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Year : 2014  |  Volume : 5  |  Issue : 3  |  Page : 117-118

Bilirubin crystallization in neutrophils in cases of neonatal unconjugated hyperbilirubinemias

Depatrment of Pathology, Maulana Azad Medical College, Bahadur Shah Zafar Marg, New Delhi, India

Date of Web Publication30-Sep-2014

Correspondence Address:
Richa Gupta
Department of Pathology, Maulana Azad Medical College, Room 263, Bahadur Shah Zafar Marg, New Delhi
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1658-5127.142004

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How to cite this article:
Dange P, Desai P, Gupta R, Singh T. Bilirubin crystallization in neutrophils in cases of neonatal unconjugated hyperbilirubinemias. J Appl Hematol 2014;5:117-8

How to cite this URL:
Dange P, Desai P, Gupta R, Singh T. Bilirubin crystallization in neutrophils in cases of neonatal unconjugated hyperbilirubinemias. J Appl Hematol [serial online] 2014 [cited 2022 Oct 6];5:117-8. Available from: https://www.jahjournal.org/text.asp?2014/5/3/117/142004


Several morphological changes in neutrophils are useful clues to underlying disorders. The common ones being hypersegmentation in case of folate or B12 deficiency and toxic changes in septicemia. In addition, rarely pelger huet anomaly and chediak higashi granules may be noticed. Presented hereby are two cases showing presence of bilirubin crystals in the cytoplasm of neutrophils in the setting of unconjugated hyperbilirubinemia.

The first case is of a 3-day-old infant presented with poor feeding, jaundice, and abnormal posturing of the body for 10 h. The baby was born to a second gravida with a previous history lower segment cesarean section. There was Rh incompatibility between the child (Rh positive) and his mother (Rh negative). On examination, baby was icteric up to palms and soles. Liver and spleen were palpable 3 cm below the costal margin. The total bilirubin level was 30 mg/dl while direct bilirubin was 5.5 mg/dl.

The second case was of a premature (31 weeks) infant with poor respiratory effort. He was intubated from day 1 of life. Subsequently, he became febrile. Blood cultures were positive for  Escherichia More Details coli. The bilirubin levels were found to be 10.5 mg/dl. There was no history of Rh incompatibility. Thus, a diagnosis of hyperbilirubinemia secondary to E. coli sepsis was made.

Smears made from ethylenediaminetetraacetic acid (EDTA) anticoagulated blood showed golden-yellow refractile bilirubin crystals in the cytoplasm of many of the neutrophils and its precursors. The crystals were needle shaped to rhomboid or irregular in shape [Figure 1]. Such crystals were not seen in other white blood cells including lymphocytes and monocytes. Heel prick smears could be obtained in one of the case, and it did not show such crystals.

Neonatal jaundice is one of the common disorders that affects newborns and have diverse etiology. The most common ones are hemolytic disease of the newborn, septicemia, physiological jaundice, and neonatal hepatitis. In all these conditions, there is an increase in bilirubin levels in the blood, which is deposited in tissues such as skin, sclera, and basal ganglia in brain causing organ dysfunction. High bilirubin levels have been associated with neutrophil dysfunction. [1] However, it does not normally crystallize in the neutrophils as evidenced by absence of these crystals in fresh heel prick smears. In fact, only the sample collected in EDTA vial that is allowed to stand for 30-40 min show crystallization. Such crystals are not found in blood collected in heparin, oxalate or citrate vials. [2],[3],[4],[5]
Figure 1: Bilirubin crystals in neutrophil and myelocyte (Giemsa stain, ×400)

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Some authors have previously reported similar findings in neutrophils specifically in ethylene glycol tetra acetic acid (EGTA) anticoagulated blood. Sandoval et al. have noted that platelet-activating factor (PAF), a primary chemoattractant, simultaneously increases cytosolic free Ca 2+ , intracellular pH, extracellular signal-regulated kinase (ERK) 1/2 and Akt/protein kinase B (PKB) phosphorylation and that the presence of EGTA reduced the intracellular alkalinization and ERK1/2 phosphorylation induced by PAF, apparently via store-operated calcium entry influx inhibition. [6] Thus, it is possible that, by reducing calcium availability, it prevents alkalinization of the cytoplasm of the neutrophil leading to crystallization of bilirubin in acidic medium. This is also evident by the fact that bilirubin crystals may be seen in acidic urine. EDTA having a similar mechanism of action like EGTA reduce the calcium availability to the neutrophils and thus prevents alkalinization of cytoplasm leading to crystallization of bilirubin. However, why oxalate or citrate (calcium chelators) do not form crystals is not known.

  Conclusion Top

The finding of such crystals in neutrophils is specific to blood anticoagulated with calcium chelators and may be missed when other anticoagulants are used. Such a finding may be an indicator of bilirubin toxicity in unsuspected cases of hyperbilirubinemias due to several causes.

Further study needs to be done to see the cutoff level of indirect bilirubin at which these crystals start appearing in the cytoplasm of neutrophils.

The case was presented to re-emphasize the importance of a careful peripheral blood smear examination to avoid missing subtle but useful findings in the era of automation.

  References Top

1.Arai T, Yoshikai Y, Kamiya J, Nagino M, Uesaka K, Yuasa N, et al. Bilirubin impairs bactericidal activity of neutrophils through an antioxidant mechanism in vitro. J Surg Res 2001;96:107-13.  Back to cited text no. 1
2.Sen Gupta PC, Ghosal SP, Mukherjee AK, Maity TR. Bilirubin crystals in neutrophils of jaundiced neonates and infants. Acta Haematol 1983;70:69-70.  Back to cited text no. 2
3.Marwaha N, Sarode R, Marwaha RK, Sharma S, Yachha S, Narang A. Bilirubin crystals in peripheral blood smears from neonates with unconjugated hyperbilirubinaemia. Med Lab Sci 1990;47:278-81.  Back to cited text no. 3
4.Basu D, Solo S, Nilkund J. Bilirubin crystals in peripheral blood neutrophils in neonatal hyperbilirubinaemia. Br J Haematol 2005;131:141.  Back to cited text no. 4
5.Shenoi UD, Nandi GK. Bilirubin crystals in neutrophils in neonatal hyperbilirubinaemia. Indian J Pediatr 1997;64:93-6.  Back to cited text no. 5
6.Sandoval AJ, Riquelme JP, Carretta MD, Hancke JL, Hidalgo MA, Burgos RA. Store-operated calcium entry mediates intracellular alkalinization, ERK1/2, and Akt/PKB phosphorylation in bovine neutrophils. J Leukoc Biol 2007;82:1266-77.  Back to cited text no. 6


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