Can the Afinion HbA1c Point-of-Care instrument be an alternative method for the Tosoh G8 in the case of Hb-Tacoma?

BACKGROUND: Hb-variant interference when reporting HbA1c has been an ongoing challenge since HbA1c was introduced to monitor patients with diabetes mellitus. Most Hb-variants show an abnormal chromatogram when cation-exchange HPLC is used for the determination of HbA1c. Unfortunately, the Tosoh G8 generates what appears to be normal chromatogram in the presence of Hb-Tacoma, yielding a falsely high HbA1c value. The primary aim of the study was to investigate if the Afinion HbA1c point-of-care (POC) instrument could be used as an alternative method for the Tosoh G8 when testing for HbA1c in the presence of Hb-Tacoma. METHODS: Whole blood samples were collected in K2EDTA tubes from individuals homozygous for HbA (n = 40) and heterozygous for Hb-Tacoma (n = 20). Samples were then immediately analyzed with the Afinion POC instrument. After analysis, aliquots of each sample were frozen at -80 °C. The frozen samples were shipped on dry ice to the European Reference Laboratory for Glycohemoglobin (ERL) and analyzed with three International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and National Glycohemoglobin Standardization Program (NGSP) Secondary Reference Measurement Procedures (SRMPs). The Premier Hb9210 was used as the reference method. RESULTS: When compared to the reference method, samples with Hb-Tacoma yielded mean relative differences of 31.8% on the Tosoh G8, 21.5% on the Roche Tina-quant Gen. 2 and 16.8% on the Afinion. CONCLUSIONS: The Afinion cannot be used as an alternative method for the Tosoh G8 when testing for HbA1c in the presence of Hb-Tacoma.

Validation of a Fast, Robust, Inexpensive, Two-Tiered Neonatal Screening Test algorithm on Dried Blood Spots for Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is one of the leading genetic causes of infant mortality with an incidence of 1:10,000. The recently-introduced antisense oligonucleotide treatment improves the outcome of this disease, in particular when applied at an early stage of progression. The genetic cause of SMA is, in >95% of cases, a homozygous deletion of the survival motor neuron 1 (SMN1) gene, which makes the low-cost detection of SMA cases as part of newborn screening programs feasible. We developed and validated a new SALSA MC002 melting curve assay that detects the absence of the SMN1 exon 7 DNA sequence without detecting asymptomatic carriers and reliably discriminates SMN1 from its genetic homolog SMN2 using crude extracts from newborn screening cards. Melting curve analysis shows peaks specific for both the SMN1 gene and the disease modifying SMN2 homolog. The detection of the SMN2 homolog, of which the only clinically relevant difference from the SMN1 gene is a single nucleotide in exon 7, was only used to confirm a correct reaction in samples that lacked the SMN1 gene, and not for SMN2 quantification. We retrieved 47 DBS samples from children with genetically-confirmed SMA, after informed consent from parents, and 375 controls from the national archive of the Dutch National Institute for Public Health and the Environment (RIVM). The assay correctly identified all anonymized and randomized SMA and control samples (i.e., sensitivity and specificity of 100%), without the detection of carriers, on the three most commonly-used PCR platforms with melting curve analysis. This test's concordance with the second-tier 'golden standard' P021 SMA MLPA test was 100%. Using the new P021-B1 version, crude extracts from DBS cards could also be used to determine the SMN2 copy number of SMA patients with a high level of accuracy. The MC002 test showed the feasibility and accuracy of SMA screening in a neonatal screening program.

Adult-onset congenital thrombotic thrombocytopenic purpura caused by a novel compound heterozygous mutation of the ADAMTS13 gene.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease, characterized by microangiopathic hemolytic anaemia and thrombocytopenia, resulting in neurologic and/or renal abnormalities. We report a 49-year-old patient with a history of thrombotic events, renal failure, and thrombocytopenia. Blood analysis demonstrated no ADAMTS13 activity in the absence of antibodies against ADAMTS13. The complete ADAMTS13 gene was sequenced, and two mutations were identified: one mutation on exon 24 (Arg1060Asp), which had previously been described, and a mutation on exon 27 (Met1260IlefsX34), which has not been reported. For these mutations, compound heterozygosity appears to be necessary to cause TTP, as family members of the patient display only one of the mutations and all displayed normal ADAMTS13 activity.

ZAP70 in B-CLL cells related to the expression in NK cells is a surrogate marker for mutational status.

The strongest prognostic factor in chronic B-cell lymphocytic leukemia (CLL) is the mutational status of the immunoglobulin heavy chain variable region (IGHV) genes. Determination of this mutational status is laborious and therefore not applied in routine diagnostics. A search for "surrogate markers" has been conducted over the past few years. One of the most promising surrogate markers is ZAP70, but standardization of the measurement of ZAP70 has proven to be difficult. Conventionally, ZAP70 expression in CLL cells is related to ZAP70 expression in T cells. We propose a new method in which ZAP70 expression in NK cells is used as reference (new NK-MFI method). We have measured ZAP70 expression in samples of 45 previously untreated CLL patients. ZAP70 in CLL cells related to ZAP70 in NK cells correlated better to cytogenetic risk profile and mutational status than the conventional methods. Negativity of both ZAP70 (new NK-MFI method) and CD38 resulted in a probability of 90% for mutated IGHV genes. In conclusion, ZAP70 expression in CLL cells related to ZAP70 expression in NK cells is a better surrogate marker for mutational status than the conventional T cell related methods.

ZAP70 in B-CLL cells related to the expression in NK cells is a surrogate marker for mutational status.

The strongest prognostic factor in chronic B-cell lymphocytic leukaemia (CLL) is the mutational status of the immunoglobulin heavy chain variable region (IGHV) genes. Determination of this mutational status is laborious and therefore not applied in routine diagnostics. A search for 'surrogate markers' has been conducted over the past few years. One of the most promising surrogate markers is ZAP70, but standardisation of the measurement of ZAP70 has proven to be difficult. Conventionally, ZAP70 expression in CLL cells is related to ZAP70 expression in T cells. We propose a new method in which ZAP70 expression in NK cells is used as reference (new NK-MFI method). We have measured ZAP70 expression in samples of 45 previously untreated CLL patients. ZAP70 in CLL cells related to ZAP70 in NK cells correlated better to cytogenetic risk profile and mutational status than the conventional methods. Negativity of both ZAP70 (new NK-MFI method) and CD38 resulted in a probability of 90% for mutated IGHV genes. In conclusion, ZAP70 expression in CLL cells related to ZAP70 expression in NK cells is a better surrogate marker for mutational status than the conventional T cell related methods. © 2013 Clinical Cytometry Society.

Novel molecular defect in the platelet ADP receptor P2Y12 of a patient with haemorrhagic diathesis.

BACKGROUND: The platelet adenosine 5'-diphosphate (ADP) receptor P2Y(12) plays a crucial role in haemostasis. Only a few patients with haemorrhagic diathesis due to molecular defects in the P2Y(12) receptor have been described so far. We report a novel molecular defect in the gene coding for P2Y(12) in a patient with a history of epistaxis, easy bruising and excessive posttraumatic blood loss. METHODS: Platelet aggregation studies, perfusion studies, in which patient blood was perfused over collagen surfaces at arterial shear rates, and PCR and sequencing were used. RESULTS: Platelet aggregation studies showed impaired ADP and collagen-induced aggregation for patient G.S. Perfusion of patient blood over collagen surfaces showed small thrombi consisting of spread platelets overlayered with non-spread platelets. These thrombi were identical to control thrombi formed in the presence of a P2Y(12) antagonist. DNA analysis of the P2Y(12) gene revealed a novel heterozygous base pair C-->A substitution in exon 3, changing codon 258 from proline to threonine in the third extracellular loop of the P2Y(12) receptor. CONCLUSIONS: We conclude that perfusion studies with patient blood are of added value in the diagnostic process, which resulted in identification of a novel molecular defect in the P2Y(12) gene of a patient with haemorrhagic diathesis.