Age dependent discrepancy between SARS-CoV-2 anti-nucleoprotein antibody and anti-RBD spike protein antibody in children reflects vaccine coverage.

BACKGROUND: COVID-19 has become widespread in Japanese children. However, the impact of varying immunization coverage on the seroprevalence of SARS-CoV-2 in children is unknown. METHODS: We examined the SARS-CoV-2 antibody in children aged 0 to 18 who were hospitalized at a university hospital from June 2020 through May 2023. The SARS-CoV-2 anti-nucleoprotein (N) antibody and anti-RBD spike (S) protein antibody was measured. RESULTS: A total of 586 cases were enrolled. The median age was 4 years old (interquartile range 1-9), and 362 (61.8 %) were male. The seroprevalence of anti-S antibodies gradually increased from October 2021 and reached 60 percent by early 2023. The anti-N antibody increased starting in January 2022 and reached 50 percent in May 2023. There was a discrepancy in the seroprevalence of anti-S and N antibodies in children 0 years of age or 12 years and older until the fall of 2022. This discrepancy was minimal for children 1-4 years of age and relatively small in the 5-11-year-old group. DISCUSSION: The data suggests that approximately half of the children in our cohort had been infected with SARS-CoV-2 by May 2023. The discrepancy in seropositivity between the anti-S and N antibodies corresponded to the reported vaccine uptake of each target age group, which suggested protective effects of immunization. However, this effect appeared to diminish after early 2023. CONCLUSION: Age dependent discrepancy between SARS-CoV-2 anti-N and anti-S antibody in children reflected differences in vaccine coverage.

Cycle Threshold (Ct) Values of SARS-CoV-2 Detected with the GeneXpert® System and a Mutation Associated with Different Target Gene Failure.

SARS-CoV-2 nucleic acid detection tests enable rapid virus detection; however, it is challenging to identify genotypes to comprehend the local epidemiology and infection routes in real-time qRT-PCR. At the end of June 2022, our hospital experienced an in-hospital cluster of COVID-19. When examined using the GeneXpert® System, the cycle threshold (Ct) value of the N2 region of the nucleocapsid gene of SARS-CoV-2 was approximately 10 cycles higher than that of the envelope gene. Sanger sequencing revealed a G29179T mutation in the primer and probe binding sites. A review of past test results revealed differences in Ct values in 21 of 345 SARS-CoV-2-positive patients, of which 17 cases were cluster-related and 4 were not. Including these 21 cases, 36 cases in total were selected for whole-genome sequencing (WGS). The viral genomes in the cluster-related cases were identified as BA.2.10, and those in the non-cluster cases were closely related and classified as being downstream of BA.2.10 and other lineages. Although WGS can provide comprehensive information, its use is limited in various laboratory settings. A measurement platform reporting and comparing Ct values of different target genes can improve test accuracy, enhance our understanding of infection spread, and be applied to the quality control of reagents.

[Evaluation of the Fundamental Performance of 4 Latex Agglutination Reagents to Measure Anti-TP Antibody Concentration and Detailed Investigation of Decision-Mismatched Samples].

Serological diagnosis of syphilis can be made by using the serological test for syphilis (STS) method for detecting a lipid antibody and Treponema pallidum (TP) method for detecting the anti-TP-specific antibody. In STS and TP methods, the basis using latex agglutination reaction has been used in many facilities. However, in latex agglutination, false-positive results due to non-specific reaction have sometimes been obtained in reactions of a routine laboratory test reagent detecting the anti-TP antibody used in our medical laboratory. We evaluated the fundamental performance of 4 reagents to measure anti-TP antibody concentration using latex agglutination: Reagents A, B, C and D produced by SEKISUI MEDICAL, FUJI REBIO, DENKA SEIKEN and SHINO TEST, respectively. We examined the correlations between Reagent A (routine laboratory test reagent) and Reagents B, C, and D in sera from 68 patients, and we performed additional investigation by using a neutralization test, immunochromatography, Western blotting, FTA-ABS (IgG), and STS method by an automatic analyzer for 13 decision-mismatched samples. The fundamental performance of each reagent was as good as that previously reported. Eight of the 13 decision-mismatched samples were false positives due to non-specific reaction of Reagent A. In latex agglutination non-specific reaction is inevitable. However, this study strongly suggests that using a neutralization test and immunochromatography that can be performed quickly is sufficient to verify whether positive reactions are true or false.

[Evaluation of Basic Performance of "Point Strip ferritin-3000" for Simple and Rapid Quantification of Serum Ferritin].

Serum ferritin is an excellent marker for total iron content in the body and is essential for the diagnosis of iron deficiency or iron overload. Recently, a simple and rapid method, which utilizes immunochromatography for the quantification of serum ferritin, was developed. However, the range of measurement in previous reagents was limited (10-500 ng/mL). This range is rather narrow and is not fully helpful for the diagnosis of iron overload which sometimes occurs as a result of prolonged transfusions, or for monitoring iron contents during iron chelation therapy against iron overload. In the present study we evaluated the basic performance of the newly developed "Point Strip ferritin-3000", which can measure serum ferritin in the range of 300-3,000 ng/mL. Coefficient of variation (CV) s of within and inter-day assays were in the ranges of 7.3-11.1% and 2.1-5.2%, respectively. Using 87 serum samples obtained from the patients with written informed consents, the correlation coefficient was calculated to be 0.93 compared to the control method. In addition, the quantification of serum ferritin by "Point Strip ferritin-3000" was not influenced by bilirubin, hemoglobin, chyle, rheumatoid factor, or ascorbic acid. From our data, "Point Strip ferritin-3000" is reliable reagent in the range of 300-3,000 ng/mL, and is therefore considered to be useful for the diagnosis of iron overload, as well as for monitoring iron contents during iron chelation therapy. In addition, this quantification method can be easily performed using a small desktop equipment without any special technique, making this system applicable for epidemiological surveys and clinical studies.

Development of a highly sensitive three-dimensional gel electrophoresis method for characterization of monoclonal protein heterogeneity.

Three-dimensional gel electrophoresis (3-DE), which combines agarose gel electrophoresis and isoelectric focusing/SDS-PAGE, was developed to characterize monoclonal proteins (M-proteins). However, the original 3-DE method has not been optimized and its specificity has not been demonstrated. The main goal of this study was to optimize the 3-DE procedure and then compare it with 2-DE. We developed a highly sensitive 3-DE method in which M-proteins are extracted from a first-dimension agarose gel, by diffusing into 150 mM NaCl, and the recovery of M-proteins was 90.6%. To validate the utility of the highly sensitive 3-DE, we compared it with the original 3-DE method. We found that highly sensitive 3-DE provided for greater M-protein recovery and was more effective in terms of detecting spots on SDS-PAGE gels than the original 3-DE. Moreover, highly sensitive 3-DE separates residual normal IgG from M-proteins, which could not be done by 2-DE. Applying the highly sensitive 3-DE to clinical samples, we found that the characteristics of M-proteins vary tremendously between individuals. We believe that our highly sensitive 3-DE method described here will prove useful in further studies of the heterogeneity of M-proteins.

Evaluation of glycated hemoglobin and fetal hemoglobin-adjusted HbA1c measurements in infants.

BACKGROUND: As the presence of fetal hemoglobin (HbF) affects the accuracy of hemoglobin A1c (HbA1c) analysis methods, HbA1c measurement may not be a good indicator for patients with neonatal diabetes mellitus, whereas glycated albumin (GA) may be a good indicator. OBJECTIVE: To investigate whether total glycated hemoglobin (GHb) or HbF-adjusted HbA1c (adj-HbA1c) can act as a glycemic control marker in infants. SUBJECTS AND METHODS: Plasma glucose (PG), GA, HbF, GHb measured using the affinity method, and HbA1c measured using the latex-immunoturbidimetry (LA) or the high-performance liquid chromatography (HPLC) methods were determined in 26 full-term newborn infants aged 4-234 d. Adj-HbA1c was calculated as HbA1c/(total Hb - HbF). RESULTS: GHb, adj-HbA1c measured using the LA and the HPLC methods were 4.8 ± 0.5%, 4.5 ± 0.5%, and 4.7 ± 0.6%, respectively. GA was most positively correlated with PG (r = 0.696, p < 0.0001). GHb was positively correlated with both PG (r = 0.479, p = 0.013) and GA (r = 0.727, p < 0.0001). Adj-HbA1c measured using the LA method was positively correlated with GA (r = 0.465, p = 0.017), but not PG (r = 0.304, p = 0.132). Adj-HbA1c measured using the HPLC method was correlated with neither PG (r = -0.077, p = 0.710) nor GA (r = 0.360, p = 0.071). CONCLUSIONS: GHb measured using the affinity method may be a useful glycemic control marker in infants. Although adj-HbA1c measured using the LA method was correlated with GA, it may not be a practical measure because it was not correlated with PG and determining HbF levels using HPLC method can be troublesome. Adj-HbA1c measured using the HPLC method should not be used as a glycemic marker in infants.

Glycated albumin is lower in infants than in adults and correlated with both age and serum albumin.

BACKGROUND: Glycated albumin (GA) reflects glycemic control in patients with neonatal diabetes mellitus (NDM). However, GA in NDM patients is apparently low in relation to glycemia. OBJECTIVE: To establish the reference intervals for GA in healthy infants. SUBJECTS AND METHODS: Fifty-eight healthy, full-term newborn infants were used to define the GA reference values and to investigate its relationship to plasma glucose (PG) and serum albumin. The infants were categorized into three groups according to age: group A, 5 (4-6) median (range) d: n = 18; group B, 33 (30-38) d: n = 19; and group C, 181 (50-352) d: n = 21. We also studied 212 non-diabetic adults [group D, 53 (28-78) yr old] and the 5 NDM patients previously reported for GA comparisons. RESULTS: In the infants, GA was strongly positively correlated with logarithmic transformation of age [log (age)] (p = 0.831, p < 0.0001). The GA in groups A, B, C, and D were 7.3 ± 1.0%, 8.6 ± 1.1%, 10.9 ± 0.8%, and 14.0 ± 1.1%, respectively. The GA was more strongly positively correlated with serum albumin (r = 0.768, p < 0.0001) than with PG (r = 0.596, p < 0.0001). When GA levels were compared with the age-dependent reference values, GA in the transient NDM patient was normalized although GA in the four permanent NDM patients decreased but remained high after insulin therapy. CONCLUSIONS: This study showed that the reference range for GA in infants is lower than that of adults and increases with age, with which we confirmed that GA in the NDM patients reflected the clinical course. Consequently, GA in NDM patients should be compared with the age-based reference values to assess the accurate glycemic status.

Analytical Performance of a Novel Latex Turbidimetric Immunoassay, "Nanopia TARC", for TARC/CCL17 Measurement: A Retrospective Observational Study.

Thymus- and activation-regulated chemokine (TARC, also known as CCL17) is used as a biomarker for atopic dermatitis. The methods currently used for its measurement are complex, time-consuming, and require large machinery, warranting the need for a method that is simple, has a quick turnaround time, and requires less complex machinery. We evaluated the analytical performance of a novel latex turbidimetric immunoassay method, "Nanopia TARC", on 174 residual serum samples from patients with skin or allergic diseases. This evaluation included the assessment of the limit of blank/detection/quantification (LOB/D/Q), precision, accuracy, linearity, interference, and commutability between Nanopia TARC and "HISCL TARC", based on the chemiluminescent enzyme immunoassay (CLEIA) method. The LOB/D/Q values were 13, 57, and 141 pg/mL, respectively. The coefficient of variation of the repeatability was 0.9-3.8%, and that of the intermediate precision was 2.1-5.4%. The total error of the accuracy was 1.9-13.4%. The linearity was 141 and 19,804 pg/mL for TARC. The correlation coefficient between Nanopia TARC and HISCL TARC determined using the Passing-Bablok regression analysis was 0.999. Furthermore, the concordance of diagnostic criteria with AD was 92%. Nanopia TARC was confirmed to have the same analytical performance for TARC measurement as the existing CLEIA method.

Differential Dynamics of Humoral and Cell-Mediated Immunity with Three Doses of BNT162b2 SARS-CoV-2 Vaccine in Healthcare Workers in Japan: A Prospective Cohort Study.

Vaccines against SARS-CoV-2 with good efficacy are now available worldwide. However, gained immunity diminishes over time. Here, we investigate the course of both humoral and cell-mediated immunity in response to three doses of the Pfizer mRNA BNT162b2 SARS-CoV-2 vaccine in healthcare workers in Japan. SARS-CoV-2 anti-receptor-binding domain (RBD) antibodies (total Ig, IgG), neutralizing antibodies (NAb), and ELISpot were measured in serum and whole blood samples collected after each vaccine dose. ELISpot numbers were higher than the cutoff values in most participants at all times. It was suggested that the difference in behavior between humoral immunity and cell-mediated immunity with age is complementary. Anti-RBD total Ig, IgG, and NAb indicated a high correlation at each time point after vaccine doses. Total Ig was retained long-term after the second dose and increased significantly faster by the booster dose than IgG. Nab levels of all subjects were ≤20% six months after the second dose, and the correlation coefficient was greatly reduced. These are due to the avidity of each antibody and differences among commercial kits, which may affect the evaluation of immunokinetics in previous COVID-19 studies. Therefore, it is necessary to harmonize reagents categorized by the same characteristics.

[Role of the clinical laboratory for patients with massive transfusion undergoing elective surgery].

The microvascular bleeding resulting from the dilutional coagulopathy can occur when patients with massive blood loss are treated by infusing a lot of crystalloids, colloids, and red blood cell concentrates. For the management of dilutional coagulopathy and the appropriate replacement therapy of with coagulation factors and platelets, we usually monitor the patient's course of with platelet count, conventional coagulation tests such as the prothrombin time, the activated partial prothrombin time, and the fibrinogen concentration. The central clinical laboratory has a responsibility for an accurate and quick report of these test results of patients with massive transfusion. Furthermore, use of point care testing is of clinical value to fulfill a clinical demand in case with dilutional coagulopathy.

Reticulocyte hemoglobin equivalent as a potential marker for diagnosis of iron deficiency.

Evaluation of parameters relating to serum ferritin and iron is critically important in the diagnosis of iron deficiency anemia (IDA). The recent development of automated systems for hematology analysis has made it possible to measure reticulocyte hemoglobin equivalent (RET-He), which is thought to reflect iron content in reticulocytes, in the same sample used for complete blood count tests. If RET-He is, indeed, capable of evaluating iron deficiency (ID), it would be useful for immediate diagnosis of IDA. In the present study, we examined the usefulness of RET-He for diagnosis of ID. Blood samples were obtained from 211 patients. Anemia was defined as hemoglobin (Hb) level of <12 g/dL. Iron deficiency was defined as serum ferritin level of <12 ng/mL. Patients were classified into four groups: IDA, ID, control, and non-ID with anemia. Patients in the IDA group had significantly lower RET-He levels than those in the control group. RET-He correlated with serum ferritin in the IDA and ID groups. The area under the curve for RET-He was 0.902, indicating that RET-He facilitates the diagnosis of ID with high accuracy. RET-He changed in parallel with changes in Hb during iron administration for 21 IDA patients. Our results indicate that RET-He may be a clinically useful marker for determining ID in the general population.

In vivo behavior of NTBI revealed by automated quantification system.

Non-Tf-bound iron (NTBI), which appears in serum in iron overload, is thought to contribute to organ damage; the monitoring of serum NTBI levels may therefore be clinically useful in iron-overloaded patients. However, NTBI quantification methods remain complex, limiting their use in clinical practice. To overcome the technical difficulties often encountered, we recently developed a novel automated NTBI quantification system capable of measuring large numbers of samples. In the present study, we investigated the in vivo behavior of NTBI in human and animal serum using this newly established automated system. Average NTBI in healthy volunteers was 0.44 ± 0.076 µM (median 0.45 µM, range 0.28-0.66 µM), with no significant difference between sexes. Additionally, serum NTBI rapidly increased after iron loading, followed by a sudden disappearance. NTBI levels also decreased in inflammation. The results indicate that NTBI is a unique marker of iron metabolism, unlike other markers of iron metabolism, such as serum ferritin. Our new automated NTBI quantification method may help to reveal the clinical significance of NTBI and contribute to our understanding of iron overload.

Age-adjusted glycated albumin: a more robust parameter to establish glycaemic control in neonatal diabetes mellitus.

BACKGROUND: We previously reported that glycated albumin (GA) levels increased in an age-dependent manner in infancy. In order to determine whether this phenomenon is true from infancy to adulthood, we investigated the GA levels in non-diabetic subjects of a wide range of age. METHODS: GA levels of 376 non-diabetic subjects [average age, 31.8 ± 23.8 years (4 days-78 years)] were determined. A relationship between GA and logarithmically transformed age [log(age)] was analysed. RESULTS: GA levels were significantly positively correlated with log(age) [R = 0.865, P < 0.0001, GA = 1.77 × log(day) + 6.55]. Based on a regression line, we established the formula for adjusting GA levels according to age. CONCLUSION: We showed that GA increases with age from infancy to adulthood and that normal GA levels are demonstrated as a simple regression formula with log(age). This formula allowing us to use the adult reference range has the potential for treatment monitoring of diabetic patients regardless of age.

Non-transferrin-bound iron assay system utilizing a conventional automated analyzer.

BACKGROUND: Iron is an essential metal in the body, but its excessive accumulation causes damage in various organs through free radical production. Iron homeostasis is therefore tightly regulated. However, when iron balance collapses, such as in prolonged transfusion, transferrin (Tf) is fully saturated and non-Tf-bound iron (NTBI) appears in the serum. Monitoring serum NTBI levels is therefore crucial in the assessment of the clinical status of patients with iron overload, since NTBI is associated with cellular and organ damage. Several methods for NTBI determination have been reported, but these are extremely complicated and very few laboratories can quantify NTBI at present. METHODS: We established a novel assay system utilizing automated analyzers that are widely used in clinical laboratories for diagnostic testing. In this assay, NTBI is chelated by nitrilotriacetic acid (NTA), after which the iron is reduced and transferred to nitroso-PSAP, a chromogen. RESULTS: The assay shows excellent linearity, reproducibility, and compatibility with HPLC, one of the most reliable conventional methods for NTBI quantification. CONCLUSIONS: Our novel method for NTBI measurement is high-throughput and may be a useful and powerful tool in the study of the physiological and clinical importance of NTBI.

Treatment of climacteric symptoms with herbal formulas of traditional Chinese medicine.

Hormone replacement therapy (HRT) is not successful or is contraindicated for the treatment of climacteric symptoms in some patients. To investigate whether certain herbal formulas of traditional Chinese medicine (Kampo in Japanese) could be used as an alternative treatment, a longitudinal 'before and after' comparative study was carried out in 18 Japanese women, and the results were compared with those of 16 women who underwent HRT. Kampo improved all the climacteric symptoms. In contrast, improvement of cold limbs, sleeping disorders, shoulder stiffness/lumbago, and fatigue in the HRT group was either not significant or of limited extent. In addition, the serum level of estradiol in postmenopausal women was raised by the combined use of two Kampo formulas. These results suggest that Kampo may be considered an alternative to HRT for the treatment of climacteric symptoms, but vigorous monitoring for potential side effects of increased estrogen levels in some postmenopausal patients is needed.