Diagnostic efficacy of hand-held digital refractometer for determining total serum protein in indigenous sheep of Pakistan

Madiha Sharif; Mushtaq Hussain Lashari; Umer Farooq; Musadiq Idris; Muhammad Abrar Afzal

doi:10.1371/journal.pone.0295107

. 2024 Mar 28;19(3):e0295107. doi: 10.1371/journal.pone.0295107

Diagnostic efficacy of hand-held digital refractometer for determining total serum protein in indigenous sheep of Pakistan

Madiha Sharif ¹, Mushtaq Hussain Lashari ^1,^*, Umer Farooq ², Musadiq Idris ², Muhammad Abrar Afzal ²

Editor: Mahmud Iwan Solihin³

PMCID: PMC10977740 PMID: 38547098

Abstract

The study was designed to ascertain the diagnostic efficacy of hand-held digital refractometer in determining total protein (TP). The Sipli sheep (n = 128) were grouped as per gender (females = 99, males = 29) and age (G1 = up till 1 year, n = 35; G2 = from 1 to 2 years, n = 63; G3 = above 2 years, n = 30). The results regarding the overall mean (±SE) values for the TPs attained through serum chemistry analyzer (TP1) and hand-held digital refractometer (TP2) were non-significantly (P≥0.05) different (59.2±1.6g/L and 59.8±0.5g/L, respectively). However, the reference intervals (RIs) were quite different for the two TPs being 45.1–95.7g/L and 57.0–67.0g/L for TP1 and TP2, respectively. Similar results were seen for gender-wise and group-wise results. On the contrary, the results regarding correlation coefficient and logilinear regression showed a negative correlation between the two TPs (r = -0.0244) with an adjusted r-square of 0.059 (5.9% probability). Furthermore, the three tests implied to assess the level of agreement between the two methods (Cronbach alpha, Intraclass correlation coefficient, and Bland & Altman test) revealed least agreement between the two methods. In a nutshell, the results of TP through digital refractometer were not in concordance with those attained through serum chemistry analyzer. However, it can cautiously be used if these results are compared with relevant corrected RIs.

Introduction

The diagnostic/prognostic tests performed out of the laboratory and near/at the site of patient are termed as point-of-care-tests (POCTs) as per the definition provided by the International Standard ISO 22870 [1]. These tests have a rapid turnaround time, and assist in rapid decision-making and faster care of the patient. In human medical practice, they are dubbed as bed-side tests, near-patient testing, and patient-focused testing; whereas in veterinary medical diagnostics, they are termed as cow-side tests, on-farm tests, barn-side tests or flock-side tests [2, 3]. On human medical side, there are various POCTs presently in vogue for detecting glucose, hemoglobin, blood gases, electrolytes, cardiac enzymes and drug metabolites. In order to attain reliable results of surveillance, outbreak or analytic testing from the POCTs, it is vital that they are kept under strict regulatory oversight, quality control, and periodic comparison/validation through gold-standard techniques. Regarding veterinary medical diagnostics, there is still a paucity of devising and validating POCT devices for field use. It has been estimated that the market of veterinary POCTs was around 2.15 billion dollars with a prediction of 12.3% growth during 2021 to 2030 [4]. Evolving technologies and increased market needs are the key reasons behind this escalatory pattern of POCTs in order to efficiently respond the new and emerging diseases.

Since their introduction in 1960s, the hand-held digital refractometers are being used extensively as POCT devices for determining Brix%, salinity, specific gravity, and total proteins (TP) in various bodily fluids. These instruments measure the angle of refraction between air and an aqueous solution, providing rapid and inexpensive determination of solutes in the fluids. These devices have widely and successfully been used in veterinary medical diagnostics for assessing failure in passive transfer (FPT) through determining IgGs in sows [5], dogs [6] and cattle calves [7, 8]. Similarly, they have also been used for determining TP in various species with varying sensitivity and specificity as compared to other gold-standard techniques [9–11]. A study conducted with an aim of determining serum TP in sheep through biuret method and refractometrically has reported statistical accuracy of 93.05% [11]. However, to the best of knowledge, the diagnostic efficacy of hand-held digital refractometers in comparison to the serum chemistry analyzers, for determining TP in indigenous sheep of Pakistan, has not yet been unearthed.

There are about 17 indigenous sheep breeds being reared in Pakistan in different geo-ecological zones. It has been claimed that these breeds probably originated from urial (Ovisvignei), the wild sheep of Afghanistan, Baluchistan and Central Asia [12]. Sipli breed of sheep is a thin-tailed indigenous sheep breed of Pakistan with a relatively long tail. Small number of this breed (n = 260) is being maintained at two institutional farms in South Punjab, Pakistan. It is a medium-sized sheep breed with an average body weight of 32.8kg for males and 29.2kg for females, and has a daily milk yield of 0.2–0.4 L [12, 13]. It has white body coat with white or light brown head/ears. Its head is medium sized and has a flat nose with ears reaching about 15 cm long [14]. It is mostly reared for mutton and wool purposes by the nomadic herders of Bahawalpur, Bahawalnagar and Rahim-Yar-Khan- the three cities which lay in the middle of the Cholistan desert, (Southern Punjab) Pakistan. As it is reared within the domains of the desert, distances far flung from metropole laboratories, hence their diseases mostly go unchecked as the herders cannot bring their sick animals/samples to the laboratories in time. Considering this constraint, the present research work focuses on the use of on-field, hand-held, digital refractometers for determination of TP- an analyte which is highly indicative of sheep health. The research work is the first of its kind being reported for this sheep breed with an objective to ascertain the diagnostic efficacy of hand-held digital refractometer in determining TP as compared to that attained through serum chemistry analyzer. It is hypothesized that the digital refractometer will give satisfactory results at par with those of serum chemistry analyzer.

Materials and methods

Geo-location of study

The present study was carried out simultaneously at the Livestock Farm, Faculty of Veterinary and Animal Sciences (FV&AS), The Islamia University of Bahawalpur (IUB), and Post-graduate Lab of Physiology, IUB, Pakistan. Both of these study venues are located close to each other (at distance of 1.5 km) within the premises of the university. The climate of Cholistan desert is arid and semi-arid tropical; the average temperature of Cholistan desert is 28.33°C, average rainfall of Cholistan desert is up to 180mm [15].

Experimental animals

The Sipli breed of sheep (n = 128) being reared at the Livestock Farm of FV&AS, IUB, Pakistan under intensive farming system were incorporated in the present study. The animals under study were grouped as per gender (females = 99, males = 29) and age (G1 = up till 1 year, n = 35; G2 = from 1 to 2 years, n = 63; G3 = above 2 years, n = 30). The animals are sent for grazing early morning. In the evening the feeding of animals includes fresh-cut and chopped seasonal fodder along with concentrate ration containing about 15% crude protein. In addition, maize silage and wheat straw is offered depending on need as and when required. The fresh clean drinking water remains available all the time. The animals have been assigned tag numbers in order to collect data.

Ethics statement

The research work was approved by the Departmental Research Ethics Committee, Department of Physiology, IUB, Pakistan vide Letter No PHYSIO-77/2023-104 dated 13-11-2023.

Blood collection and analyses

Approximately 5mL blood sample were collected from each experimental animal. Bleeding was conducted once with a total of 128 blood samples. The blood was collected aseptically from the jugular vein using a 5mL disposable syringe in yellow-capped vacutainers containing silica and a polymer gel for serum separation. The vacutainers were centrifuged at 3000rpm for 15 minutes by centrifuge machine (Centrifuge 800, China) for serum extraction. Serum was extracted in Eppendorf tubes, appropriately labeled, and transported in ice-packs to the Post-graduate Lab Physiology, IUB (situated at 1.5km from the sample collection site) for further analyses. Samples were visually inspected and confirmed for the level of hemolysis.

The serum TP from attained serum samples was analyzed within 2–3 hrs by the same person, using following two methods:

Through serum chemistry analyzer: The TP was determined through commercially available TP kit (Bioactive Diagnostic Systems, JTC, Cat No. 5–172, Germany) using semi-automated serum chemistry analyzer (Rayto-9600, China). The analyzer works on two basic principles i.e. optical and electrochemical techniques. As per the instruction manual of the commercial kit, the analyzer was set at 25°C. The commercial kit had a sensitivity/limit of quantification 0.17g/dL (7.3g/L) and Linearity of up to 15g/dL (150g/L), and implied the working principle of biuret reaction according to which the protein form colored complex with cupric ions in alkaline medium. The TP thus attained was termed as TP1 in this study.
Through digital refractometer: Determination of TP was carried out using a temperature-compensated hand-held digital refractometer (Serum Protein Tester, DR503, China) as per the instructions of the manufacturer. Briefly, after turning on the device, it was cleared with the distilled water and the sample plate was dried. Distil water was used to confirm the functioning of the refractometer before the measuring of TP for the samples. A 0.2–0.3 mL sample was dripped on the plate, and the cover/lid was closed. Pressing the ‘read’ button one time provided the TP in g/dL. The set of readings was attained at room temperature viz. 25-26°C. All the samples were analyzed twice and mean was determined as a final measure. The device had a performance range of 2.0–14.0g/dL, accuracy of ±0.2, and a resolution of 0.1. The TP thus attained was termed as TP2 in this study.

Statistical analyses

Statistical Package for Social Sciences (Windows Version 12, SPSS Inc, Chicago, IL, USA) was used for data analyses. Normality of the data and homogeneity of variance were tested through Shapiro-wilk test and Levene’s test, respectively. Mean (±Standard Error, SE) values for serum TP attained through serum chemistry analyzer (TP1) and through digital hand-held refractometer (TP2) were analyzed for difference through independent t-test. Pearson’s correlation coefficient and linear regression were implied to assess the level of relation, and to deduce the regression prediction equation between the two values. Three tests were implied to check the level of agreement between the two types of tests viz. Bland & Altman, Cronbach Alpha and Intraclass Correlation Coefficient (ICC). The mean (±SE), median, range and reference intervals (RIs) (25^th to 95^th percentile) were deduced for the data (n = 128) keeping in view the guidelines provided by the American Society for Veterinary Clinical Pathology [16] using the Reference Value Advisor (freeware v.2.1: http://www.biostat.envt.fr/reference-value-advisor).

Results

The results regarding overall as well as group-wise data for RIs of the TP1 (attained through serum chemistry analyzer) and TP2 (attained through hand-held digital refractometer) are given in Table 1. Both the mean (±SE) values (for TP1 and TP2) were non-significantly (P≥0.05) different (as ascertained through independent t-test) with mean values of 59.2±1.6g/L and 59.8±0.5g/L, respectively. However, the RIs were quite different between the two TPs being 45.1–95.7g/L and 57.0–67.0g/L for TP1 and TP2, respectively. Similar results were seen for gender-wise and group-wise results being non-significantly (P≥0.05) different between all study groups, but with wide range of RIs.

Table 1. Reference intervals for serum total protein (g/dL) attained through serum chemistry analyzer (TP1) and through digital refractometer (TP2) as affected by sex and age in Sipli sheep (n = 128).

Groups	Mean±SE	Median (IQR)	Range (Min-Max)	RI (25^th to 95^th)	95% CI
TP1
Females (n = 99)	59.3±1.8	58.2(28.2)	78.3(21.2–99.5)	44.7–95.8	55.6–63.1
Males (n = 29)	58.7±3.2	58.9(21.7)	70.6(25.0–95.7)	45.7–93.1	52.1–65.4
G1 (n = 35)	58.1±3.1	59.1(29.2)	76.5(21.2–97.7)	44.6–96.5	51.6–64.6
G2 (n = 63)	58.9±2.4	57.8(26.6)	77.2(22.3–99.5)	43.1–95.7	54.0–63.8
G3 (n = 30)	61.0±2.9	58.5(28.8)	61.3(34.4–95.8)	45.8–91.7	54.9–67.1
Overall (n = 128)	59.2±1.6	58.5(26.3)	78.3(21.2–99.5)	45.1–95.7	56.0–62.5
TP2
Females (n = 99)	59.4±0.6	60.0(7.0)	47.0(34.0–81.0)	56.0–67.0	58.2–60.6
Males (n = 29)	60.9±0.7	61.0(6.0)	17.0(51.0–68.0)	58.5–67.5	59.4–62.4
G1 (n = 35)	59.8±1.3	61.0(8.0)	47.0(34.0–81.0)	57.0–72.2	57.1–62.5
G2 (n = 63)	59.3±0.6	60.0(8.0)	22.0(47.0–69.0)	56.0–66.8	58.1–60.5
G3 (n = 30)	60.6±0.6	60.0(4.0)	17.0(51.0–68.0)	58.9–67.4	59.2–62.0
Overall (n = 128)	59.8±0.5	60.0(6.7)	47.0(34.0–81.0)	57.0–67.0	58.8–60.7

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The results regarding correlation coefficient and logilinear regression (Fig 1) between TP1 and TP2 showed a negative correlation between the two attributes (r = -0.0244) and an adjusted r-square of 0.059 (5.9% probability) with following regression prediction equation:

y = - 0.073x + 64.17.

Results for Cronbach alpha and ICC between TP1 and TP2 are given in Table 2. Both the values for single measure and average values were lower between TP1 and TP2 being -0.135 and -0.313, respectively.

Table 2. Cronbach alpha and intraclass correlation between TP1 (attained through serum chemistry analyzer) and TP2 (attained through hand-held digital refractometer).

TP1 vs TP2
Intraclass Correlation		95% CI	Cronbach Alpha
Single Measure	-0.135	-0.301–0.038	-0.313
Average Measures	-0.313	-0.862–0.074	-0.313

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Similarly, Bland and Altman chart between TP1 and TP2 (Fig 2) showed a weak level of agreement. A proportional bias on the distribution of data around the mean difference line was noticed between TP1 and TP2 (Mean = 0.5; 95% CI = 39.8 to -40.9) with an S.D. of biasness being 20.58.

Discussion

The present work is the first of its kind being reported for indigenous Sipli breed of sheep from Pakistan which was conducted with an aim to assess diagnostic efficacy of hand-held digital refractometer for determining serum TP, as compared to the serum chemistry analyzer which is considered as the gold-standard technique. The digital hand-held refractometer is globally acclaimed and in vogue, and its efficacy for determining TP has been elucidated. However, it failed to reveal satisfactory results for determining serum TP of the sheep in the present study as compared to those attained through serum chemistry analyzer which is obvious through the three tests of agreement implied in this study (Bland & Altman, Cronbach Alpha and ICC).

In the present study, the overall mean values of 59.2±1.6g/L and 59.8±0.5g/L attained through serum chemistry analyzer using commercial kit, and through hand-held digital refractometer, respectively are in line with those reported for three indigenous sheep of Iraq being 62.0g/L [17]. Similar values of 58.2g/L have been reported for White Dorper and Suffolk sheep using Biuret method of TP determination [18]. Another study has also reported similar value of 60.0g/L using Biuret method for Dorper sheep of Brazil [19]. Higher values of 72.0g/L have been reported for free-ranging desert big-horn sheep [20] and Merino sheep [21] using automated serum chemistry analyzer. Similarly, a study from Greece has reported higher values of 83.3g/L and 79.1g/L for serum TP in sheep as determined through biuret method and through refractometry, respectively. Variation in breed, methods of sample handling and methods of TP determination may be attributed to these higher values.

Regarding the RIs (25^th and 95^th percentile) for the TP of sheep serum in the present study attained through two methods i.e. serum chemistry analyzer and hand-held digital refractometer, it was noticed that the RIs were quite different. They were 45.1–95.7g/L and 57.0–67.0g/L for TP1 and TP2, respectively. Comparing these RIs with those of prior studies, it was revealed that the RIs of the present study were though within the range provided in earlier studies [11, 21–23], yet different. As the RIs are different for two methods in our study, it seems inevitable that relevant RIs for each method may be considered while determining TP in sheep serum. The different RIs could be due to the different devices used in our study, their efficacy, their internal validation, and variability in temperature under which the samples were assayed.

Literature is rife with studies in which the digital refractometers (especially Brix refractometers) have successfully been used for determining serum immunoglobulins (IGs) in various species such as canines [6], bovines [7, 24, 25], porcine [26], ovine [27], and equine [28]. All these prior studies have validated hand-held digital refractometers for determining IGs in serum and have reported satisfactory sensitivity, specificity, negative predictive value and positive predictive values for this device dubbin it a reliable on-farm, cow-side POCT in contrast to our results. However, it is to be noted that the analyte(s) in these studies have been different (IGs) from that reported in present study (TP). Hence, the difference in used devices/equipment, methodology, method sampling, processing protocols and species under consideration could be possible factors for this difference.

In the present study, though the independent t-test failed to reveal statistical difference between the two methods of TP determination, however other statistical tests of association/agreement implied in this study presented a rather different picture. The correlation coefficient and regression showed a weak association between the two methods of TP determination (r = -0.0244, adjusted r-square = 0.059/5.9% probability) in the present study. This is in contrast to a previous study which has shown strong linear relation for both methods as ascertained through concordance correlation coefficient being 68.7% for sheep [11]. On similar lines, using Pearson’s correlation coefficient, higher correlation has been reported for both methods in dogs (r = 0.632) and cats (r = 0.826) while determining TP [29].

The present study utilized three additional tests (Bland & Altman, Cronbach Alpha and ICC) for ascertaining level of agreement between the two methods using for determining TP i.e. through serum chemistry analyzer and hand-held digital refractometer. All these tests also indicated a poor level of agreement between the two studied tests. The Bland & Altman test is considered as a gold-standard test for checking level of agreement between two different techniques measuring one similar variable [30–32]. For the present study, this test revealed a proportional bias on the distribution of data around the mean difference line between TP1 and TP2 (Mean = 0.5; 95% CI = 39.8 to -40.9) with an S.D. of biasness being 20.58. These results are also not in line with those reported for sheep [11], dogs and cats [29]. Similarly, the ICC revealed negative values (-0.135 and -0.313 for single and average measures, respectively) for both tests in the present study. The difference in level of association between two methods of determining TP in our study could plausibly be attributed to the difference in temperature at which the samples were assayed, sensitivity and specificity of refractometers, and validation of analyzer.

Considering the results of the present study, it seems inevitable to conclude that the hand-held digital refractometer does not have substantial concordance to the results of a serum chemistry analyzer, and they cannot be used interchangeably. However, being an inexpensive and quick POCT device, its use cannot be overruled. Hence, it is recommended that while using a digital hand-held refractometer, comparison of results should be made with relevant corrected RIs. Further studies may be carried out with stricter sample handling and analyses protocols such as fixed temperature for both devices. Instead of visual assessment of hemolysis, the samples maybe assessed for hemolysis through appropriate chemistry analyzers in future.

Data Availability

All relevant data are within the manuscript.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0295107.r001

Decision Letter 0

Mahmud Iwan Solihin

15 Feb 2024

PONE-D-23-37822Diagnostic efficacy of hand-held digital refractometer for determining total serum protein in indigenous sheep of PakistanPLOS ONE

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Reviewer #1: Partly

Reviewer #2: Partly

**********

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Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Overview

This paper has the basis of a good publication, but needs significant adjustment to be publishable. More detail is needed about the materials and methods, including the sample handling, analysis implementation and statistical methods used, see Katsoulos et al (2017) and the references they cite for a demonstration of what is required.

The results differ significantly from those published by other authors, given that refractometer results appear to decrease with increasing serum chemistry TP results. While these results could be correct for this particular dataset, where such major deviation to expected outcomes occur, rigorous checking of the data and it’s construction is needed by the authors. This needs to include a review of the sample handling processes - timings and temperatures, and data handling. It would be recommended that the authors return to the raw data records (paper or whichever format they were first recorded in) and to check them against the final dataset and ensure that there hasn’t been a mistake in the sorting of data which has resulted in a loss of connection between the TP1 results and their relevant TP2 results.

As TP1 and TP2 are acquired by different methods, I would not expect them to give the same numerical result, but would expect them to be correlated, so that in clinical practice different reference ranges would be needed depending on which methodology was being used, and an theoretical equation can be produced to simulate the relationship between the outputs of these 2 methods (Katsoulos et al., 2017). Also, the main comparison of interest is how well correlated each pair of results are for each individual sample. Therefore I think that the data in table 1 and statistical comparison undertaken between the summary statistics for the whole group of results should moved to the end of the results section and given less attention in the results and a abstract, or removed altogether.

Also, the English language used needs to be reviewed before resubmission.

All abbreviations need to be given in full and then the abbreviation in parentheses the first time they are used in the abstract and in the full text (TP, RI, POCT, etc …). For example, ‘total protein (TP)…’

Abstract

The abstract is difficult to follow, it needs simplifying, giving the interpretation of the results and then results that support these interpretations in brackets.

M&M

More detail is needed about the sample handling (including timings and temperatures etc) and assessment of test precision, see for Katsoulos et al (2017) for examples.

Also, the method used by the serum chemistry analyser to test TP is not given. The authors need to find out from the manufacturers which method is used (biuret, refractometry, other) and state this.

How many times was each test run on each sample? Duplicate testing is advised, triplicate is better, but not necessary.

Were all samples checked for haemolysis etc.? Were any excluded for this?

Statistical analysis

Outliers - what was considered to be an outlier? Why were these removed? Any samples with TP levels that are physiologically viable should be included, because it is often the animals outside of the reference ranges that we are interested in.

More detail is needed in the statistical methods, to give more detail about how each of the tests were implemented

Results

Tables 2 and 3 should be combined into one table.

Discussion

Lines 180 and 181 - check the units for the TP values given here, the magnitude is x10 different to the other results given but the units (g/L) are the same.

Lines 188-190 - I do not understand what the authors are trying to say in this sentence. The values of TP1 and TP2 given are of very similar in magnitude and are also similar in magnitude to those reported from other studies, contrary to what this sentence appears to be saying.

Line 211 - these results cannot be generalised to all sheep, as this study only included 1 breed in specific circumstances and differs significantly from results from other studies, and these other studies need to be referenced.

Panagiotis D. Katsoulos, Labrini V. Athanasiou, Maria A. Karatzia, Nektarios Giadinis, Harilaos Karat-zias, Constantin Boscos, Zoe S. Polizopoulou. Comparison of biuret and refractometry methods for the serumtotal proteins measurement in ruminants. Vet Clin Pathol46/4 (2017) 620–624 DOI:10.1111/vcp.12532

Reviewer #2: The study presents a pertinent exploration of point-of-care testing (POCT) in veterinary medicine, particularly in remote or field settings, highlighting its significance. While commendable, the manuscript would benefit from a more robust interpretation of statistical analysis to strengthen its conclusions. Furthermore, expanding upon the interpretation of findings and discussing their clinical relevance would augment the study's impact on the veterinary medicine community. Addressing inherent limitations and suggesting avenues for future research are essential for a comprehensive understanding of the results. On top of that, enhancing the results and discussion section organization would augment readability and strengthen the scholarly integrity of the manuscript. With several adjustments (described below), the study holds promise for publication and warrants consideration.

Abstract:

The manuscript's abstract on the diagnostic efficacy of a hand-held digital refractometer for determining total serum protein in indigenous sheep of Pakistan presents the study's objectives, methods, and critical findings. However, it could benefit from a more straightforward presentation, interpretation of results, and simplification of the statistical analyses described. In addition, a more concise and focused presentation of the most significant results and a more precise interpretation of their implications for veterinary practice or research would enhance the abstract's readability and effectiveness.

Introduction:

Line 35–50: The introduction provides a general overview of POCTs in both human and veterinary medicine and briefly discusses hand-held digital refractometers for assessing various parameters in bodily fluids. However, it lacks a more thorough review of the existing literature on refractometers for determining TP in sheep. Incorporating relevant studies that have explored similar topics could help contextualize the current research within the broader scientific landscape and highlight any gaps or inconsistencies in the existing literature that the study aims to address.

Line 61–71: The introduction briefly mentions the indigenous Sipli breed of sheep in Pakistan and its significance for the local agricultural economy. However, providing a more detailed rationale for selecting this specific breed for the study would be beneficial. Why is it essential to assess TP levels in this particular breed, and what unique challenges or opportunities does studying indigenous sheep present compared to other breeds or species?

Line 71–73: A concise statement outlining the anticipated outcomes or hypotheses of the study would be beneficial to enhance the introduction. This would help orient readers and provide a roadmap for understanding the significance of the study findings.

Material and methods:

Line 112: The manuscript states that 13 outliers were removed from the data before analysis. However, the methodology for outlier removal is not clearly described. It is essential to provide details on how outliers were identified and justify their removal from the dataset. In addition, the impact of outlier removal on the results should be addressed to ensure transparency and reproducibility of the findings.

Line 113–117: Abbreviations like RI (Reference Interval) and SE (Standard Error) should be spelled out upon first use in the text. On line 113, it's mentioned: "Hence, RIs were deduced for remaining data (n=128) keeping in view the guidelines provided by the American Society for Veterinary Clinical Pathology". On line 117, it states: "Mean (±SE) values for serum TP attained through serum chemistry analyzer (TP1) and digital hand-held refractometer (TP2) were analyzed for difference through independent t-test". However, there's no initial explanation for RIs and SE.

Results:

Line 126–131: It states that the mean values of TP1 and TP2 were "non-significantly (P≤0.05) different." This statement is contradictory because it suggests that the difference between TP1 and TP2 is non-significant, but then it specifies a p-value threshold of 0.05, which is commonly used to determine significance. The P≤0.05 implies that the p-value is less than or equal to 0.05. In statistical interpretation, a p-value less than the significance level (0.05) indicates statistical significance, suggesting evidence to reject the null hypothesis (i.e., a significant difference between the groups being compared). Please explain regarding this condition.

Discussion:

Line 176: Despite finding non-significant differences in mean TP values between the two methods (TP1 from serum chemistry analyzer and TP2 from hand-held digital refractometer), it's essential to consider the clinical significance of these findings. While statistical significance is a crucial measure of whether differences observed in a study are likely due to chance, it doesn't necessarily reflect the practical importance or impact of those differences. Even minor differences in TP values can have significant clinical implications in veterinary diagnostics. Total protein levels are crucial indicators of an animal's health status, reflecting nutritional status, hydration status, and potential underlying medical conditions. Therefore, even if the differences in mean TP values between the two methods are statistically non-significant, they may still be clinically relevant if they fall outside an acceptable range or result in misclassification of animals' health status. Additionally, the discussion could explore potential factors contributing to the lack of significant differences in mean TP values between the two methods. This could include factors such as the sensitivity and specificity of the hand-held digital refractometer, variations in sample handling and processing, or inherent limitations of the analytical techniques employed.

Line 187: The wide discrepancies in reference intervals (RIs) between TP1 and TP2 raise significant concerns about the clinical utility and reliability of the hand-held digital refractometer for determining TP in sheep serum. Furthermore, the discussion should consider the limitations of the hand-held digital refractometer in accurately determining TP in sheep serum, particularly in comparison to the serum chemistry analyzer, considered the gold standard. Factors such as instrument calibration, sample volume requirements, and interference from other constituents in the serum could contribute to the discrepancies observed in reference intervals.

Line 194: It has been mentioned that digital refractometers (especially Brix refractometers) have successfully been used to determine serum immunoglobulins (IGs) in various species. Nevertheless, to provide a nuanced understanding of the diagnostic efficacy of hand-held digital refractometers, the discussion should explore potential factors contributing to variability in study findings across different studies. This could include differences in instrument calibration, sample handling, processing protocols, population demographics, and analytical methodologies. By examining these factors, the discussion can offer insights into the sources of variability in study outcomes and the broader implications for veterinary diagnostics.

Line 200–209: The discussion on agreement analysis, encompassing Bland & Altman, Cronbach Alpha, and Intraclass Coefficient, warrants a more thorough interpretation. Specifically, elucidating the implications of observed proportional bias in the Bland & Altman analysis and the significance of negative values in the Cronbach Alpha and Intraclass Correlation coefficients is crucial. While acknowledging potential biases in data collection, a deeper exploration of this issue and its potential impact on study findings is warranted. Addressing potential sources of bias or confounding factors that may have influenced results could offer valuable insights for interpreting findings comprehensively. Integrating these elements into the discussion enables a more robust understanding of the agreement analysis outcomes and their implications, enhancing the overall coherence and depth of the discussion section.

Line 210: The conclusion begins with a clear statement regarding the inadequacy of the hand-held digital refractometer for determining serum TP in sheep. However, the subsequent statement suggesting the potential use of other refractometer models with higher sensitivity and specificity is somewhat ambiguous. It's unclear whether the conclusion is definitive or speculative. To address this issue, the conclusion should clarify the rationale behind suggesting the exploration of other refractometer models. It should specify whether this recommendation is based on limitations identified in the current study, such as specific technical shortcomings of the hand-held digital refractometer used, or if it is merely speculative and based on the possibility that different models may yield different results. Providing additional context and justification for this recommendation would help readers better understand the implications of the study findings and the potential avenues for future research.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2024 Mar 28;19(3):e0295107. doi: 10.1371/journal.pone.0295107.r002

Author response to Decision Letter 0

17 Feb 2024

Dated: 17th of February, 2024

Dear Editor(s)

Kindly find below point-wise justification and actions taken on Manuscript # PONE-D-23-37822 as per the reviewer’s comments:

Reviewer’s Comments Action Taken/Justification

REVIEWER 1

More detail is needed about the materials and methods, including the sample handling, analysis implementation and statistical methods used, see Katsoulos et al (2017) and the references they cite for a demonstration of what is required. Keeping in view the reference provided by the esteemed reviewer (Katsoulos et al. 2017) and the instructions, details have been added in all relevant chapters of the amended draft, please.

Where such major deviation to expected outcomes occur, rigorous checking of the data and its construction is needed by the authors. Done and reconfirmed, please.

It would be recommended that the authors return to the raw data records (paper or whichever format they were first recorded in) and to check them against the final dataset and ensure that there has been no mistake in the sorting of data which has resulted in a loss of connection between the TP1 results and their relevant TP2 results. The data has been reconfirmed and it is reiterated that that the final data was soundly sorted and had no outliers or errors in it. Statistical analysis part of the amended draft elaborates the procedure, please.

As TP1 and TP2 are acquired by different methods, I would not expect them to give the same numerical result, but would expect them to be correlated, so that in clinical practice different reference ranges would be needed depending on which methodology was being used, and an theoretical equation can be produced to simulate the relationship between the outputs of these 2 methods (Katsoulos et al., 2017). - The Pearsons correlation coefficient was implied which showed r-square of 0.059 (5.9% probability). This has been mentioned in the article, please.

Furthermore, regression was implied and regression prediction equation was also deduced which has been incorporated in the amended draft, please.

The English language used needs to be reviewed before resubmission. Revised accordingly, please.

All abbreviations need to be given in full and then the abbreviation in parentheses the first time they are used in the abstract and in the full text Corrected at all relevant places in the amended draft, please.

Abstract

The abstract is difficult to follow, it needs simplifying, giving the interpretation of the results and then results that support these interpretations in brackets. Abstract has been redone, please.

M&M

- More detail is needed about the sample handling (including timings and temperatures etc) and assessment of test precision, see for Katsoulos et al (2017) for examples.

- Also, the method used by the serum chemistry analyzer to test TP is not given. The authors need to find out from the manufacturers which method is used (biuret, refractometry, other) and state this.

- How many times was each test run on each sample? Duplicate testing is advised, triplicate is better, but not necessary.

- Were all samples checked for hemolysis etc.? Were any excluded for this?

- Appropriate details regarding the serum extraction, timing, and TP determination have been added, please.

- The optical and electrochemical techniques utilized by the analyzers has been mentioned in amended draft, please.

- Yes, the samples were visually assessed for the level of hemolysis. None of the samples were hemolyzed enough to be discarded. Mentioned in M&M chapter accordingly, please.

Statistical analysis

- Outliers - what was considered to be an outlier? Why were these removed? Any samples with TP levels that are physiologically viable should be included, because it is often the animals outside of the reference ranges that we are interested in.

- More detail is needed in the statistical methods, to give more detail about how each of the tests were implemented Initially, data of 141 sheep was attained and outliers were visually inspected as well as with Shapiro-wilk test. Outliers (values far beyond normal range) were deleted.

However, in amended draft we have omitted the mention of outliers and the whole draft has been redevised with a data of 128 sheep/samples, please. Corrections have been made in appropriate sections.

Statistical details have been made robust through appropriate addition in the amended draft, please.

Results

Tables 2 and 3 should be combined into one table. - Combined as Table 1 in amended draft, please.

Discussion

- Lines 180 and 181 - check the units for the TP values given here, the magnitude is x10 different to the other results given but the units (g/L) are the same.

- Lines 188-190 - I do not understand what the authors are trying to say in this sentence. The values of TP1 and TP2 given are of very similar in magnitude and are also similar in magnitude to those reported from other studies, contrary to what this sentence appears to be saying.

- Line 211 - these results cannot be generalized to all sheep, as this study only included 1 breed in specific circumstances and differs significantly from results from other studies, and these other studies need to be referenced.

- The units have accordingly been corrected.

- The RIs in our study were different for both methods (TP1 and TP2). Though they were within the range reported in earlier studies. Corrections have accordingly been made in the amended draft, please.

- Agreed that our conclusion was not definitive and clear. It has been rewritten with clarity in amended draft, please.

REVIEWER 2

The manuscript would benefit from a more robust interpretation of statistical analysis to strengthen its conclusions. The discussion and conclusion have been redevised, please.

Expanding upon the interpretation of findings and discussing their clinical relevance would augment the study impact on the veterinary medicine community.

Addressing inherent limitations and suggesting avenues for future research are essential for a comprehensive understanding of the results. On top of that, enhancing the results and discussion section organization would augment readability and strengthen the scholarly integrity of the manuscript.

Abstract

A more concise and focused presentation of the most significant results and a more precise interpretation of their implications for veterinary practice or research would enhance the abstracts readability and effectiveness. Abstract has been rewritten, please.

Introduction

Line 35&50: The introduction provides a general overview of POCTs in both human and veterinary medicine and briefly discusses hand-held digital refractometers for assessing various parameters in bodily fluids. However, it lacks a more thorough review of the existing literature on refractometers for determining TP in sheep. Incorporating relevant studies that have explored similar topics could help contextualize the current research within the broader scientific landscape and highlight any gaps or inconsistencies in the existing literature that the study aims to address.

Line# 58-60

Literature regarding TP determination in sheep serum has been added in Introduction, please.

Line 61&71: The introduction briefly mentions the indigenous Sipli breed of sheep in Pakistan and its significance for the local agricultural economy. However, providing a more detailed rationale for selecting this specific breed for the study would be beneficial. Why is it essential to assess TP levels in this particular breed, and what unique challenges or opportunities does studying indigenous sheep present compared to other breeds or species? Line# 75-82

Te Sipli sheep breed is being reared under pastoral nomadism in the Cholistan desert, Pakistan. Its herds are mostly far away from the metropole laboratories and this constraint makes it inevitable that on-field POCT devices (such as hand-held refractometers) may be validated. The reason has clearly been justified in amended draft, please.

Line 71&73: A concise statement outlining the anticipated outcomes or hypotheses of the study would be beneficial to enhance the introduction. This would help orient readers and provide a roadmap for understanding the significance of the study findings. The statement of hypothesis is added in amended draft, as directed, please.

M & M

- Initially, data of 141 sheep was attained and outliers were visually inspected as well as with Shapiro-wilk test. Outliers (values far beyond normal range) were deleted.

However, in amended draft we have omitted the mention of outliers and the whole draft has been redevised with a data of 128 sheep/samples, please. Corrections have been made in appropriate sections.

Statistical details have been made robust through appropriate addition in the amended draft, please.

Line 113&117: Abbreviations like RI (Reference Interval) and SE (Standard Error) should be spelled out upon first use in the text. there's no initial explanation for RIs and SE. - Corrected and the the mean (±SE), median, range and reference intervals (RIs) (25th to 95th percentile) were deduced for the data (n=128) keeping in view the guidelines provided by the American Society for Veterinary Clinical Pathology using the Reference Value Advisor. Similar has been mentioned in amended draft, please.

Results

Line 126&131: It states that the mean values of TP1 and TP2 were non-significantly (P0.05) different. This statement is contradictory because it suggests that the difference between TP1 and TP2 is non-significant, but then it specifies a p-value threshold of 0.05, which is commonly used to determine significance. The P0.05 implies that the p-value is less than or equal to 0.05. In statistical interpretation, a p-value less than the significance level (0.05) indicates statistical significance, suggesting evidence to reject the null hypothesis (i.e., a significant difference between the groups being compared). Please explain regarding this condition.

- We apologize that the symbol of ≤ was inserted instead of ≥.

In fact, as per the independent t-test, the mean values of both TPs were non-significantly different (P≥0.05) indicating that both methods can be used interchangeably.

However, interestingly when the tests of agreement were implied (Cronbach alpha, Intraclass coefficient and Bland-Altman), less agreement between two methods was revealed.

Corrections have accordingly been made in amended draft, please.

Discussion

The discussion could explore potential factors contributing to the lack of significant differences in mean TP values between the two methods. This could include factors such as the sensitivity and specificity of the hand-held digital refractometer, variations in sample handling and processing, or inherent limitations of the analytical techniques employed.

The discussion has been made stronger with appropriate justifications and additions of newer references, please.

Line 194: It has been mentioned that digital refractometers (especially Brix refractometers) have successfully been used to determine serum immunoglobulins (IGs) in various species. Nevertheless, to provide a nuanced understanding of the diagnostic efficacy of hand-held digital refractometers, the discussion should explore potential factors contributing to variability in study findings across different studies. This could include differences in instrument calibration, sample handling, processing protocols, population demographics, and analytical methodologies. The discussion has been rewritten keeping in view these factors mentioned by the esteemed reviewer, please.

Line 200&209: The discussion on agreement analysis, encompassing Bland & Altman, Cronbach Alpha, and Intraclass Coefficient, warrants a more thorough interpretation. Specifically, elucidating the implications of observed proportional bias in the Bland & Altman analysis and the significance of negative values in the Cronbach Alpha and Intraclass Correlation coefficients is crucial. While acknowledging potential biases in data collection, a deeper exploration of this issue and its potential impact on study findings is warranted. Addressing potential sources of bias or confounding factors that may have influenced results could offer valuable insights for interpreting findings comprehensively. Integrating these elements into the discussion enables a more robust understanding of the agreement analysis outcomes and their implications, enhancing the overall coherence and depth of the discussion section.

Line 210: The conclusion begins with a clear statement regarding the inadequacy of the hand-held digital refractometer for determining serum TP in sheep. However, the subsequent statement suggesting the potential use of other refractometer models with higher sensitivity and specificity is somewhat ambiguous. It is unclear whether the conclusion is definitive or speculative. To address this issue, the conclusion should clarify the rationale behind suggesting the exploration of other refractometer models. It should specify whether this recommendation is based on limitations identified in the current study, such as specific technical shortcomings of the hand-held digital refractometer used, or if it is merely speculative and based on the possibility that different models may yield different results. Providing additional context and justification for this recommendation would help readers better understand the implications of the study findings and the potential avenues for future research. The conclusion has been rewritten keeping in view the directions, please.

DR. MUSHTAQ HUSSAIN LASHARI

(Corresponding Author)

PLoS One. doi: 10.1371/journal.pone.0295107.r003

Decision Letter 1

Mahmud Iwan Solihin

6 Mar 2024

PONE-D-23-37822R1Diagnostic efficacy of hand-held digital refractometer for determining total serum protein in indigenous sheep of PakistanPLOS ONE

Dear Dr. Lashari,

==============================Kindly provide the reviewers' comments and respective authors' responsein tabular form in such a way that the revisions were clear.==============================

Please submit your revised manuscript by Apr 20 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Mahmud Iwan Solihin

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

The authors have made quite significant response to the reviewers' comments. However, authors need to put in a neater way in such a

way that is easier to read and understand, e.g. in tabular format

-per comment of reviewer (left column) and respective response of the author (right column).

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PLoS One. 2024 Mar 28;19(3):e0295107. doi: 10.1371/journal.pone.0295107.r004

Author response to Decision Letter 1

7 Mar 2024

The document titled 'Response to Reviewers' contained tabulated form of comment-wise amendments made in the draft, please. And the said document has been uploaded separately please.

Attachment

Submitted filename: Response to reviewers.pdf

pone.0295107.s001.pdf^{(196.7KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0295107.r005

Decision Letter 2

Mahmud Iwan Solihin

13 Mar 2024

Diagnostic efficacy of hand-held digital refractometer for determining total serum protein in indigenous sheep of Pakistan

PONE-D-23-37822R2

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PLoS One. doi: 10.1371/journal.pone.0295107.r006

Acceptance letter

Mahmud Iwan Solihin

18 Mar 2024

PONE-D-23-37822R2

PLOS ONE

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Diagnostic efficacy of hand-held digital refractometer for determining total serum protein in indigenous sheep of Pakistan

Madiha Sharif

Mushtaq Hussain Lashari

Umer Farooq

Musadiq Idris

Muhammad Abrar Afzal

Roles

Abstract

Introduction

Materials and methods

Geo-location of study

Experimental animals

Ethics statement

Blood collection and analyses

Statistical analyses

Results

Table 1. Reference intervals for serum total protein (g/dL) attained through serum chemistry analyzer (TP1) and through digital refractometer (TP2) as affected by sex and age in Sipli sheep (n = 128).

Fig 1. Scatterplot for logilinear regression between TP1 (attained through serum chemistry analyzer) and TP2 (attained through hand-held digital refractometer).

Table 2. Cronbach alpha and intraclass correlation between TP1 (attained through serum chemistry analyzer) and TP2 (attained through hand-held digital refractometer).

Fig 2. Scatterplot of Bland and Altman test between difference of total protein determined through serum chemistry analyzer and through hand-held digital refractometer (TP1-TP2) and average of both TPs (TP1+TP2/2) in Sipli sheep (n = 128).

Discussion

Data Availability

Funding Statement

References

Decision Letter 0

Mahmud Iwan Solihin

Roles

Author response to Decision Letter 0

Decision Letter 1

Mahmud Iwan Solihin

Roles

Author response to Decision Letter 1

Decision Letter 2

Mahmud Iwan Solihin

Roles

Acceptance letter

Mahmud Iwan Solihin

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Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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