ABSTRACT
The measurement of free hemoglobin (free Hb) in blood is crucial for assessing the risk of organ damage in patients with hemolytic diseases. However, the colorimetric method, commonly used in clinical practice, does not distinguish between free Hb and the hemoglobin-haptoglobin complex (Hb-Hp) in the blood, instead reflecting the total Hb level. Although size-exclusion high-performance liquid chromatography (SEC-HPLC) can specifically measure free Hb, its clinical use is limited by long assay times. Here, we developed a novel assay method for the rapid quantification of free Hb in serum, distinguishing it from Hb-Hp, using a latex agglutination immunoturbidimetric assay (LATIA). This method could be used to measure free Hb in sera in the range of 1-100 µg/mL in approximately 15 min using an automatic biochemistry analyzer. Using Hb-spiked serum samples from healthy adults, there was a high correlation with Hb levels determined using the newly developed method and SEC-HPLC, indicating a high specificity for free Hb. This novel assay can be used to monitor levels of free Hb in patients with various hemolytic diseases and to design therapeutic strategies based on measured values. However, further studies are required to assess its clinical performance.
ABSTRACT
The fecal immunochemical test for hemoglobin (FIT), which detects lower gastrointestinal bleeding, is widely accepted for population-based colorectal cancer (CRC) screening programs. However, the FIT screening process has not been standardized yet, and standardizing the pre-analytical phase and establishing an external quality assurance (EQA) program compliant with ISO requirements is urgently needed. Although there have been various attempts to establish EQA materials suitable for FIT, no materials have yet been reported to have sufficient uniformity and acceptable immunochemical stability of hemoglobin (Hb). The Health Care Technology Foundation (HECTEF; Tokyo Japan) is now developing a ready-to-use artificial stool containing Hb and an internal standard, glycerol. Accordingly, we verified the adaptability and efficacy of this material for the evaluation of the specimen collection phase of FIT. This material uniformly contained both Hb and glycerol. The glycerol allowed us to estimate the weight of the collected artificial stool and to correct the Hb concentration with the estimated weight. Furthermore, the stability of both Hb and glycerol were confirmed to be sufficient for an EQA material under appropriate storage, in-use, repeated freeze-thaw, and heated conditions. These in-house performance characteristics suggest that HECTEF artificial stool is acceptable as an EQA material for FIT.
Subject(s)
Clinical Chemistry Tests/standards , Feces/chemistry , Immunochemistry/standards , Colorectal Neoplasms/diagnosis , Occult Blood , Quality Control , Reference StandardsABSTRACT
Hepatitis B virus (HBV) infections are sometimes overlooked when using commercial kits to measure hepatitis B virus surface antigen (HBsAg) due to their low sensitivities and reactivities to mutant strains of various genotypes. We developed an ultrasensitive bioluminescent enzyme immunoassay (BLEIA) for HBsAg using firefly luciferase, which is adaptable to a variety of HBsAg mutants, by combining four monoclonal antibodies with a polyclonal antibody against HBsAg. The measurement of seroconversion panels showed trace amounts of HBsAg during the early infection phase by the BLEIA because of its high sensitivity of 5 mIU/ml. The BLEIA detected HBsAg as early as did PCR in five of seven series and from 2.1 to 9.4 days earlier than commercial immunoassay methods. During the late infection phase, the BLEIA successfully detected HBsAg even 40 days after the disappearance of HBV DNA and the emergence of antibodies against HBsAg. The HBsAg BLEIA successfully detected all 13 recombinant HBsAg and 45 types of HBsAg mutants with various mutations within amino acids 90 to 164 in the S gene product. Some specimens had higher values determined by the BLEIA than those by a commercial chemiluminescent immunoassay; this suggests that such discrepancies were caused by the dissociation of preS1/preS2 peptides from the particle surface. With its highly sensitive detection of low-titer HBsAg, including various mutants, the HBsAg BLEIA is considered to be useful for the early diagnosis and prevention of HBV infection because of the shorter window of infection prior to detection, which facilitates early prediction of recurrence in HBV-infected individuals.
