Community based hematological reference intervals among apparently healthy adolescents aged 12-17 years in Mekelle city, Tigrai, northern Ethiopia: A cross sectional study.

BACKGROUND: Hematological reference intervals are important in clinical and diagnostic management for the assessment of health and disease conditions. Hematological reference intervals are better to be established based on gender and age differences as these are among the main affecting factors.
OBJECTIVE: The aim of this study was to establish hematological reference intervals among apparently healthy adolescents aged 12-17 years in Mekelle City, Tigrai, Northern Ethiopia, 2019.
METHOD: A community-based cross-sectional study was conducted in 249 adolescents aged 12-17 years from December 2018 to May 2019. About 4ml of blood sample was collected from each study participant using vacutainer tube containing K2EDTA. Hematological parameters were analyzed using Sysmex KX-21N hematology analyzer (Sysmex Corporation Kobe, Japan). Data were entered and analyzed using SPSS version 23. Both parametric and non-parametric analyses were used to calculate the median and 95% of reference intervals. The 97.5th and 2.5th percentiles were calculated using descriptive statistics for the upper and lower reference limits of the study participants. Differences in reference intervals between male and female participants were evaluated using the Mann-Whitney U test. RESULT: Among the 249 participants 122 (49%) were males and 127 (51%) were females with the median age of 14.5 (range 12 to 17) years were recruited in this study. The median and the 95% reference intervals of hematological parameters were determined. The 95% RIs were: Red blood cells (1012/Liter) 4.6-5.9 (Males) and 4.3-5.6 (Females), White blood cells (109/Liter) 2.9-9.6 (Males) and 3.4-10.2 (Females), Hemoglobin (g/dl) 12.6-17.1 (Males) and 12-15.4 (Females), Platelets (109/Liter) 138-364 (Males) and 151-462 (Females). Almost all of the hematological parameters showed significant differences (p<0.05) across gender.
CONCLUSION: The hematological reference intervals established in this study showed a difference based on gender. We suggest preparing and using distinct local reference intervals for males and females separately.

Background

Reference interval (RI) is a standard component of reporting laboratory results and important to transform a numerical value into clinically meaningful information. It is intended to inform the clinical care provider that laboratory values within the interval indicate a non-diseased condition. The most common approach is to base RI on the central 95% of laboratory test values observed for a reference population that is free of disease that influence the laboratory test result, as many diseases are asymptomatic [1].

Human beings are characterized by a dynamic period of growth and development in their lives. The different periods of life possess special hematological development. The chemical makeup of the circulating red blood cells and fetal hemoglobin content in the first days and months is not identical to in later life [2]. In old age, there is increased replacement with fatty marrow [3]. This may affect the hematological parameters and result in different reference intervals for the difference among age groups [4] and gender [5]. Thus, knowledge of the reference values during the dynamic period of growth and development in an individual is important for correct interpretation of a disease condition.

Hematological reference intervals are important in clinical practice for the assessment of health and disease, underscoring the importance of establishing population-appropriate values. Such parameters are important for measuring disease progression, response to therapy, and in the assessment of adverse reactions to therapy [6]. Due to the prevailing disease epidemics in sub-Saharan Africa, the normal hematological indices for these populations become critical in supporting decisions concerning treatment initiation and disease management [7]. Establishment of well-controlled, reliable RI is an important mission for all clinical laboratories. The clinical laboratory standards institute (CLSI) [6, 8] guideline recommends each laboratory to establish its own RIs from the local population or validate them if derived from a different setting. RIs need to be verified periodically every 5 years, to capture changes in the community over time [8].

Most reference values of hematological tests currently being used in Africa are derived the data collected from populations living in developed countries [9]. The RIs adopted from people living in developed countries differ from the people living in sub-Saharan countries which are endemic to different diseases [10]. Most blood specimens of Africans have been found to have lower Hemoglobin (Hgb), Hematocrit (Hct), Red blood cell count (RBC), mean corpuscular volume (MCV), neutrophil and platelet counts compared to the developed countries [11].

The hematological reference intervals which are used for clinical trials in sub-Saharan Africa follow the WHO reference standard guidelines which are different from the Ethiopian guidelines. However, typical Laboratory parameters in different communities may vary based on race, age, gender, diet, local disease patterns and environmental characteristics [12]. Here, we present our efforts to generate RIs in adolescents separately for males and females for the local community.

Methods

Study area

A community based cross-sectional study was conducted from December 2018 to May 2019 in Mekelle City, Tigrai, and northern Ethiopia. Mekelle is the capital city of Tigrai regional state. It is located 780 kilometers north of Addis Ababa (the capital city of Ethiopia) at an altitude of 2,254 meters above sea level characterized by temperate climate [13]. Based on the 2007 Census conducted by the Central Statistical Agency) of Ethiopia (CSA), Mekelle has a total population of 215,914 (104,925 men and 110,989 women) [13, 14].

Eligibility criteria

The apparently healthy individuals aged 12–17 years and lived at least for 5 years in the study area were included. Individuals with any chronic and acute illnesses, taking antibiotic treatment, recent history of blood loss, blood transfusion in the last one year, immunization in the last 6 months, major surgical procedures in the past 6 months, who have any intestinal and hemoparasites were excluded during data analysis.

Sample size determination

The CLSI guideline, which was developed through the clinical and laboratory standards institute consensus processes, was employed to determine the sample size. These guidelines recommended that the best means to establish a reference interval is to collect samples from a sufficient number of reference individuals to yield a minimum of 120 samples for analysis, by non-parametric means for each partition (e.g. Gender, age range) [6, 8]. Therefore starting from the age of 12 years, males and females must separate for the establishment of reference intervals (thus, 240 participants are needed for both sexes). According to previous studies in other African countries, in large scale studies about 30% of apparently healthy individuals [7] do not qualify for RI determination for various reasons [7]. Considering a 30% withdrawal rate from data analysis, to reach the recommended sample size of 240, 344 individuals were enrolled (i.e. assuming 30% of 344 around 104 of the participants may withdraw during data collection).

Reference population selection technique

Three sub-cities (Ayder, Hawelti and Semen) were selected from the total of seven sub-cities through random sampling techniques then the total sample (344) was categorized based on the relative house hold size in each sub-city using Probability Proportional to Size (PPS). The 3 sub-cities have a total of 68477 households (18266, 33319 and 16892 in Semen, Hawelti and Ayder respectively).The total study participants were proportionally distributed to each sub-cities based on their number of households. Accordingly, 92 from Semen, 167 from Hawelti and 85 from Ayder adolescents were recruited. In each sub-city, there were 5 Kebelles (kebelle-is part of a sub city which is a small administrating part of the city), and then the numbers were distributed to recruit participants from each Kebelles based on the number of house hold. Finally the study participants were selected using the systematic sampling techniques (Kth = 199). If the Kth (199th) house hold is not fulfilled the eligible criteria or does not have adolescent aged participant they were passed to the nearby household either to the next or former which fulfilled the criteria. Once volunteering participants fulfilling the eligibility criteria were identified by the health extension workers, they were invited to go to nearby health facilities for sample collection.

Data collection

From the study participants who give assent and family consent, demographic data and a brief medical history were collected. The physical examination was performed by physicians. Blood specimens were collected for analyzing hematological parameters and hemoparasites. Stool specimens were collected for intestinal parasites examination and urine samples for urinalysis. The Laboratory results were shown to the participants upon their request through the health extension workers. But when their results show abnormal findings, they were linked to nearby health institutes for management and treatment according to the facilities guidelines. Socio-demographic and clinical data were collected using a structured questionnaire by translating to the local language. Data were collected by trained data collectors, physical examinations and anthropometric measurements were carried out by physicians.

Sample collection and laboratory analysis

About 4ml of venous blood sample was collected from each participant using K2EDTA anticoagulated test tubes using multisampling needle from 8 am to 11 am. During the blood collection time participants were seated on a comfortable chair and tourniquet was applied for less than one minute until the vein is visible and anchored with the syringe. One’s the vein is visible and anchored tourniquet was removed. All samples were labeled with unique identification number. The hematological samples were transported within 2-8oc cooled icebox and analyzed within 5 hours of collection. Before analysis the 2-8oc preserved sample was put in the room temperature for 30 minutes. Whole blood samples were used for analyzing hematological tests, blood morphology and hemoparasites identification. Complete blood counts were analyzed using Sysmex KX-21N an automated 3-part differential hematology analyzer (Sysmex Corporation Kobe, Japan). Both thick and thin blood film was performed for the detection and identification of hemoparasites as well as for blood morphology using giemsa staining methods. Even if the sample was collected from the apparently healthy participants by following stringent criteria and family folder of the health extension workers before sample collection but after sample collection left over plasma was screened for HBsAg, HCV, HIV and RPR in Tigrai blood bank using an ELISA technique.

Leak proof clean containers were used to collect urine and stool samples. Urinalysis was performed using both the reagent strip and microscopic analysis methods. Stool examination was performed using wet mount, Formol Ether sedimentation, kato-katz and modified acid fast staining techniques. Wet mount was performed on the collection sites immediately within 10 minutes after delivering the sample. But Formol Ether sedimentation, kato-katz and modified acid fast staining techniques was performed at Tigrai health research institution after collecting the necessary samples.

Data quality control

The questionnaire was pre-tested with 5% (18) of individuals other than the study subjects. This pre-testing of a research instrument was entailed a critical examination of each question as to its clarity, understanding, wording, and meaning as understood by potential respondents to remove possible problems with the questions. Besides, adequate training was given to the data collectors before the data collection period. Participants were also being adequately oriented on how to collect specimens. The quality of laboratory analysis was maintained by following standard operating procedures of the pre analytical, analytical and post analytical stages. The Hospital was on the process of accreditation. Its maintenances protocol was controlled according to its standard procedures continuously. Accuracy was done by using the daily QC tests (Low, Normal and High values) and also reproducibility was checked. Between run and within run also checks to avoid carry over between the former and current samples.

Data analysis

Data were cleaned, entered and analyzed using SPSS version 23. Both parametric and non-parametric analyses were performed since the data was not normally distributed when checked its normality. Data lower than first quartile (Q1-1.5 × IQR), or higher than third quartile (Q3+1.5 × IQR) [15] were considered as outliers and the outliers was excluded. The median and 95th percentile reference intervals were determined by using 2.5th and 97.5th percentiles of each hematological parameter with descriptive statistics based on gender. When comparing the data of the three sub-cities and also the kebelle levels there was no significant difference in the RIs. Differences between males and females were evaluated using the Mann–Whitney U test. "P-value < 0.05" was considered as clinically significant at 95% confidence intervals.

Ethical consideration

Ethical clearance was obtained from the research and ethical review committee of Department of Medical Laboratory Sciences of Addis Ababa University, Ethiopia. Before starting the study, permission was obtained from Tigrai regional health bureau and the selected sub-cities. Also, after explaining the purpose and relevance of the study, written consent was obtained from each guardian of study participant and assent from the participants before data collection. Confidentiality of information (results) was kept between the study participant and the investigators. All participants who were diagnosed positive for intestinal parasites were linked to nearby health institutions for treatment immediately. Cooperation letter was obtained from Wukro General Hospital to do the hematological tests.

Results

Socio demographic characteristics

In this study, 344 adolescents were participated and 249 study participants were eligible for final analysis. Out of the 249 eligible participants, 127 (51%) were females). The median age of study participants was 14.5 (SD 1.09) years. The overall exclusion rate was 27.6%. Out of the total samples collected, 50 (14.5%) were excluded due to the presence of intestinal parasites (ova of S.mansoni (33), ova of H.nana (12) and ova of E.vermicularis (5)), and 45(13.1%) were excluded due to incomplete information (15), due to outliers (20) and due to the presence of hemolysis (10).

Hematological parameters of the study participants

The median values of RBCs, Hgb, HCT, absolute and percentile of mixed values (basophiles, Monocytes and Eosinophils) of the WBC differential and MCHC in females were lower than males (p<0.05). Statistically significant higher values in MCV, platelets, RDW-CV, absolute Lymphocyte and Neutrophil counts were found in females (p<0.05). The Median and 95% RIs were as follows: RBC (1012/L) 5.2, 4.6–5.9; Hgb (g/dl) 14.8,12.6–17.1; HCT (%) 44.8, 40–55; MCHC (g/dl) 32.5, 30–35.8; WBC (109/L) 5.4, 2.9–9.6; Platelet (109/L) 261, 138–364 for males; RBC (1012/L) 4.9, 4.28–5.61; Hgb (g/dl) 14,12–15.4; HCT (%) 43.3, 38–47; MCHC (g/dl) 32.2, 30.4–34.2; WBC (109/L) 5.9, 3.4–10.2; Plt (109/L) 288,151–448 for females. There is no significant difference between males and females on the MCH, absolute Neutrophil count, percentile of lymphocytes and MPV values Table 1. When p value is <0.05 among the gender there is a significance difference (clinically difference) between Males and Females needs a separate reference interval based on the median value.

Table 1

Median and 95% RI of hematological parameters of the study participants in Mekelle, Tigrai, Northern Ethiopia, 2019 (n = 249).

Parameter	Gender	N	Median	RI (95%)	2.5th centile	97.5th centile	p-value
					(90% CI)	(90% CI)
WBC(109/L)	M	122	5.4	2.9–9.6	2.6–3.3	9–9.7	0.027*
	F	127	5.9	3.4–10.2	2.7–3.6	9.2–10.7
RBC(1012/L)	M	122	5.2	4.59–5.91	4.45–4.63	5.88–5.98	<0.001*
	F	127	4.9	4.28–5.61	4.13–4.42	5.4–5.76
Hgb(g/dl)	M	122	14.8	12.6–17.1	12.1–13	17–18.6	<0.001*
	F	127	14	12–15.4	11.4–12.4	15–16
HCT (%)	M	122	44.8	40–55	38–40.8	51–58	<0.00*
	F	127	43.3	38–47	36–39	46–50
MCV(fl)	M	122	86.7	76–94	74–80	93–95	0.019*
	F	127	88	80–98	73–82	94–99
MCH(pg)	C	249	28.3	24–31	23–24.8	30.8–31.8	0.085
MCHC(g/dl)	M	122	32.5	30–35.8	29.7–30.8	34.3–41.3	0.027*
	F	127	32.2	30.4–34.2	29–30.9	33.8–34.3
PLT(109/L)	M	122	261	138–364	133–188	353–391	<0.001*
	F	127	288	151–448	140–181	413–467
RDW-CV (%)	M	122	14.1	13–16	12.8–13.2	16–16.6	0.002*
	F	127	13.7	12.5–15.6	12.4–12.8	15–17.6
Neutrophil(109/L)	C	249	3	0.9–6.8	0.8–1	6.3–7.2	0.113
Mixed value(109/L)	M	122	0.8	0.3–3.8	0.2–0.4	2.- 4.2	<0.001*
	F	127	0.6	0.2–1.6	0.1–0.3	1.2–2.4
Lymphocyte(109/L)	M	122	2	1.2–3.3	0.9–1.3	3.1–3.7	0.004*
	F	127	2.3	1.1–3.7	1–1.3	3.5–3.8
Neutrophil (%)	M	122	45.5	23.7–64.7	21–24.9	62–74.5	0.036*
	F	127	50.6	24.8–71	22–28.5	66–89.5
Mixed value (%)	M	122	14.5	7.2–36.8	5.5–9.4	29–43.5	<0.001*
	F	127	10.5	4.8–22.7	1–5.1	19–37.7
Lymphocyte (%)	C	249	39	19.8–61.5	17–23	59–64	0.48
MPV(fl)	C	249	10.6	8.8–13.1	8.6–8.9	(12.6–13.6)	0.567

WBC: White Blood Cell; RBC: Red Blood Cell; Hgb: Hemoglobin; Hct: Hematocrit; MCV: Mean Corpuscular Volume; MCH: Mean Corpuscular Hemoglobin; MCHC: Mean Corpuscular Hemoglobin Concentration; PLT: Platelet; MPV: Platelet Mean Volume M: Male; F: Female; C: Combined N: Number of participants. Mixed values: are values which includes Basophiles, Monocytes & Eosinophiles displaying as one value as the machine is three differential *P < 0.05 by (Mann–Whitney U test) for comparison of medians between males and females.

Comparing the upper and lower limits of our study to the currently in use reference intervals (company values) (Table 2) shows a higher proportions of out of range values observed for RBCs 19(15%), WBC 33(27%), MCHC 31(25.4%), Hgb 30(24%), Hct 33(24%), Mixed value 35(28%) and percentile neutrophils 39(31%) in males. In females, a higher proportion with out of range values were observed for RBCs 12(9%), WBC 23(18%), Hct 25 (19%), MCHC 49(38%) and percentile neutrophils 28(21%). The combined value out of range when comparing with currently in use reference intervals (company ranges) were Lymphocyte percentile 104 (42%) MPV 95(38.5%) and absolute neutrophils 69(27%). The greatest proportion of the study participants with values outside the lower reference limits of reference intervals were observed for absolute neutrophil 68 (27%) while Lymphocyte percentile 104 (42%) had the greatest proportion of participants with values above the upper reference limits of the currently in use reference intervals. When comparing between our study reference intervals and the currently in use reference interval for WBC test parameters, 51(20%) out of the 249 participants were considered leucopenia while 5(2%) were considered as leukocytosis.

Table 2

Comparison of out of range values between new and old reference intervals for hematological parameters in Mekelle city, Tigrai, Northern Ethiopia, 2019 (n = 249).

Parameter	Gender	Current value	Instruments value	Lower range	Upper range	Total out of range
				Frequency (%)	Frequency (%)	Frequency (%)
WBC(109/L)	M	2.9–9.6		31(25)	2(1)	33(27)
	F	3.4–10.2	4.5–13	20(16)	3(2)	23(18)
RBC(1012/L)	M	4.6–5.9	4.2–5.6	2(1)	17(14)	19(15)
	F	4.3–5.6	4.1–5.3	3(2)	9(7)	12(9)
Hgb(g/dl)	M	12.6–17.1	12.5–16.1	2(1)	28(23)	30(24)
	F	12–15.4	Dec-15	0	4(3)	4(3)
HCT (%)	M	40–55	36–47	5(4)	28(23)	33(27)
	F	38–47	35–45	3(2)	22(17)	25(19)
MCV(fl)	M	76–94	78–95	1(0.8)	1(0.8)	2(1)
	F	80–98	78–95	2(1)	0	2(1)
MCH(pg)	C	24–31	26–32	13(5)	2(0.8)	15(6)
MCHC(g/dl)	M	30–36	32–36	31(25)	0	31(25.4)
	F	30.4–34		46(36)	3(2)	49(38)
PLT(109/L)	M	138–364	140–385	0	1(0.8)	1(0.8)
	F	151–462		2(1.6)	11(8)	13(2.4)
Neutrophil(x109/L)	C	0.9–6.8	02-Jul	68(27)	1(0.4)	69(27)
Mixed value(109/L)	M	0.3–3.8		0	7(2.8)	7(2.8)
	F	0.2–1.6	0.24–1.6	0	0	0(0)
Lymphocyte(109/L)	M	1.2–3.3	01-Mar	0	3(2.4)	3(2.4)
	F	1.1–3.7		0	16(12.6)	16(12.6)
Neutrophil (%)	M	24–65	40–80	37(30)	2(1.6)	39(31)
	F	24.78–71		26(20)	2(1.6)	28(21)
Mixed value (%)	M	Jul-37	Apr-18	2(1.6)	33(27)	35(28)
	F	May-23		2(1.6)	6(4.7)	8(6)
Lymphocyte (%)	C	20–62	20–40	0	104(42)	104(42)
MPV(fl)	M		7.2–10.4
	F		7.5–11.5
	C	8.8–13.1		4(1.6)	91(36.5)	95(38.5)

WBC: White Blood Cell; RBC: Red Blood Cell; Hgb: Hemoglobin; Hct: Hematocrit; MCV: Mean Corpuscular Volume; MCH: Mean Corpuscular Hemoglobin; MCHC: Mean Corpuscular Hemoglobin Concentration; PLT: Platelet; MPV: Platelet Mean Volume M: Male; F: Female; C: Combined N: Number of participants. Mixed values: are values which includes Basophiles, Monocytes & Eosinophiles displaying as one value as the machine is three differential.

Finally, our study was compared with others studies. As shown in Table 3, variations among the difference parameters were noted. For example, the white cell count of the study participants as well as the values from other Africans was lower compared to the instrument or American/European values. RBC parameters were higher in the lower range than southwest Ethiopia. Some inconsistencies were also noted in Platelet counts.

Table 3

WBC, Hgb, Hct, MCV, Plt and WBC subsets for apparently healthy Mekelle city children compared to previously published Tanzanian, Ugandan, Zimbabwe and industrialized country reference intervals.

Parameter	Gender	Current	Instruments	South west Ethiopia [17]	Tanzanian	Uganda	United States/Europe(95% RI) [20]	Zimbabwe (95% RI) [21]
		Study (95% RI)	Values [16]		(95% RI) [18]	(95% RI) [19]
	M	2.9–9.6		4.0–11.7				3.25–8.64
WBC(109/L)	F	3.4–10.2		3.7–11.4				3.3–9.8
	C		4.5–13		3.2–10.3	4.1–10.7	4.5–13
RBC(1012/L)	M	4.6–5.9	4.2–5.6	4.06–6.57	-	-	-	4.47–6.47
	F	4.3–5.6	4.1–4.5	4.32–5.63				4.8–5.83
Hgb(g/dl)	M	12.6–17.1	12.5–16.1	12–19.6	10.8–17	11.2–15.9	13–16	12.1–17.4
	F	12–15.4	12–15.4	11.6–15.9	10–14.9	9.9–14.5	Dec-16	11.1–15.7
HCT (%)	M	40–55	36–47	35.6–55.2	33–48.1	32.3–45.5	37–49	36.1–49.7
	F	38–47	35–45	36–47	30.8–44.7	28.1–42.4	36–46	34.6–46.7
MCV(fl)	M	76–94	78–95	75–93	63.2–91	65–89.5	78–98	70.1–93.2
	F	80–98	78–95	74.5–91	62.2–94.5	67.4–89.9	80–100	68.7–96.9
MCH(pg)	M		26–32	25–31	-	-	-	22.5–30.9
	F		26–32	25–30.8				22.1–32
	C	24–31
MCHC(g/dl)	M	30.1–35.8	32–36	32–36	-	-	-	30.3–36.1
	F	30.4–34.2	32–36	32–35				29.8–35.8
PLT(109/L)	M	138–364	140–385	158.5–470	119–458	110–327	150–400	186–415
	F	151–462		198–460.4	107–482	124–353	150–400	214–476
Neutrophil(109/L)	M			1.3–7.4				1.03–3.9
	F			01-Jul				1.13–5.7
	C	0.9–6.8	02-Jul		0.9–4.6	0.9–3.5	1.5–6
Mixed value(109/L)	M	0.3–3.8			0.2–2.5	0.5–3.6	0.6–1.5	0.41.5
	F	0.2–1.6	0.24–1.6	-				0.3–1.5
Lymphocyte(109/L)	M	1.2–3.3	01-Mar	-				1.4–3.9
	F	1.1–3.7			1.4–4.2	1.7–4.7	1.5–4.5	1.4–3.9
Neutrophil (%)	M	23.7–64.7	40–80	-	-	-	-	23.3–58.5
	F	24.78–71						24–61.2
Mixed value (%)	M	7.2–36.8	Apr-18	-	-	-	-	5.4–67.3
	F	4.8–22.7						5–73.4
Lymphocyte (%)	M		20–40	-	-	-	-	27.7–62.6
	F							28.4–65
	C	19.8–61.5
MPV(fl)	M		7.2–10.4	-	-	-	-	8.6–12.3
	F		7.5–11.5					8.4–11.2
	C	8.8–13.1

WBC: White Blood Cell; RBC: Red Blood Cell; Hgb: Hemoglobin; Hct: Hematocrit; MCV: Mean Corpuscular Volume; MCH: Mean Corpuscular Hemoglobin; MCHC: Mean Corpuscular Hemoglobin Concentration; PLT: Platelet; MPV: Platelet Mean Volume M: Male; F: Female; C: Combined N: Number of participants. Mixed values: are values which includes Basophiles, Monocytes & Eosinophiles displaying as one value as the machine is three differential.

Discussion

Reference intervals are essential for decision making in clinical diagnosis, to initiate and monitor therapeutic actions, or to provide accurate data for epidemiological purposes. Several factors including age, gender, race, environment, socio-economic conditions, dietary pattern influence laboratory parameters. RIs also depend on the type of instrument, reagents and methods used. Hematological parameters tested in this study were WBC and its differential, RBC with indices and platelets counts, analyzed using the automated Sysmex KX 21-N 3 diff hematology analyzer. Most of the parameters showed a significant difference among gender, which is similar to studies done in Western Kenya [11], Tanzania [18], Southwest Ethiopia [19], African American [22], Kuwaiti [23] and Port Harcourt Nigeria [24]. The RBC, Hgb and HCT were significantly higher in males than females (p< 0.001). The reason for the differences among gender may be due to hormonal variations among males and females. For example, erythropoietin release is different in response to the hormonal production among males and females [25] and also progressive maturation increase in muscle mass resulting in increased RBCs production in males but females having lower level may be due to menstrual blood loss [26].

In contrast in our study the mean value of RBCs and hemoglobin results were significantly different from mean values of studies reported in Kuwaiti [23] and African American [22] among children. The differences may be due to ethnicity [22], altitude variation since Kuwaiti have an average mean altitude of 108m above sea level and nutritional differences [23].

There is also a clinical difference among gender for WBC RI in our study which is higher in females than males similar with studies done in Kuwaiti [23], African American [22], Zimbabwe [21], and western Kenya [27]. This gender difference in the total WBC counts is almost in all ethnic groups. This difference may be due to a genuine biological difference [28], and also due to body composition difference between men and women because women have proportionally more fat mass than men [5, 29]. So, fatty mass is the critical component that influences leukocyte counts [30]. However, Kenyan study did not find significant differences between males and females [11].

When comparing WBC results with similar studies, which are done in African American [22], Kuwaiti [23], Zimbabwe [21] results were higher than our study population. The reason may be as the peoples living in Sub-Saharan countries are exposed to chronic viral and bacterial infections [7] lowers their white blood cells.

The current finding also showed that the mean, median and 95th percentile reference intervals for the platelet counts varies significantly between females and males (p<0.05). This study indicated that the platelet value is higher in females than males similar to other studies done in Kuwaiti [23], Zimbabwe [21], Port Harcourt Nigeria [24] and Southwest Ethiopia [19]. The gender difference in the platelets count is in all ethnic groups. It may be due to biological difference [28], due to proteomic variability as males have higher cytokine and growth factor levels in platelet rich plasma when compared with females [31]. Additionally, due to the differences on serum estrogen level in females may play a role in the increasement of platelet counts. As many studies have revealed that estrogen favorably benefits platelet production [31, 32]. Thrombopoietin also increases in response to regular menstruation [33, 34], due to this the total body iron storage is generally lower in women. Therefore, iron depletion is a well-known factor to stimulate platelet production [35, 36]. However, the Kenyan study does not find a significant differences between males and females [11].

The platelet counts are also lower in our study population than similar studies done in Southwest Ethiopia [19], and Zimbabwean adolescents [21].The cause of platelet count differences among different study areas is unknown [24].

Though limited studies are available on children and adolescents, the neutrophil range in the current study which is lower than the currently in use company derived ranges, is consistent with the findings from Zimbabwean adolescents [21]. In addition, the upper limit of the newly established lymphocyte RI is higher than the company derived value but again consistent with values generated from Zimbabwean adolescents [21]. Comparing to the reference interval generated in southwest Ethiopia from a total of 334 children, the WBC count of both lower and upper limit was higher than the current study especially in males. On the other hand, both the lower and upper limit of RBC count was slightly higher in the current study whereas slightly higher upper limit for Hgb was recorded in male children of the Southeast Ethiopian study. Similarly higher lower & upper limit for PLT count in males and higher lower limit only in Females was recorded as compared to the current study [17].

Taken together, when comparing our values with the currently available RI almost all tests have a variation. In our study WBC results were lower than the currently available RI the reason may be, as we know Africans are more exposed to chronic infections [7]. But the RBC and RBC indices are higher than the currently in use RI, the reason for the increment may be due to the temperate climatic conditions of the study area.

Finally the study checked how much misclassification could have happened as a result of utilizing company derived RIs, which is widely being practiced in our country as in most resource limited settings. The reported result showed such misclassification is noted in most of the hematological parameters by the RI established by manufacturer of the instrument Sysmex KX-21N as compared to the RI established by this study. This may be due to nutritional difference, climate, parasitic and viral infections, ethnic background differences between the current study population and the population used for the company derived values, mostly Caucasians. So, it is better to use the locally established RI for the local populations.

Conclusion

The hematological parameters for apparently healthy adolescents in this study almost differ from other African countries and western countries. There was a significant difference in Red Blood Cells, MCHC, Hemoglobin, Hematocrit, White Blood Cells and Platelets. All reported results were higher in males than females except platelet and WBC counts were higher in females. The established reference interval in this study will help to diagnosis, treat, and follow up of the clients in comparing to the locally healthy adolescents.

(DOCX)

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(SAV)

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27 Sep 2019

PONE-D-19-22528

Establishment of community based hematological reference intervals among apparently healthy adolescents aged 12-17 years in Mekelle city, Tigrai, northern Ethiopia: a cross sectional study

PLOS ONE

Dear Mr. Haileslasie,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands.

Specifically both reviewers expressed concern over the quality of writing and significant editing and re-wording is required. Also of concern is the fact that the manuscript refers to Figure 1, yet no figure is provided. In addition, concerns over how many adolescents did not give assent to participate and it is unclear if  ethical clearance for listing religion has been obtained. If you choose to submit a revised manuscript, please address all concerns raised by both reviewers.

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

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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: Thank you for the opportunity to review “Establishment of community based hematological reference intervals among apparently healthy adolescents aged 12-17 years in Mekelle city, Tigrai, northern Ethiopia: a cross sectional study.” This is a well-designed study that adds to the literature and describes an important task: generating locally relevant reference intervals for safety labs. It was a little hard to follow at times, but with a review of grammar and spelling, should make a good contribution to the literature.

Major comments:

Please review the manuscript throughout with a fluent English speaker. There are many grammatical errors which make reading the paper challenging, particularly in the results section (see page 10 – very hard to follow). Too many to summarize here.

Please include a STROBE style flow diagram to go through numbers screened, enrolled, and included in analysis, showing the numbers and reasons for excluding volunteers at each step. For more information, see: Vandenbroucke JP, von Elm E, Altman DG, Gøtzsche PC, Mulrow CD, Pocock SJ, et al. (2007) Strengthening the Reporting of Observational Studies in Epidemiology (STROBE): Explanation and Elaboration. PLoS Med 4(10): e297. https://doi.org/10.1371/journal.pmed.0040297

Minor comments:

Median age (abstract): report as XX.X, the extra level of precision (15.31) is unnecessary.

Methods, data analysis: how many outliers did you exclude? Should you be doing this? The 95% RI should control for this. Please make sure this is clarified in your results and STROBE flow diagram

Table 2: please clarify where and why you combined values. I presume this was because they were not significantly different? Is this mentioned in the methods?

Page 12: what are “the company’s reference intervals (Table 3)”?

I recommend ending your introduction with a quick statement about what you propose to present in this paper. The last sentence of the introduction should be something like “Here, we present our efforts to generate reference intervals in adolescents .,..” etc.

Reviewer #2: Haileslaslie et al: Hematological reference intervals among adolescents

A prospective community-based study defining hematological reference intervals in apparently healthy adolescents in Ethiopia. A major strength is that participants were randomly selected from the community in the area studied and would be suitable to use for the local lab, at least in the short run. However, the presentation of data is complex and difficult to follow, and it is difficult to assess if these figures are generalizable to similar populations.

I assume that presently RIs from the manufacturer is used. Company values are stated in several tables. Where these figures for adults, or for adolescents, or both? This is needed to evaluate how these new RIs compare to the presently RIs used.

Key words: suggest that standardized expressions are used, e.g. not CBC.

Abstract: could be shortened, omit spelling out EDTAs chemical name, what statistical software was used. Also, numbers for RIs could be omitted, but hematology parameters measured should be included, along with a brief description of gender differences.

Background, page 3, last paragraph. I assume studies on pharmaceuticals are meant when stating “toxicity grading scales”, but these sentences are unclear.

Methods: 3.1 could be shortened, e.g. “Mekelle was formerly the capital”, “Mekelle is considered a special zone”. Weyna-Dega climate and altitude is relevant, but preferably state something about this climate instead of using a local term.

3.2 CLSI guidelines should be accompanied by a reference.

3.3 incl table could be shortened.

3.4 How many adolescents did not give assent to participate? Such figures are needed to know how representative this population was when using this random enrollment design.

3.6 Samples were transported ice-cooled within “allowable time”? What was the maximal time allowed until analysis? And during how long period was the collection of samples performed? Hematology systems sometimes drift over time. Where there any internal or external controls used to assure stability over time for the measurements, and comparability to other labs?

3.8 CLSI suggest Harris Boyd (or Lahti) statistics for partitioning. In the study Mann Whitney U-test was employed. What considerations was behind that decision?

Ethical considerations. Some countries do not allow recording of religious belief in scientific studies, as could be seen in Table 2. Was religion covered by the ethical clearance?

Operational definitions could be omitted, and the information in that section integrated into the Methods section.

Results: Figure 1 shows age, however I cannot find any fig 1.

The 1st paragraph relating figures for numerous RIs could be shortened, figures could be omitted or substantially shortened since they also appear in Table 3. Now table 3 shows combined only for those three parameters where the Mann Whitney U-test turned out with insignificant differences between genders (MCH, Neutrophils, lymphocytes in %). However, even though lymphocyte% is insignificant (0.48), lymphocyte in absolute numbers is highly significant (0.004). Thus, I suggest the authors show all data for males and females as well as combined.

Page 12 starts with “Comparing the upper and lower limits….”. I assume the authors refer to Table 4, not Table 3?

Table 5 is a comparison of RIs from different sources. The United States/Europe lack reference/s. Also, information about ages is relevant for these studies, e.g. in the Ethiopian stud (ref 20) children from the age of 5 years are compared to the present study. This is a major limitation in the presentation, comparisons to gender and age in other studies, and to RIs presently used are difficult to follow. Thius, the practical use even in the area where the study was conducted is unclear.

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Reviewer #1: No

Reviewer #2: No

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4 Dec 2019

Point by point response for reviewers

Response for reviewer 1

Comment 1: Please review the manuscript throughout with a fluent English speaker.

Response: The grammatical and writing errors are corrected. The manuscript was drafted and rewrites again.

Comment 2: Median age (abstract): report as XX.X, the extra level of precision (15.31) is unnecessary

Response: The decimal number are corrected in the manuscript and rewritten again

Comment 3: How many outliers did you exclude? Should you be doing this?

Response: Yes, 20(5.8%) of the participants were excluded due to its outlierness. Please, see on page 9 on the result section.

Comment 4: Please clarify where and why you combined values. I presume this was because they were not

Significantly different? Is this mentioned in the methods?

Response: No, We included in the new revised manuscript in the result part page 10.

Comment 5: what are “the company’s reference intervals (Table 3)”?

Response: The value which is inserted on the machine using the values of the test leaflet of the specific machine. When a new machine come to install in a Laboratory the machine comes with its own specific leaflets. Inserted the values to the machine to use as a reference

Comment 6: I recommend ending your introduction with a quick statement about what you propose to present in this paper

Response: We accept your comment and the manuscript is modified. You can see at the last of the introduction part.

Response for reviewer 2

Comment 1: I assume that presently RIs from the manufacturer is used. Company values are stated in several tables. Where these figures for adults, or for adolescents, or both? This is needed to evaluate how these new RIs compare to the presently RIs used.

Response: The inserted values on the CBC machine were for both adolescents and adults. There was no separated value on the machine but I compare the result with the leaflet which I have got separate values for adolescents and adults in the manual CBC machine.

Comment 2: Abstract: could be shortened, omit spelling out EDTAs chemical name, what statistical software was used. Also, numbers for RIs could be omitted, but hematology parameters measured should be included, along with a brief description of gender differences.

Response: We abbreviate the long terms of EDTA and numbers out of the hematological parameters. The data were entered and analyzed by SPSS

version 23 statistical software.

Comment 3: Background, page 3, last paragraph. I assume studies on pharmaceuticals are meant when stating “toxicity grading scales”, but these sentences are unclear.

Response: During the treatment of hematological cases for the patients it is based on the WHO guideline which prepared based on the developed countries. So, it is better to follow clinical cases of the patients in relation to the local population.

Comment 4: Methods: 3.1 could be shortened, e.g. “Mekelle was formerly the capital”, “Mekelle is considered a special zone”. Weyna-Dega climate and altitude is relevant, but preferably state something about this climate instead of using a local term

Response: We tried to shortened and local terms are avoided

Comment 5: CLSI guidelines should be accompanied by a reference.

3.3 including table could be shortened.

Response: Tables are slightly modified and CLSI reference is putting on the fifth line of the 3.2 sample size determination part [2, 4].

Comment 6: 3.4 How many adolescents did not give assent to participate? Such figures are needed to know how representative this population was when using this random enrollment design

Response: All of the participants who were present at study area during data collection time and who fulfilled the criteria were voluntarily participated after oriented the aim of the research.

Comment 7: 3.6 Samples were transported ice-cooled within “allowable time”? What was the maximal time allowed until analysis?

Response: The maximum allowable time was 5 hours. I have corrected on the manuscript part

Comment 8: During how long period was the collection of samples performed? Hematology systems sometimes drift over time.

Response: The data collection was performed from the middle of December to the last of April within 4 months.

Comment 9: Where there any internal or external controls used to assure stability over time for the measurements, and comparability to other labs?

Response: Yes, We have used both internal and external controls. Comparability was done between Ayder referral Hospital hematology CBC value and Wukro General Hospital. The result was similar related to each other by transporting the sample within 5 hours like that of participant’s sample.

Comment 10: 3.8 CLSI suggests Harris Boyd (or Lahti) statistics for partitioning. In the study Mann Whitney U-test was employed. What considerations were behind that decision?

Response: Harris Boyd (or Lahti) is important for partitioning of the study participants but Mann Whitney U-test is used for analysis of the data to compare the gender difference between males and females based on their median values.

Comment 11: Ethical considerations. Some countries do not allow recording of religious belief in scientific studies, as could be seen in Table 2. Was religion covered by the ethical clearance?

Response: No, because in Ethiopia many research are done without religion ethical clearance. There is no problem participating a religion belief in scientific studies.

Comment 12: Operational definitions could be omitted, and the information in that section integrated into the Methods section.

Response: we have omitted and merge to the methodology parts

Comment 13: Results: Figure 1 shows age, however I cannot find any fig 1.The 1st paragraph relating figures for numerous RIs could be shortened; figures could be omitted or substantially shortened since they also appear in Table 3. Now table 3 shows combined only for those three parameters where the Mann Whitney U-test turned out with insignificant differences between genders (MCH, Neutrophils, lymphocytes in %). However, even though lymphocyte% is insignificant (0.48), lymphocyte in absolute numbers is highly significant (0.004). Thus, I suggest the authors show all data for males and females as well as combined.

Page 12 starts with “Comparing the upper and lower limits….” I assume the authors refer to

Table 4 not to Table3

Response: Figure and Tables rearranged and corrected the problem was due to typical errors.

Comment 14: Table 5 is a comparison of RIs from different sources. The United States/Europe lack reference/s Also, information about ages is relevant for these studies, e.g. in the Ethiopian children from the age of 5 years are compared to the present study. This is a major limitation in the presentation; comparisons to gender and age in other studies, and to RIs presently used are difficult to follow. Thus, the practical use even in the area where the study was conducted is unclear.

Response: We put the reference for United States/Europe on the appropriate space on Table5.

Submitted filename: Response to Reviewers.docx

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27 Jan 2020

PONE-D-19-22528R1

Establishment of community based hematological reference intervals among apparently healthy adolescents aged 12-17 years in Mekelle city, Tigrai, northern Ethiopia: a cross sectional study

PLOS ONE

Dear Mr. Haileslasie,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’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.

Specifically, the Reviewer thought that the manuscript is of value to the research community, and that if their concerns are addressed, the manuscript should be published. Thus, I have decided a minor revision of the manuscript should be made.Specifically, the comments by the reviewer should be addressed, including the need to incorporate a laboratory analysis section for hemoparasite examination.

We would appreciate receiving your revised manuscript by Mar 12 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

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Colin Johnson, Ph.D.

Academic Editor

PLOS ONE

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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 #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

**********

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

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). 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 #3: Yes

**********

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

PLOS ONE 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 #3: Yes

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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 #3: The manuscript covers a topic of scientific significance as it generally described the problem and need for establishing reference intervals for the populations of interest.

However, the following observations need some clarifications.

1. I suggest the title to be adjusted as “Community based hematological reference intervals among apparently healthy adolescents aged 12-17 years in Mekelle city, Tigrai, Northern Ethiopia: a cross-sectional study”

2. Topographical errors throughout the manuscript: spelling, capitalization of proper nouns including author names, sentence form (present Vs past) and grammatic problems

3. There are sentences that start with numbers here and there. It is not recommended to start a sentence with numbers please correct.

4. Information which are not related to the manuscript should be removed e.g. in the background section paragraph 2 last 3 sentences have low value to the manuscript.

“Growth is an extremely complex and non-linear biological process, driven by hormonal mechanisms, characterized by an intrinsic variability reflecting environmental, genetic influences and individual adaptive responses. Growth charts are very helpful as reference tools for pediatricians, in order to monitor individual growth and provide therapeutic interventions. Most female’s puberty starts around 12 years but males from 14 years [6].”

5. Repetitions: e.g. last two sentences of Paragraph 4 of Background: please merge them

Adult Africans have been shown to have lower levels of hemoglobin, red blood cells, platelets, and neutrophil counts compared to adults in the developed countries [12-15]. Further, most blood specimens of Africans have been found to have lower Hemoglobin (Hgb), Hematocrit (Hct), Red blood cell count (RBC), mean corpuscular volume (MCV), neutrophil and platelet counts [16].

6. “Sex” should be replaced by “Gender”

7. “Sampling technique” should be “Reference population selection technique”

8. “K” should be specified: what is the value of “K” it should be mentioned

9. Words such as “doesn’t” should be written in full term as “did not”

10. Table 1 is not cited

11. Were there significant differences in RIs of the three kebeles? And also, please clarify kebele? What do you mean by Kebele?

12. Blood sample for hemoparasites, Stool specimens for intestinal parasites examination and urine samples for urinalysis were collected but there is no lab analysis section. There must be laboratory analysis section for hemoparasite examination, Stool examination and urinalysis. A more complete description of the laboratory methods, especially metrological traceability, is required.

13. How do you exclude the influence of infectious diseases such as HIV, Hepatitis on RIS?

14. How representative is the population for their values to be used for reference intervals?

15. Quality control: What was the validation status of the equipment and what QA protocols (maintenance, reproducibility, accuracy, between run, within run, etc) used to validate the analyser used? These establish how accurate the analysers used produce results. How long did samples stay before analysis and how were they kept in case samples were not analyzed immediately?

16. A comment should be made on the pre-analytical factors. Especially given the dependence of cell counts on haemoconcentration, it is necessary to indicate whether subjects are seated or lying, how long a tourniquet was applied for and also how long between collection and analysis.

17. Data analysis: The statement “Data lower than first quartile (Q1-1.5 × IQR), or higher than third quartile (Q3+1.5 × IQR) (Whisker and blot method) were considered as outliers and the outlier was excluded.” Needs reference

18. Data analysis: Do you check normality of data? Was the data normally distributed?

19. Results: Socio demographic characteristics: The use of mean and SD to describe the age distribution is inappropriate as the population age is clearly not Gaussian. I would recommend median, range and interquartile ranges or similar.

20. Result: The numbers in the statement “(15) were outliers (20) and presence of hemolysis (10”) are not clear

21. Table 2 should be deleted. It has no value.

22. Mean and 95% CI should be deleted from table 3 because it is inappropriate as the parameters value are clearly not Gaussian. I recommend the use of Median and Interquartile range.

23. Table 5: should also include other studies, and also check reference 22, is it for US/Europe or Tanzanian?

24. Result and Discussion: Please remove comparisons that made with adult population. Some studies used for comparison are adult population RIs.

25. Discussion: Reference 22 and 23 are about RIs but how they could be a reference for the last statements in the first paragraph of the background?

26. Discussion: paragraph 4 and 5: The references should be revised…..

Overall Recommendation

The manuscript is potentially acceptable for publication if the investigators were able to exhaustively address the suggested changes above.

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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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: Yes: Bamlaku Enawgaw

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4 Mar 2020

Comment 1: I suggest the title to be adjusted as “Community based hematological reference intervals among apparently healthy adolescents aged 12-17 years in Mekelle city, Tigrai, Northern Ethiopia: a cross-sectional study”

Response 1: We have changed the Title to “Community based hematological reference intervals among apparently healthy adolescents aged 12-17 years in Mekelle city, Tigrai, Northern Ethiopia: a cross-sectional study”

Comment 2: Topographical errors throughout the manuscript: spelling, capitalization of proper nouns including author names, sentence form (present Vs past) and grammatical problems

Response 2: We have corrected the topographical errors

Comment 3: There are sentences that start with numbers here and there. It is not recommended to start a sentence with numbers please correct.

Response 3: We have corrected it (Example: Summary Result part)

Comment 4: Information which is not related to the manuscript should be removed e.g. in the background section paragraph 2 last 3 sentences have low value to the manuscript.

“Growth is an extremely complex and non-linear biological process, driven by hormonal mechanisms, characterized by an intrinsic variability reflecting environmental, genetic influences and individual adaptive responses. Growth charts are very helpful as reference tools for pediatricians, in order to monitor individual growth and provide therapeutic interventions. Most female’s puberty starts around 12 years but males from 14 years [6].”

Response 4: We have tried to remove the unwanted part and tried to rearrange it.

Comment 5: Repetitions: e.g. last two sentences of Paragraph 4 of Background: please merge them. Adult Africans have been shown to have lower levels of hemoglobin, red blood cells, platelets, and neutrophil counts compared to adults in the developed countries [12-15]. Further, most blood specimens of Africans have been found to have lower Hemoglobin (Hgb), Hematocrit (Hct), Red blood cell count (RBC), mean corpuscular volume (MCV), neutrophil and platelet counts [16].

Response 5: The unwanted repetition tried to merged and rearranged on Paragraph 4 the Background part

Comment 6: “Sex” should be replaced by “Gender”

Response 6: We tried to replace Sex by Gender (Summary background part, background section paragraph 2, discussion parts and also in other parts)

Comment 7: “Sampling technique” should be “Reference population selection technique”

Response 7: We have tried to replace “Sampling technique” by “Reference population selection technique”

Comment 8: “K” should be specified: what is the value of “K” it should be mentioned

Response 8: “K” is the interval of data taking households from the first household to the next (second) household. Kth=total number of households in the 3 sub cities divided by total number of participants. Kth=199 the participants data were collected every 199th household.

Comment 9: Words such as “doesn’t” should be written in full term as “did not”

Response 9: We tried to corrected such like terms (discussion Paragraph 3)

Comment 10: Table 1 is not cited

Response 10: The data was obtained from the municipality of Mekelle city (unpublished data).

Comment 11: Were there significant differences in RIs of the three kebeles? And also, please clarify kebele? What do you mean by Kebele?

Response 11: There was no significance difference in RIs among the three kebeles. Kebele is part of a sub city which is a small administrating part of the government.

Comment 12: Blood sample for hemoparasites, Stool specimens for intestinal parasites examination and urine samples for urinalysis were collected but there is no lab analysis section. There must be laboratory analysis section for hemoparasite examination, Stool examination and urinalysis. A more complete description of the laboratory methods, especially metrological traceability, is required.

Response 12: We have tried to incorporate the Lab analysis section

Comment 13: How do you exclude the influence of infectious diseases such as HIV, Hepatitis on RIS?

Response 13: The health extension professionals have a family folder which follows the health status continuously. So, by following stringent criteria for exclusion before sample collection and those apparently healthy individuals sample was screened in the Tigrai blood bank using an ELISA technique.

Comment 14: How representative is the population for their values to be used for reference intervals?

Response 14: The participants were 100% representative for the city because of their systematically selection methods. The three sub cities were selected randomly from the total of seven sub cities. According to CLSI guideline in order to say representative it is better to cover more than 30% of the total subjects.

Comment 15: Quality control: What was the validation status of the equipment and what QA protocols (maintenance, reproducibility, accuracy, between run, within run, etc) used to validate the analyzer used? These establish how accurate the analyzers’ used produce results. How long did samples stay before analysis and how were they kept in case samples were not analyzed immediately?

Response 15: The Hospital was on the process of accreditation. Its maintenances protocol was controlled according to its standard procedures continuously. Accuracy was done by using the daily QC tests (Low, Normal and High values) and also reproducibility was checked. Between run and within run also checks to avoid carry over between former and later samples. The sample was preserved within 2-8oc cold chain box until analysis. The CBC sample was done with in five hours of collection. The 2-8oc preserved sample was put in the room temperature for 30 minutes before analysis.

Comment 16: A comment should be made on the pre-analytical factors. Especially given the dependence of cell counts on haemoconcentration, it is necessary to indicate whether subjects are seated or lying, how long a tourniquet was applied for and also how long between collection and analysis.

Response 16: During the blood collection time participants was seated on a comfortably chair before collection and tourniquet was applied for less than one minute until the vein is visible and anchored with the syringe. One’s the vein is visible and anchored with the syringe the tourniquet was removed. The collected sample was analyzed with in five hours.

Comment 17: Data analysis: The statement “Data lower than first quartile (Q1-1.5 × IQR), or higher than third quartile (Q3+1.5 × IQR) (Whisker and blot method) were considered as outliers and the outlier was excluded.” Needs reference

Response 17: We have putting the reference for the outliers on the data analysis part

Comment 18: Data analysis: Do you check normality of data? Was the data normally distributed?

Response 18: Yes, We have checked the normality before doing the analysis. But, the data was not normally distributed.

Comment 19: Results: Socio demographic characteristics: The use of mean and SD to describe the age distribution is inappropriate as the population age is clearly not Gaussian. I would recommend median, range and interquartile ranges or similar.

Response 19: We take the comments and corrected it.

Comment 20: Result: The numbers in the statement “(15) were outliers (20) and presences of hemolysis (10”) are not clear

Response 20: We have tried to make clear and easily understandable

Comment 21: Table 2 should be deleted. It has no value.

Response 21: Table 2 is removed completely

Comment 22: Mean and 95% CI should be deleted from table 3 because it is inappropriate as the parameters value are clearly not Gaussian. I recommend the use of Median and Interquartile range.

Response 22: Ok, Mean is removed from the Table but 95% CI is important for RIs than the Inter Quartile range

Comment 23: Table 5: should also include other studies, and also check reference 22, is it for US/Europe or Tanzanian?

Response 23: It is for Tanzanian it is a typical error we have corrected it

Comment 24: Result and Discussion: Please remove comparisons that made with adult population. Some studies used for comparison are adult population RIs.

Response 24: We have tried to reject the comparison with adults

Comment 25: Discussion: Reference 22 and 23 are about RIs but how they could be a reference for the last statements in the first paragraph of the background?

Response 25: We have replaced and rearranged the unnecessary references

Comment 26: Discussion: paragraph 4 and 5: The references should be revised…..

Response 26: Discussion: paragraph 4 is tried to rearranged with other references

Submitted filename: Response to Reviewers 3.docx

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17 Mar 2020

PONE-D-19-22528R2

Community based hematological reference intervals among apparently healthy adolescents aged 12-17 years in Mekelle city, Tigrai, northern Ethiopia: a cross sectional study

PLOS ONE

Dear Mr. Haileslasie,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that the manuscript is much improved and invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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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 #3: (No Response)

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

**********

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

Reviewer #3: Yes

**********

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Reviewer #3: Yes

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Reviewer #3: Yes

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Reviewer #3: My previous comments are addressed in the line by line response but some responses especially responses for my comments in the method section should be included in the main manuscript. Before publication the following minor comments should be addressed.

1. Table 1 is not cited in the text

2. My previous comment in method section is addressed but it is not included in the manuscript. The responses given for my comments should be included in the manuscript. (Comments 11, 13, 15, 16, 18)

3. Table needs modification. The RI is not clearly indicated. The table column is better to be rearranged as:

Parameter

Gender

N

Median

RI (95th percentile)

2.5th Percentile 90% CI

2.5th Percentile 90% CI

P value

4. Table 2: Partition in gender is not needed for parameter which have no significant difference between male and female. (MCH. Neutrophil, Lymphocyte and MPV)

5. Table 2: “mixed value” needs definition.

6. Discussion: As indicated in the result section, most of the parameters show a statistically significant difference between male and Female. There should be a comment on statistically Vs Clinically significant difference.

7. Mean should be removed

8. Sex should be replaced by gender

**********

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Reviewer #3: Yes: Bamlaku Enawgaw

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28 Apr 2020

Especially reviewer 3 asses the manuscript in short period of time in detail we appreciate him

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

29 Apr 2020

PONE-D-19-22528R3

Community based hematological reference intervals among apparently healthy adolescents aged 12-17 years in Mekelle city, Tigrai, northern Ethiopia: a cross sectional study.

PLOS ONE

Dear Mr. Haileslasie,

Thank you for submitting your manuscript to PLOS ONE. After careful review, we feel that the revised manuscript is significantly improved, but should include the responses you provided the referee in the method section of the main manuscript. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Jun 13 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

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Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Colin Johnson, Ph.D.

Academic Editor

PLOS ONE

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[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 to be viewed.]

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18 May 2020

We appreciate for the reviewers for responding the activity in short period of time

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

20 May 2020

Community based hematological reference intervals among apparently healthy adolescents aged 12-17 years in Mekelle city, Tigrai, northern Ethiopia: a cross sectional study.

PONE-D-19-22528R4

Dear Dr. Haileslasie,

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Academic Editor

PLOS ONE

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Reviewers' comments:

31 Aug 2020

PONE-D-19-22528R4

Community based hematological reference intervals among apparently healthy adolescents aged 12-17 years in Mekelle city, Tigrai, northern Ethiopia: a cross sectional study.

Dear Dr. Haileslasie:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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PLOS ONE