Henna: a potential cause of oxidative hemolysis and neonatal hyperbilirubinemia.

OBJECTIVE: To evaluate the in vitro oxidation potential of lawsone (2-hydroxy-1,4 naphthoquinone). Lawsone is a chemical present in henna, the crushed leaves of which are used worldwide as a cosmetic agent to stain the hair, skin, and nails. METHODOLOGY: Venous blood from glucose-6-phosphate dehydrogenase (G6PD)-normal and G6PD A- subjects were incubated with various amounts of lawsone for 2 hours at 37 degrees C. Reduced glutathione and methemoglobin (MHb) levels were measured before and after incubation. RESULTS: Final molar concentrations of lawsone in normal blood of 1.4, 2.8, 5.7, and 8.6 x 10-3 mol/L increased MHb percentages from 0.5% to 2.2%, 8.3%, 9.5% and 12.5%, respectively. In a C6PD A- blood, MHb percentages were 19.8%, 32.2%, 44.9%, and 53.9%. At a lawsone concentration of 2.8 x 10-3 mol/L, blood from 15 healthy adults formed MHb percentages of 7.4% +/- 3.3% (+/- 1 SD); in blood from 4 G6PD A- adults, percentages were 44.5%, 40.6%, 41.3%, and 42.8%. Simultaneous measurements of reduced glutathione revealed preincubation values of greater than 40 mg/100 mL of red cells in blood of healthy and G6PD A- subjects. Postincubation values were greater than 40 in blood of healthy subjects and less than 40 in blood of G6PD A- subjects. CONCLUSIONS: These in vitro observations indicate that lawsone is an agent capable of causing oxidative hemolysis. In regions of the world where there is a high incidence of G6PD deficiency and unexplained hyperbilirubinemia, oxidative hemolysis secondary to the cutaneous application of henna could be the initiating event.

Effect of erucic acid on platelets in patients with adrenoleukodystrophy.

In a clinical trial for the management of adrenoleukodystrophy, we analyzed the effect of erucic acid (a component of Lorenzo's oil) on platelet number, fatty acid composition, and function. Analysis of variance was performed to compare platelet counts before starting treatment with Lorenzo's oil and at 6 and 12 months. We measured platelet fatty acid composition in subjects and control patients and correlated these values with their platelet counts using discriminant analysis. After 6 months, the mean platelet count decreased from 247,000/mm3 to 169,000/mm3 (+/- 1 standard deviation 58,000,n =39), P < 0.0001 compared to 18 subjects on a control diet having a mean baseline platelet count of 259,000/mm3 (+/- 1 standard deviation 67,000, n = 19) and at 6 months 267,000/mm3 (+/- 1 standard deviation 71,000). We found at P < 0.05 that the platelet counts showed a strong inverse relationship with erucic acid levels and other omega 9 fatty acids that form from the administration of the erucic acid component of Lorenzo's oil. Morphologic and platelet sizing measurements suggest that the physical properties of platelets may also be affected by erucic acid. Our studies show that the ingestion of erucic acid affects platelet biology. This indicates that platelet counts and properties are influenced by monounsaturated fatty acids, in addition to the well-known effects of polyunsaturated fatty acids. In areas of the world where erucic acid is widely ingested, the biology of platelets in these populations may be affected.

Methylene blue-induced Heinz body hemolytic anemia.

OBJECTIVE: To describe the manifestations of methylene blue toxicity, with a review of the literature. DESIGN: A descriptive analysis of physical findings and significant laboratory tests in patients with methylene blue toxicity. SETTING: A pediatric referral center. PATIENTS: Two infants, one a neonate with trisomy 21 exposed to methylene blue as an intraoperative diagnostic marker and the other a neonate treated with methylene blue for type II glutaric acidemia. INTERVENTIONS: Laboratory tests to define the occurrence of methylene blue toxicity, phototherapy for hyperbilirubinemia, and transfusions for anemia. MEASUREMENTS AND RESULTS: Within hours after exposure to methylene blue, the infants voided green-blue urine, followed by hyperbilirubinemia, recurrent anemia requiring transfusions, and red blood cell dysmorphology, including the appearance of blister cells and Heinz bodies visible in both Wright's- and supravital-stained peripheral blood smears. After the initiation of phototherapy, both infants exhibited cutaneous bullae followed by desquamation. CONCLUSION: Significant neonatal morbidity may occur following postpartum administration of methylene blue. Toxic manifestations include hyperbilirubinemia, Heinz body hemolytic anemia, and possibly desquamation of the skin. In our infants toxicity was secondary to an overdose of methylene blue, as is true for most of the previously reported cases. Methods for defining the mechanism of dye-related hemolysis and simple screening tests for elucidating the unique sensitivity of certain individuals to dye toxicity are suggested.

Variable degrees of suppression of hemoglobin S synthesis in subjects with hemoglobin SS disease on a long-term transfusion regimen.

The objectives of this study were to quantify the amount of blood required to suppress synthesis of hemoglobin S (HbS) in patients with hemoglobin SS on a long-term transfusion regimen and to evaluate factors that might contribute to variations in transfusion-induced patterns of responsiveness. Eleven patients with hemoglobin SS (age range, 2 years 4 months to 19 years 9 months) who had had a cerebrovascular accident were monitored during a period of 1 1/2 to 4 years for HbS percentages, reticulocyte percentages, the amount of erythrocytes infused, and weight. From these data the amount of blood necessary to maintain the HbS concentration at less than 30% was expressed as units of packed erythrocytes administered per week per kilogram of body weight. Percentage of HbS were significantly lower in three subjects than in the other eight (6.1 +/- 0.6 vs 23.0 +/- 2.1; p = 0.0009) as were the reticulocyte percentages (2.9 +/- 0.3 vs 7.9 +/- 0.7; p = 0.0021). However, there were no significant differences between pretransfusion hematocrit (0.278 +/- 0.012 vs 0.281 +/- 0.01; p = 0.90) and units of erythrocytes given per week per kilogram (0.0147 +/- 0.0008 vs 0.0156 +/- 0.0009; p = 0.58). Factors explored to define the reason that HbS synthesis was more easily suppressed in some patients than in others included measurements of serum chemistry values and erythropoietin, identification of erythrocyte alloantibodies, and a survey for Howell-Jolly bodies. No significant differences were seen. Although the reasons for the marked variation in transfusion-induced depression of HbS synthesis are unclear, this study emphasizes the importance of determining the units of packed erythrocytes needed per week per kilogram and correlating this value with the pretransfusion HbS percentage. By doing so, one can select the minimal amount of blood necessary to achieve the desired HbS percentage and thereby decrease the risks of transfusion.

Twenty-nail dystrophy associated with hematologic abnormalities.

This report describes a 15 1/2 year old white male with twenty-nail dystrophy who has had recurrent episodes of immune thrombocytopenic purpura, autoimmune hemolytic anemia, and mild depression of immunoglobulin levels. The concurrence of these events suggests that each shares a common pathophysiologic mechanism, possibly an autoimmune process.

Hb Catonsville (glutamic acid inserted between Pro-37(C2)alpha and Thr-38(C3)alpha). Nonallelic gene conversion in the globin system?

Hb Catonsville is an unstable variant in which glutamic acid is inserted into the alpha-globin chain between Pro-37(C2) and Thr-38(C3). The peptide sequence data are consistent with the DNA sequence of the polymerase chain reaction-amplified fragment of the variant globin gene, which shows the insertion of the triplet codon--GAA--into the mutant alpha-globin gene. In the normal alpha-globin gene cluster the codon for glutamic acid is GAG rather than GAA. Thus, there are two features unique to Hb Catonsville, one the insertion of a single residue into the interior of the alpha-globin chain, and two the presence of the alternate codon for glutamic acid. The experimental evidence suggests that Hb Catonsville may be an example of nonhomologous nonallelic gene conversion, an observation not previously reported in this gene family. The mutation occurs in the critical alpha 1 beta 2 interface of the hemoglobin tetramer and leads to a variant with high oxygen affinity, a reduced cooperativity, and Bohr effect.

Unstable hemoglobins: influence of environment on phenotypic expression of a genetic disorder.

Unstable hemoglobins account for approximately one fifth of the abnormal hemoglobins identified to date. Clinical expression ranges from a continuing and severe hemolytic anemia which may or may not be abated by splenectomy to a compensated hemolytic state with exacerbations of hemolysis after exposure to drugs or during febrile episodes. Other clinical indicators of the disorder include the voiding of urine darkened by the presence of dipyrroles, as well as intermittent episodes of scleral icterus. Recognition and definition of the unstable hemoglobins can be difficult. Approximately one third are spontaneous mutations; many are neutral substitutions, so that routine electrophoretic methods are of limited value; and in some the molecule is so unstable that it is lost during preparative techniques employed for its identification. Most informative are those methods designed to demonstrate the instability of the molecule, including the exposure of red cells or hemolysate to heat, redox dyes, or isopropanol. Each of these tests is easily performed and should be employed in the evaluation of any patient with an unexplained hemolytic disorder, and especially in those patients with drug-or hyperthermic-induced hemolysis. A review of red cell indices of our patients, together with those reported in the literature, indicates the subjects with moderate to severe forms of the unstable hemoglobinopathies have macrocytosis with abnormally low MCHCs. Incubating normal or Hb Zürich red cells in vitro at 37 degrees and 41 degrees C effects similar changes in the red cells, the phenomenon occurring more rapidly in Hb Zürich than in normal cells. The phenotypic expression of the unstable hemoglobinopathies may be influenced by a variety of environmental factors, including smoking, pyrexia, exposure to oxidants, and the presence or absence of splenic function. Especially noteworthy is the effect of smoking on the clinical and laboratory findings in subjects with Hb Zürich. As a result of the 65-fold increase in the affinity of the abnormal beta-globin chain of Hb Zürich for CO, the molecule is stabilized, followed by resistance to oxidative injury, a decreased rate of hemolysis, and a marked increase in oxygen affinity.

Homozygous protein C deficiency: observations on the nature of the molecular abnormality and the effectiveness of warfarin therapy.

An infant with severe homozygous protein C deficiency was brought to medical attention because of purpura fulminans and severe bilateral vitreous hemorrhages in the neonatal period. Infusions of fresh frozen plasma were given for 8 months. On two occasions, attempts to decrease the frequency of fresh frozen plasma infusions to less than twice a day led to episodes of microangiopathic hemolysis, fibrinolysis, and acute renal failure. Infarction of skin and subcutaneous tissues did not recur. Both episodes were controlled after reinstitution of fresh frozen plasma. Complications of therapy with fresh frozen plasma included hyperproteinemia and hypertension. Warfarin therapy was instituted when the baby was 8 months of age, followed by a gradual withdrawal of fresh frozen plasma therapy. The dose of warfarin required to maintain the prothrombin time in a range of 1.8 to 2.2 times normal varied considerably during short periods, a phenomenon that may have been due to several factors: hypercatabolism of the drug with prolonged administration, abnormality of liver function, variation in levels of serum albumin, fluctuations in drug dosage secondary to oral administration, and variations in dietary vitamin K. Protein C determinations by immunologic and functional assays consistently showed detectable but reduced protein C antigen levels with undetectable activity levels, suggesting that a dysproteinemia rather than a deficiency of synthesis is responsible for the child's coagulopathy.

Kasabach-Merritt syndrome: therapeutic considerations.

During the past 15 years we have managed six children with capillary hemangiomas in association with consumptive coagulopathy--the Kasabach-Merritt syndrome. Their ages when first seen ranged from 2 weeks to 4 years with a mean of 19 months. In three of the patients, the hemangiomas remained small for many months and then suddenly enlarged and hemorrhagic diatheses appeared. The duration of the thrombocytopenia ranged from 5 to 20 months, a time span that in some of the patients was influenced by the type of treatment used. Each patient received a variety of therapies including the following: medications to control the coagulopathy; mechanical, cytolytic, and pharmacologic treatment to eradicate the lesion; and blood product support with platelets and cryoprecipitate for severe bleeding. A prompt elevation of the platelet count occurred in three of the patients after the lesion was biopsied. Subtotal resection of the lesion resulted in an immediate increase of the platelet count in one patient. A transient increase in platelet counts and fibrinogen levels was observed in another patient following several embolizations of a portion of the hemangioma. Other modes of therapy were difficult to evaluate, especially because the same therapy applied at different times in the same patient effected a different type of response. Eventually, all of the patients experienced resolution of their lesions and, with this, concomitant reversal of the coagulopathy.

Farber disease: pathologic diagnosis in sibs with phenotypic variability.

We describe new pathologic findings in two sibs with Farber lipogranulomatosis. The first child, a 3-month-old boy, presented with only hepatosplenomegaly and had a fulminant clinical course suggestive of malignant histiocytosis. The second child, a 5 1/2-month-old girl, had the typical clinical presentation of Farber disease, with hoarseness and painful swollen joints. At autopsy, storage material was demonstrated in the second child at laryngeal and periarticular subcutaneous sites. Visceral involvement was prominent in both sibs, although not typical of the disease, and included a newly described nephropathy with elevated urine ceramide levels. Liver and spleen contained massive histiocytic infiltrates in association with elevated ceramide levels. Lymph nodes also contained histiocytic infiltrates but without the sinusoidal involvement typical of proliferative histiocytic disorders. These two cases demonstrate new pathologic anomalies in Farber disease, indicating that biochemical analyses of biopsy specimens may be necessary to establish the diagnosis of Farber disease when atypical clinical and morphologic anomalies are present.

Generalized venocentric lesions in the virus-associated hemophagocytic syndrome.

Clinical and autopsy findings in a 41/2-year-old boy who had been healthy until the onset of virus-associated hemophagocytic syndrome are presented. In addition to findings previously reported in association with the syndrome, widely disseminated perivenular expansile nodules of bland necrosis were seen. These nodules were suggestive of vessel injury, with extravasation of vascular contents and focal destruction of normal elements. The morphologic similarities of these nodules to early lesions of pulmonary veno-occlusive disease are discussed.

Partial deficiency of erythrocyte spectrin in hereditary spherocytosis.

Hereditary spherocytosis (HS) is a common, clinically heterogeneous haemolytic anaemia in which the primary erythrocyte defect is believed to be some abnormality in the spectrin-actin membrane skeleton, leading to loss of surface membrane. Recessively inherited spectrin deficiency with extreme erythrocyte fragility and spherocytosis has been identified in certain mutant mice and two severely anaemic humans. Although suspected, deficiency of spectrin has not been demonstrated in less severe forms of human HS. We not report the quantitation of erythrocytes spectrin by radioimmunoassay. We found that normal erythrocytes contained 240,000 copies of spectrin heterodimer, whereas erythrocytes from 14 patients with a variety of types of HS were all partially deficient in spectrin (range 74,000-200,000 copies), the magnitude of the deficiency correlating with the severity of the disease. Spectrin deficiency of varying degrees is common in HS and probably represents the principal structural defect leading to loss of surface membrane.

Gonadal function in two siblings with Fanconi's anemia.

2 siblings with Fanconi's anemia, 1 male and 1 female, aged 22 and 24 years, respectively, were evaluated at the Johns Hopkins Hospital because of short stature and hypogonadism. Plasma levels of somatomedin-C were normal in both patients, suggesting that the production of biologically active growth hormone was normal in these subjects. In addition, measurements of serum gonadotropins and plasma androgens in our patients, along with data accumulated from previous studies in the literature, show that abnormal sexual development in patients with Fanconi's anemia is due to hypergonadotropic hypogonadism.

Unique sensitivity of Hb Zürich to oxidative injury by phenazopyridine: reversal of the effects by elevating carboxyhemoglobin levels in vivo and in vitro.

Phenazopyridine (PAP) causes a hemolytic anemia in normal individuals who receive an overdose or in patients with decreased renal function given therapeutic doses. There are no reports of PAP-induced hemolysis in individuals with unstable hemoglobins. Therapeutic doses of PAP administered to a subject with Hb Zürich (His E7 (63) beta leads to Arg) caused a severe hemolytic anemia with many large Heinz bodies appearing in the red cells. Incubation of whole blood from three asymptomatic Hb Zürich subjects with PAP at a molar ratio of PAP/Hb of 1.3/1 produced a moderate to marked increase in methemoglobin (MHb) and Heinz body formation accompanied by a slight to moderate decrease in levels of reduced glutathione. The rates of MHb formation were proportional to the concentration of PAP. In two of the subjects the rates of formation of Heinz bodies and MHb were substantially higher than in the third subject. Red cells from five normal adults were not affected. In Hb Zürich red cells the affinity of the abnormal beta chain for carbon monoxide is much greater than that of the normal alpha and beta chains. The two subjects with higher rates of MHb and Heinz body formation were nonsmokers with in vivo carboxyhemoglobin (HbCO) levels of 4-6%. The third subject with low rates of MHb and Heinz body formation was a smoker with in vivo HbCO levels of 15-18%. Increasing levels of HbCO from 8.2% to 14.3% by the in vitro addition of CO caused a marked reduction in the rate of Heinz body formation and a moderate decrease in MHb formation. Rates of MHb formation similar to those observed in normal red cells occurred at HbCO percentages of 89.2 and 99.2. Red cells containing Hb Zürich are extremely sensitive to oxidative injury by PAP, either in vivo or in vitro. The degree of oxidative injury diminishes as the level of HbCO increases, a phenomenon that is enhanced by preferential binding of CO to the abnormal beta subunit of Hb Zürich.

Hemoglobin Cheverly: an unstable hemoglobin associated with chronic mild anemia.

The evaluation of a family with chronic mild anemia led to the identification of a new unstable hemoglobin (Hemoglobin Cheverly). Modest anemia and reticulocytosis, normal to slightly increased mean corpuscular volume (MCV), and normal mean corpuscular hemoglobin concentration (MCHC) were present in the affected family members. Electrophoresis of blood samples on cellulose acetate and on citrate agar revealed normal patterns. Globin chain analysis and isoelectric focusing data were also normal. After incubation for 3 h at 41 degrees C, Heinz bodies were detected in 95-100% of erythrocytes from affected individuals. Positive heat and isopropanol tests confirmed the initial observation of the Heinz body preparation and indicated that an unstable hemoglobin was present. Structural analysis showed an amino acid substitution of Phe-Ser at position 45 (CD4) in the beta chain. Hemoglobin Cheverly has a reduced affinity for oxygen and a reduced Bohr effect, properties that can be rationalized on the basis of the x-ray crystallographic structure of normal hemoglobin. Despite structural and functional similarities between Hb Cheverly and Hb Hammersmith, beta 42 (CD1) Phe-Ser, the clinical manifestations of Hb Cheverly are mild in contrast to the severe disease observed with Hb Hammersmith. Reasons for the apparently silent clinical expression of Hb Cheverly are not known. We discuss the implications of unstable hemoglobins in the evaluation of chronic anemia in pediatric patients.

Abnormalities of the spleen and liver in patients with hemophilia.

Previous studies have demonstrated functional and histologic abnormalities of the liver, and, more recently, splenomegaly in patients with hemophilia. Since these observations usually were derived from hemophiliacs who had received intensive replacement therapy, the question was posed as to whether the frequency of splenic and hepatic abnormalities was secondary to the amount of therapy utilized. In this study, a variety of tests were employed to evaluate spleen and liver size and function to determine if abnormalities in these organs correlated with the intensity of the transfusion program. The study group was comprised of 25 hemophiliacs (mean factor replacement-18,361 U/year; median factor replacement-12,920 U/year). Over 70% of our patients had elevations of aspartate and alanine aminotransferase. Immunoglobulin and complement levels were normal in most subjects. Ninety-six percent had evidence of exposure to hepatitis B virus. Liver-spleen imaging suggested significant hepatic abnormalities in most of the patients as evidenced by inhomogeneity of tracer uptake in the liver in 33% and relatively increased colloid uptake in the spleen in 90%. Splenomegaly (palpable spleen or enlargement on liver-spleen imaging) was detected in 40% of our patients, and tended to occur in the more frequently transfused patients. These findings indicate that significant abnormalities of the spleen and liver can occur in hemophiliacs who have received moderate amounts of replacement therapy and that liver-spleen imaging may be a useful method for monitoring the development of hepatic and splenic abnormalities.

Relation between variations in the phenotypic expression of an unstable hemoglobin disorder (hemoglobin Zürich) and carboxyhemoglobin levels.

Longitudinal studies in a group of 15 asymptomatic subjects with hemoglobin Zürich from two unrelated families indicated marked variation in the clinical and hematologic expression of the hemoglobinopathy, even within the same family. Carbon monoxide bound to normal hemoglobin stabilized the molecule when exposed to a variety of denaturing procedures. The marked increase in the affinity of hemoglobin Zürich for carbon monoxide, approximately 65 times that of normal hemoglobin, led to a study of carboxyhemoglobin levels in smoking and nonsmoking subjects with hemoglobin Zürich and their relation to the phenotypic expression of the hemoglobinopathy. The carboxyhemoglobin content in whole blood from persons with hemoglobin Zürich ranged from 3.9 to 6.7 percent in nine nonsmokers and from 9.8 to 19.7 percent in six smokers. Rates of hemolysis as determined by values for hematocrit, reticulocyte count, and haptoglobin and hemopexin levels were less in smokers than in nonsmokers. Also, rates of Heinz body formation in intact red cells and isopropanol-induced precipitation of the abnormal hemoglobin in hemolysates were less in smokers than in non-smokers. These observations suggest that variability in the phenotypic expression of hemoglobin Zürich is a post-translation event secondary to stabilization of the abnormal hemoglobin by carbon monoxide.

Prognostic role of the intravenous urogram in children with acute leukemia.

We retrospectively studied intravenous urograms (IVU) performed at presentation on 108 children with acute leukemia in order to determine whether any association existed between an abnormal IVU and known prognostic factors at presentation, initial renal function, and outcome. Sixteen patients (14%) had abnormal IVUs. These patients were compared to the remainder of the group and found to be comparable in respect to known prognostic factors at diagnosis (P greater than 0.05). No correlation was found between abnormal renal function and an abnormal IVU. Renal enlargement was associated with shorter survival only in patients who had both adenopathy and hepatosplenomegaly (P less than 0.01). This effect did not persist in any other group of patients examined. When subjected to multivariate analysis the IVU did not significantly influence relapse or survival in these patients. Renal enlargement may be a prognostic feature in some patients with childhood leukemia. Nevertheless routine evaluation by IVU is discouraged because of potential adverse affects of urographic contrast media and the availability of noninvasive ultrasound techniques.