**1.4 Laboratory evaluation of jaundice**

Laboratory evaluation of jaundice is directed by the age of the newborn. The first step in evaluation, for a newborn jaundiced in the first 24 hours of life, is to perform total serum bilirubin (TSB) or transcutaneous bilirubin (TcB) measurement [2, 4, 10]. Transcutaneous bilirubin (TcB) can be a powerful and noninvasive screening tool for bilirubin estimation with reported close correlation to TSB measurement in different populations [10]. When jaundice appears excessive for newborn's age, a TSB should be obtained. In infants under phototherapy and TSB above the 75th percentile or rising rapidly (i.e., crossing percentiles), it is recommended to extend the diagnostic workout by performing additional tests such as complete blood count and smear, reticulocyte count, blood grouping, and Coombs

test as well as end-tidal carbon monoxide levels. In cases of specific ethnic origin or positive family history, analysis of G6PD and pyruvate kinase deficiencies is considered. Once direct (or conjugated) bilirubin level is elevated, urinalysis, urine culture, and evaluation for sepsis are recommended. As per jaundice persisting beyond the third week of life, a diagnostic protocol for identification of cholestasis causes should be followed [2, 4, 10].

### **1.5 Treatment of jaundice**

#### *1.5.1 Phototherapy*

It is a standard method for treatment of hyperbilirubinemia that is applied when bilirubin levels exceed gestation and hour-specific treatment thresholds [1, 4, 9, 25–27]. It is effective through photoisomerization of bilirubin to a water-soluble product that is readily excreted via bile or urine. The efficacy depends on the wavelength and the dose of the delivered light, as well as on the illuminated skin surface area [9, 27]. Specific phototherapy treatment graphs have been developed to address the phototherapy needs in term and in preterm babies [10, 26, 27]. General measures are involved concurrently to phototherapy such as maintenance of fluids and treatment of underlying disease cause such as infection [27].

#### *1.5.2 Exchange transfusion (ECT)*

It is a method for rapid elimination of the bilirubin and the circulating antibodies from the circulation, therefore most beneficial in cases of ongoing hemolysis. Small amount of blood are removed through a central venous catheter and replaced with the same amount of donor red blood cells suspended in plasma. The procedure is repeated until twice the blood volume of the newborn is replaced with the donor blood. This procedure involves multiple complications among which most pronounced are graftversus-host disease, necrotizing enterocolitis, and portal thrombosis [1, 9]. Although being the first therapy for severe jaundice, this intervention is becoming virtually obsolete and reserved only for cases of severe hyperbilirubinemia that could not be managed by intensive phototherapy. Likewise phototherapy, exchange transfusion treatment threshold graphs have been devised for term and preterm gestations to serve as clinical guidance for initiation of therapy [10, 26, 27]. Exchange transfusion should only be performed in highly developed neonatal intensive care units (NICU) adequately equipped for monitoring and resuscitation as well as with trained personnel [4].

#### *1.5.3 Pharmacologic therapies*

These drugs interfere with variable effectiveness at different stages of the bilirubin metabolism. For example, phenobarbital has been used to improve the conjugation and excretion of bilirubin. Tin mesoporphyrin inhibits heme oxygenase thereby acting on the production of bilirubin. Other drugs are involved with the enterohepatic circulation of bilirubin [9, 10]. Intravenous immunoglobulin has been shown to significantly reduce the need for exchange transfusion in Rh or ABO hemolytic disease [4].

### **2. Neonatal hyperbilirubinemia in the Republic of North Macedonia**

Our study group performed an extensive retrospective study for the purpose of evaluation of the etiology and management of indirect hyperbilirubinemia at the

**7**

*Neonatal Hyperbilirubinemia in Newborns of the Republic of North Macedonia*

University Pediatric Clinic in Skopje (UPCS), now Republic of North Macedonia (RNM). The study group included 284 newborns who had been admitted to the neonatology department at the University Pediatric Clinic in Skopje with the diagnosis of neonatal indirect hyperbilirubinemia during the period of 2 years [28]. They represented one quarter of the total number of 1126 hospitalized patients during this period in a tertiary level university teaching clinical hospital setting. Relevant history, clinical data, laboratories, and the type of therapy applied were retrieved from the medical records, recorded on questionnaires, and statistically analyzed. Perinatal history data of relevance were birth parameters, Apgar scores, and delivery mode. Clinical presentations that could potentially influence duration and intensity of jaundice had been searched for such as hematomas, cephalohematoma, intracranial hemorrhages, hypothyroidism, impaired intestinal motility, and infection. All laboratories and investigations of relevance were recorded, as well as the therapies applied. Moreover, the day of the bilirubin peak was noted, as well as two subsequent bilirubin measurements. Standard techniques for analysis of blood count and smear, bilirubin and fractions, serum aminotransferases, and G6PD, as well as infants' and mothers' blood group and direct antiglobulin Coombs test (DAT), were applied as described elsewhere [12, 28]. Statistical Package for the Social Sciences (SPSS) for Windows (SPSS Inc., Chicago, IL, USA) was used for the statistical analyses. Absolute numbers and percentages were used to present the categorical variables, whereas mean, standard deviation, minimum, maximum, median, and rang were used to present the quantitative variables. Testing of significance between groups was performed using Kruskal-Wallis test, student *t*-test, Mann–Whitney *U* test, and analysis of variance. The result was considered significant if probability value (*p*) was <0.05 and <0.01 for high significance. Nine types of jaundice had been identified and grouped as follows: (1) ABO incompatibility, (2) rhesus (Rh) incompatibility, (3) cephalohematoma and bruising, (4) sepsis, (5) prematurity, (6) intracranial hemorrhage, (7) hemolysis (neither ABO nor Rh incompatibility), (8) Down syndrome, and (9) undefined. The highest prevalence was found for jaundice of undefined etiology (44.37%), and the second most prevalent was etiology of neonatal infection represented with 19.37%. Hemolytic etiologies were represented with 8.45% for ABO incompatibility, 5.63% for Rh incompatibility, and 0.35% for hemolysis neither ABO nor Rh or in total 14.43%. Least prevalent were cephalohematoma and bruising due to birth trauma (2.82%), intracranial hemorrhage (2.46%), and Down syndrome (0.70%) (**Table 2**) [28]. Etiology of sepsis was assigned to newborns with a positive blood/cerebrospinal fluid culture or clinically relevant infection requiring antibiotic therapy. Subjects that had sepsis and elevated direct bilirubin were not included [2, 28]. Prematurity, defined as less than completed 37 weeks of gestational age, was considered a distinct etiology of jaundice. Our group of undefined etiology included cases of early- and late-onset breast-milk jaundice, exaggerated physiological jaundice, [2, 9, 28], and no identifiable

etiology. Cephalohematoma and bruising were representatives of birth trauma.

We found a high percentage of jaundice of undefined etiology (44.37%). Another study reported higher prevalence of undetermined etiology (75.8%) [16]. Clinical evaluation of severe neonatal hyperbilirubinemia in a resource-limited setting similarly showed highest prevalence of idiopathic jaundice (33.3%) [24]. No cause for the extreme hyperbilirubinemia of ≥25 mg per dL (428 μmol per L) could be identified in 65.6% of cases admitted for treatment at the NICU in Southern Turkey [29]. Etiology was unknown in 11 of the 79 ECT cases (13.9%) in the Eastern Mediterranean region of Turkey as reported by Davutoğlu et al. [30]. Dissimilarly,

**2.1 Undefined etiology of jaundice**

*DOI: http://dx.doi.org/10.5772/intechopen.87045*

#### *Neonatal Hyperbilirubinemia in Newborns of the Republic of North Macedonia DOI: http://dx.doi.org/10.5772/intechopen.87045*

University Pediatric Clinic in Skopje (UPCS), now Republic of North Macedonia (RNM). The study group included 284 newborns who had been admitted to the neonatology department at the University Pediatric Clinic in Skopje with the diagnosis of neonatal indirect hyperbilirubinemia during the period of 2 years [28]. They represented one quarter of the total number of 1126 hospitalized patients during this period in a tertiary level university teaching clinical hospital setting. Relevant history, clinical data, laboratories, and the type of therapy applied were retrieved from the medical records, recorded on questionnaires, and statistically analyzed. Perinatal history data of relevance were birth parameters, Apgar scores, and delivery mode. Clinical presentations that could potentially influence duration and intensity of jaundice had been searched for such as hematomas, cephalohematoma, intracranial hemorrhages, hypothyroidism, impaired intestinal motility, and infection. All laboratories and investigations of relevance were recorded, as well as the therapies applied. Moreover, the day of the bilirubin peak was noted, as well as two subsequent bilirubin measurements. Standard techniques for analysis of blood count and smear, bilirubin and fractions, serum aminotransferases, and G6PD, as well as infants' and mothers' blood group and direct antiglobulin Coombs test (DAT), were applied as described elsewhere [12, 28]. Statistical Package for the Social Sciences (SPSS) for Windows (SPSS Inc., Chicago, IL, USA) was used for the statistical analyses. Absolute numbers and percentages were used to present the categorical variables, whereas mean, standard deviation, minimum, maximum, median, and rang were used to present the quantitative variables. Testing of significance between groups was performed using Kruskal-Wallis test, student *t*-test, Mann–Whitney *U* test, and analysis of variance. The result was considered significant if probability value (*p*) was <0.05 and <0.01 for high significance. Nine types of jaundice had been identified and grouped as follows: (1) ABO incompatibility, (2) rhesus (Rh) incompatibility, (3) cephalohematoma and bruising, (4) sepsis, (5) prematurity, (6) intracranial hemorrhage, (7) hemolysis (neither ABO nor Rh incompatibility), (8) Down syndrome, and (9) undefined. The highest prevalence was found for jaundice of undefined etiology (44.37%), and the second most prevalent was etiology of neonatal infection represented with 19.37%. Hemolytic etiologies were represented with 8.45% for ABO incompatibility, 5.63% for Rh incompatibility, and 0.35% for hemolysis neither ABO nor Rh or in total 14.43%. Least prevalent were cephalohematoma and bruising due to birth trauma (2.82%), intracranial hemorrhage (2.46%), and Down syndrome (0.70%) (**Table 2**) [28].

Etiology of sepsis was assigned to newborns with a positive blood/cerebrospinal fluid culture or clinically relevant infection requiring antibiotic therapy. Subjects that had sepsis and elevated direct bilirubin were not included [2, 28]. Prematurity, defined as less than completed 37 weeks of gestational age, was considered a distinct etiology of jaundice. Our group of undefined etiology included cases of early- and late-onset breast-milk jaundice, exaggerated physiological jaundice, [2, 9, 28], and no identifiable etiology. Cephalohematoma and bruising were representatives of birth trauma.

#### **2.1 Undefined etiology of jaundice**

We found a high percentage of jaundice of undefined etiology (44.37%). Another study reported higher prevalence of undetermined etiology (75.8%) [16]. Clinical evaluation of severe neonatal hyperbilirubinemia in a resource-limited setting similarly showed highest prevalence of idiopathic jaundice (33.3%) [24]. No cause for the extreme hyperbilirubinemia of ≥25 mg per dL (428 μmol per L) could be identified in 65.6% of cases admitted for treatment at the NICU in Southern Turkey [29]. Etiology was unknown in 11 of the 79 ECT cases (13.9%) in the Eastern Mediterranean region of Turkey as reported by Davutoğlu et al. [30]. Dissimilarly,

*Update on Critical Issues on Infant and Neonatal Care*

causes should be followed [2, 4, 10].

*1.5.2 Exchange transfusion (ECT)*

*1.5.3 Pharmacologic therapies*

hemolytic disease [4].

**1.5 Treatment of jaundice**

*1.5.1 Phototherapy*

test as well as end-tidal carbon monoxide levels. In cases of specific ethnic origin or positive family history, analysis of G6PD and pyruvate kinase deficiencies is considered. Once direct (or conjugated) bilirubin level is elevated, urinalysis, urine culture, and evaluation for sepsis are recommended. As per jaundice persisting beyond the third week of life, a diagnostic protocol for identification of cholestasis

It is a standard method for treatment of hyperbilirubinemia that is applied when bilirubin levels exceed gestation and hour-specific treatment thresholds [1, 4, 9, 25–27]. It is effective through photoisomerization of bilirubin to a water-soluble product that is readily excreted via bile or urine. The efficacy depends on the wavelength and the dose of the delivered light, as well as on the illuminated skin surface area [9, 27]. Specific phototherapy treatment graphs have been developed to address the phototherapy needs in term and in preterm babies [10, 26, 27]. General measures are involved concurrently to phototherapy such as maintenance of fluids

It is a method for rapid elimination of the bilirubin and the circulating antibodies from the circulation, therefore most beneficial in cases of ongoing hemolysis. Small amount of blood are removed through a central venous catheter and replaced with the same amount of donor red blood cells suspended in plasma. The procedure is repeated until twice the blood volume of the newborn is replaced with the donor blood. This procedure involves multiple complications among which most pronounced are graftversus-host disease, necrotizing enterocolitis, and portal thrombosis [1, 9]. Although being the first therapy for severe jaundice, this intervention is becoming virtually obsolete and reserved only for cases of severe hyperbilirubinemia that could not be managed by intensive phototherapy. Likewise phototherapy, exchange transfusion treatment threshold graphs have been devised for term and preterm gestations to serve as clinical guidance for initiation of therapy [10, 26, 27]. Exchange transfusion should only be performed in highly developed neonatal intensive care units (NICU) adequately

equipped for monitoring and resuscitation as well as with trained personnel [4].

These drugs interfere with variable effectiveness at different stages of the bilirubin metabolism. For example, phenobarbital has been used to improve the conjugation and excretion of bilirubin. Tin mesoporphyrin inhibits heme oxygenase thereby acting on the production of bilirubin. Other drugs are involved with the enterohepatic circulation of bilirubin [9, 10]. Intravenous immunoglobulin has been shown to significantly reduce the need for exchange transfusion in Rh or ABO

**2. Neonatal hyperbilirubinemia in the Republic of North Macedonia**

Our study group performed an extensive retrospective study for the purpose of evaluation of the etiology and management of indirect hyperbilirubinemia at the

and treatment of underlying disease cause such as infection [27].

**6**


### **Table 2.**

*Causes of neonatal indirect hyperbilirubinemia in the republic of North Macedonia.*


*Min, minimum; Max, maximum; M, median; GW, gestation weeks; BW, birth weight; BL, birth length; CS, cesarean section; AS, Apgar score. Information from Ref. [28].*

#### **Table 3.**

*Basic characteristics of the undefined etiology group at UPCS, RNM.*

we did not find undefined etiology among our ECT cases. It could be assumed that the variable prevalence of "undefined etiology" reported in different studies was a result of diverse classification of the causes of neonatal jaundice and also of different levels of TSB considered (pathologic or extreme). We described undefined etiology as such, where intensive workout could not provide an identifiable cause or contributing factor for jaundice. Through a careful selection process, a homogenous group of clinically stable patients was obtained that had normal birth parameters and required treatment with phototherapy (**Table 3**). We speculated that an imbalance between bilirubin production and conjugation was the primary concept of jaundice in this group since no history, clinical, and laboratory data existed to indicate another mechanism of jaundice [28, 31].

**9**

*Neonatal Hyperbilirubinemia in Newborns of the Republic of North Macedonia*

bruising, intracranial hemorrhage), and (5) undefined etiology.

The basic characteristics of the undefined etiology group are presented in **Table 3**. Newborn infants of this group were generally delivered spontaneously (90.47%) with normal birth parameters [birth weight (BW) and birth length (BL)] and did not suffer major perinatal hypoxia. The median (interquartile range) age of presentation of jaundice was at day 3 (3–4) (**Table 3**). In the group of undefined etiology, the median (interquartile range) day at which bilirubin reached its peak was 9 (6–17). The median (interquartile range) of the peak TSB level was 324 (270–394) μmol/L, whereas the mean ± standard deviation (SD) peak serum bilirubin concen-

Statistical analyses included comparison of laboratory parameters between five etiological groups: (1) hemolytic etiology of jaundice including ABO incompatibility, Rh incompatibility, and hemolysis (neither ABO nor Rh incompatibility), (2) neonatal infection/sepsis, (3) prematurity, (4) hematomas (cephalohematoma,

To summarize the analyzed laboratory parameters, mean peak bilirubin levels in newborns with hemolysis (group 1) were shown to be statistically significantly higher than levels in the groups with neonatal infection, prematurity, and hematomas (groups 2, 3, and 4). The first control serum bilirubin level was significantly higher in newborns with hemolysis (group 1) than prematurity and undefined etiology (groups 3 and 5). No statistically significant differences were found in the second control bilirubin measurement; also levels of hepatic transaminases (AST and ALT) were not found to depend significantly on the etiology of jaundice. Estimation of hepatic transaminases has not proven of substantial influence on

Premature newborns, due to physiological characteristics, associated risk factors, and proneness to development of pronounced jaundice, were assigned a separate etiological group contrary to assignment of these patients into the "undetermined etiology" performed by other authors [16]. We were able to show slower increase toward the peak bilirubin level in the group of premature newborns than in groups of hemolysis, hematomas, and infection. Levels of erythrocytes (Er), hemoglobin (Hb), and hematocrit (Hct) in premature newborns were statistically

We did not find cases of G6PD deficiency in the studied group. Although no cases of G6PD deficiency were confirmed, a standard was set for a new quantitative spectrophotometric assay for G6PD detection, thereby overcoming the uncertainties connected with the previously used qualitative methods. Previous qualitative studies of the G6PD deficiency in Macedonia are those of Fraser et al. [32] and of Andreeva et al. [33]. The first group of authors assessed the average prevalence of the G6PD deficit in Yugoslavia from 1% [32] based on tests carried out on 144 samples from then Republic of South Macedonia and 512 samples from the region of Dalmatia. The second group of authors in 1974 examined the prevalence of the G6PD deficiency in 3263 male school children from the area of Southeastern Macedonia (territory of nowadays Republic of North Macedonia) and showed a frequency of 1–2% of the G6PD deficit in that part of the republic. In the second examination of the same group of authors, realized on samples of 1196 male school children from the territory of Skopje, when processing the enzyme, it was concluded that it was a Mediterranean variant and the prevalence of the

significantly lower than the groups of undefined etiology and infection.

*DOI: http://dx.doi.org/10.5772/intechopen.87045*

tration was 333.4 ± 91.1 μmol/L.

jaundice workout and management.

**2.2 Prematurity and jaundice**

**2.3 G6PD deficiency**

*Neonatal Hyperbilirubinemia in Newborns of the Republic of North Macedonia DOI: http://dx.doi.org/10.5772/intechopen.87045*

The basic characteristics of the undefined etiology group are presented in **Table 3**. Newborn infants of this group were generally delivered spontaneously (90.47%) with normal birth parameters [birth weight (BW) and birth length (BL)] and did not suffer major perinatal hypoxia. The median (interquartile range) age of presentation of jaundice was at day 3 (3–4) (**Table 3**). In the group of undefined etiology, the median (interquartile range) day at which bilirubin reached its peak was 9 (6–17). The median (interquartile range) of the peak TSB level was 324 (270–394) μmol/L, whereas the mean ± standard deviation (SD) peak serum bilirubin concentration was 333.4 ± 91.1 μmol/L.

Statistical analyses included comparison of laboratory parameters between five etiological groups: (1) hemolytic etiology of jaundice including ABO incompatibility, Rh incompatibility, and hemolysis (neither ABO nor Rh incompatibility), (2) neonatal infection/sepsis, (3) prematurity, (4) hematomas (cephalohematoma, bruising, intracranial hemorrhage), and (5) undefined etiology.

To summarize the analyzed laboratory parameters, mean peak bilirubin levels in newborns with hemolysis (group 1) were shown to be statistically significantly higher than levels in the groups with neonatal infection, prematurity, and hematomas (groups 2, 3, and 4). The first control serum bilirubin level was significantly higher in newborns with hemolysis (group 1) than prematurity and undefined etiology (groups 3 and 5). No statistically significant differences were found in the second control bilirubin measurement; also levels of hepatic transaminases (AST and ALT) were not found to depend significantly on the etiology of jaundice. Estimation of hepatic transaminases has not proven of substantial influence on jaundice workout and management.

#### **2.2 Prematurity and jaundice**

*Update on Critical Issues on Infant and Neonatal Care*

we did not find undefined etiology among our ECT cases. It could be assumed that the variable prevalence of "undefined etiology" reported in different studies was a result of diverse classification of the causes of neonatal jaundice and also of different levels of TSB considered (pathologic or extreme). We described undefined etiology as such, where intensive workout could not provide an identifiable cause or contributing factor for jaundice. Through a careful selection process, a homogenous group of clinically stable patients was obtained that had normal birth parameters and required treatment with phototherapy (**Table 3**). We speculated that an imbalance between bilirubin production and conjugation was the primary concept of jaundice in this group since no history, clinical, and laboratory data existed to

*Min, minimum; Max, maximum; M, median; GW, gestation weeks; BW, birth weight; BL, birth length; CS, cesarean* 

Age 4 ± 2.5 2 14 3 3–4 GW 39 ± 1.2 37 42 39 38–40 BW 3247.1 ± 437.4 2200 4500 3245 2980–3500 BL 50.2 ± 1.8 46 56 50 49–51

*Causes of neonatal indirect hyperbilirubinemia in the republic of North Macedonia.*

**Etiology Number Percentage (%)** Undefined etiology 126 44.37 Neonatal infection 55 19.37 Prematurity 45 15.85 ABO incompatibility 24 8.45 Rh incompatibility 16 5.63 Cephalohematoma and bruising due to birth trauma 8 2.82 Intracranial hemorrhage 7 2.46 Hemolysis (neither ABO nor Rh incompatibility) 1 0.35 Down syndrome 2 0.70 Total 284 100

**Mean Min Max** *M* **Interquartile range**

indicate another mechanism of jaundice [28, 31].

Mode of delivery N % Spontaneous 114 90.47 CS 9 7.14 Vacuum extraction 2 1.59 Forceps 1 0.8 Perinatal hypoxia N % No 109 86.51 AS 7 16 12.7 AS 4–6 1 0.79

*Information from Ref. [28].*

**Table 2.**

*section; AS, Apgar score. Information from Ref. [28].*

*Basic characteristics of the undefined etiology group at UPCS, RNM.*

**8**

**Table 3.**

Premature newborns, due to physiological characteristics, associated risk factors, and proneness to development of pronounced jaundice, were assigned a separate etiological group contrary to assignment of these patients into the "undetermined etiology" performed by other authors [16]. We were able to show slower increase toward the peak bilirubin level in the group of premature newborns than in groups of hemolysis, hematomas, and infection. Levels of erythrocytes (Er), hemoglobin (Hb), and hematocrit (Hct) in premature newborns were statistically significantly lower than the groups of undefined etiology and infection.

#### **2.3 G6PD deficiency**

We did not find cases of G6PD deficiency in the studied group. Although no cases of G6PD deficiency were confirmed, a standard was set for a new quantitative spectrophotometric assay for G6PD detection, thereby overcoming the uncertainties connected with the previously used qualitative methods. Previous qualitative studies of the G6PD deficiency in Macedonia are those of Fraser et al. [32] and of Andreeva et al. [33]. The first group of authors assessed the average prevalence of the G6PD deficit in Yugoslavia from 1% [32] based on tests carried out on 144 samples from then Republic of South Macedonia and 512 samples from the region of Dalmatia. The second group of authors in 1974 examined the prevalence of the G6PD deficiency in 3263 male school children from the area of Southeastern Macedonia (territory of nowadays Republic of North Macedonia) and showed a frequency of 1–2% of the G6PD deficit in that part of the republic. In the second examination of the same group of authors, realized on samples of 1196 male school children from the territory of Skopje, when processing the enzyme, it was concluded that it was a Mediterranean variant and the prevalence of the

deficit of 1.02% was reported among the children of Macedonian nationality and 6.63% for Roma children [33]. Quantitative testing for G6PD deficiency has been recommended to be performed, thus avoiding partially G6PD-deficient patients such as heterozygous females to be missed [34, 35]. As much as 1/3 higher levels of G6PD in the neonatal period can be encountered due to the presence of physiologic polycythemia in this period [35]. Therefore, it is reasonable to schedule for another subsequent test in cases of borderline normal results and a specific ethnic origin.

In a subsequent neonatal jaundice study, we showed an incidence of 8.57% of G6PD-deficient infants in a strictly prospectively selected group of infants with jaundice of undetermined etiology (own unpublished results). From this study, a population-specific range of normal values for the G6PD quantitative spectrophotometric assay will be derived.

#### **2.4 Extravasation of blood and jaundice**

A separate group of patients with hematomas was developed, encompassing patient with extravascular collections of blood where an increased bilirubin load was presumed the fundamental mechanism of hyperbilirubinemia [2, 9, 16, 18, 28]. No statistically significant hematological correlations between this group and the other four groups of patients were found.

#### **2.5 Infection-associated jaundice**

Sepsis is a known perinatal risk factor for both unconjugated and conjugated jaundice [2, 9, 18] and is also listed as a risk factor for hyperbilirubinemia neurotoxicity [17]. Analysis of prevalence rates in different regions of the world showed varying importance of infection in connection with jaundice. Highest variability of prevalence rates was reported in Asia (from 9.7 to 31.2%). In Africa infection was related with over 13.9% of the hyperbilirubinemia or kernicterus cases, whereas in Europe and North America, infection was related with 14.3% of the kernicterus cases [1].

In our study, the group of infection-associated jaundice was represented with 19.37%. On the contrary, sepsis was found in almost twice as much (35.3%) severe hyperbilirubinemia cases in South East Nigeria [24]. Similar to the North Macedonian study, sepsis was present in 15.7% indirect hyperbilirubinemia cases at Zanjan Province of Iran [16]. We assumed our figure an overrepresentation due to the fact that not only culture positive cases were included but also newborns with clinical or biochemical markers of sepsis. Reliable discrimination between culture positive and culture negative cases was not possible due to the variety of processing of initial hemoculture between the tertiary level and the referral hospitals. Therefore, the term "infection" rather than "sepsis" was used for more accurate reflection on this group of patients. Statistically significant higher levels of hematological parameters (Er, Hb, and Hct) were shown for this group than the hemolytic group and the premature newborns.

#### **2.6 Hemolytic jaundice**

We have established a group of hemolytic jaundice according to the mechanism of the hyperbilirubinemia employed in cases of ABO and Rh isoimmunization. According to the 2009 update on the management of newborn infants ≥35 weeks' gestation, isoimmune and other hemolytic diseases (e.g., G6PD deficiency) were included in two important of risk factors' categories: severe hyperbilirubinemia and hyperbilirubinemia-induced neurotoxicity [17]. Furthermore, it has been

**11**

(**Table 5**) [38].

*Neonatal Hyperbilirubinemia in Newborns of the Republic of North Macedonia*

postulated that DAT-positive isoimmune hemolytic disease and severe hyperbilirubinemia exert synergistic effect in potentiating the bilirubin-induced neurotoxicity [36]. Lower phototherapy and exchange transfusion threshold levels have been recommended in isoimmune hemolytic disease in order to prevent the acute manifestations of bilirubin toxicity that might evolve into chronic neurological condition, kernicterus, also a pre-discharge risk assessment and early post-discharge follow-up [4, 17, 25–27]. Tiker et al. report isoimmunization in 19 out of 93 (20.43%) patients admitted for treatment of extreme hyperbilirubinemia in Southern Turkey [29]. ABO isoimmunization was reported the most common cause of hyperbilirubinemia requiring ECT in two other studies performed in Turkey; the reported rates were 38% and 27.8%, respectively [30, 37]. ABO incompatibility was present in 8.45% of our study cases. Rh incompatibility was represented with 5.63% of all hyperbilirubinemia cases. We found one hemolysis positive patient who had neither ABO nor Rh incompatibility. The pooled prevalence of all hemolytic etiology cases in our study was 14.43%. When compared to the groups of neonatal infection, prematurity, and hematomas, the group of hemolytic etiology presented with significantly higher peak bilirubin levels. A statistically significant higher level of bilirubin in hemolytic etiology than prematurity and undefined etiology was also noted on the first control bilirubin level estimation. This observation pointed out a slower tendency of reduction of bilirubin under phototherapy in hemolysis than undefined etiology. However, the majority of cases with hemolytic etiology (97.89%) were managed conventionally by phototherapy using double-surface blue light phototherapy lamps at wavelength of 460 nm, and only 2.11% Coombs-positive ABO/Rh incompatibility

A comparison of hematological and biochemical parameters was performed between groups of patients with undefined (unspecified) etiology (126 patients, 74.5%) and 41 patients with ABO or rhesus-type hemolytic disease of the new-

Despite the fact that we did not show statistically significant higher peak levels of bilirubin in the hemolytic etiology group than the other group of jaundice, a propensity toward faster elevation of bilirubin and more pronounced level of jaundice was noted. The peak bilirubin level showed significantly longer duration

The group of newborns with ABO/Rh incompatibility presented with significantly lower mean values of all analyzed hematological parameters than the group of jaundice with unspecific etiology [hemoglobin (*p* = 0.038), erythrocytes (*p* = 0.0023), and hematocrit (*p* = 0.037)] (**Table 4**) [38]. Mean reticulocyte count was significantly higher (*p* = 0.000036) in the group of ABO/Rh incompatibility (27.88 ± 26.4 vs. 11.94 ± 7.4). The group of hemolytic etiology was characterized by significantly earlier jaundice appearance (*p* < 0.01) than the group of jaundice of unspecified etiology. The mean ± standard deviation (SD) for hemolysis group was 2.63 ± 2.4 days versus 4.02 ± 2.5 days for the unspecified etiology group. The peak bilirubin level (mean ± SD) in hemolysis group of 379.76 ± 133.5 μmol/L was higher than unspecified etiology (333.44 ± 91.1 μmol/L) although the differences were not statistically significant (*p* = 0.052). Statistically insignificantly higher levels at the first (*p* = 0.062) and second (*p* = 0.448) control bilirubin measurements were registered for the hemolytic etiology group than the unspecific jaundice group. On the other hand, duration of the bilirubin peak was significantly lengthier (*p* = 0.036) for the group of unspecific jaundice. The mean ± SD for this group was 15.03 ± 25.7 days versus 10.22 ± 9.02 days for the ABO/Rh incompatibility group

*DOI: http://dx.doi.org/10.5772/intechopen.87045*

patients were treated by exchange transfusion.

**2.7 Laboratory analyses**

born (24.6%).

*Neonatal Hyperbilirubinemia in Newborns of the Republic of North Macedonia DOI: http://dx.doi.org/10.5772/intechopen.87045*

postulated that DAT-positive isoimmune hemolytic disease and severe hyperbilirubinemia exert synergistic effect in potentiating the bilirubin-induced neurotoxicity [36]. Lower phototherapy and exchange transfusion threshold levels have been recommended in isoimmune hemolytic disease in order to prevent the acute manifestations of bilirubin toxicity that might evolve into chronic neurological condition, kernicterus, also a pre-discharge risk assessment and early post-discharge follow-up [4, 17, 25–27]. Tiker et al. report isoimmunization in 19 out of 93 (20.43%) patients admitted for treatment of extreme hyperbilirubinemia in Southern Turkey [29]. ABO isoimmunization was reported the most common cause of hyperbilirubinemia requiring ECT in two other studies performed in Turkey; the reported rates were 38% and 27.8%, respectively [30, 37]. ABO incompatibility was present in 8.45% of our study cases. Rh incompatibility was represented with 5.63% of all hyperbilirubinemia cases. We found one hemolysis positive patient who had neither ABO nor Rh incompatibility. The pooled prevalence of all hemolytic etiology cases in our study was 14.43%. When compared to the groups of neonatal infection, prematurity, and hematomas, the group of hemolytic etiology presented with significantly higher peak bilirubin levels. A statistically significant higher level of bilirubin in hemolytic etiology than prematurity and undefined etiology was also noted on the first control bilirubin level estimation. This observation pointed out a slower tendency of reduction of bilirubin under phototherapy in hemolysis than undefined etiology. However, the majority of cases with hemolytic etiology (97.89%) were managed conventionally by phototherapy using double-surface blue light phototherapy lamps at wavelength of 460 nm, and only 2.11% Coombs-positive ABO/Rh incompatibility patients were treated by exchange transfusion.

### **2.7 Laboratory analyses**

*Update on Critical Issues on Infant and Neonatal Care*

tometric assay will be derived.

**2.4 Extravasation of blood and jaundice**

other four groups of patients were found.

**2.5 Infection-associated jaundice**

group and the premature newborns.

**2.6 Hemolytic jaundice**

deficit of 1.02% was reported among the children of Macedonian nationality and 6.63% for Roma children [33]. Quantitative testing for G6PD deficiency has been recommended to be performed, thus avoiding partially G6PD-deficient patients such as heterozygous females to be missed [34, 35]. As much as 1/3 higher levels of G6PD in the neonatal period can be encountered due to the presence of physiologic polycythemia in this period [35]. Therefore, it is reasonable to schedule for another subsequent test in cases of borderline normal results and a specific ethnic origin. In a subsequent neonatal jaundice study, we showed an incidence of 8.57% of G6PD-deficient infants in a strictly prospectively selected group of infants with jaundice of undetermined etiology (own unpublished results). From this study, a population-specific range of normal values for the G6PD quantitative spectropho-

A separate group of patients with hematomas was developed, encompassing patient with extravascular collections of blood where an increased bilirubin load was presumed the fundamental mechanism of hyperbilirubinemia [2, 9, 16, 18, 28]. No statistically significant hematological correlations between this group and the

Sepsis is a known perinatal risk factor for both unconjugated and conjugated jaundice [2, 9, 18] and is also listed as a risk factor for hyperbilirubinemia neurotoxicity [17]. Analysis of prevalence rates in different regions of the world showed varying importance of infection in connection with jaundice. Highest variability of prevalence rates was reported in Asia (from 9.7 to 31.2%). In Africa infection was related with over 13.9% of the hyperbilirubinemia or kernicterus cases, whereas in Europe and North America, infection was related with 14.3% of the kernicterus

In our study, the group of infection-associated jaundice was represented with 19.37%. On the contrary, sepsis was found in almost twice as much (35.3%) severe hyperbilirubinemia cases in South East Nigeria [24]. Similar to the North Macedonian study, sepsis was present in 15.7% indirect hyperbilirubinemia cases at Zanjan Province of Iran [16]. We assumed our figure an overrepresentation due to the fact that not only culture positive cases were included but also newborns with clinical or biochemical markers of sepsis. Reliable discrimination between culture positive and culture negative cases was not possible due to the variety of processing of initial hemoculture between the tertiary level and the referral hospitals. Therefore, the term "infection" rather than "sepsis" was used for more accurate reflection on this group of patients. Statistically significant higher levels of hematological parameters (Er, Hb, and Hct) were shown for this group than the hemolytic

We have established a group of hemolytic jaundice according to the mechanism of the hyperbilirubinemia employed in cases of ABO and Rh isoimmunization. According to the 2009 update on the management of newborn infants ≥35 weeks' gestation, isoimmune and other hemolytic diseases (e.g., G6PD deficiency) were included in two important of risk factors' categories: severe hyperbilirubinemia and hyperbilirubinemia-induced neurotoxicity [17]. Furthermore, it has been

**10**

cases [1].

A comparison of hematological and biochemical parameters was performed between groups of patients with undefined (unspecified) etiology (126 patients, 74.5%) and 41 patients with ABO or rhesus-type hemolytic disease of the newborn (24.6%).

The group of newborns with ABO/Rh incompatibility presented with significantly lower mean values of all analyzed hematological parameters than the group of jaundice with unspecific etiology [hemoglobin (*p* = 0.038), erythrocytes (*p* = 0.0023), and hematocrit (*p* = 0.037)] (**Table 4**) [38]. Mean reticulocyte count was significantly higher (*p* = 0.000036) in the group of ABO/Rh incompatibility (27.88 ± 26.4 vs. 11.94 ± 7.4). The group of hemolytic etiology was characterized by significantly earlier jaundice appearance (*p* < 0.01) than the group of jaundice of unspecified etiology. The mean ± standard deviation (SD) for hemolysis group was 2.63 ± 2.4 days versus 4.02 ± 2.5 days for the unspecified etiology group. The peak bilirubin level (mean ± SD) in hemolysis group of 379.76 ± 133.5 μmol/L was higher than unspecified etiology (333.44 ± 91.1 μmol/L) although the differences were not statistically significant (*p* = 0.052). Statistically insignificantly higher levels at the first (*p* = 0.062) and second (*p* = 0.448) control bilirubin measurements were registered for the hemolytic etiology group than the unspecific jaundice group.

On the other hand, duration of the bilirubin peak was significantly lengthier (*p* = 0.036) for the group of unspecific jaundice. The mean ± SD for this group was 15.03 ± 25.7 days versus 10.22 ± 9.02 days for the ABO/Rh incompatibility group (**Table 5**) [38].

Despite the fact that we did not show statistically significant higher peak levels of bilirubin in the hemolytic etiology group than the other group of jaundice, a propensity toward faster elevation of bilirubin and more pronounced level of jaundice was noted. The peak bilirubin level showed significantly longer duration


*t, student t-test; N, number of patients; SD, standard deviation; p, probability value. Information from Ref. [38].*

#### **Table 4.**

*Hematological parameters in neonatal jaundice, comparison between ABO/Rh incompatibility and unspecified etiology groups.*


*\* p < 0.05. \*\*p < 0.01.*

*Z, (Mann-Whitney U test); N, number of patients; SD, standard deviation; p, probability value; NS, not significant. Information from Ref. [38].*

#### **Table 5.**

*Reticulocytes and bilirubin analyses in neonatal jaundice, comparison between ABO/Rh incompatibility and unspecified etiology groups.*

**13**

*Neonatal Hyperbilirubinemia in Newborns of the Republic of North Macedonia*

applied, or other influences such as diverse stringency to phototherapy.

pathological causes presents constant clinical challenge.

The authors declare no conflict of interest.

and were candidates for subsequent neurodevelopmental follow-up.

in the group of unspecific jaundice. It remains speculative whether this was due to different mechanisms of jaundice involved, different responses to the phototherapy

According to an evidence-based review on neonatal hyperbilirubinemia, the majority of kernicterus cases occurred in infants with a bilirubin level higher than 20 mg/dL (342 μmol/L) [39]. It was obvious that our hemolysis cases with mean peak bilirubin levels of 379.8 ± 133.5 μmol/L were eligible for the neurotoxic effects of the hyperbilirubinemia, especially the ones toward the higher end of the spectrum and candidates for long-term neurodevelopmental follow-up. Therefore, clinicians' awareness of potential treats and harms that might be associated with

Neonatal indirect hyperbilirubinemia is a common phenomenon during the first week of postnatal life affecting almost two thirds of term newborns. The mechanism of neonatal jaundice is multifactorial, involving delicate balance between processes that potentiate bilirubin production and the ones that diminish bilirubin clearance. Although etiology of jaundice has been widely studied, identification of

Hyperbilirubinemia was found to be a common clinical presentation at the neonatology department of the University Pediatric Clinic in Skopje, Republic of North Macedonia, and encompassing one quarter of the hospitalized patients. Most cases suffered from a less severe jaundice of undefined etiology that had tendency to longer duration. Almost 15% of the hyperbilirubinemia cases presented with hemolytic causes of jaundice that had earlier and more severe peak of the bilirubin level. Those required immediate clinicians' attention and prompt management plan

*DOI: http://dx.doi.org/10.5772/intechopen.87045*

isoimmunization is vital.

**3. Conclusions**

**Conflict of interest**

#### *Neonatal Hyperbilirubinemia in Newborns of the Republic of North Macedonia DOI: http://dx.doi.org/10.5772/intechopen.87045*

in the group of unspecific jaundice. It remains speculative whether this was due to different mechanisms of jaundice involved, different responses to the phototherapy applied, or other influences such as diverse stringency to phototherapy.

According to an evidence-based review on neonatal hyperbilirubinemia, the majority of kernicterus cases occurred in infants with a bilirubin level higher than 20 mg/dL (342 μmol/L) [39]. It was obvious that our hemolysis cases with mean peak bilirubin levels of 379.8 ± 133.5 μmol/L were eligible for the neurotoxic effects of the hyperbilirubinemia, especially the ones toward the higher end of the spectrum and candidates for long-term neurodevelopmental follow-up. Therefore, clinicians' awareness of potential treats and harms that might be associated with isoimmunization is vital.
