Study site: The study was conducted at the post-graduate laboratory of the Department of Physiology, The Islamia University of Bahawalpur (IUB), Pakistan from May to July 2023 located at the outskirts of the Cholistan desert. The Cholistan desert is about 112m above sea level. It is sprawling in 26,000 km² area is located in latitudes 27°42′ and 29°45′ North, longitudes 69°52′ and 75°24′ East [25]. Its arid and semi-arid tropical climate has an average temperature of 28.33°C. Month of June is considered as being the warmest month showing a temperature of up-to 45°C. The recorded annual rainfall of this desert is up to 180mm. From November till January, the weather is at its coldest when the temperature is about 13°C.

Study animals: Cholistani cattle (n=63) being harbored under intensive farming system at the University Livestock Farm were incorporated in the study. Similar management and feeding patterns are being maintained for them at the farm. In order to ascertain the general health status of the animals, focal group discussions (FDGs) were carried out with the personnel of the farm. In addition, clinical examinations including temperature, pulse, respiratory rate, and behavioral patterns were also carried out. Resultantly, only apparently healthy animals were incorporated in the study. Animals showing signs of lethargy, anorexia, off-feed and segregation from their herds were excluded from the present study. Under the nomadic pastoralism livestock system of the desert, all the young animals are kept in their pens near the water sources (Tobas) whereas the adult animals are sent for grazing, and this is termed as split-herding [25].

Ethics statement: Apropos to PHYSIO-77/2023 (10-03-2023), this MPhil research work was approved by the Departmental Research Ethics Committee, Department of Physiology, IUB, Pakistan.

Blood collection: Blood samples were drawn from the jugular vein of the cattle using a disposable syringe as per prescribed protocol, in EDTA-containing purple-topped vacutainers (Becton Dickinson, USA). They were gently inverted, and transferred in an icebox to the laboratory where they were tested for hemoglobin estimation within 2 hrs.

Hemoglobin estimation: In this study, three methods for the estimation of Hb concentration in Cholistani cattle blood were implied viz. one through hematology analyzer and two through indirect cyanmethemoglobin method using DBS, as given below:

1. Automated veterinary hematology analyzer: Before analysis by an automated hematology analyzer, each blood sample was gently mixed by keeping it on a Roller Mixer (MixR-40, Daihan Scientific, Korea). Analysis for Hb concentration (HbA) was conducted using a multi-species, off-hand validated veterinary hematology analyzer (Rayto RT-7600, China). This HbA was considered as the gold standard technique for Hb determination for the present study.
2. Indirect cyanmethemoglobin methods using dried blood spot: For this method, priorly prescribed protocol was implied [20,27], with minor modifications, and two methods were implied for measuring Hb in two different ways using DBS on the filter paper (Whatman Grade 42, Sigma Aldrich, Germany). This filter paper has a maximum ash of 0.007%, pore size of 2.5µm, and Herzberg filtration speed of 1870S. In first method, 20µL of measured blood was placed on the filter paper, and the paper was allowed to dry on a non-absorbent surface. This paper was then placed in a test tube containing 5mL of Drabkin’s solution (Drabkin’s Reagent, CAT D5941, Sigma Aldrich, Germany). After the incubation period of 1hr, the solution containing the eluted blood spot was vortexed for 2mins, reincubated for another half hr, and finally analyzed for O.D through spectrophotometer (Irmeco GmbH & Co, Germany, Model U2020 UV-VIS) at 540nm and Hb was estimated through standard curves (HbIC) using Hb standard solution. In second method, using the similar filter paper, an unmeasured drop of blood was placed on it, allowed to dry and a 5mm hole (~5µL of blood) was punched with an office hole-puncher. It was transferred to 1.25mL of Drabkin’s solution, and after the incubation of 1hr, O.D was taken spectrophotometrically, and Hb was ascertained as given for first method (HbICX) [28].

Statistical analysis: Statistical Package for Social Science (SPSS for Windows version 12, SPSS Inc., Chicago, IL, USA) was used for data analyses. Data was analyzed for normality visually, as well as through Shapiro-wilk test. Means (±SE) and 95% CI for the three Hb values (HbA, HbIC and HbICX) attained in this study were computed using prescribed formulae. To attain difference between the three Hb values for overall data as well as for various study groups viz. age-wise (G1 up till 1 year, n=20; G2 from 1 to 2 years, n=20; G3 above 3 years, n=23) and sex-wise (males, n=26 and females, n=37), ANOVA with Duncan’s as post-hoc test was implied. Pearson’s correlation coefficient and linear regression were implied to deduce the level of correlation between the three Hb values. Accordingly, regression prediction equations were computed. Keeping a cut-off value of 9.6g/dL, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated for HbIC and HbICX [29]. Level of agreement between the three methods assessed through Bland Altman Agreement Analysis [30]. Similarly, Cronbach alpha and intraclass correlation coefficient were also implied on overall data as tests of agreement between HbA and HbIC, and between HbA and HbICX [31,32].

The overall mean (±SE) values for HbA, HbIC and HbICX obtained through hematology analyzer and through two indirect cyanmethemoglobin methods using DBS in the present study were 9.2±0.2, 9.3±0.3 and 15.8±0.8g/dL, being significantly (P≤0.05) higher for HbICX. The group-wise and overall results revealed that HbA and HbIC were statistically (P≥0.05) non-significant whereas, they both were statistically (P≤0.05) different from HbICX (Fig 1). The HbICX gave overestimated Hb values as compared to those attained by other two methods. The results of regression analyses and the regression prediction equations for overall as well as for all the study groups (age- and sex-wise) have been given in Table 1. The HbA and HbIC had a positive correlation (P≤0.01) (r= 0.734; adjusted r-square= 0.528). Similarly, the correlation coefficient between HbA and HbICX was r= 0.324 with an  adjusted r-square= 0.083) being higher between HbA and HbIC.

The sensitivity, specificity, PPV, and NPV for HbIC were higher being 86.1%, 88.3%, 88.0%, and 86.0%, respectively as compared to the lower values of 45.0%, 12.0%, 12.0% and 45.0%, for HbICX.

Regarding the level of agreement, Bland and Altman chart between HbA and HbIC (Fig 2) showed a better level of agreement between HbA and HbIC having a relational bias of data distribution around the mean difference line (Mean = -0.16, 95% CI = 0.34 to -0.67). Regarding the other tests of level of agreement viz. Cronbach alpha and intraclass correlation coefficient,  both the values for single measure and average values were higher between HbA and HbIC being 0.703 and 0.825 as compared to the values of 0.200 and 0.333 between HbA and HbICX (Table 2).

Figures & Tables

Discussion

Blood profiling and analyses is one of the vital diagnostics/prognostic tools both in human and veterinary medical practice. Clinical hematology has resultantly attained substantial advancement globally especially for the diagnosis/prognosis of blood-borne disorders and novel sampling methods are being tested for their diagnostic efficacy. For veterinary medical practice, blood sampling is a major issue of concern due to restraint of animals, and appropriate transport of the sample to distantly-located laboratories. Cholistani breed of cattle are reared in the desert under pastoralism and split-herding is implied. According to this livestock pastoral system, the natural or man-made water reservoirs (‘Tobas’ in local dialect) are selected. Young animals are confined in their pens near these ‘Tobas’. However, the adult animals are led to grazing along with Cholistani herders. Remotely located laboratories for these livestock make it difficult for the desert livestock herders to get their animals timely screened or tested for diseases. It, hence, seems inevitable to devise and validate alternate sampling protocols for livestock being least stressful, needing least animal, are feasible to be transported, and can bear temperature fluctuations within a certain limit. The present work is a novel one being reported for diagnostic efficacy of DBS using filter paper for Hb estimation in Cholistani cattle blood. And it reveals that a quantified drop of blood (20µL) on filter paper provides reliable results of Hb as compared to the Hb attained through gold standard method. There is scarcity of research work on the diagnostic accuracy and efficacy of DBS using filter paper for Hb estimation for livestock blood, hence the comparisons for the sake of discussion have been made with the studies conducted on human blood, wherever needed. 

In the present study, results revealed that HbA and HbIC were non-significantly different whereas, they both were statistically different and higher from HbICX. These results were the same group-wise and overall data of the present study. This indicates that the measured drop of blood (20µL) taken on a filter paper in present study gives reliable results for Hb, comparable to those by the gold standard method (hematology analyzer for the present study). However, in our results Hb estimated through DBS using 5mm punch of the filter paper gave higher results. Results similar to ours have been reported in various previous studies using human blood. From India, while assessing anemia in pregnant women, 20µL of blood on Whatman filter paper was used. And this BDS technique was endorsed to be a reliable technique for Hb estimation [33]. Similar to the results of our study attained for Hb using 5mm filter paper punch, yet two other studies from India which were aimed to assess the diagnostic efficacy of indirect cyanmethemoglobin method in adolescent girls have also reported that the punch method gives overestimated values for Hb [20,27]. They endorsed that the values of Hb, being overestimated, needed a correction factor for their appropriateness. On the contrary, certain other studies have successfully detected Trypanosoma congolense and T. brucei in cattle blood using both the Whatman Grade 41 and 42 filter papers [10, 34]. 

The sensitivity, specificity, PPV, and NPV for HbIC were higher being 86.1%, 88.3%, 88.0%, and 86.0%, respectively as compared to the lower values of 45.0%, 12.0%, 12.0% and 45.0%, for HbICX. While using human blood in research work, an even higher sensitivity of 99.2% has been reported through indirect cyanmethemoglobin method [20]. The overall sensitivity (86.1%) and specificity (88.3%) of HbIC in the present study seems quite satisfactory for Hb estimation in cattle. Though future further validations of this technique may render these values even higher.

In the present study, three tests to assess the level of agreement viz. Bland Altman, Cronbach alpha and intraclass coefficient were implied between HbA and HbIC, and between HbA and HbICX. A better level of agreement was noticed between HbA and HbIC. The relational bias was not noticed regarding the data distribution around the mean difference line (Mean = -0.16, 95% CI = 0.34 to -0.67). Similarly, the Cronbach alpha and intraclass correlation coefficient between HbA and HbIC also revealed that both for single measure and average, the values were higher between HbA and HbIC being 0.703 and 0.825 as compared to the values of 0.200 and 0.333 between HbA and HbICX. Comparing these results of the tests of agreement with the sensitivity and specificity of the present study, it seems obvious that HbIC is a reliable and accurate test for Hb estimation in cattle blood using DBS.

Summing up, the present study concludes that the indirect cyanmethemoglobin method for Hb estimation is reliable and accurate for cattle blood, if a measured quantity of blood drop is taken on a filter paper. Furthermore, if a standardized 5mm punch is holed in the filter paper having the DBS, then a correction factor (Hb=0.12(HbICX)+7.4) attained from the regression prediction equation of present study between HbA and HbIC could be implied for accurate results. We recommend this DBS technique for Hb estimation in cattle blood for resource-poor settings and for livestock herds being reared distantly from the laboratories. For future, it is recommended that DBS technique with various other modifications and for other hematochemical biomarkers may be validated for livestock blood. 

Author Contributions

All the authors contributed equally.

Conflict of Interest

The author declare that there is no conflict of interest regarding the publication of this paper.

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