Eur J Pediatr DOI 10.1007/s00431-014-2374-7

CASE REPORT

Neonatal hemolytic anemia due to pyknocytosis Michel J. Vos & Daniëlle Martens & Sjef J. van de Leur & Richard van Wijk

Received: 21 May 2014 / Revised: 25 June 2014 / Accepted: 27 June 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract A newborn boy was referred to our hospital because of hemolytic anemia and severe hyperbilirubinemia. Extensive investigations aimed at determining the cause of hemolysis was initiated at the time of admission and 3 months after blood transfusion. Notably, no intrinsic erythrocyte abnormalities could be detected. The only possible cause explaining the progressive anemia and unconjugated hyperbilirubinemia was the finding of pyknocytes, severely distorted erythrocytes, on the blood film at hospital admission. We propose a role for an increased free fraction of plasma unconjugated bilirubin in the formation of pyknocytes through bilirubin membrane toxicity with subsequent anemia and progressive hyperbilirubinemia. Conclusion: Pyknocytosis is a transitory erythrocyte-related condition which can result in severe anemia and hyperbilirubinemia. Recognition of pyknocytes by microscopic analysis of a blood film is essential for a correct diagnosis. Treatment consists of correction of the anemia by top-up blood transfusion and light therapy to prevent toxic bilirubin buildup. High levels of free

Communicated by Jaan Toelen M. J. Vos (*) : S. J. van de Leur Department of Clinical Chemistry, Isala Hospital, Vlinder 1 kamer V1.2.606 (103) Dr. Van Heesweg 2, 8025 AB Zwolle, Postbus 10400, 8000 GK Zwolle, Netherlands e-mail: [email protected] S. J. van de Leur e-mail: [email protected] D. Martens Department of Pediatrics, Isala Hospital, Zwolle, The Netherlands e-mail: [email protected] R. van Wijk Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands e-mail: [email protected]

unconjugated bilirubin could be the underlying cause for the formation of pyknocytes. Keywords Pyknocytosis . Hyperbilirubinemia . Hemolytic anemia

Introduction Anemia and hyperbilirubinemia during the fetal and neonatal period can be caused by a broad range of conditions. Apart from transient physiologic jaundice due to increased erythrocyte breakdown, intrinsic erythrocyte disorders like enzyme deficiencies, hemoglobinopathies and membrane disorders can pose a risk for developing severe hyperbilirubinemia. In addition, anemia and hyperbilirubinemia can result from immune-mediated degradation of erythrocytes caused by maternal antibodies directed against fetal blood group antigens which are able to cross the placenta. Subsequent buildup of unconjugated bilirubin, due to immaturity of the neonatal liver, can be a life-threatening condition resulting in possible damage to brain basal ganglia and brainstem nuclei [3]. Due to the multifactorial nature of neonatal anemia and hyperbilirubinemia, which includes genetic susceptibility, environmental factors and variation in hepatic bilirubin clearance, the etiology behind individual cases can be diverse. One cause of anemia and severe hyperbilirubinemia in which the etiology is largely unknown is infantile pyknocytosis. This rare transitory disorder was originally described as an erythrocyte abnormality with the characteristic finding of irregularly distorted, dense, and contracted erythrocytes, termed pyknocytes [8, 9]. Here, we present a case of severe hyperbilirubinemia and progressive anemia in which the underlying cause was unknown. The diagnosis pyknocytosis was eventually made based on microscopic analysis of blood films made at hospital admission.

Eur J Pediatr

Case report A full-term newborn Caucasian boy (blood group A-positive) presented with anemia and jaundice at the age of 16 days. The patient was born after 37 weeks and 4 days of gestation. The mother (32 years old, G5 P3, blood group O-positive) was diagnosed during her pregnancy with cholestasis and associated liver function disorder (aspartate aminotransferase 227 U/

Fig. 1 Hemoglobin concentration (a), total bilirubin concentration (b), and percentage of pyknocytes (c) were determined during (days 0–5) and after (dag 40, 130) hospitalization. Microscopic analysis before blood transfusion (D1) shows the presence of pyknocytes (indicated by arrows). At 130 days (D2) and 3 months (D3) after blood transfusion, no pyknocytes were present during microscopic analysis. The possibility of a red blood cell membrane disorder was investigated by osmotic gradient

L, alanine aminotransferase 247 U/L). There was no history of newborn hyperbilirubinemia associated with previous pregnancies. Four days postpartum, the patient showed signs of jaundice after which the mother placed the newborn in direct daylight. The jaundice disappeared after 4 days, but at the age of 13 days, the jaundice returned and rapidly progressed in combination with increasing paleness. At 16 days of age, the patient was admitted to our hospital.

ektacytometry by Laser-assisted Optical Rotational Cell Analyzer (LoRRca, Mechatronics, The Netherlands). The observed pattern showed no abnormalities (e). (For microscopic analysis of pyknocyte percentage, five fields of 200–300 erythrocytes each were counted, depicted is the mean with standard deviation; asterisk indicates after blood transfusion; bar represents 20 μm)

Eur J Pediatr

On examination, the patient was pale and extremely icteric, but he was alert and reactive and his vital signs were normal. Auscultation of heart and lungs and abdominal investigation were normal. He had no hepatosplenomegaly. At the day of hospitalization (day 0), laboratory investigation showed anemia (hemoglobin 5.4 mmol/L (8.7– 13.0 mmol/L); 8.7 g/dL (14–21 g/dL)) (Fig. 1a) and unconjugated hyperbilirubinemia (490 μmol/L (0–17 μmol/L); 28.7 mg/dL (0–1 mg/dL)) (Fig. 1b) with a direct reacting fraction of 18 μmol/L (1 mg/dL). Reticulocytes were 19%. Within 24 h, the patient became more anemic (hemoglobin 4.1 mmol/L (8.7–13.0 mmol/L); 6.6 g/dL (14–21 g/dL)) while the bilirubin concentration decreased due to phototherapy and fluid supplementation. Active hemolysis was suspected, and additional laboratory tests to investigate the nature of the hemolytic process were initiated. Notably, morphological examination of the patient’s peripheral blood film displayed numerous abnormalities. In particular, a large number of dense, contracted erythrocytes with highly distorted membrane characteristics were visible (Fig. 1d1). The dense, contracted erythrocytes resembled pyknocytes and increased in number at day 1 (Fig. 1c). Since the direct antiglobulin test was negative, an immunological origin of hemolysis was excluded. Pre-transfusion hemoglobin analysis by capillary electrophoresis displayed no aberrations, making a hemoglobin disorder unlikely. This was later confirmed by DNA sequence analysis of the α- and βglobin genes which showed no abnormalities. Treatment On admission, the patient received intensive phototherapy and fluid supplementation to reduce the high bilirubin blood concentration. On the third day of hospitalization, two top-up transfusions with packed red cells were given to correct the anemia. The patient stabilized and bilirubin further decreased (Fig. 1b). After 5 days, the phototherapy could be discontinued, and 1 day later the patient was discharged. The patient received another top-up transfusion 22 days after admission after which the hemoglobin concentration stabilized at 6.3 mmol/L (8.7–13.0 mmol/L); 10.2 g/dL (14– 21 g/dL). Post-stabilization laboratory tests At day 130 and 3 months after hospitalization, a blood film was made. At this point, pyknocytes were no longer present (Fig. 1c, d2–3). Additional laboratory investigations aimed at identifying the cause of hemolysis were performed 3 months after blood transfusion to allow for the complete removal of donor erythrocytes. Hemoglobin concentration at that time (5 months

of age) was 8.1 mmol/L (5.9–9.5 mmol/L); 13.1 g/dL (9.5–14.5 g/dL). Red blood cell membrane abnormalities w e r e c o n s i d e r e d u n l i k e l y as os m o t i c g r a d i e n t ektacytometry showed no abnormalities (Fig. 1e). Red blood cell pyruvate kinase and glucose-6-phosphate dehydrogenase enzyme activities were normal, thereby again excluding the most common red blood cell enzyme deficiencies (Table 1, hexokinase activity was included to correct for the mean age of the red blood cell population). Furthermore, the glutathione stability test was normal (Table 1), indicating no aberrations in the capability of the red blood cells to withstand oxidative stress. In conclusion, no intrinsic erythrocyte abnormalities could be identified which could be responsible for the observed progressive anemia experienced during the first weeks of life.

Discussion Although the appearance of pyknocytes seems a non-harmful physiological event, ranging from 0.3–1.9 % in full-term infants and 0.3–5.6 % in premature infants [8], the development of pyknocytosis can result in severe anemia and (unconjugated) hyperbilirubinemia and as such should be regarded as a potential life-threatening condition. Careful microscopic analysis of a blood film made before top-up transfusion is essential to correctly identify cases of pyknocytosis. As donor erythrocytes have been described to have a decreased lifespan and resemble pyknocytes after transfusion in an infant with severe pyknocytosis [5], a role of a plasma factor for pyknocyte induction seems reasonable to assume. A possible candidate to contribute to pyknocytosis is an increased free fraction of unconjugated bilirubin (UCB), which can increase during the first days post-birth due to enhanced erythrocyte breakdown and immaturity of the liver. Table 1 Erythrocyte enzyme and glutathione measurements Enzyme test

Patient

Reference range

PK activity (U/gHb) PK activity, low [S] (%)

12.6 37

6.1–12.3 32–67

G6PD activity (U/gHb) HK activity (U/gHb) GSH (μmol/ml RBCs) GSH instability (%) 6

10.4 1.8 2.2 6

6.4–10.5 0.8–1.5 1.5–2.9 0–30

PK pyruvate kinase, G6PD glucose-6-phosphate dehydrogenase, HK hexokinase, GSH glutathione, RBC red blood cell, gHb gram hemoglobin

Eur J Pediatr

Binding of UCB to neuronal cells is considered the main cause of bilirubin encephalopathy [3]. However, UCB not only affects neuronal cells but it also interacts with erythrocyte membranes [2]. Interestingly, UCB binding to the erythrocyte membrane can cause rapid appearance of echinocytes, crenation, and altered cell membrane integrity in vitro, resembling pyknocytes [1, 4]. Altered cell membrane integrity of erythrocytes is probably promoting hemolysis in vivo which further increases UCB concentration. Toxic binding of UCB to the erythrocyte membrane would only occur when the UCB concentration exceeds the albumin-binding capacity for UCB, increasing the free UCB fraction. Such an event can occur at low albumin concentrations, displacement of UCB from albumin by medication, or altered binding affinity during, for example, acidosis. In addition, neonatal erythrocytes are more prone to oxidative damage and experience membrane damage due to denaturing fetal hemoglobin, resulting in a shorter lifespan and increasing the risk of hemolysis [6, 7]. Our patient suffered high unconjugated bilirubin concentrations for several days. Although we are not aware of underlying factors influencing UCB binding to albumin in this patient, the sharp increase in pyknocytes and anemia within 24 h could be explained by an abrupt increase of free UCB-induced membrane toxicity. In conclusion, pyknocytosis is a transitory condition easily diagnosed by careful microscopic analysis of a blood film and after exclusion of other erythrocyte disorders associated with hemolysis. We hypothesize that elevated free UCB concentrations due to liver immaturity could be the yet unknown

factor which contributes to the formation of pyknocytes due to bilirubin-induced membrane toxicity.

Conflict of interest The authors declare that there are no conflicts of interest.

References 1. Brito MA, Silva R, Tiribelli C, Brites D (2000) Assessment of bilirubin toxicity to erythrocytes. Implication in neonatal jaundice management. Eur J Clin Invest 30:239–247 2. Chen LX, Lu JF, Wang K (1992) The influence of bilirubin on fluidity and rotational correlation times of human erythrocyte membrane. Cell Biol Int Rep 16:567–573 3. Hansen TW (2002) Mechanisms of bilirubin toxicity: clinical implications. Clin Perinatol 29:765–78, viii 4. Kaul R, Bajpai VK, Shipstone AC, Kaul HK, Krishna Murti CR (1981) Bilirubin-induced erythrocyte membrane cytotoxicity. Exp Mol Pathol 34:290–298 5. Keimowitz R, Desforges JF (1965) Infantile pyknocytosis. N Engl J Med 273:1152–1154 6. Kleihauer E, Braun H, Betke K (1957) Demonstration of fetal hemoglobin in erythrocytes of a blood smear. Klin Wochenschr 35:637–638 7. Pearson HA (1967) Life-span of the fetal red blood cell. J Pediatr 70: 166–171 8. Tuffy P, Brown AK, Zuelzer WW (1959) Infantile pyknocytosis; a common erythrocyte abnormality of the first trimester. AMA J Dis Child 98:227–241 9. Zannos-Mariola L, Kattamis C, Paidoucis M (1962) Infantile pyknocytosis and glucose-6-phosphate dehydrogenase deficiency. Br J Haemato 8:258–265

Neonatal hemolytic anemia due to pyknocytosis.

A newborn boy was referred to our hospital because of hemolytic anemia and severe hyperbilirubinemia. Extensive investigations aimed at determining th...
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