An infant with chronic hemolytic anemia.

Case

A 7-month old male patient was referred to our clinic because of mild anemia and icteric sclerae. The patient had a history of intensive phototherapy which was applied for three days because of neonatal jaundice which developed on the second day of life. In the follow-up performed in the external center where the delivery took place, moderate reticulocytosis, increased indirect bilirubin and decreased haptoglobulin which suggested chronic hemolysis were found. In the familial history, it was learned that the parents were third degree relatives. A history of chronic jaundice and anemia was present in the patient's aunt's daughter who was one year old. On physical examination, the conjunctivae were slightly pale, the sclerae were icteric; the spleen was nonpalpable and percussion of the Traube's space was found to be normal. The neuromotor development of the patient was appropriate for age. The necessary laboratory tests were performed.

Diagnosis : Glucose phosphate isomerase deficiency

Detailed biochemical tests including complete blood count, peripheral blood smear, indirect Coomb test, direct-indirect bilirubin and lactate dehydrogenase (LDH) levels were performed in the patient. Mild hypochomia, anisocytosis and poikilocytosis were observed on peripheral blood smear. There was no erythrocyte deformity which would suggest membrane disease. Hemogram revealed the following: Hb: 9 g/dL, HCT: 27%, MCV: 81 fL, MCHC: 32 g/dL, reticulocyte count: 3%. Direct and indirect Coombs tests were found to be negative, indirect bilirubin value: 3.5 mg/dL (normal: <2 mg/dL), LDH: 800 IU/L (normal: <600 IU/L). Other biochemical variables were found to be normal. The blood type of the mother and children was O Rh (+). Serum haptoglobulin value was found to be low (8 mg/dL) (normal: 16-200 mg/dL). The complete blood counts and peripheral blood smears of the parents were found to be normal. No abnormal band was found in hemoglobin electrophoresis of the patient (Hb A: 98%, Hb A2: 2%). In the incubated osmotic fragility test, the resistance of the erthyrocytes was normal. Glucose 6 phosphate dehydrogenase (G6PDH) and pyruvate kinase (PK) enzyme levels were within the normal limits. Physical examination revealed no pathological finding and no finding suggesting hypersplenism. There was no history of drug usage. The level of glucose phosphate isomerase (GPI) which is observed more rarely in the population was measured in the patient who had values which supported chronic hemolysis. The GPI level was found to be low (32%) (normal: 85-100%).

Discussion

Anemia is a very common health problem in children. Especially in communities in developing countries, 80% of the people present to a hospital because of anemia at least for one time during their childhood (1). In hemolytic anemia, erythrocytes which have a normal life span of 100-120 days are eliminated from the circulation in a shorter time (2). Hemolytic anemia may be observed in a healthy individual because of immune-mediated mechanisms, drugs and infections. In hemolytic anemies caused by erythrocyte enzyme defects, membrane defects or hemoglobinopathies, the course is chronic. Exceptions of this include G6PD and gluthatione synthetase deficiency which are characterized with acute hemolytic attacks. The classification of hemolytic anemies is shown in Table 1.

In hemolytic anemia, the bone marrow which is healthy synthesizes more erythrocytes than normal as a response to increased erythropoetin; the reticulocyte count increases above 2%. When the hemolytic process becomes chronic, the myeloid/erythroid ratio which is 3/1 increases in favour of the erythroid cells. In conditions where there is deep anemia including major thalassemia, enlargement in some bones including mainly the facial bones and bones in the hands is observed as a result of extension of increased activity in the bone marrow to the medullary area. The hemolytic process is demonstrated directly by determining the life span of the erythrocytes (with radioactive sodium chromate; Cr-51) or indirectly by measurement the metabolites of hemolysis (increased indirect bilirubin and LDH, decreased haptoglobulin). Even if the indirect bilirubin values are higher than normal, marked jaundice may not be observed. If there is no accompanying hepatic disease, the total bilirubin level rarely increases above 5 mg/dL. Urinary and fecal urobilinogen level is increased because of destruction of bilirubin. Increased excretion of bilirubin pigments due to chronic hemolysis causes to formation of pigmented gallstones in the childhood. In any condition where hemolysis occurs, hemoglobin released into the plasma irreversibly binds to serum haptoglobulin. This complex which is formed is eliminated from the circulation in the liver. Since the level of haptoglobulin is limited, it is measured to be low during hemolysis (<20 mg/dL) or is not found in the serum. However, a low haptoglobulin level is not always a reliable indicator for hemolysis; haptoglobulin level is found to be low without hemolysis in haptoglobulin deficiency which is observed with a rate of 30% in the population in Western Africa. Since haptoglobulin is an acute phase reactant, it is found to be increased in cases of infection and inflammatory conditions. It should be kept in mind that the haptaglobulin level can not be measured accurately in infants younger than 2-3 months. In patients with chronic hemolytic anemia, the spleen is slightly-moderately enlarged excluding sickle cell anemia in which the spleen is enlarged to a great degree. Although leg ulcer may be observed specifically in patients with hereditary spherocytosis and sickle cell anemia, it may also be observed in other hemolytic diseases with a much lower frequency.

In the differential dignosis of chronic hemolytic anemia, the time and severity of the symptoms are important. Patients present with jaundice in the neonatal period in hereditary spherocytosis, alpha thalassemia and G6PD and PK deficiency, whereas the first findings generally appear after the 4-[6.sup.th]month in beta thalassemia. While patients with thalassemia major need chronic blood transfusion, some of the patients with hereditary xerocytosis and stomatocytosis may be asymptomatic for a life time. While glutathione synthetase and G6PD enzyme deficiency generally progress with acute attacks, hemolysis is chronic in all the remaining anemias. Presence of additional signs including mainly neurological findings is directive especially in the diagnosis of erythrocyte membrane defects (Table 2). The main laboratory tests include evaluation of a well-prepared peripheral blood smear by an experienced hematologist in addition to complete blood count. The primary tests include osmotic fragility test, hemoglobin electrophoresis, G6PD and PK enzyme levels. The levels of enzymes deficiencies of which occur more rarely are measured, if the previous tests are inconclusive. Familial history is valuable in the diagnosis of hemolytic anemies. Since many erythrocyte enzyme deficiencies and thalassemias are autosomal recessive diseases, patients should be interrogated in terms of consanguineous marriage and history in the siblings. Osmotic fragility and hemoglobin electrophoresis tests do not give an accurate result after erythrocyte suspension transfusion or in the neonatal period. In such cases, performance of these tests in the mother and father are helpful in the diagnosis. Table 2 shows the characteristics of chronic hemolytic anemias.

Glucose phosphate isomerase is an enzyme which reversibly catalyses transformation of glucose 6 phosphate and fructose 6 phosphate to each other which is the second step of anaerobic glycolysis in the Embden Meyerhof pathway. Therefore, it is an essential part of carbonhydrate metabolism. It is observed considerably rarely, although it is the second most common glycolytic enzyme disease following piruvate kinase deficiency (3). About 50 cases have been reported in the literature, though its frequency has not been clarified exactly (4). Glucose 6 phosphate which is increased because of deficiency of the enzyme inhibits hexokinase enzyme which transforms glucose to glucose 6 phosphate with negative feedback and thus the glycolysis and hexose monophosphate pathway is supressed. The synthesis of gluthatione, 2-3 diphosphoglycerate and adenozine triphosphate decreases. In this condition, the erythrocyte membrane loses its stabilization and a chronic non-spherocytic anemia develops. In case of exposure to oxidants including viral infections or antibiotherapy, acute hemolytic attacks may be observed as a result of decreased synthesis of antioxidant substances (5). Hydrops fetalis may even be observed, when the disease has a severe course (6). Glucose phosphate isomerase plays a role as a neurotrophic factor for spinal and sensory neurons in its monomeric form in addition to its enzymatic activity. Thus neurodegenerative diseases associated with muscle weakness, ataxia, dysarthria and mental retardation may be observed in addition to hemolytic anemia in its deficiency (7). In addition, granulocyte disorders and splenomegaly have been reported in some patients (8). Since it is an autosomal recessive diseases, women and men are affected equally. There is no clinical finding in carriers. Clinical findings are observed in homozygous or combined heterozygous individuals. The diagnosis of the disease is made by excluding other anemias causing to hemolysis and with an enzyme activity lower than 85% of the normal value. The parents of our patient were third-degree relatives. The fact that the same disease was observed in the aunt's daugther who was born from a consanguineous marriage (third degree relatives) was directive for us.

In this article, a patient with chronic hemolytic anemia caused by GPI deficiency which starts in the neonatal period and which is observed rarely was presented and chronic hemolytic anemies were reviewed hereby. The diagnosis of glucose phosphate isomerase deficiency should be kept in mind in the follow-up of a patient with hemolytic anemia. In this article, it was emphasized that the cause may be GPI deficiency in chronic hemolytic anemia.

Informed Consent: Written informed consent was obtained from patient's parents.

Peer-review: Externally peer-reviewed.

Author Contributions: Concept - G.N.O.; Design - T.C.; Supervision -T.C.; Funding - G.T.; Materials - G.T.; Data Collection and/or Processing - G.T.; Analysis and/or Interpretation - G.T., G.N.O.; Literature Review - G.N.O., G.T.; Writer - G.T.; Critical Review - T.C., G.N.O.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study has received no financial support.

References

(1.) Recht M, Pearson H. The hemolytic anemias. In: McMillan JA, Deangelis CD, Feigin RD, Warshaw JB, (eds). Oski's Pediatrics. Philadelphia: Lippincott Williams and Wilkins, 1999: 1453.

(2.) Oski FA, Brugnara C, Nathan DG. In: Nathan DG, Orkin SH, Ginsberg D, Look AT, (eds). A diagnostic approach to the anemic patient. Nathan and Oski's hematology of infancy and childhood. 6th. Philadelphia: WB Saunders, 2003: 409.

(3.) Kugler W, Lakomek M. Glucose-6-phosphate isomerase deficiency. Baillieres Best Pract Res Clin Haematol 2000; 13: 89-101. [CrossRef]

(4.) Climent F, Roset F, Repiso A, et al. Red cell glycolytic enzyme disorders caused by mutations: an update. Cardiovasc Haematol Disord Drug Targets 2009; 9: 95-106. [CrossRef]

(5.) Rossi F, Ruggiero S, Gallo M, Simeone G, Matarese SMR, Nobili B. Amoxicillin-induced hemolytic anemia in a child with glucose 6- phosphate isomerase deficiency. Ann Pharmacother 2010; 44: 1327-9. [CrossRef]

(6.) Ravindranath Y, Paglia DE, Warrier I, Valentine W, Nakatani M, Brockway RA. Glucose phosphate isomerase deficiency as a cause of hydrops fetalis. N Engl J Med 1987; 316: 258-61. [CrossRef]

(7.) Kugler W, Breme K, Laspe P, et al. Molecular basis of neurological dysfunction coupled with haemolytic anaemia in human glucose-6-phosphate isomerase (GPI) deficiency. Hum Genet 1998; 103: 450-4. [CrossRef]

(8.) Kugler W, Lakomek M. Glucose-6-phosphate isomerase deficiency. Baillieres Best Pract Res Clin Haematol 2000; 13: 89-101. [CrossRef]

Gulen Tuysuz, Gul Nihal Ozdemir, Tiraje Celkan

Division of Pediatric Hematology and Oncology, Istanbul University Cerrahpasa Faculty of Medicine, Istanbul, Turkey

Address for Correspondence: Gulen Tuysuz, Division of Pediatric Hematology and Oncology, Istanbul University Cerrahpasa Faculty of Medicine, Istanbul, Turkey. E-mail: gulentuysuz@hotmail.com

Received: 30.12.2013

Accepted: 24.02.2014

Table 1. Causes of hemolytic anemia in children

Causes related with erythrocytes

*  Hemoglobinopathiesr
  - Thalassemias, alpha thalassemia, beta thalassemia (major/intermedia)
   - Sickle cell anemia
   - Abnormal hemoglobins; HbC, Hb Lepore, Hb D Los
     Angeles, Hb O Arab, Hb Constant Spring, HB E
*  Erythrocyte membrane diseases
   -  Hereditary spherocytosis
   -  Hereditary eliptocytosis
   -  Hereditary piropiokilocytosis
   -  Hereditary stomatositosis
   -  Hereditary xerocytosis
*  Erythrocyte enzyme deficiencies
   -  Hexose monophosphate pathway deficiencies; G6PD
      deficiency
   -  Other gluthatione metabolism disorders; gluthatione
      synthetase and gluthatione peroxidase deficiency
   -  Glycolytic pathway deficiencies; PK, GPI, PPK, TPI, PGK,
      aldolase deficiency
   -  Erythrocyte nucleotide metabolism disorder; P-5'-N
      deficiency
*  Paroxysmal nocturnal hemoglobinuria
Extra-erythrocyte causes
*  Hemolytic anemias which develop with antibodies
   -  Warm antibodies
   -  Cold antibodies
   -  Paroxysmal cold hemoglobinuria
   -  Incompatible blood transfusion
   -  Neonatal hemolytic disease
*  Hemolysis related with drugs, chemical substances
*  Hemolysis caused by infections
*  Hypersplenism

G6PD: glucose 6phosphate dehydrogenase; PK: piruvate kinase; GPI:
glucose phosphate isomerase; PPK: phoosphofructokinase; TPI: triose
phosphate isomerase; PGK: phosphoglycerate kinase; P-5-N: Pyrimidine
5'-nucleotidase

Table 2. Properties of chronic hemolytic anemies

Disease                 Inheritance, pathology

Beta-thalassemia major  Autosomal recessive
                        Beta-chain synthesis defect
Beta-thalassemia        Autosomal recessive
intermedia              Beta-chain synthesis defect
Alpha-thalassemia       Autosomal recessive
(HbH disease)           alpha-chain synthesis defect
Sickle cell             Autosomal co-dominant
anemia                  hemoglobinopathy
Hereditary              Autosomal dominant (75%)
spherocytosis           Non-dominant inheritance
                        (%25)
                        erythrocyte membrane
                        defect
Hereditary              Autosomal dominant
eliptocytosis/HPP       Erythrocyte membrane defect
Hereditary              Autosomal dominant
stomatocytosis          erythrocyte membrane defect
Hereditary              Autosomal dominant
xerocytosis             erythrocyte membrane defect
G6PD deficiency         X-linked recessive
                        erythrocyte membrane defect
PK deficiency           Autosomal recessive
                        erythrocyte membrane defect
GPI deficiency          Autosomal recessive
                        erythrocyte membrane defect
Hexokinase              Autosomal recessive
deficiency              erythrocyte membrane defect
PPK deficiency          Autosomal recessive
                        erythrocyte membrane defect
TPI deficiency          Autosomal dominant
                        erythrocyte enzyme deficiency
PGK deficiency          Autosomal dominant
                        erythrocyte enzyme deficiency
Aldolase deficiency     Autosomal recessive
                        erythrocyte membrane defect
P-5'-N deficiency       Autosomal recessive
                        erythrocyte membrane defect
Gluthatione synthetase  Autosomal recessive
deficiency              erythrocyte membrane defect

Disease                 Clinical picture

Beta-thalassemia major  The clinal findings appear after the
                        [6.sup.th]month; deep anemia, Hb:<7 g/dL,
                        dependent on regular transfusion
                        Hepatosplenomegaly, modification in facial bones
Beta-thalassemia        The diagnosis is made after the age of 2 years
intermedia              Moderate anemia, Hb: 7-10 g/dL
                        Clinical variable: there may be no need for
                        transfusion- transfusion may be needed
Alpha-thalassemia       Findings appear in the neonatal period
(HbH disease)           Moderate anemia, Hb: 7-10 g/dL
                        Hepatosplenomegaly, gallstone, leg ulcer
Sickle cell             Clinical findings appear in the [4.sup.th]
anemia                  month at the  earliest Chronic anemia
                        progressing with acute attacks; Vasoocclusive
                        (*), aplastic and splenic sequestration crisis
Hereditary              Different clinical course; mild-moderate-deep
spherocytosis           anemia (Hb<6-12 g/dL)
                        Dependent on transfusion-asymptomatic course
                        Neonatal jaundice, rarely deep anemia
                        Splenomegaly
Hereditary              Clinical course is variable;
eliptocytosis/HPP       In utero mortality, hydrops fetalis,
                        chronic transfusion-asymptomatic course
                        Anemia; mild-moderate-deep
Hereditary              Clinical course is variable;
stomatocytosis          chronic hemolytic anemia-asymptomatric course
Hereditary              Clinical course is variable;
xerocytosis             Perinatal ascites, hydrops fetalis,
                        pseudohyperkalemia-asymptomatic course
G6PD deficiency         Neonatal jaundice
                        Acute hemolytic anemia (**)
                        Chronic anemia[dagger]
PK deficiency           Clinical picture varies with enzyme activity;
                        In utero/neonatal anemia,
                        kernicterus-anemis in adulthood
GPI deficiency          Clinical picture varies with enzyme activity;
                        Hydrops fetalis/neonatal anemia,
                        kernicterus-anemis in adulthood
                        Neurodegenerative disease, granulocyte disorders
Hexokinase              Clinical picture is variable;
deficiency              Neonatal jaundice, need for regular
                        erythrocyte transfusion-asymptomatic course
PPK deficiency          If only erythrocyte enzyme is deficient,
                        anemia in adolescence If muscle enzyme
                        is also deficient,
                        blood lactate [down arrow] after exercise
                        Myopathy, neonatal mortality
TPI deficiency          The degree of anemia is variable
                        Sever neuromuscular disease accompanies
                        (progrssive)
PGK deficiency          The degree of anemia is variable
                        Neurological finding may accompany
Aldolase deficiency     Clinical picture is variable;
                        Severe chronic hemolytic anemia-asymptomatric
                        course
                        Neurological finding may accompany
P-5'-N deficiency       The degree of anemia is variable
                        Growth retardation may be observed
Gluthatione synthetase  Hemolysis in oxidative stres
deficiency              Intermittant neutropenia, metabolic acidosis

                         Peripheral blood smear,
Disease                  Laboratory findings

Beta-thalassemia major   Anisocytosis, polychromasia,
                         target cell, normoblast
                         Hb electrophoresis; HbF >50%,
                         HbA2:< 4%
Beta-thalassemia         Anisocytosis, polychromasia, target
intermedia               cell, normoblast
                         Hb electrophoresis;
                         Hb F: 10-50%, Hb A2:> 4%
Alpha-thalassemia        Hypochromia, microcytosis, normoblast
(HbH disease)            HbH inclusion (with supravital staining)
                         Hb electrophoresis: 0.8%-40 HbH (beta4)
Sickle cell              Sickle cell, target cell
anemia                   Hb electrophoresis; Hb S and Hb F are
                         found
Hereditary               Spherocyte
spherocytosis            MCHC [up arrow]
                         OF [up arrow] (incubated test)
Hereditary               Elyptocyte (>5%), spherocyte, poikilocytosis
eliptocytosis/HPP
Hereditary                Stomatocyte (5-50%), macrocytosis
stomatocytosis
Hereditary                Xerocytosis
xerocytosis               MCV and MCH [up arrow]
                          OF [down arrow]
G6PD deficiency           Bite cells,
                          Heinz body in erythrocytes
                          (blister cell with supravital staining)
                          Erythrocyte G6PD activity [down arrow]
PK deficiency             Spiculated pyknocytes
                          Erythrocyte PK activity [down arrow]
GPI deficiency            Erythrocyte GPI activity [down arrow]
                          (below 85% of the normal level)
Hexokinase                Target cell, echinocyte
deficiency                Erythrocyte HK activity [down arrow]
PPK deficiency            Basophilic stippling
                          Erythrocyte PPK activity [down arrow]
TPI deficiency            Macrocytosis, polychromasia
                          Erythrocyte TPI activity [down arrow]
PGK deficiency            Erythrocyte PGK activity [down arrow]
Aldolase deficiency       Erythrocyte aldolase activity [down arrow]
P-5'-N deficiency         Basophilic stippling
                          Erythrocyte P-5'-N activity [down arrow]
Gluthatione synthetase    Eryhtrocyte gluthatione synthase level
deficiency                [down arrow]

G6PD: glucose 6 phosphate dehydrogenase; GPI: glucose phosphate; KH:
hexokinase; HPP: hereditary propoikilocytosis, OF: osmotic fragility;
PGK: phosphoglycerate kinase; TPI: triose phosphate isomerase
(*) Vaso-occlusive crisis; painful crisis, acute chest syndrome, acute
abdominal pain, acute central nervous system event and priapism
(**) Acute hemolytic anemia; triggered by drugs, naphtaline and
infection, causes Hb to drop to 2.5 g/dL and intravascular hemolysis
[dagger]If the enzyme level is below 1%, life-long dependence to
transfusion.