Detlef Ritter

Black children deficient in galactose 1-phosphate uridyltransferase: correlation of activity and immunoreactive protein in erythrocytes and leukocytes.

A recent study found a high prevalence of a missense mutation (S135L) in the gene for galactose 1-phosphate uridyltransferase (GALT) in black children with galactosemia (J Pediatr 1996; 128:89-95). In the present study, GALT activity and GALT protein content were measured in erythrocytes and leukocytes of eight black and seven white galactosemic (GALT-deficient) children, for correlation with the presence of the S135L and Q188R (highly prevalent in white galactosemic children) missense mutations. The S135L mutation was found in 9 of 16 alleles of black children but not in white children; the Q188R mutation was found in 10 of 14 alleles examined in white galactosemic children and in 4 of 16 alleles in black galactosemic children. The GALT activity was near zero in the erythrocytes of white and black galactosemic children (0.26 +/- 0.28 vs 0.33 +/- 0.25 mumol/hr per gram of hemoglobin, respectively; p = 0.61) (normal 17 to 26 mumol/hr per gram), and no correlation of erythrocyte activity with genotype was observed. The GALT activity was higher in the leukocytes of black galactosemic children compared with white children (5 +/- 6 vs 1 +/- 2 mumol/hr per gram, respectively) (normal 172 to 374 mumol/hr per gram), but the difference was not statistically significant (p = 0.11). Analysis by genotype revealed that the two S135L homozygotes had much more leukocyte activity (9 and 17 mumol/hr per gram) than Q188R homozygotes or than all non-S135L allelic genotypes. Compound heterozygotes (S135L/G) had intermediate activity. The GALT protein was not detectable by Western blot in the erythrocytes of either white or black galactosemic children, as determined by antibodies specific for both C- and N-terminal sequences. The GALT protein was undetectable in the leukocytes of white galactosemic children, but leukocytes from black galactosemic children with the S135L mutation contained reduced but readily detectable GALT protein. Erythrocyte galactose 1-phosphate levels were significantly lower in galactosemic children with an S135L mutant allele (1.1 +/- 0.2 gm/dl) compared with children who had other mutations (3.1 +/- 0.9 mg/dl; p = 0.0001). The correlation of protein content data with activity levels in the blood cells suggests that the S135L missense mutation affects the stability of GALT protein to produce a deficiency state.

Clinical Significance of Low-Positive Troponin I by AxSYM and ACS:180

We compared troponin I (TnI) assays (AxSYM [Abbott]; ACS:180 [Bayer]) in blood samples with concentrations less than 10 ng/mL (< 10 micrograms/L). Discordant results were evaluated by linearity studies and by testing for rheumatoid factor. Patients with discordant TnI results were compared with patients with concordant results and patients with negative TnI who had a new myocardial infarction or died within 2 months of initial testing. Positive TnI cutoffs by AxSYM and ACS:180 were 0.7 ng/mL (0.7 microgram/L) and 0.13 ng/mL (0.13 microgram/L), respectively. We identified 173 specimens that were repeatedly positive by at least 1 assay; 143 specimens were positive by both assays. Twenty samples positive for TnI by AxSYM were negative by ACS:180, while 10 samples positive by ACS:180 were negative by AxSYM. The discordant samples showed no evidence of interfering substances, including rheumatoid factor. Clinical follow-up showed that 26% of patients with elevated TnI by both assays, 33% with TnI positive only by AxSYM, 22% with TnI positive only by ACS:180, and 8% with negative TnI by AxSYM encountered at least 1 clinical end point. Variable detection rates by these assays for low-positive TnI represent a clinically significant problem.

Diagnosis of Early HIV-1 Infection

The diagnosis of acute infection with human immunodeficiency virus (HIV) presents a challenge for the primary care provider. We present a case of early HIV infection and discuss the limitations of the currently established diagnostic algorithm for HIV infection. We conclude that alternative diagnostic testing for human immunodeficiency virus type 1 (HIV-1) RNA is warranted in certain clinical settings. Appropriate, early diagnosis of HIV infection may improve the patients outcome and provide additional public health benefits by reducing transmission of disease.

Polyethylene glycol-induced immunoglobulin precipitation may cause invalidation of antiglobulin tests.

Volume 39, May 1999 TRANSFUSION 537 Mild hemolytic disease of the newborn due to anti-Goa Goa is a low-frequency blood group antigen detected in 1.9 to 2.8 percent of blacks; it is absent in whites1,2 and present on red cells (RBCs) of the partial D category IVa phenotype.1,3 Go(a–) persons may become alloimmunized to their “missing” epitopes in response to transfusion with D+ RBCs or by pregnancy with a D+ fetus. We report a case of mild hemolytic disease of the newborn (HDN) due to anti-Goa in a black male of Senegalese parents. The infant was born after an uncomplicated 36-week gestation and delivery to a gravida II, para I woman. The mother had never received a transfusion. At birth, the infant’s bilirubin and hemoglobin concentrations were 3.6 mg per dL and 18.5 g per dL, respectively. Twenty-four hours later, bilirubin concentration had increased to 4.6 mg per dL, and the hemoglobin was 17.6 g per dL. No other clinical or laboratory abnormalities were detected. A direct antiglobulin test (DAT) performed on the infant’s RBCs was positive (IgG, 3+; C3d,–). The newborn’s blood was group O Rh-positive (cDe), and the antibody screening test with the infant’s serum and several commercial reagent RBCs was negative. The mother’s blood was group O Rh-positive (cDe), and no alloantibody was detected. Tests of the mother’s serum, as well as eluates (Elu-Kit II, Gamma Biologicals, Houston, TX) from the RBCs against the father’s RBCs were positive (2+) by indirect antiglobulin test. The results did not change when the serum was treated with dithiothreitol, which suggested that the antibody was IgG. The DAT using mouse monoclonal antibodies to human IgG subclasses (PeliClass, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, the Netherlands) showed the antibody in mother’s serum to be IgG4. Blood samples from the infant and his parents were sent to a reference laboratory (American Red Cross Reference Services, Los Angeles, CA.) These results showed that the mother was Go(a–) with anti-Goa in her serum. The father and the infant were Go(a+), and eluates from infant’s RBCs contained anti-Goa. No therapy was needed, and the patient was discharged from the hospital 7 days after birth with a hemoglobin concentration of 16 g per dL and in good clinical status. One month later, the DAT was still weakly positive (IgG, +/–), and anti-Goa was still present in his eluate without any sign of hemolysis. Anti-Goa has been reported, although rarely as a cause of HDN1,4,5 or transfusion reactions.6 Severe HDN caused by anti-Goa has been reported in a mother immunized to Goa by prior pregnancies.5 In this case, although the Gonzales phenotype of her first infant is unknown, the mother was probably immunized to Goa in her previous pregnancy. However, unlike the patients reported by Leschek et al.,5 this infants clinical course was benign, and he required no specific therapy. This clinical outcome may be explained because the implicated antibody was of the IgG4 subclass. The greater hemolytic activity of anti-Goa reported by other authors may be explained by the fact that different subclasses of IgG were responsible for the hemolytic episodes. Unfortunately, the IgG subclasses were not included in these reports.1,5,6 The variable clinical activity of anti-Goa suggests that pregnant women known to be immunized to Goa should be closely followed, including testing for IgG subclass, serial antibody titers, amniotic fluid bilirubin, or fetal hemoglobin levels, to optimally determine the hemolytic activity of the antibody.

Diagnostic testing for HIV type 1 RNA in seronegative blood.

We studied the feasibility of routine diagnostic testing for HIV-1 RNA at a publicly funded testing site. HIV-1 RNA was determined with a commercial polymerase chain reaction assay in pooled seronegative blood samples submitted for HIV testing to a public health laboratory. Recovery of HIV-1 RNA from the samples was estimated as at least 8% of viral RNA that was found in freshly prepared plasma. We estimated that screening for HIV-1 RNA in serum pools would result in the identification of blood specimens from more than 95% of acutely infected patients. The frequency of HIV-1 RNA in seronegative blood samples was estimated to be between 19 and 601 per 10(6) submitted specimens. The ratio of HIV-1 RNA positive and seronegative samples to specimens with HIV-1 antibodies confirmed by Western blot was estimated to be between 0.2% and 6.6%. The reagent costs for identifying 1 HIV-infected blood sample were 10-fold higher with the commercially available HIV-1 RNA assay compared with the HIV antibody enzyme-linked immunosorbent assay. Diagnostic testing for HIV-1 RNA may be warranted in high-risk populations since acutely infected patients may benefit most from anti-retroviral therapy and are thought to contribute disproportionately to the HIV epidemic.

BIOCHEMICAL AND MOLECULAR BASIS FOR THE AFRICAN AMERICAN VARIANT OF GALACTOSEMIA. |[dagger]| 873

Early studies suggested that African American children (AA) with galactosemia developed fewer symptoms than white galactosemic children (W). The biochemical and molecular basis for this disparity has not been clarified. Galactosemia is caused by deficiency of galactose-1-phosphate uridyl transferase (GALT). In this study, GALT activity was measured in the RBC and WBC of 10 W and 8 AA. WBC activities were measured because WBC unlike RBC make GALT protein and may therefore more accurately reflect GALT activity in other tissues. GALT protein content was determined by Western blot. Mutational analysis of the gene was undertaken to determine the presence of Q188R (common mutation) and S135L (found only in AA) alleles. S135L was present in 9/16 AA alleles.