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10.1371/journal.pgph.0003715
PGPH-D-23-02498
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Seasonal variations in haematological and biochemical parameters of healthy Gambian adults: Retrospective study 2018–2022
Seasonal and yearly variations in the routine haematological and biochemical parameters
https://orcid.org/0000-0002-8524-2521
Dibbasey Mustapha Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Resources Validation Visualization Writing – original draft Writing – review & editing 1 2 *
https://orcid.org/0000-0002-9046-5279
Dahaba Mamudou Data curation Methodology Writing – review & editing 1
Sarfo Francess Data curation Methodology Writing – review & editing 1
Begum Rosyna Data curation Methodology Writing – review & editing 1
Kanteh Mustapha Data curation Methodology Writing – review & editing 1
Sumareh Nyima Data curation Methodology Writing – review & editing 1
Bakare Mustapha Data curation Methodology Writing – review & editing 1
Umukoro Solomon Data curation Methodology Writing – review & editing 1
Amambua-Ngwa Alfred Conceptualization Data curation Formal analysis Supervision Validation Visualization Writing – original draft Writing – review & editing 1 *
1 Medical Research Council Unit The Gambia at the London School of Hygiene and Tropical Medicine, Banjul, The Gambia
2 Department of Biochemistry, Cell and Molecular Biology, School of Biological Science, University of Ghana, Accra, Legon, Ghana
Robinson Julia Editor
PLOS: Public Library of Science, UNITED STATES OF AMERICA
The authors have declared that no competing interests exist.

* E-mail: lshmd4@lshtm.ac.uk, mdibbasey@mrc.gm (MD); Alfred.Ngwa@lshtm.ac.uk (AA-N)
17 9 2024
2024
4 9 e000371512 12 2023
23 8 2024
© 2024 Dibbasey et al
2024
Dibbasey et al
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The objectives of this study were to determine the presence and effect of seasonal variations and provide insights into trend from 2018 to 2022 in a comprehensive set of routine haematological indices and biochemical measurements in Gambian adults with no known underlying health condition. We retrieved five years of data from an electronic database and analysed 493 full blood counts and 643 biochemical data from different individuals. In this study, we focused on data from individuals with no known underlying health condition who visited the clinical diagnostic laboratory for routine medical examinations or assessments.Our study found a positive association between seasonality (wet season as the reference) and Hb (HB: 0.014(0.015), P<0.05), White blood cells (WBC) (WBC: 0.243(0.163), p = 0.0014), and neutrophils (neutrophils: 0.271(0.131), P<0.05) with exception to red blood cells (RBC) (RBC: - 0.184(0.061), P< 0.003) that showed negative association. Despite the association, the seasonal effects on our derived reference intervals for haematological indices and biochemical measurements from wet season to dry season were not statistically significant (P>0.05). In addition, we observed in our heatmap result that some laboratory parameters, including HB, RBC, haematocrit (HCT), urea, liver enzymes, and potassium, showed seasonal variation patterns throughout the year, with median levels being normal to slightly low during the dry season and normal to high during the wet season. We also found no significant difference (P>0.05) among the median values for all parameters from 2018 to 2022. Additionally, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) parameters showed a consistent declining trend from 2018 to 2022. Our study found no seasonal effects on the derived reference intervals of haematological indices and biochemical measurements. However, we observed changes in patterns for certain parameters particularly HB, RBC, liver enzymes, and potassium based on seasonality.

Medical Research Council the Gambia at London School of Hygiene This project received no specific funding for this work. Data AvailabilityThe data for the study is fully available and submitted with manuscript.
Data Availability

The data for the study is fully available and submitted with manuscript.
==== Body
pmcIntroduction

Routine haematological and biochemical parameters are essential for assessing an individual’s health and diagnosing various medical conditions [1–4]. Seasonal and yearly variations in these parameters can be influenced by environmental factors such as temperature, humidity, and rainfall. Changes in environmental conditions can impact an individual’s dietary preferences and physical activity levels [5–8], which in turn affect their metabolic processes and overall health. Additionally, certain diseases, including respiratory infections, malaria, and parasitic infestations, often exhibit seasonal patterns that can impact haematological and biochemical parameters, particularly total white cell count and differentials. Moreover, the COVID-19 pandemic has caused significant lifestyle changes, such as physical inactivity and limited access to a healthy diet, which can also affect the general health of populations or countries [9–11].

Investigating seasonal variations and yearly trends in routine parameters over the past five years is of clinical and epidemiological significance. Few studies conducted in sub-Saharan Africa have considered the seasonal effects on haematological and biochemical parameters reference intervals and have shown to affect the reference intervals [1–3,12]. However, an earlier study conducted in Rwanda, Uganda, and Zambia reported minimal impact of seasonality on adult clinical laboratory parameter values in these populations, suggesting that seasonal variation may not be important in evaluating adult clinical laboratory parameters [13]. Despite these contradictory findings, seasonal influences on haematological and biochemical parameters can potentially impact the interpretation of laboratory results, disease diagnosis, and patient care management. In the ongoing country-tailored reference interval study project, this retrospective study has the potential to contribute to refining geographically and genetically tailored reference ranges for routine parameters in the context of seasonality, ensuring more accurate and context-specific interpretation of laboratory results.

This study aimed to evaluate the presence and extent of seasonal variations in a comprehensive set of haematological and biochemical routine parameters in Gambian adults with no known underlying health condition and their effects on our defined reference intervals. We also examined the trending pattern of the main haematological and biochemical parameters over the past five years. By utilizing the retrospective data captured in the electronic medical record system of the Medical Research Council Unit The Gambia at LSHTM (MRCG@LSHTM) clinical laboratories, we provided a description of these variations and their potential clinical implications.

Method

Study setting

The study was conducted in the ISO15189:2012 and good clinical laboratory practice accredited clinical diagnostic laboratory hosted at the Kuyateh Building at MRCG@LSHTM, which is located in the Kanifing municipality in the greater Banjul area of The Gambia [14–16]. The diagnostic laboratory is responsible for providing standard and quality-oriented diagnostic services to MRCG@LSHTM staff, clinical trial projects, as well as government and private hospitals. Staff employed by MRCG@LSHTM underwent routine medical examinations at the clinical diagnostic laboratory, and their haematological and biochemical results data were captured in an electronic medical record system database.

Study design and data extraction

This retrospective study retrieved haematological full blood count results data and biochemical results data from 1st January 2018 to 31st December 2022 from the MRCG@LSHTM electronic medical record system database. In this study, we focused on data from individuals with no known underlying health condition who visited the clinical laboratory department for routine medical examinations and have their haematological and biochemical tests performed. After completing the retrieval process and conducting data cleaning, a total of 493 haematological full blood count results and 643 biochemical results were retrieved from the electronic database (Fig 1 and S1 Data). Out of the 643 biochemical results, 150 were identified as lacking corresponding haematological full blood count results in the electronic database. However, these 150 results were still included in the final analyses to increase the sample size. The extracted haematological full blood count dataset included Hb, RBC, platelets, and WBC and differentials results. The extracted biochemical dataset comprised serum Gamma-glutamyl transpeptidase (GGT), Sodium, Potassium, Urea, Creatinine, AST, ALT, Total Protein (TP), Albumin (ALB), and Globulin results.

10.1371/journal.pgph.0003715.g001 Fig 1 Flow chart demonstrating the data retrieval process and excluded records.

During the data extraction, we considered the age range of 18–63 years, in accordance with the employment age range outlined in MRCG@LSHTM policies. Based on clinical information available in the electronic system, we excluded data from the study if the electronic medical records showed positive serological results for human immunodeficiency virus and hepatitis B virus as well as positive malaria and pregnancy test (Fig 1).

Ethics approval and consent to participate

This study was approved by the Gambia Government/MRCG Joint Ethics Committee (S1 Text). Since all data retrieved from the database were anonymized and coded, informed consent was waived by the Ethics Committee. All methods were performed in accordance with the Declaration of Helsinki.

Statistics

The data were retrieved in Microsoft Excel and analysed using the R package to obtain summary statistics, including the mean, median, and plots. The Shapiro test was validated by Q-Q plot, and a normal distribution bell-shaped curve was used to study the distribution pattern of our data across the parameters. A box plot was used to assess the presence of outliers in datasets. Reference intervals were determined based on the 2.5th and 97.5th percentiles after eliminating outliers in our data using interquartile range calculations (25th—1.5 * IQR; 75th—1.5 * IQR). The comparison between the dry and wet season medians was performed by nonparametric Wilcox Rank test in R. Multivariate analysis using a generalized linear modelling approach in R was used to model the association between explanatory variables (season, age, gender and year) and haematological and biochemical parameters. We further compared the medians of 2018 to 2021 using the Dunn test in R to generate Kruskal-Wallis chi-square and p-value results. P value (P) of less than 0.05 was used to determine statistical significance.

Results

From this study, 493 haematological full blood count data (N = 493) from haematology laboratory department that comprises haematological indices such as RBC, HB, PLT, and WBC and differentials were analysed (Table 1). Additionally, 643 biochemistry laboratory data retrieved from clinical biochemistry laboratory department that comprises biochemical parameters such as liver enzymes, sodium, potassium, urea, and creatinine were analysed (Table 1).

10.1371/journal.pgph.0003715.t001 Table 1 Summary table of characteristics of sample population.

Explanatory variables	Characteristics	Summary N(%)	
Haematology laboratory	
Season (N = 493)	Dry (n)	271 (55.0)	
Wet (n)	222 (45.0)	
Gender (N = 493)	Male (n)	300 (60.85)	
Female (n)	193 (39.15)	
Dry Season (N = 271)	Male (n)	175 (64.58)	
Female (n)	96 (35.42)	
Wet Season (N = 222)	Male (n)	125 (56.31)	
Female (n)	97 (43.69)	
Biochemistry Laboratory	
Season (N = 643)	Dry (n)	373(58.01)	
Wet(n)	270(41.99)	
Dry Season (N = 373)	Male(n)	183(49.06)	
Female (n)	190(50.94)	
Wet Season (N = 270)	Male(n)	148(54.81)	
Female(n)	122(45.19)	
Gender (N = 643)	Male (n)	339 (52.72)	
Female(n)	304(47.28)	
Age profile of the two datasets retrieved	
Datasets	Age Range	Median	Mean	
Haematological Dataset	18–63	28.0	30.5	
Biochemical Dataset	18–63	33.0	36.2	

Over the five-year period from 2018 to 2022, the seasonality (dry and wet seasons) was defined based on the visit date for sample collection at the clinical laboratory department. The wet season was represented by visits from June to October, while the dry season was represented by visits from November to May. Based on the visit data, 55% of the full blood count data and 58% of the biochemistry laboratory data were generated in the dry season (Table 1).

Unlike the biochemistry laboratory data, which showed almost equal gender proportions, there was a gender disparity in the haematological laboratory data, with 60% of the routine medical examination full blood count data came from the male population (Table 1).

The age range for both haematological and biochemistry datasets was 18 to 63 years. The mean age of the haematological dataset was 31 years, and the median age was 28 years (Table 1). For biochemistry data, the mean age was 36 years, and the median age is 33 years. However, there was no significant difference between the mean and median ages of the two datasets (Table 1).

Seasonal and yearly effects and reference intervals

To assess the seasonal effects on the haematological indices and biochemical parameters, we analysed association between seasonality (i.e. whether the sample was collected in wet or dry season), and the haematological indices and biochemical parameters (Table 3). Using generalised linear modelling (GLM), we found that seasonality was associated with various haematological indices (Hb: 0.014(0.015), P<0.05; RBC: -0.184(0.061), P<0.003; PLTs: 17.47 (6.86), p<0.01; WBC: 0.243(0.163), p = 0.0014; neutrophils: 0.271(0.131), P<0.05; and lymphocytes: -0.003(0.066), P = 0.01). However in the biochemical dataset, the biochemical measurements were not affected by the explanatory variables of dry and wet seasons.

In the haematological dataset, age was found to have an association with certain parameters, including haemoglobin (0.028(0.011); P = 0.011), platelets (-0.918(0.44): P = 0.039). In the biochemical dataset, only creatinine (1.202(0.472); P<0.01), and total protein (-0.117(0.048); P<0.05) were found to have an association with age (Table 2). Furthermore, several haematological indices, except for monocytes and eosinophils, were associated with gender. In the biochemical dataset, only sodium (0.680(0.323); P = 0.04) was found to have significant association with gender.

10.1371/journal.pgph.0003715.t002 Table 2 Association study of explanatory variables season, age and gender, and all the haematological and biochemical parameters.

The association was determined by estimated effects on the median (SE) value of the parameters. P value <0.05; Standard Error of the estimate intercept (SE).

Laboratory parameters	Seasona
Estimate (Std. Error);
P value <0.05	Age
Estimate (Std. Error);
P value <0.05	Genderb
Estimate (Std. Error);
P value <0.05	Year
Estimate (Std. Error);
P value <0.05	
HB (g/dL)	0.014(0.015);
P<0.05	0.028(0.011); P = 0.011	2.355(0.126); P<0.0001	0.156(0.079): P<0.05	
RBCa(106/uL)	-0.184(0.061);
P<0.05	0.004(0.003); P = 0.287	0.750(0.049); P<0.0001	0.080(0.034): P<0.05	
PLT (103/uL)	17.47 (6.86);
P<0.01	-0.918(0.44): P = 0.039	-60.19(6.276); P<0.0001	7.861(3.16); P<0.05	
WBC (103/uL)	0.243(0.163);
P<0.01	-0.006(0.011); P = 0.566	-0.929(0.158); P<0.0001	0.030(0.071) P>0.67	
Neutrophils (103/uL)	0.271(0.131);
P<0.05	-0.009(0.008); P = 0.295	-0.624(0.129); P<0.0001	-0.027(0.05); P = 0.61	
Lymphocytes (103/uL)	-0.003(0.066);
P< 0.01	0.006(0.004); P = 0.169	-0.288(0.0654); P<0.0001	0.038(0.03); P = 0.22	
Monocytes (103/uL)	-0.004(0.165); P = 0.798	-0.002(0.001); P = 0.092	-0.027(0.017); P = 0.11	0.037(0.007); P<0.05	
Eosinophils (103/uL)	-0.021(0.023); P = 0.359	-0.001(0.001); P = 0.380)	0.011(0.023); P = 0.623	-0.01(0.01); P = 0.139	
Sodium
(mmol/L)	-0.238(0.330);P = 0.471	0.0188(0.013); P = 0.156	0.680(0.323); P = 0.04	-0.93(0.31); P<0.05	
GGT (U/L)	0.213(7.31);
P = 0.977	0.227(0.241); P = 0.347	10.696(7.27); P = 0.142	-0.43(1.39); P = 0.76	
Potassium
(mmol/L)	0.020(0.039);
P = 0.6143	0.001(0.001): P = 0.424	0.065(0.039); P = 0.098	-0.11(0.04); P = 0.013	
Urea (mmol/L)	-4.128(0.259);
P = 0.112	0.064(0.040); P = 0.113	1.225(1.301); P = 0.347	-0.11(0.136); P = 0.42	
Creatinine
(umol/L)	-18.13(14.88);
P = 0.224	1.202(0.472); P<0.01	28.15(15.23); P = 0.066	0.62(2.00); P = 0.76	
AST (U/L)	-2.045(2.256);
P<0.344	-0.066(0.069); P = 0.597	0.566(0.256); P = 0.798	-0.56(0.65); P = 0.40	
ALT (U/L)	-1.619(2.60);
P = 0.534	0.031(0.089); P = 0.729	1.244(2.846); P = 0.662	-1.69(1.12); P = 0.14	
Albumin (g/dL)	-1.663(0.863);
P = 0.06	-0.052(0.029); P = 0.08	2.028(0.814); P = 0.07	-0.89(0.43); P = 0.04	
Total Protein (g/dL)	-2.142(1.465); P = 0.148	-0.117(0.048 = 9); P<0.05	1.017(1.408); P = 0.472	-0.51(0.64); P = 0.43	
AST (U/L)	-0.536(1.228); P = 0.663	-0.064(0.041);
P = 0.12	-1.057(1.168); P = 0.367	1.33(0.50); P = 0.01	
aWet Season(reference) is compared to Dry Season

cMale (reference) is compared to Female gender.

Next, we assessed the association of the year of visit on various parameters using the GLM approach (Table 3). We found significant associations between the year of visit and haematological parameters such as haemoglobin (0.156(0.079): P<0.05), RBC (0.080(0.034): P<0.05), and platelets (7.861(3.16); P<0.05). In the biochemical dataset, biochemical measurements such as sodium (-0.93(0.31); P<0.05), potassium (-0.107(0.041); P = 0.013), albumin (-0.89(0.43); P = 0.04), and globulin (1.33(0.50); P = 0.01) were found to be associated with the year explanatory variable.

10.1371/journal.pgph.0003715.t003 Table 3 The effect of exploratory seasonal factor on the reference ranges of the haematological indices and biochemical measurements (P>0.05).

Laboratory Parameters	Seasons	Dry Season	Wet Season	
n	Missing
Data
(n)	Median (Reference Range)	n	Median (Reference Range)	n	Median (Reference Range)	
HB (g/dL)	461	32	13.90(11.0–16.5)	258	14.0(11.34–16.36)	206	13.70
(10.70–16.29)	
RBCa(106/uL)	444	49	4.87(3.90–5.85)	238	4.95(4.06–5.89)	201	4.77(3.77–5.76)	
PLT (103/uL)	433	60	244.5(158.6–346.2)	238	237(156.9–338.2)	197	248(163.5–361.1)	
WBC (103/uL)	437	56	5.40(3.52–7.65)	245	5.34(3.51–7.60)	194	5.34(3.52–7.80)	
Neutrophils (103/uL)	426	67	2.55(1.34–4.05)	235	2.47(1.32–3.81)	194	2.49(1.40–4.55)	
Lymphocytes (103/uL)	418	75	2.15(1.33–2.96)	232	2.14(1.30–3.065)	192	2.14(1.33–2.93)	
Monocytes (103/uL)	416	77	0.43(0.25–0.64)	230	0.42(0.25–0.62)	190	0.43(0.25–0.64)	
Eosinophils (103/uL)	335	158	0.13(0.13–0.31)	201	0.14(0.02–0.36)	138	0.11(0.01–0.26)	
Basophils (103/uL)	350	143	0.04(0.01–0.07)	185	0.03(0.02–0.06)	146	0.04(0.02–0.07)	
Sodium (mmol/L)	384	259	140(136–144)	215	140(137–144)		140(136–144)	
Potassium (mmol/L)	498	145	4.12(3.6–4.7)	235	4.20(3.6–4.7)	152	4.10(3.70–4.50)	
GGT (U/L)	240	403	26(11.00–52.025)	134	26(10.23–51.03)	106	26(12.78–53.00)	
Creatinine (umol/L)	500	143	75.2(50.48–107.05)	289	75(49.00–107.08)	211	77(52.93–106.68)	
Urea (mmol/L)	507	136	3.1(1.7–4.9)	381	3.1(1.80–4.7)	217	3.1(1.70–4.9)	
AST (U/L)	354	289	20(12–32)	211	20(12–34.5)	138	20(12.43–29.00)	
ALT (U/L)	234	409	17(7.0–33.0)	130	17(6.23–33.78)	104	17(8.15–31.43)	
Albumin (g/dL)	236	407	40(34–47)	150	40(34.00–47.00)	82	40(34.03–45.00)	
Total Protein (g/dL)	336	307	76(67.00–83.63)	195	76(68.00–85.00)	147	76(67.00–83.35)	
Globulin (g/dL)	203	440	35(27.0–42.00)	116	35(27–42.13)	73	35(29.8–40.0)	
aMean value was used for RBC parameter because the data was normally distributed.

We determined reference intervals nonparametrically based on the 2.5th and 97.5th percentiles for both haematological indices and biochemical measurements (Table 3). To assess the effects of seasonality, we compared the median values between the dry season and the wet season for each year from 2018 to 2022. Although the differences were not statistically significant across all haematological indices (P>0.05), the median values for platelets, as well as WBC and differentials, were slightly higher in the wet season compared to the dry season. Conversely, the median values for Hb and RBC were lower in the wet season. Similarly, the median values for all the biochemical measurements were not statistically significant (P>0.05). However, potassium had a slightly higher median in the dry season (median = 4.20) compared to the wet season (median = 4.10).

We also evaluated the effects of seasonality on the reference intervals derived from our haematological and biochemical study datasets. In haematological data, we observed changes in reference intervals from the dry season to the wet season were not significant (P>0.05). However, we observed a slight reduction in the upper limit of reference intervals in the wet season for HB and RBC haematological indices, and a slight increase in the upper limit of WBC and differentials. Similarly, in biochemical measurements, we also found the changes in reference intervals from the dry season to the wet season not statistically significant (P>0.05).

Monthly and yearly pattern in parameters

We further analysed patterns based on changes in the monthly and yearly median of haematological and biochemical parameters (Figs 2, S1 and S2). These figures demonstrated a change in the pattern of our monthly median of haematological indices and biochemical measurements from January to December over five-year period. From these our study results (Figs 2, S1 and S2), we observed that laboratory parameters, including HB, RBC, HCT/PCV showed seasonal patterns throughout the year, with levels being normal to slightly low during the dry season and normal to high during the wet season. Likewise, we observed a seasonal pattern for total WBC, neutrophils, lymphocytes, and platelets from November to March with normal to slightly high median. In terms of biochemistry parameters, liver enzymes (AST, ALT, GGT, total protein) and potassium were generally high in the wet season (June to October) but moderately low in the dry season.

10.1371/journal.pgph.0003715.g002 Fig 2 Monthly pattern of haematological and biochemical parameters from 1st January 2018 to 31st December 2022 in five year period.

Hb; RBC; WBC; PLT; Neutrophils (NEUTRO); Lymphocytes (LYMPH); Mean cell volume (MCV); Packed cell volume (PCV); Eosinophils (EOS); Basophil (BASO); AST; ALT; GGT.

Finally, we assessed the changes in median values of the parameters from 2018 to 2022 using the boxplots in Figs 3 and 4. We followed this with a statistical significance test using the non-parametric Kruskal-Wallis test (H). Overall, no statistically significant difference was found (H = 0.1148, P>0.05) among the median values of all the haematological indices and biochemical measurements in the last five years (2018 to 2022). In haematological dataset, the median values for HB and RBC parameters decreased in 2020, while the median values for total WBC count, absolute neutrophils count, and platelets increased slightly. In the biochemical dataset, there was no clear pattern except for median values of AST and ALT, which showed a downward trend from 2018 to 2022.

10.1371/journal.pgph.0003715.g003 Fig 3 Boxplot showing pattern/trend of haematological parameters in the last five years.

The results displayed in medians (Kruskal-Wallis chi-squared (H) = 0.063, P > 0.05).

10.1371/journal.pgph.0003715.g004 Fig 4 Boxplot showing pattern/trend of biochemical parameters in the last five years.

(Kruskal-Wallis chi-squared (H) = 0.8433, P > 0.93).

Discussion

The study examined the effects of seasonality on haematological indices, biochemical measurements and the reference ranges derived from our study datasets. Seasonal variations can be influenced by factors such as changes in environmental temperature and humidity, diet, physical activity, and seasonality of certain diseases like malaria. Overall, we found no statistically significant differences between the median values generated in the dry and wet seasons as well as reference intervals generated between the dry and wet season, indicating that there was no seasonal effect on the reference intervals derived from our study data. The reference intervals derived from our study data were comparable to the results reported by Adetifa et al in 2009, which focused on Gambian adults without any known underlying health conditions during a TB contact tracing study [17]. Notably, there was a slight difference in the upper limit of neutrophils reference intervals between the dry season (1.32–3.81) and wet season (1.40–4.55), suggesting the need for more informative reference intervals that consider seasonality. These seasonal variations should be taken into consideration, especially in research studies or clinical trials where patients are monitored at different times of the year. Being aware of these subtle seasonal variations can help prevent erroneous conclusions based on study data.

To further investigate the seasonal variation pattern of these parameters from January to December, we observed moderately normal to slightly higher median pattern for haemoglobin and RBC during the wet season. Haemoglobin and RBC parameters are crucial for diagnosing anaemia in clinical settings. It has been reported that haemoglobin levels in individuals without any known underlying health conditions can vary due to seasonal factors such as changes in diet, including increased consumption of iron-rich foods during certain seasons, as well as asymptomatic infection [18]. In the Gambia, the wet season is characterized by the abundant availability of iron-rich foods, which could explain the higher levels of haemoglobin and RBCs during this period [13]. Besides, the moderately normal to high HCT seasonal variation patterns in the wet season commensurate with the high haemoglobin and RBC median values in the wet season, which could be attributed to low fluid intake during the wet season as a result of low temperature and humidity [13].

The seasonal variations in WBC and platelets were observed in our study from November to March. In the Gambia, changes in the environment during the wet season, such as increased rainfall and cold weather from November to March, favour the transmission of malaria parasites, which typically peaks in November, as well as respiratory infections [19–25]. The finding from our study supports previous findings regarding the seasonal effects on WBC and differentials [1]. Infectious diseases like malaria tend to flourish during the wet season, likely due to environmental factors such as heavy rainfall leading to stagnant water and waterlogged areas [26–29]. This can potentially lead to an increase in mean total WBC and differentials in the general population due to low-grade systemic inflammation in the community caused by malaria transmission and viral illnesses [3,10]. The seasonal variation in total WBC and differentials could be impacted by malaria parasite infection. However, our study records showed malaria microscopy was negative for all the haematological and biochemical datasets retrieved electronically.

Similar to haemoglobin and RBCs, the monthly pattern of liver enzymes and potassium changed based on seasonality. Our findings are aligned with earlier studies [30] that have demonstrated how seasonal changes in diet and fluid intake can impact biochemical measurements, especially electrolytes. The seasonal effects on these biochemical measurements may be attributed to the temperature difference between the wet and dry seasons [30–32]. A reference interval study conducted in Guinea also reported seasonal effects on biochemical parameters, particularly liver enzymes [27]. Despite reports of dehydration being more common during the dry season and potentially affecting biochemical measurements like serum creatinine and urea levels, our study found that median values for urea and creatinine remained relatively normal across both seasons [33]. Alarmingly, our study also revealed a consistent decline in the median values of AST and ALT from 2018 to 2022, warranting further investigation.

Limitations

As observed in numerous retrospective studies, our study is also subject to limitations that restrict our control over certain factors. These factors include haemoglobin genotype status, body size, smoking, alcohol consumption, exercise, and contraceptive pill use, all of which have the potential to influence the outcome [34]. Additionally, our sample size was constrained by the available data, and we were unable to obtain additional data to enhance statistical power [35,36]. These limitations could be addressed through the implementation of a well-designed prospective study conducted on a general population, following the approach employed by Okebe et al. in 2016. Despite these limitations, we maintain that the results of our study are accurate, as our sample size was considerable. Therefore, our study remains representative and supports the current prospective reference intervals study that will provide a more comprehensive sampling and data stratification reflecting both dry and wet seasons.

Conclusion

Our study found no significant seasonal effects on the reference intervals for haematological and biochemical parameters, despite the identification of certain parameters that exhibited some association with seasonality. However, we did observe seasonal variations in specific parameters such as haemoglobin, red blood cells, liver enzymes, and potassium levels from January to December. Furthermore, we did not observe significant yearly variations in the median values of all the haematological indices and biochemical measurements.

Recommendations

Our findings emphasize the importance of considering seasonal variations in haematological indices and biochemical measurements when interpreting laboratory results and making diagnostic decisions, as well as when recruiting participants for clinical trial projects. Our study should be followed by well-designed context-specific studies that employ standardized sampling and analytical methods within our geographical setting.

Supporting information

S1 Data Study raw data.

(XLSX)

S1 Text L2023.E03 Dibbasey Ethics Waiver 1.

(PDF)

S1 Fig Haematological indices from January to December in five year period.

The point showed monthly medians in five year period.

(TIF)

S2 Fig Biochemical measurements from January to December in five year period.

The point showed monthly medians in five year period.

(TIF)

10.1371/journal.pgph.0003715.r001
Decision Letter 0
Gallo Valentina Academic Editor
© 2024 Valentina Gallo
2024
Valentina Gallo
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
9 Apr 2024

PGPH-D-23-02498

Seasonal and yearly variations in haematological and biochemical parameters of healthy Gambian adults: a retrospective study

PLOS Global Public Health

Dear Dr. Dibbasey,

Thank you for submitting your manuscript to PLOS Global Public Health. After careful consideration, we feel that it has merit but does not fully meet PLOS Global Public Health’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by May 24 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 globalpubhealth@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pgph/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Kind regards,

Valentina Gallo

Academic Editor

PLOS Global Public Health

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Reviewers' comments:

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Comments to the Author

1. Does this manuscript meet PLOS Global Public Health’s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS Global Public Health does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The study requires major modification. The use of the term/word healthy people is problematic. If someone came to the laboratory for routine check-up/testing does not make such individual healthy person. Perhaps, individual with no known underlying health condition could suffice. Although the study design was retrospective, reading through the entire write-up appears more prospective.

Reviewer #2: R1:

Title:

-if possible; the title can be changed by remove of the word (yearly), and addition of the year to (retrospective study 2018-2022)

Abstract:

-Divide background rom the aim of the study

-Better to write the objective of the study.

-in abstract give brief idea about study design, study population and area, sampling, methods of data collection and the measurments (not more than 5 rows).

-briefly give the main results of the study with the accurate numbers.

-there is some confusion rgarding the effect of seasonal differences in the measurement of hematological index and biochemical measurements.

-write only the conclusion of the result. interpretation of result can be discussed in the section of discussion.

-use mean full and brief key words

Methods:

- some texts were not needed

- regarding exclusion and inclusion criterias, what about the other seasonal infections e.g. influenza

- what is the final diagnose of the healthy participants who participate in the current study.

- the biomedical history of the participants is absent from the current study.

- inclusion and exclusion criteria should minimize the un-suspected results or errors.

Results:

-493 participant for haematological indexes.

-643 participant for biochemical measurements.

-the measured parameters was for separated individuals?

-643 and 493 individuals measured for different parameters.

-the current study have 2 study population.

-the study must divide into two studies.

In the results section its clearly that there was 2 study population:

-in the first one the researcher examine the seasonal haematological changes.

-in the second one the researcher examine the biochemical changes.

Then:

-there was a comparisons between the two populations.

So:

-the researcher must find the haematological indexes and biochemical measurement change in one population.

OR:

-compare the change in hematological indexes in the first population which occurs as a result of seasonal change as separate work.

-then find the seasonal changes in biochemical measurements in the second population

-finally the researcher can compare the results of the two studies

Discussion:

-in discussion there is unacceptable justification regarding the personal, socio- demographic and life style data which didn't appear in the methods of data collection in form of questionnaire or study objectives.

**********

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Reviewer #1: Yes: David Larbi Simpong

Reviewer #2: No

**********

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Attachment Submitted filename: Comment.pdf

Attachment Submitted filename: PGPH-D-23-02498.pdf

10.1371/journal.pgph.0003715.r002
Author response to Decision Letter 0
Submission Version1
19 Apr 2024

Attachment Submitted filename: Authors Response Letter.docx

10.1371/journal.pgph.0003715.r003
Decision Letter 1
Gallo Valentina Academic Editor
© 2024 Valentina Gallo
2024
Valentina Gallo
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
22 May 2024

PGPH-D-23-02498R1

Seasonal variations in haematological and biochemical parameters of healthy Gambian adults: Retrospective Study 2018-2022

PLOS Global Public Health

Dear Dr. Dibbasey,

Thank you for submitting your manuscript to PLOS Global Public Health. After careful consideration, we feel that it has merit but does not fully meet PLOS Global Public Health’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please consider carefully the notes from one of the reviewers and double check your figures, and transparently disclose the missing values leading to different totals in your tables. Consider adding a flowchart if needed.

Please submit your revised manuscript by Jun 21 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 globalpubhealth@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pgph/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Valentina Gallo

Academic Editor

PLOS Global Public Health

Journal Requirements:

1. 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.

2. Please amend your detailed Financial Disclosure statement. This is published with the article. It must therefore be completed in full sentences and contain the exact wording you wish to be published.

a. State the initials, alongside each funding source, of each author to receive each grant.

b. State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

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If you did not receive any funding for this study, please simply state: “The authors received no specific funding for this work.”

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Does this manuscript meet PLOS Global Public Health’s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS Global Public Health does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: All the comments have been addressed

Reviewer #2: Unacceptable data analysis.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: David Larbi Simpong

Reviewer #2: Yes: Nahla Ahmed Mohammed Abderhman

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment Submitted filename: PGPH-D-23-02498_R1.pdf

10.1371/journal.pgph.0003715.r004
Author response to Decision Letter 1
Submission Version2
28 May 2024

Attachment Submitted filename: Authors Response Letter v2.docx

10.1371/journal.pgph.0003715.r005
Decision Letter 2
Landenmark Hanna Staff Editor
© 2024 Hanna Landenmark
2024
Hanna Landenmark
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version2
5 Aug 2024

PGPH-D-23-02498R2

Seasonal variations in haematological and biochemical parameters of healthy Gambian adults: Retrospective Study 2018-2022

PLOS Global Public Health

Dear Dr. Dibbasey,

Thank you for submitting your manuscript to PLOS Global Public Health. After careful consideration, we feel that it has merit but does not fully meet PLOS Global Public Health’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Reviewer 2 has provided some additional comments in the attached file - the comments are also found below, but in the attachment you can see the location where they are making the comments and suggestions.

Please submit your revised manuscript by Sep 01 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 globalpubhealth@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pgph/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Hanna Landenmark

Staff Editor

PLOS Global Public Health

Journal Requirements:

1. 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 (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Does this manuscript meet PLOS Global Public Health’s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS Global Public Health does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The concerns raised have been addressed by authors

Reviewer #2: R2:

1. Regarding study participants, grouping and measurements; are still not clear

2. no need to mention type of analysis in the results in abstract

3. not important (ignore this and focus on the sample that meet research criteria)

4. focus on the objectives

5. in the first group the researcher measure hematological parameters

6. in the first group the researcher measure hematological parameters

7. mention number of participant who were in the median

8. mention number of participant who were in the mean

9. table 2 is for un-desirable date please no need for table 2 as mentioned above

10. a lot of repeated data and information

11. add column for the missing data in all hematological and biochemical measurements

12. how you compare the hematological measurements with biochemical measurements

13. use only the abbreviations for all the define term

discussion:

14. you mean (using reference interval as standared?)

15. add the subtitle (limitations and recommondations)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

Do you want your identity to be public for this peer review? If you choose “no”, your identity will remain anonymous but your review may still be made public.

For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Nahla Ahmed Mohammed Abderhman

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment Submitted filename: PGPH-D-23-02498_R2.pdf

10.1371/journal.pgph.0003715.r006
Author response to Decision Letter 2
Submission Version3
9 Aug 2024

Attachment Submitted filename: Author Response to Reviewer v3.docx

10.1371/journal.pgph.0003715.r007
Decision Letter 3
Robinson Julia Staff Editor
© 2024 Julia Robinson
2024
Julia Robinson
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version3
26 Aug 2024

Seasonal variations in haematological and biochemical parameters of healthy Gambian adults: Retrospective Study 2018-2022

PGPH-D-23-02498R3

Dear Dibbasey,

We are pleased to inform you that your manuscript 'Seasonal variations in haematological and biochemical parameters of healthy Gambian adults: Retrospective Study 2018-2022' has been provisionally accepted for publication in PLOS Global Public Health.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they'll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact globalpubhealth@plos.org.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Global Public Health.

Best regards,

Julia Robinson

Executive Editor

PLOS Global Public Health

***********************************************************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Does this manuscript meet PLOS Global Public Health’s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS Global Public Health does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: All concerns raised has been addressed by authors

Reviewer #2: R3:

Thank you for your patience.

General comments:

� Great effort is needed to accomplish this work.

� Please see the enclosed document for more information

� *avoid to use pronouns like (we, you, our…etc)

� The action you must be use is in past participle

� Avoid repeated information

� Please down load scientific peer reviewed published paper related to your current research to mimic their scientific language.

Abstract:

� abstract is presented in clear way. fine.

� key words are too much. (suggestion)=seasonal variation, hematological indices, biochemical measurements, Gambia

Methodology:

� study setting: please mention the name of the lab first then its quality and type of routine work.

� author can give brief description of lab quality without evidence.

� mention the source of ethical approval and consent only.

� Study design: missing date will appear in analysis. no need for justification

Results:

� Some references and researchers express p-value<=0.05

Suggestion to results section:

1. * write all results regarding hematological parameters, then in the next paragraph write about biochemical parameters

2. *then compare all the results(if needed)

3. *comparison become uninteresting.

� please mention the same source (eg. table 1) in one paragraph one time. delete repeated information

Another suggestion:

Seasonal and Yearly Effects and Reference Intervals:

1. compare hematological parameters only in wet and dry seasons using different methods of data analysis.

2. compare biochemical parameters only in wet and dry seasons using different methods of data analysis.

Monthly and Yearly Pattern in Parameters:

suggestions to this section:

1. *the author write about all changes that occurs over all year per month, So, this results is a base of the over-mentioned results.

2. *write this paragraph firstly then put the comparisons of wet and dry seasons of hematological and biochemical parameters by different methods of analysis.

3. *that mean you need to rearrange results section.

Discussion:

� repeated information: author can justify and present the results in different style.

� Repetition of same information by similar words is so dull.

� Justification of variation on hematological indices as result of affecting by malaria parasite which showed negative results means :

1. previous effect with malaria parasite result in TWBCs, or

2. test for malaria parasite is false, or

3. affect with malaria parasite did not result in the seasonal variation of TWBCs

limitations: re- write in points. put limitations in points without comparing with other study.

Recommendations: re- write in points

Conclusion: need revision

**********

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Reviewer #1: No

Reviewer #2: Yes: Nahla Ahmed Mohammed Abdelrahman

**********

Attachment Submitted filename: PGPH-D-23-02498_R3_reviewer.pdf
==== Refs
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