Danish Hasan Qaiser  ( Department of Physiology, Ziauddin University,Karachi. )
Mohammad Perwaiz Sandila  ( Department of Physiology, Liaquat Medical and Dental College,Karachi. )
Syed Tousif Ahmed  ( Department of Physiology, Ziauddin University,Karachi. )
Tahseen Kazmi  ( Department of Community Health Sciences, Liaquat National Medical College,Karachi. )

Health of an individual is known to vary in different countries, in the same country at different times, and in same individuals at different ages. It is thus a relative and not an absolute stage. This means that the condition of individuals must be related to or compared with reference data. On comparing the individuals' data collected through the medical interview, clinical examination and supplementary investigations with the reference data, the condition of individuals can be interpreted.¹ Reference value is defined as the value obtained by observation or measurement of a particular type of quality on an individual belonging to the reference group.¹ Investigation of an individual should entirely depend on the normal ranges, which have been established to an individuals locality.² Newborn haematological parameters differ in infants and adults.³ A study was done which revealed that various blood indices vary in the newborns as compared to older children or adults. It depends on the gestational age, day of life, maternal factors, mode of delivery and site of blood collection.⁴ The first study on the haematology of the newborns was published in 1924 and since then many studies have been conducted that have examined babies at different gestational ages and of varying birth weights.⁵ Furthermore, determination of reference range of healthy, term neonate is clinically important in terms of various complete blood count parameters for example, haemoglobin concentration which is an important clinical measurement used in decisions regarding clinical diagnosis, treatment and public health interventions for anaemia.⁶ Haemoglobin and haematocrit have been used routinely in the diagnosis of neonatal anaemia and polycythaemia.⁷ White blood cells and platelet count have proved helpful in the assessment of neonatal sepsis and haemostatic status of the infant respectively.⁸ Manroe et al showed that the use of absolute blood neutrophil count has improved the sensitivity in screening for neonatal bacterial diseases.⁹ Haematological values are also frequently determined in the newborns for diagnostic purposes in suspected infections and in bleeding disorders.¹⁰ It is now widely accepted that there are no universal or international standard haematological parameters and all reference values are affected to some extent by factors such as age, race, diet, drug intake, method employed for determination etc. It is thus important that standard reference values of local population should be established.² However, haematological reference values which are in use in Pakistan are derived from studies done on Caucasoid populations and to the best of our knowledge, there is no reference value of neonatal haematological parameters available in Pakistan, using umbilical cord blood. The objective of this study was to establish reference hematological values (complete blood count) in healthy full term newborns using umbilical cord blood, either after vaginal delivery or elective caesarean section.

This is a cross sectional study design based on cord blood sample of newborns and their mothers. Total 897 mothers and their neonates were recruited initially, from four hospitals (Sindh Government Qatar Hospital, Ziauddin University hospital, gole market campus, Sindh Government hospital, Liaquatabad and Chinniot maternity and child hospital.) of Karachi from July 2006 to April 2008. A total of 404 newborns were included in the study (100 from Sindh Government Qatar Hospital, 85 from Ziauddin University hospital, goal market campus, 92 from Sindh Government hospital, Liaquatabad and 127 from Chinniot maternity and child hospital.). Excluded were 493 patients due to multiple pregnancy (101), maternal anaemia (120), 73 due to the PIH, 59 due to the diabetes, 42 due to antipartum haemorrhage, 61 due to the emergency c/section and 37 due to the birth asphyxia. The inclusion criteria for the mother were, age between 15 to 45 years, booked case, uneventful pregnancy and haemoglobin of 10 grams and above. For Neonates it was full term (37 to 42 weeks) with normal birth weight (2.5 to 4.0 Kg). The exclusion criteria for the mother were, multiple pregnancy, diseases complicating pregnancy (anaemia, antepartum haemorrhage, pregnancy induced hypertension, eclampsia, diabetes (gestational or insulin dependent), heart, kidney or lung disease, malaria, disseminated intravascular coagulation), Thalasemia / Sickle cell disease, drug or alcohol abuse, immediate shock like state after post partum period, and emergency caesarian section. The exclusion criteria for neonates were: Abnormal partogram, perinatal blood loss, hydrops foetalis, birth asphyxia, low Apgar score (<8 at 5 minutes), obvious congenital / chromosomal abnormality, and pathologic jaundice (within 24 hours of birth).
Blood sampling:
Mother: Three ml venous blood was collected in EDTA containing tube for complete blood count of mother when she came in labour or for elective caesarean section.
Baby: After delivery of the baby, umbilical cord was immediately clamped; 5 ml cord blood was taken from the umbilical vein and transferred into an EDTA containing tube. The sample was then transported as early as possible (maximum 3 to 6 hours) to the laboratory for analysis.
Laboratory analysis: For haematological parameters a standard coulter gram was done on the Beckman Coulter Counter Max M. u The differential leukocyte count was done under the light microscope using Leishman's stained smears. u Recording of nucleated RBCs was done as per 100 leukocytes using Leishman's stained smear and the WBC count expressed as corrected for the presence of nucleated RBCs if the number was 10 or more.  Procedure of cord blood sampling was explained to the mothers and written informed consent was also taken from them. The data entry and analysis was done on computer package SPSS (Statistical Packages of Social Sciences) version 12.0. The results were given in the text as mean, standard deviation and 95% confidence intervals of haematological values (complete blood count).  Reference range has been calculated by using the formula of X ± 2Sd.

A total of 206 male and 198 female neonates were included in the study. Of these neonates 271 were delivered by spontaneous vaginal delivery and 133 by elective caesarian section. The average weight of the babies was 3.03 ± 0.385 kg. The mean haemoglobin of the newborns were 14.99 ± 1.47 g/dl with reference range of 12.05 - 17.93 g/dl whereas the total WBC count was 13.61 ± 4.23 9/L with reference range of 5.15 - 22.07.The mean platelet count was found to be

256.25 ± 76.54 9/L with a reference range of 103.17-409.33 9/L (Table-1).

Interpretation of values obtained in an individual baby depends on the knowledge of the normal values for the locality.¹⁰ Hence, a study was conducted to determine the reference ranges of complete blood count of term, healthy newborns. A comparison between our study and the results of others in different countries are projected in Table-2. It was observed that the mean haemoglobin of our population was comparable with the figures of Abidjan, but were lower than the European and Indian results, probably due to varying numbers of newborns,8 low socioeconomic status,2,3 poor nutrition,2 maternal factors such as low iron7,11 high gravidity and time between birth and clamping of the cord.^8,10 Reference range of haemoglobin in our study was 12.057 to 17.93 gm/dl, which means that less than 12.0 gm/dl should be considered anaemic and more than 18 gm/dl as polycythaemic, whereas

the Abidjan study set the cut off value for anaemia as less than 16 gm/dl, and Zimbabwean study suggested this level below 13.5 gm/dl. This warrants a need for local reference values. Mean Corpuscular Volume (MCV) value was higher in Iranian, Italian and Malawian studies compared to our study. Stress during pregnancy, maternal factors, low socioeconomic condition and geographical distribution may have played a role in this. Total leukocyte count was higher in the Italian study than rest of the studies including our results. In all of the studies, neutrophils12 were the predominant cells followed by lymphocytes. The wide range of leukocyte count was observed in all studies. Platelet count showed wide variation in different countries. Abidjan, Nigeria and India have low platelet values than us, Italy and Malawi, possibly because of the following four reasons. First are environmental factors such as medicinal herbs widely used in Africa, acute or chronic malaria and poverty with malnutrition in African countries. ¹³ Secondly, contamination with Wharton's jelly,10 thirdly the mechanical trauma of severing the cord10 and fourthly the procedure to estimate the platelets. We found clumping of platelets on peripheral smear, with false positive results when compared to the results when platelet count is done by machine. Hence, it is recommended that platelets be counted manually. Nucleated RBCs indicate degree of hypoxia during labour, which were more in Malawian and less in Indian studies as compared to our study. Our study is based on healthy, term newborns having normal birth weight, haemoglobin of mother equal to or more than 10 gm/dl and having good sample size. This study may help in developing a reference range of Karachi (the urban area). This method can also be applied to the rural areas, urban areas of Sindh province and other provinces of Pakistan and a comparison can be done. Our study on reference ranges of full term, healthy newborns may thus provide clinicians the normal values for our community, which they can use to compare with their patients.

1.Ashavaid TF, Todur SP, Dherai AJ. Establishment of reference intervals in Indian population. Indian J Clin Biochem 2005; 20: 110-8. 
2.Mukiibi JM, Mtimavalye LA, Broadhead R, Mzula E, Dzinyemba WE, Merrick R, et al. Some hematological parameters in Malawian neonates. East Afri Med J 1995; 72: 10-4. 
3.Noguera NI, Detarsio G, Perez SM, Bragos IM, Lanza O, Rodriguez JH, et al. Hematologic study of newborn umbilical cord blood. Medicina (B Aires) 1999; 59: 446-8. 
4.Siddiqui MA, Saxena H, Srivasta JR. A study of hematological values in newborns. Indian Pediatr 1972; 9: 90-4. 
5.Stancheva VP, Sherman GG, Avent M, Cory BJ, Ballot DE, Cooper PA, et al. Hematological reference ranges in black very low birth weight infants. Pediatr Hematol Oncol 2002; 19: 91-4. 
6.Mcelory PD, Lal AA, Hawley WA, Bloland PB, Ter Kulie FO, Oloo AJ, et al. Analysis of repeated hemoglobin measures in full term, normal birth weight Kenyan children between birth and four years of age III. The asembo bay cohort project. Am J Trop Med Hyg 1999; 61: 932-40. 
7.Mamoury GH, Hamedy AB, Alkhlaghi F. Cord hemoglobin in newborn in correlation with maternal hemoglobin in Northeastern Iran. Iran J Med Sci 2003; 28: 166-8. 
8.Marwaha N, Marwaha RK, Narang A, Thusu K, Garewal G, Bhakoo ON. Routine hematological values in term newborns. Indian Pediatr 1992; 29: 1095-9. 
9.Manroe BL, Weinberg AG, Rosenfeld CR, Browne R. The neonatal blood count in health and disease. I. Reference values for neutrophilic cells. J Pediatr 1979; 95: 89-98. 
10.Abdurrahman MB, Adekoje MA. Hematological values in Northern Nigerian neonates. Trans R Soci Tropi Med Hyg 1983; 77: 786-8. 
11.Mukiibi JM, Nkrumah FK, Kaur M, Pollard R, Akino V, Nhembe. Neonatal hematology in Zimbabwe-II. The red cell and white cell parameters. Cent Afr J Medi 1995; 41: 76-82. 
12.Aneja S, Mandranda R, Patwari A, Sagreiya K, Bhargava SK. Normal Hematological values in Newborns. Indian Pediatr 1979; 16: 781-6. 
13.Mukiibi JM, Nkrumah FK, Kaur M, Pollard R, Akino V, Nhembe M. Neonatal hematology in Zimbabwe-I. The platelet parameters. Cent Afr J Med 1994; 40: 80-3. 
14.Christensen RD. Hematopoiesis in the fetus and neonate. Pediatr Res 1989; 26: 531-5. 
15.Hasan R, Inoue S, Baneriee A. Higher White blood cells counts and band forms in newborns delivered vaginally compared with those delivered by caesarean section. Am J Clin Pathol 1993; 100: 116-8. 
16.Huch A, Huch R, Rooth G. Guidelines for cord blood sampling and blood gas values in obstetrics. Based upon a workshop held in Zurich, Switzerland, March 19, 1993 by an Ad Hoc Committee. Eur J Obstet Gynecol Reprod Biol 1994; 54: 165-75. 
17.Javed MT, Abbas N, Hussain R, Zahoor T. A study on iron deficiency anemia and hematological differences around delivery in women of different socio-economic and age groups. Pediatr 2001; 14: 151-60. 
18.Maconi M, Rolfo A, Cardropoli S, Brini M, Danise P. Hematological values in healthy and small for gestational age newborns. Lab Hematol 2005; 11: 152-6. 
19.Van den Hof MC, Nicolaides KH. Platelets count in normal, small and anemic fetuses. Am J Obstet Gynecol 1990; 162: 735-9. 
20.Nikischin W, Peter M, Oldigs HD. The influence of mode of delivery on hematologic values in the umbilical vein. Gynecol Obstet Invest 1997; 43: 104-7. 
21.Reinhardt MC, Marti HR. Haematological data of African newborns and their mothers in Abidjan. Helv Paediat Acta Suppl 1978; 41: 85-99. 
22.Sheffer-Mimouni G, Mimouni FB, Lubetzky R, Kelpfermine M, Deutsch V, Dollberg S. Labour does not affect the neonatal absolute nucleated red blood cell count. Am J Perinatol 2003; 20: 367-71.

Objective: To establish reference haematological values (complete blood count) in healthy full term newborns of Karachi.
Methods: This is a cross sectional study design based on cord blood sample of 404 newborns and their mothers recruited from Sindh Government Qatar Hospital, Ziauddin University Hospital gole market campus, Sindh Government hospital, Liaquatabad and Chinniot maternity and child hospital. Karachi from July 2006 to April 2008. Both mother's and the new born baby's blood was collected in EDTA containing tubes and analyzed for different haematological parameters. The data entry and analysis was done on computer package SPSS (Statistical Packages of Social Sciences) version 12.0. The results were given in the text as mean, standard deviation and 95% confidence intervals of haematological values (complete blood count).
Results: The mean haemoglobin was found to be (14.99±1.47 gm), which is lower than the standard value described in literature. However the total WBCs count and platelet count were found to be (13.61±4.23) and (256. 25±76. 54) respectively, which were similar to the other studies.
Conclusion: Our values provide reference range for haematological values in healthy newborns of Karachi, for clinical use by our physicians (JPMA 59:618; 2009).