Anemia is a well-known complication of IBD occurring in both UC [10] and CD [11–17]. Anemia is generally defined as a hemoglobin value <120 g/L or hematocrit <0.4. With respect to IBD, severe anemia is defined as a hemoglobin level <100 g/L. For reasons that are not well characterized, many patients with IBD are intolerant of oral iron replacement therapy or their anemia is refractory to such supplementation [17]. The reported prevalence of anemia is variable in IBD, but anemia appears to be more prevalent in CD compared to UC [18]. In one more recent population-based adult Scandinavian study from Denmark, Norway, and Sweden, the overall prevalence of anemia was 19% with iron deficiency and anemia of chronic disease being the primary etiologies [18]. Additionally, anemia may be more common in children compared to adolescents and adults [19]. Using the WHO age-adjusted definitions of anemia, Goodhand et al., [19] assessed the prevalence, severity, type, and response to treatment of anemia in patients attending pediatric, adolescent, and adult IBD clinics at a single center. These authors observed the prevalence of anemia to be 70% (41/59) in children, 42% (24/54) in adolescents, and 40% (49/124) in adults (p  <  0.01). Overall, children (88% (36/41)) and adolescents (83% (20/24)) were more often iron-deficient than adults (55% (27/49)) (p  <  0.01). In one recent Saudi Arabian study, anemia was found in 86% of children affected by either UC or CD [20]. In other studies, anemia has been described occurring in 16–77% of patients with CD (16, 58, 70, and 77% reported in pediatric cohorts) [14–17, 19, 21–24] and 9–67% of patients with UC (30% reported in one pediatric cohort) [17, 21, 24].

The cause of iron deficiency with or without frank anemia is likely multifactorial in both CD and UC [25]. In CD, anemia may result from iron, folate, or vitamin B12 micronutrient deficiencies from under or malnutrition, which commonly accompanies extensive small bowel disease, particularly if the ileum is involved [25]. In addition, anemia may result from gross or occult gastrointestinal blood loss due to underlying intestinal inflammation. Finally, iron deficiency and/or anemia may be due to decreased overall iron stores due to chronic disease, and lack of appropriate dietary intake to replace iron stores [25]. The anemia observed in UC is generally the result of iron losses from chronic intestinal bleeding, but as with CD can be due to anemia of chronic disease. The assessment of iron status in IBD in many cases is rather difficult due to coexistent inflammation secondary to chronic disease. For this assessment several indices and markers have been suggested. Ferritin seems to play a central role in the definition and diagnosis of anemia in IBD and transferrin, transferrin saturation (Tsat), and soluble transferrin receptors have also been found to useful markers in clinical practice. All these biochemical markers have limitations because they may be influenced by factors other than changes in iron balance. In addition, the iron metabolism regulators, hepcidin and prohepcidin, are still under investigation in IBD. Erythrocytes parameters like the red cell distribution width (RDW) and the percentage of hypochromic red cells as well as reticulocyte parameters such as hemoglobin concentration of reticulocytes, red blood cell size factor, and reticulocyte distribution width could be useful markers for the evaluation of anemia.

Anemia of chronic disease that can be seen in IBD is also believed to be multifactorial in its etiopathogenesis. Three potential mechanisms leading to the anemia associated with chronic disease have been recently postulated, namely (1) anemia results as a consequence of cytokine activation and subsequent alteration of iron homeostasis, (2) anemia occurs due to the inhibition of erythropoiesis, and (3) a shortened red blood cell half life is associated with chronic disease and thereby results in the anemia [16, 26]. Additionally, the anemia of chronic disease such as that found in IBD involves erythropoiesis disturbance due to circulating inflammation mediators. In one study by Tsitsika et al., erythropoietin (Epo) levels in children and adolescents with IBD were investigated and correlated to disease activity [27]. In this particular study [27], 33 patients with IBD were evaluated (18 boys, 15 girls) ages 4–15 years (median 11 years). Patients were separated into two study groups related to their disease activity; those with active disease (n  =  21), and those in remission (n  =  12). Chronic disease-associated anemia was present only in patients with active disease, and, those patients also had a significantly higher possibility of low, altered Epo levels than expected compared with patients with inactive disease. Thus, it appears that impaired Epo production is another mechanism of anemia of chronic disease development.

Once the diagnosis of anemia is established, the etiology should be further investigated so that treatment can be initiated. For macrocytic anemias, folate, vitamin B12, and methylmalonic acid levels should be obtained. Iron studies including ferritin, total iron binding content (TIBC), and iron levels should be evaluated in cases of microcytic anemia. However, the results of these studies may be difficult to interpret, as ferritin, a measure of iron stores, is also an acute-phase reactant and may be falsely elevated in inflammatory conditions. Thus, in patients with a microcytic anemia, obtaining a soluble transferrin receptor in addition to standard iron studies may be helpful in differentiating iron deficiency anemia and anemia of chronic disease [28–30]. Soluble transferrin receptor concentration, which is not affected by inflammation, is elevated in iron deficiency anemia, but remains normal in anemia of chronic disease [28–30]. In addition to soluble transferring receptor, intestinal ferroportin expression should be considered as a marker of anemia in relationship to IBD and, particularly, CD in children. In a recent study performed by Burpee et al., [31] intestinal iron exporter ferroportin expression was studied in subjects with and without CD. In this investigation, the authors evaluated duodenal mucosal biopsies from 29 pediatric subjects, 19 of whom had CD (n  =  19) and ten were without CD. The authors observed that intestinal ferroportin protein was higher in anemic CD subjects than in non-anemic CD subjects, while ferroportin mRNA levels were not significantly different. Thus, intestinal ferroportin protein is upregulated in anemic CD subjects, suggesting yet another pathway for the iron deficiency and the anemia observed in children with CD [31].

In inflammatory conditions such as CD and UC, there is a rise in acute-phase reactant proteins as a result of chemokine stimulation. The assessment of acute-phase reactants has been employed as laboratory tests in the standard work-up of the child with suspected IBD, as well as other inflammatory conditions (e.g., juvenile rheumatoid arthritis) [32, 33]. Reactive thrombocytosis, a nonspecific marker of inflammation, is a result of this acute phase response. Since the first published paper describing the association of thrombocytosis with chronic IBD by Morowitz et al., [34] the characterization of platelet elevation in the peripheral blood has been a “standard” part of the work-up of patients for suspected IBD and in the monitoring of their disease activity. However, more recently, studies of the pathogenesis of IBD have implicated platelets in the propagation of intestinal inflammation. In a murine model of intestinal inflammation, CD40-CD40L appears to be involved in the pathogenesis of intestinal inflammation, and suggest that modulation of leukocyte and platelet recruitment by activated, CD40-positive endothelial cells in colonic venules may represent a major action of this signaling pathway. In addition, Kayo et al. [35] evaluated the role of platelets in inflammation in peripheral blood and in the mucosa of a cohort of patients with active UC. These investigators compared the group of patients with active UC to patients with inactive UC and a small cohort of healthy controls. The authors observed a close association between activated platelets and neutrophils in both the affected colonic mucosa and peripheral blood of patients with active-phase UC compared to the normal volunteers (i.e., healthy controls) and those with inactive UC. The investigators inferred from their study results that a platelet–­neutrophil association may play a role in the progression of inflammatory processes in UC [35]. There is also evidence that coagulation activation may mediate and amplify inflammatory cascades in IBD, especially via activating proteinase-activated receptor-related pathways [36]. Patients with CD and UC are at least 3–4-fold increased risk of developing thromboembolic (TE) complications compared to control patients [36]. Although the etiology is multifactorial, TE phenomena in IBD is largely attributable to coagulation activation and platelet aggregation during systemic inflammation [36]. Thus, it appears that platelets may in fact play more of a role in the propagation of intestinal inflammation and potentially some of the severe sequelae (e.g. TE processes) of the system inflammation of IBD, rather than being a simple “biomarker” of IBD [32, 36].

In children referred for endoscopy for evaluation of abdominal pain, diarrhea, rectal bleeding, weight loss, or mouth ulcerations, 85% of patients with CD and 70% of patients with UC had elevated platelet counts compared to 6% of children with normal endoscopic assessment [21]. The presence of thrombocytosis may be overestimated in this study, or a unique response in the child with IBD as a lower prevalence of increased platelets in IBD is reported in adults [37–39]. However, an elevated platelet count in a child with chronic intestinal symptoms should raise clinical suspicion of underlying intestinal inflammation. In one study evaluating pediatric patients with chronic abdominal complaints, the presence of an abnormal hemoglobin and/or elevated platelet count on a routine CBC was able to differentiate between IBD and healthy controls, with 90.8% sensitivity and 80.0% specificity [40]. Furthermore, the platelet count may help differentiate between IBD and infectious processes, as thrombocytosis is a relatively uncommon finding in diarrhea associated with enteric pathogens [37].

Mean platelet volume (MPV) is influenced by the degree and type of mucosal and system inflammation. A recent study analyzed overall accuracy of MPV in disease activity and compared MPV with other inflammatory markers in 61 UC patients and 27 healthy subjects [41]. MPV was compared to ESR, CRP, and white blood cell count. The authors found that MPV accuracy was roughly equivalent to standard acute-phase reactants and was significantly lower in UC patients and particularly in active UC patients than controls [41]. Thus MPV may be another indicator of intestinal inflammation and a useful marker in patients with symptoms concerning for IBD.

Liver function tests and electrolyte panels may add additional information to aid the clinician in differentiating IBD from non-IBD and in the determination of the IBD phenotype—i.e., presence or absence of extra-intestinal manifestations such as liver disease [54, 55]. Although severe liver disease can be the first presentation of IBD in pediatric patients, hypoalbuminemia, which may be due to liver parenchymal damage, decreased production and/or due to bowel injury accompanied by increased fecal loss, is a more frequent finding at diagnosis [55]. Hypoalbuminemia is observed in both CD and UC; however, overall decreased serum albumin appears to be present at a much higher frequency in CD. In pediatric cohorts, hypoalbuminemia has been reported in 35–64% of patients with CD and 15% of patients with UC [21, 22, 56–60]. In a more recent, relatively small-sized (N  =  57) pediatric study of children with UC from Saudi Arabia, hypoalbuminemia was observed in over half (i.e., 54%) of the cohort evaluated, with disease severity correlating with the degree of hypoalbuminemia [20]. In addition to being useful in the diagnosis of IBD compared to non-IBD, as well as a factor in the assessment of the child’s overall nutritional status, hypoalbuminemia when present may have value as a prognostic factor for surgical risk [56] as well as for osteopenia and decreased bone mineral density scores [58]. Albumin can also be used as a marker for response to therapy. In an adult multicenter clinical trial evaluating one of the newer biologics for therapy of CD, the authors investigated the effect of adalimumab on changes in laboratory values using data from CHARM trial [61]. In a total of 778 adult patients, adalimumab every-other-week (N  =  260), adalimumab weekly (N  =  257), or placebo (N  =  261), the authors observed significant improvements in nutritional, hematologic, and inflammatory markers, including and specifically albumin, in moderately to severely active CD [61].

Similar to the pathobiology of anemia associated with IBD, the etiology of hypoalbuminemia in the child or adolescent with IBD is multifactorial, with protein loss from intestinal inflammation, decreased albumin production (negative acute phase response), and long-term poor nutrition all contributing to the overall low circulating levels of this important protein [46, 57, 59].

Elevation of AST and ALT may also be present on this initial screen in the evaluation of a patient with suspected IBD. In one study by Mendes et al., [62] the prevalence of abnormal hepatic biochemistries and chronic liver disease in a cohort of IBD patients was described in a retrospective case–control fashion. Patients with normal and abnormal liver biochemistries were compared, and in the cohort of 544 patients, abnormal hepatic biochemistries were present in nearly one-third of these adult patients. Contrary to what the investigators hypothesized, abnormal liver biochemistries in this single-center cohort were not associated with IBD activity. These authors recommended that persistently abnormal hepatic biochemistries should be evaluated, but to use caution and not immediately attribute these abnormal liver biochemistries to IBD activity [62]. Abnormal liver biochemistries may also be primarily related to poor nutrition as a result of active disease, and thus spontaneous resolution of these transient elevations are common [63].

However, when AST/ALT are persistently elevated or seen in association with an elevated ALP, elevated direct bilirubin and/or γ-glutamyl transpeptidase, the extraintestinal complication of primary sclerosing cholangitis (PSC) or autoimmune hepatitis/overlap syndrome should be considered. PSC is a reported complication in 3% of children with IBD and can precede or occur coincident with diagnosis of IBD [64–66]. In a recent US population-based health maintenance organization study, the prevalence of PSC in conjunction with IBD was characterized in addition to the demographic differences between racial/ethnic groups in patients with PSC compared to non-IBD and non-liver disease controls. Using the Northern California Kaiser Permanente (KP) database, the authors identified 169 (101 males) cases fulfilling PSC diagnostic criteria with a mean age at diagnosis of 44 years (range 11–81); age-adjusted point prevalence was 4.15 per 100,000 on December 31, 2005 [67]. IBD was present in 64.5% (109/169) cases and was significantly more frequent in men than women with PSC (73.3 and 51.5%, respectively, p  =  0.005) [67]. In another small-sized single-center study (N  =  29), the incidence of IBD in PSC patients was 68.9% (20/29) [68]. The investigators showed two peaks in the age distribution of PSC with male PSC patients demonstrated a first peak and female patients a second peak. Male PSC–IBD patients were in their teens and 20s making the first peak and female PSC–IBD patients were in their 50s and 60s making the second peak. Of note, the study demonstrated that PSC–IBD patents were significantly younger than the patients without IBD (33.6 vs. 58.9 years, p  <  0.001) [68]. With regards to pediatric patients, Wilschanski et al [66]. demonstrated of 32 children with PSC, that the majority of patients were diagnosed in their second decade (median age: 13 years) and four children presented before the age of 2 years. Seventeen of the 32 patients had IBD, all with colitis, 14 UC, and 3 CD [66]. Eight patients presented with chronic liver disease before clinical onset of IBD. Thus, of the hepatic pathologies reported associated with IBD in children and adults, PSC remains the more common presentation. In one longitudinal, cohort study by Feldstein et al., [64] 52 children with cholangiography-proven PSC were followed to determine the long-term outcome (mean follow-up was 16.7 years) of children with PSC diagnosed over a 20-year period (34 boys and 18 girls; mean age 13.8  ±  4.2 years; range, 1.5–19.6 years). Two-thirds presented with symptoms and/or signs of PSC and 81% had concomitant IBD [64]. During follow-up, 11 children underwent liver transplantation for end-stage PSC and 1 child died with the median (50%) survival free of liver transplantation being 12.7 years. Compared with an age- and gender-matched US population, survival was significantly shorter in children with PSC (P  <  0.001). Using a statistical regression model for analysis, the authors determined that lower platelet count, splenomegaly, and older age were associated with shorter survival. Moreover, presence of autoimmune hepatitis overlapping with PSC (P  =  0.2) or medical therapy (P  =  0.2) did not affect survival. Thus, the authors concluded that PSC, whether associated with IBD or not, significantly decreases survival in this child population [64].

Renal as well as pancreatic disease may also be important extra-intestinal manifestations of IBD or can be adverse events associated with IBD pharmacotherapy [69–74]. In a multicenter study from Israel, both adults and children presenting with acute pancreatitis as the first symptom of IBD were retrospectively identified (10 years, 7 university hospitals). These authors demonstrated that 10 of 460 pediatric patients with IBD (2.17%), compared with only 2 in 3,500 adults (0.06%) presented with pancreatitis. Eight children had colonic disease (4 CD, 4 UC (3 pancolitis)) with the mean amylase level being 1,419 (range 100–1,370) and three children (30%) having mildly elevated transaminases. Of note was that median time between onset of first episode of acute pancreatitis in relation to onset of IBD was 24 (range 1–156) weeks and the most common presentation was abdominal pain. Similarly, renal disease may precede diagnosis of IBD. Although small in sample size, Izzedine et al. [75] described 4 patients with severe interstitial nephritis demonstrated on histopathological examination of kidney biopsy specimens. Renal failure was discovered before or simultaneously with the diagnosis of CD, and patients were not treated with mesalamine. More importantly, impairment of renal function progressed to end-stage renal failure in 3 of the 4 patients [75]. A similar small case series of 2 pediatric patients with renal disease occurring concurrently with diagnosis of IBD has been reported [76]. Thus, with respect to appropriate adjunct or complementary lab tests to obtain in the work-up of a child with suspected IBD, given the reports of interstitial nephritis in patients with CD in the absence of 5-aminosalicylate exposure, a baseline comprehensive chemistry panel, should be considered during the initial evaluation. Moreover, amylase and lipase should be obtained where clinical signs and symptoms raise suspicion of pancreatic disease; prior to or after initiation of therapy particularly those medications with a predilection (e.g., 6MP, 5-ASA) for pancreatitis as a side effect.

The above-mentioned details highlight the standard evaluation that is recommended for all children with history and physical exam findings suspicious for IBD. These diagnostic tests may aid the clinician in the differentiation UC and CD from functional bowel disorders and infectious etiologies of their presenting signs and symptoms. However, because the clinical presentation of IBD is so diverse and symptoms can be nonspecific, at times, it may be difficult to distinguish between inflammatory and functional disorders. In fact, since May 13, 1932, when Dr. Crohn and his colleagues, Oppenheimer and Ginzburg, presented a paper on terminal ileitis describing the features of CD to the American Medical Association, the average time from onset of symptoms to definitive diagnosis continues to be prolonged, ranging from 6 to 18 months [77–79].

Several other noninvasive studies have been proposed to aid in the diagnosis of IBD including IBD serologies, fecal calprotectin, and lactoferrin. The following section reviews these tests including a brief overview of the use of IBD serology and the evidence to support or disprove their use in the preliminary evaluation of the child with suspected IBD. In addition, this section will describe the stool tests which are an essential part of the initial work-up of the child with suspected IBD, and includes a discussion of more novel markers of intestinal inflammation, fecal calprotectin, and fecal lactoferrin.