non-hemolyzed plasma samples are reported as reference intervals in Table 1. Potassium, albumin:globulin A:G ratio, and albumin decreased significantly over the nesting season Table 4. This study provides a comprehensive analysis of the effect of hemolysis on various blood chemistry analytes in nesting leatherback sea turtles from Florida and reports reference intervals for these analytes using non-hemolyzed plasma.
Hemolysis effects were substantial despite being investigated using dry slide chemistry, which is generally less affected by interferents such as hemolysis and lipemia than wet chemistry [ 33 ]. Conversely, the magnitude of interference would be presumptively more pronounced with wet chemistry. The degree of hemolysis interference in leatherback turtles suggests unique species-specific RBC chemical components in this taxon in comparison to previously studied species.
Visual evaluation of serum or plasma color is an important aspect in specimen accession in any diagnostic laboratory; severe hemolysis may lead to rejection of a sample [ 2 ]. Hemolysis is considered an important economic factor in human diagnostic laboratories, since it is the most frequent reason for sample rejection and because of the costs of resampling [ 34 ].
Although the economic factor may not be as relevant for veterinary diagnostic laboratories, resampling blood may not be an option after sample collection in the field for sea turtles or other wildlife species. Semiquantitative assessment of the degree of hemolysis by automated chemistry analysis is considered superior over visual assessment in human laboratory medicine [ 3 ]. However, semiquantitative assessment is not often performed in veterinary diagnostic laboratories. Visual assessment of hemolysis is often the only available methodology in veterinary laboratories, but even that information may not be reported or considered in interpretation of data.
Table 5 provides an overview of common field or sample handling challenges and recommendations for avoidance of hemolysis during field blood sampling, handling, and processing. After excluding all potential causes of hemolysis in a given sample, the conclusion of possible in vivo hemolysis can be made when interpreted in the context of relevant clinical and other laboratory findings.
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Understanding hemoglobin concentrations can be a valuable component of the hemogram evaluation in any species. Hemoglobin measurements have traditionally been based on the cyanmethemoglobin technique which utilizes toxic reagents and also requires a centrifugation step of the blood-cyanmethemoglobin mixture to remove free nuclei before measurement of optical density [ 5 ]. Given that the comparison of frozen-thawed whole blood analyzed with and without centrifugation did not identify a statistical difference in our study or in birds [ 23 ], this finding indicates that the centrifugation step is not necessary when the hemoglobinometer is used with frozen-thawed blood from species with nucleated RBC.
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The positive and proportional correlation of PCV with hemoglobin concentration confirmed that in clinically normal leatherback sea turtles, similar to mammals and other non-mammalian species, the hemoglobin concentration is about 3 times the PCV PCV x 3. Furthermore, if only a minimal amount of blood can be obtained e. Fleming, unpublished data. This information can also be used as an additional end point when interpreting blood data from clinically normal leatherback sea turtles in future studies.
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Furthermore, the hemoglobinometer is cost-effective and easy to operate, which makes hemoglobin analysis a suitable additional endpoint for sea turtle health assessment studies. Electrolytes were altered by hemolysis to a degree that could affect proper data interpretation. Pseudohyperkalemia in mammals and humans can result from hemolysis, prolonged contact of erythrocytes with serum or plasma, thrombocytosis from potassium release of platelets after clotting for serum harvest, leukocytosis, or accidental use of EDTA plasma [ 3 , 5 ].
In our study, the use of frozen-thawed blood resulted in release of intracytoplasmic components of all blood cells, including RBC, white blood cells, and thrombocytes. Therefore, contributions of potassium from lysed WBC and thrombocytes, when present in expected numbers in a clinically normal sea turtle are likely negligible, even if they contain small amounts.
In addition, study turtles were considered clinically healthy and none of the study animals had concerns for leukocytosis or thrombocytosis data not included. Since heparinized plasma but not serum is used for chemistry analysis in sea turtles for various reasons [ 1 ], the concern for potassium release from thrombocytes does not apply to routine plasma samples. True hyperkalemia is an important laboratory interpretation as potassium is an essential electrolyte linked with regulation of blood gases, muscle tissue, kidney function, and hormonal regulation [ 5 ].
Therefore, spurious potassium increases that fall outside the reference interval can affect clinical diagnosis and treatment decisions on an individual animal level, since cardiac effects e. Concern for hyperkalemia due to exertion is one of the reasons why health monitoring for leatherback sea turtles during field responses or studies is important [ 35 ].
Correction formulas for estimation of actual potassium concentration in a sample affected by hemolysis have been developed for human laboratory medicine but are not recommended since hemolysis results in higher potassium concentrations than can be corrected by calculation [ 36 ].
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This is consistent with our attempt to develop a correction formula for potassium in leatherback sea turtles, which did not produce a useful formula Table 3. Although sodium and chloride were not affected to the degree of potassium, their spurious increases were significant, with observed concentrations outside the reference interval. This could affect laboratory data interpretation associated with hydration status and functional assessment of kidneys, gastrointestinal tract, and salt glands [ 1 , 5 ].
Minerals were variably affected by hemolysis, but overall less than electrolytes. While calcium was not significantly increased, all minerals including calcium were observed outside their established reference intervals, and significant increases were observed in phosphorus, calcium:phosphorus ratio, and magnesium.
This has the potential to alter interpretation of mineral homeostasis, vitamin D metabolism, and plasma biochemical evaluation of bones, muscles, and kidneys. Although the calcium:phosphorus ratio is not used in marine turtles as in terrestrial reptiles in the laboratory evaluation of renal function, it can provide useful information in sea turtles regarding diet, tissue growth, and life-stage class differences [ 1 ]. Hence, accurate interpretative information gets lost in the presence of sample hemolysis. Plasma protein fractions are important chemistry analytes in reptiles, since they enable evaluation of overall health and nutrition, inflammation, and immune and various organ functions such as gastrointestinal tract, liver, and kidneys, when interpreted in context of other plasma chemistry analytes [ 1 , 5 ].
All protein fractions except for pre-albumin were affected by moderate hemolysis in leatherback sea turtles.
Since total protein measurement by biuret method and plasma protein electrophoresis is the gold standard to obtain accurate plasma albumin in reptiles [ 37 ], falsely increased albumin may mask hypoalbuminemia in leatherbacks. The effect on globulins was expected, since globin chains of hemoglobin migrate into the alpha- or beta-globulin regions in various species, specifically beta in dogs, gamma in birds, beta 2 in green iguanas Iguana iguana , and alpha 1 and beta in pond sliders Trachemys scripta [ 5 , 7 , 38 ].
This finding is important since this protein fraction typically increases with inflammatory responses and would alter interpretation of electrophoretic patterns of plasma proteins. Our conversion equations could theoretically be used by leatherback researchers and clinicians to estimate non-hemolyzed plasma data using those from hemolyzed plasma samples. In practice, however, the relatively poor correlations between hemolyzed and non-hemolyzed values raise concern regarding their utility.
The lack of any significant correlations between values of phosphorous, pre-albumin, alpha 1 -globulins, and potassium is especially concerning.
This absence of model fit makes predicting non-hemolyzed values from hemolyzed ones nearly impossible and raises concern regarding the interpretation of these values from hemolyzed samples. Further research is needed to investigate red blood cell chemistry of leatherbacks and conversion factors accounting for hemolyzed plasma samples. Blood analyte data of leatherback sea turtles of various life-stage classes and from different geographical locations have been documented [ 14 — 15 , 17 , 35 ], including chemistry data from nesting leatherbacks from Equatorial Guinea, Gabon, and French Guiana [ 12 , 16 , 39 ], and protein electrophoresis data from nesting leatherbacks from Florida and St.
Croix, U. Virgin Islands [ 18 — 19 ]. Slight variations are overt in comparing our results to previous reports. These variations may be attributed to differences in sample handling, processing, and analytical methodologies, and possibly different times of sample collection during months of the nesting season over time [ 16 , 18 — 20 , 39 ]. The lower trending potassium, albumin, and A:G ratio across the nesting season in our study are consistent with other reports of nesting leatherbacks and hawksbills in which changes in these analytes were attributed to reduced food intake [ 16 , 18 — 19 , 40 ].
Potential additional trends may not have been detected in our study given the relatively low sample size. Further studies are warranted e. Packed cell volume in nesting leatherbacks from Florida in this study is similar to nesting leatherbacks from Gabon, Equatorial Guinea, and Papua New Guinea [ 12 , 15 — 16 ], which is collectively lower compared to foraging leatherbacks in the northwest Atlantic [ 17 ] and eastern Pacific [ 15 ].
Lower trending PCV is common in leatherbacks during nesting season [ 1 , 16 , 18 , 20 , 39 ]. Hemoglobin quantification has not been reported in leatherback sea turtles to date, but results are much higher than in loggerheads Caretta caretta , green turtles Chelonia mydas and hawksbill sea turtles [ 21 , 41 — 42 ]; Perrault, unpublished data.
Although methodology differences among studies may factor into the observed difference to some degree, the comparatively higher hemoglobin in addition to the observation of higher PCV in leatherback sea turtles compared to other chelonids suggest higher blood oxygen-carrying capacity in this species. This consideration is supported by differences in diving physiology e.
Non-hemolyzed plasma samples provide the best quality for chemistry analysis of electrolytes, minerals, and proteins in leatherback sea turtles.
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