The purpose of this study was to find a 488-nm excitable fluorescent dye for reticulocyte analysis by the use of fluorescence activated cell cytometry. The chemical structure of thioflavin T, a dye used for reticulocyte analysis with 457-nm excitation, was used as a model. Several dyes were synthesized and evaluated by quantum yield determination, fluorescence microscopy, and flow cytometry.The measurement of reticulocyte percentage, or percentage of immature blood cells, is a diagnostic indicator of erythropoietic activity. Several methods are currently available for the quantitative analysis of reticulocytes (5,6,9,11,12). Most methods are two-step procedures: the reticulocytes are visualized by treatment with a dye that stains the residual RNA characteristic of these cells; the stained cells are then counted by manual or instrumental techniques.Instrumental counting, e.g., flow cytometry, has the potential to be a superior method over manual counting for several reasons: less statistical error is observed owing to the larger quantity of cells analyzed, flow cytometry is usually faster than microscopic counting, and more reproducible results are obtained among different workers. The satisfactory use of flow cytometry, however, relies on the availability of suitable dyes.Pyronin Y, DiOC1[3], acridine orange, and thioflavin T have each been used as a fluorescent reticulocyte stain in flow cytometry. Although reasonable correlation between manual analysis with new methylene blue and instrumental analyses with the fluorescent dyes have been obtained, detailed mathematical analyses were necessary for the first three dyes to distinguish the reticulocyte signal from the background. Thioflavin T showed the best resolution among these dyes; however, the staining procedure was complicated, requiring staining at RT followed by dilution and rapid analysis.What is the reason for poor resolution or a complicated protocol? How can the dyes be improved? We chose to answer these questions by examining the physical properties of the dyes used for reticulocyte analysis.The best results were obtained with a dye we have named "thiazole orange"; analysis of several blood samples with thiazole orange gave a correlation coefficient of 0.97 as compared to a manual determination of reticulocyte percentage. Key terms: Flow cytometry, reticulocytes, nucleic acid dyesThe ideal dye for instrumental analysis of reticulocytes must meet several criteria: the staining procedure and data analysis should be simple, and the results should be accurate and reproducible. A priori, several likely physical criteria for fluorescent activated flow cytometry are apparent: the excitation maximum of the dye should match the instrument, the fluorescence enhancement upon binding of the dye to RNA should be large, and the bound dye should have a high quantum yield. In addition, the dye should be permeable to the cell membrane to eliminate additional steps in the sample preparation (i.e., fixing of cells).None of the currently used reticulocyte dyes meet all o...

Several new derivatives of fluorescein were synthesized. The dyes were characterized by NMR; and the absorbance, excitation, and emission spectra were measured. The fluorescence quantum yields of the dyes were determined. The pKa3 values of the dyes were measured by fluorescence titration. The characteristics of the fluorescein and sulfonefluorescein derivatives were compared. The most promising dye for use in cell analysis appeared to be compound 9, which was given the name Vita Blue. The dibutyrate ester of Vita Blue was made and the compound was given the name Vita Blue dibutyrate (VBDB, 14). The K, of VBDB with pig liver esterase was measured and found to be 4 x M. The pKa3 of Vita Blue was 7.56 f 0.03; both acidic and basic forms were fluorescent (dual fluorescence). The use of VBDB as an intramolecular esterase substrate and its utility for the discrimination between live and dead cells by flow cytometry is described.Key terms: Viable dye, fluorescent esterase substrate, flow cytometry, dual fluorescence, new xanthene dyes Xanthene dyes are among the most commonly used class of fluorescent dyes. Fluorescein (11, a dihydroxy xanthene dye, is a particularly useful dye for biological purposes for several reasons: the dianionic form of the dye is very soluble in water; its pKa3 is 6.3-6.7 and it is therefore mostly deprotonated and fluorescent at physiological pH (7.0-7.6); the excitation maximum of the dye (490 nm) is well matched to the 488-nm line of the argon ion laser; it has a high extinction coefficient (8 x lo4 M-lcm-l) and a high quantum yield (0.92). The last three properties of the dye allow the fI uorescence detection of minute quantities of the dye. In addition, derivatives of fluorescein are readily synthesized and allow utilization of the dye for biological purposes; e.g., fluorescein isothiocyanate can be used to make fluorescent derivatives of proteins (3), and fluorescein diacetate has utility as a membrane permeable esterase substrate (15).Our objective was to synthesize new fluorescent dyes for use in flow cytometry with all the desirable characteristics of fluorescein described above, but with excitation maxima to the red of fluorescein. The advantages of a dye with longer wavelength excitation and emission are several: background fluorescence from biological material (DNA, proteins) diminishes, the cost and size of the laser diminishes, and the possibility of dual laser excitation using an argon ion laser as the lower wavelength laser arises.We were interested in examining the properties of sulfonefluorescein and sulfonenaphthofluorescein derivatives as well as fluorescein and naphthofluorescein derivatives. In general, the sulfonefluorescein analogs are more difficult to synthesize than the fluorescein analogs due to the deactivating character of sulfonic acid groups in Friedel-Crafts acylations; this difficulty in synthesis may explain why these dyes have not been previously exploited. For example, there are only a handful of references to sulfonefluorescein in the literature as compare...

BackgroundThe impact of small-for-gestational-age (SGA) on very-low-birth-weight (VLBW) premature infants remains inconclusive. This study aimed to assess the effects of being born SGA status on the short-term and long-term outcomes in VLBW preterm infants.MethodsWe conducted a population-based, prospective cohort study on VLBW preterm infants born in Taiwan between 2012 and 2017. Sociodemographic, neonatal, growth and neurological data at 2 years of corrected age were collected. A total of 4243 VLBW infants born at 24 through 32 completed weeks' gestation participated in this study, of whom 1,005 had SGA status defined as a birth weight <10th percentile of gestation, and 3,238 did not (the non-SGA group).We compared the risks of short-term outcomes (neonatal mortality and morbidities), long-term outcomes (growth status, including weight, height, and head circumference <10th percentile, and neurodevelopmental impairments at 2 years of age). Subgroup analysis was performed by stratification of gestation age (GA): GA 24–26, 27–29 and 30–32 weeks.ResultsIn the analysis of short-term outcomes, the SGA group had an increased risk of neonatal mortality [adjusted odds ratio (OR) = 2.66, 2.99, and 2.19, respectively] in all GA subgroups in comparison with the non-SGA group (p < 0.05). The SGA group had a significantly increased risk of bronchopulmonary dysplasia in GA 27–29 and 30–32 weeks (adjusted OR = 2.11 and 1.86, respectively). We also found that there was an increased risk of severe retinopathy of prematurity in GA 24–26 and 27–29 weeks in the SGA group compared with the non-SGA group (adjusted OR = 1.68 and 1.59, respectively).In the analysis of long-term outcomes, the SGA group had a significantly increased risk of NDI throughout all GA subgroups (adjusted = 1.94, 1.33, and 1.35, respectively) in comparison with the non-SGA group. The SGA groups also had an increased risk of growth status <10th percentile at 2 years of age (p < 0.05).ConclusionsSGA VLBW premature infants had higher risks of neonatal death, growth status <10th percentile, and NDI at 2 years of corrected age compared with the non- SGA premature infants. Prenatal surveillance, postnatal attention, and long- term follow-up are warranted to improve the outcomes of VLBW SGA premature infants.

ObjectiveTo evaluate the association of mode of delivery (MOD) with short‐term and neurodevelopmental outcomes at 2 years of corrected age (CA) in periviable singleton infants.MethodsThis retrospective cohort study of the Taiwan Premature Infant Follow‐up Network database between 2010 and 2016 compared non‐anomalous singleton deliveries (cesarean delivery [CD] vs vaginal delivery [VD]) between 22 0/7 and 25 6/7 gestational weeks. Major morbidities, mortality, and neurodevelopmental outcomes were evaluated at 2‐year CA.ResultsThe CD and VD groups included 354 and 472 infants, respectively. The intraventricular hemorrhage (IVH) rate was lower in the CD group (54% vs 66%, P = 0.001), but severe IVH differed non‐significantly between groups (20% vs 26%, P = 0.057). In the small‐for‐gestational age subgroup, CD was associated with lower IVH (56% vs 84%, adjusted odds ratio [aOR] 0.17, 95% confidence interval [CI] 0.04–0.69) and better survival without neurodevelopmental impairment (29% vs 8%, aOR, 6.64, 95% CI 1.02–43.29) after controlling for potential confounders.ConclusionThe optimal MOD for periviable singleton birth and its impact are unclear. CD in periviable singleton births is associated with a decreased IVH risk, without improvement in severe IVH, mortality, or neurodevelopment at 2‐year CA. The small‐for‐gestational age subgroup may benefit from CD for better survival without neurodevelopmental impairment.