Plasma and Urine Hydroxyurea Levels Might be Useful in the Management of Adult Sickle Cell Disease

Hydroxyurea (HU) is useful for treating sickle cell anemia because of its ability to reduce some of the severe clinical events such as painful crises and acute chest syndrome. It may also reduce the need for blood transfusions and frequent hospitalizations and reduce mortality. Nevertheless, no consistent recommendations regarding its therapeutic schedule are defined. Our aim was to improve and validate a high performance liquid chromatography (HPLC) technique to measure HU and to study HU levels in serum and urine of sickle cell anemia patients and relate this to treatment efficacy and compliance. Thirty-seven patients received 1,128 ± 333 mg of HU per day (8.0 to 28.0 mg/kg/day). Plasma and/or urine were sampled and HU was measured using an HPLC method coupled with UV detection. We validated a specific, sensitive assay with good reproducibility and linearity, and showed a positive relationship between plasma HU concentrations and time elapsed between oral HU intake and sampling. We observed plasma HU concentrations were positively correlated with change in mean corpuscular volume (MCV) before and during the treatment. No correlation was obtained between HU concentration and Hb F level.     

Comparison Between Capillary Electrophoresis and High Performance Liquid Chromatography for Detection and Quantification of Hb Constant Spring [Hb CS; α142, Term→Gln (TAA>CAA IN α2)]

Hb Constant Spring [Hb CS; α142, Term→Gln (TAA>CAA in α2)] is often missed by routine laboratory testing since its mRNA as well as gene product are unstable and presented at a low level in peripheral blood. This study aimed to analyze the efficacy of capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) for detecting and quantifying Hb CS in 19 heterozygotes and 14 homozygotes with Hb CS as well as 10 Hb H-CS disease subjects who were detected by molecular analysis. In the CE electrophoregram, Hb CS was seen at zone 2 and was observed in all samples, while the chromatogram of Hb CS peaks was found in 26.32% heterozygotes, 42.86% homozygotes and 90% Hb H-CS disease subjects, respectively. In addition, the Hb CS levels in each group of subjects quantified by CE were significantly higher than those quantified by HPLC. Based on the CE method, the lowest Hb CS level was found in the heterozygotes, whereas the highest level was found in the Hb H-CS disease patients. Therefore, the CE method was superior to the HPLC method for detecting Hb CS. Furthermore, the level of Hb CS quantified by CE proved useful in screening heterozygotes and homozygotes with Hb CS as well as Hb H-CS disease.     

Detection of Coinherited Hb H-Constant Spring/Paksé Disease and Hb E by Capillary Electrophoresis and High Performance Liquid Chromatography

A capillary electrophoresis (CE) method has been proven to be superior to a high performance liquid chromatography (HPLC) method in the detection of Hb H-Constant Spring/Paksé [Hb H (β4) Hb CS or α142, Term→Gln, TAA>CAA (α2)]/Paksé [α142, Term→Tyr, TAA>TAT (α2)]. It also has the ability to quantify Hb Bart’s (γ4). The aim of this study is to analyze the efficacy of the CE and HPLC in the detection of Hb H (β4)-CS/Paksé-E [β26(B8)Glu→Lys, GAG>AAG] disease. The laboratory results from July 2009 to July 2012 were reviewed at the Thalassemia Laboratory of the Associated Medical Sciences Clinical Service Center, Chiang-Mai University, Chiang-Mai, Thailand. The HPLC or CE method was used for the diagnosis of β-thalassemia (β-thal) and hemoglobinopathies, and molecular analysis was used for the diagnosis of α-thalassemia-1 (α-thal-1) Southeast Asian (SEA) and Thai type deletions, Hb CS and Hb Paksé. Hb H-CS-E was found in six samples and Hb H-Paksé-E was found in one sample, respectively. On the capillary electrophoregram, peaks of Hb Bart’s and Hb CS/Paksé were observed in all samples with the mean levels at 2.4 and 1.0%, respectively. These peaks were also presented on the HPLC chromatogram. However, the Hb CS/Paksé level could be quantified in only three of these seven (43.0%) samples. Therefore, CE was proven to be superior to HPLC in the detection of Hb H-CS/Paksé-E disease, which will assist in diagnostic, counseling and prevention programs for these diseases.     

Hemoglobin Duan, α75(EF4) ASP→ALA,a New Variant Found in China

A simple isoelectric focusing technique is presented for the screening of α- and β-globin chain variants on polyacrylamide gel in the presence of 8 M urea. No detergent was used. Variants focused at discrete pH values, depending on the charge variation (-2, -1, 0, +1 or +2) introduced by the mutation. A linear relationship was evidenced between the charge variation and the shift of the apparent isoelectric point of the variants. The practical interest of this procedure is illustrated.     

A Novel Base Change Leading to Hb Vanderbilt [β89(F5)Ser→Arg,AGT>AGA]

We describe a high oxygen affinity hemoglobin (Hb) variant (Hb Vanderbilt) as a result of a heterozygous novel base change from T to A at codon 89 (AGT>AGA) leading to an amino acid change from serine to arginine.     

毛细管电泳与高效液相色谱法在Hb Q-Thailand检测中的比较研究

目的了解毛细管电泳(CE)与高效液相色谱法(HPLC)在Hb Q-Thailand检测中的差异。方法收集10例CE筛查出的可疑Hb Q-Thailand,经地贫基因分析和DNA测序确诊后,再用HPLC检测比对。结果 10例样本中CE均检出Hb Q-Thailand和Hb Q-Thailand A2(Hb QA2),而HPLC仅检出10例Hb QThailand和5例Hb QA2,而且HPLC错误检出Hb Q-H病1例存在Hb A2。CE和HPLC检出Hb Q-Thailand、Hb A2、Hb QA2的含量分别为(34.41±21.10)%、(1.68±0.57)%、(0.77±0.10)%和(31.72±21.46)%、(2.43±0.22)%、(0.24±0.24)%,两组Hb Q-Thailand差异无统计学意义(P0.05),Hb A2和Hb QA2的差异有统计学意义(P0.01)。结论 CE在检测Hb Q-Thailand中优于HPLC。     

Hb Gerland [α55(E4)Val→Ala]: A Mutation Found on the α1-Globin Gene

The Hb Gerland [α55(E4)Val→Ala] mutation has been described in the α2-globin gene. We report here the same mutation in the paralogous α1-globin gene. This variant was found in a healthy 18-month-old boy of Chinese origin. Abnormal hemoglobin (Hb) fractions were visible on isoelectric focusing (IEF) and cation exchange high performance liquid chromatography (HPLC), with elution patterns differing from one system to another. Direct sequencing of the α-globin genes revealed a GTT>GCT (Val→Ala) transversion at codon 55 of the α1-globin gene. We propose to name the variant encoded by the α1-globin gene Hb Gerland [A1], and the variant that is encoded by the α2-globin gene Hb Gerland [A2].     

Hb Sichuan [α67(E16)Thr→Ile,HBA2: c.203C>T]: A Novel Hemoglobin Variant That Can Be Detected by Glycated Hemoglobin Electrophoresis

Abstract

We report a novel α2-globin chain hemoglobin (Hb) variant in a 23-year-old female of Chinese Han nationality. The Hb variant can be detected by glycated Hb electrophoresis (CapillaryS2, Hb A_1c program). However, Hb fractions analyzed by capillary electrophoresis (CE) (CapillaryS2, Hb program) and high performance liquid chromatography (HPLC), (VARIANT II? β-Thalassemia Short Program) showed no suspicious Hb variant. Sanger sequencing revealed a novel mutation [α67(E16)Thr→Ile, HBA2: c.203C>T]. We named this novel variant Hb Sichuan after the geographic origin of the proband.     

Hb G-Waimanalo [A1] [α64(E13)Asp→Asn; HBA1: c.193 G > A] with Decreased Oxygen Affinity

Abstract

A clinically asymptomatic 12-year-old girl showed microcytosis in routine examination. Cation exchange high performance liquid chromatography (HPLC), revealed two additional peaks eluting after Hb A and DNA sequencing uncovered a novel heterozygous mutation at codon 64 of the α1-globin gene. The hemoglobin (Hb) variant was annotated as Hb G-Waimanalo [A1]. Further analyses demonstrated a decreased oxygen affinity Hb compared to the normal Hb configuration.     

Hematological Characterizations and Molecular Diagnostic Aspects of Hb Wiangpapao [α44(CE2)Pro→Ser (α1), CCG>TCG; HBA1: c.133C>T], a New α-Globin Variant Found in a Pregnant Thai Woman

We report the hematological parameters and provide a rapid molecular analysis method for detection of Hb Wiangpapao [α44(CE2)Pro→Ser, CCG>TCG; HBA1: c.133C>T], a new α-globin variant found in a pregnant Thai woman. Her red cell indices were measured by an automated blood counter. The results were: red blood cell (RBC) count 4.03?×?10¹²/L, Hb 13.1 (g/dL), packed cell volume (PCV) 0.39?L/L, mean corpuscular volume (MCV) 97.0 fL, mean corpuscular hemoglobin (Hb) (MCH) 32.5?pg, mean corpuscular Hb concentration (MCHC) 33.4?g/dL, and RBC distribution width (RDW) 9.4%. The Hb typing by high performance liquid chromatography (HPLC) showed 13.6% abnormal Hb at a retention time of 2.20?min. that was difficult to distinguish from Hb A. On the capillary electrophoresis (CE) electropherogram, this hemoglobinopathy peak did not separate from the Hb A peak. DNA sequencing showed a C>T transition at the first position of codon 44 (CCG>TCG) of the α1-globin gene that led to a substitution of proline for serine. This mutation has not been recorded in the public databases. Therefore, we named it Hb Wiangpapao as it was first discovered in the Wiangpapao District, Chiang Rai, Thailand. The multiplex allele-specific polymerase chain reaction (ASPCR) for detection of Hb Wiangpapao was developed and revealed a 510?bp specifically amplified fragment. The better understanding of hematological characterizations and the newly developed multiplex ASPCR for diagnosis of Hb Wiangpapao are useful for genetic counseling and family education.     

Hb Matera (HBB: c.167 T > A): A Second Case Detected in a Pregnant Chinese Woman by the Capillary Electrophoresis Method

Hb Matera (HBB: c.167?T?>?A) is an unstable β-globin gene variant with an ATG?>?AAG substitution at codon 55. Its coelution with Hb A₂ on high performance liquid chromatography (HPLC) makes it difficult to discriminate between Hb Matera and Hb E (HBB: c.79?G?>?A) that also coelutes with Hb A₂ in this method. However, we found that capillary electrophoresis (CE) was able to detect Hb Matera and discriminate it from Hb E, based on the quantification of the peaks and on hematological parameters.     

Identification of Hb Constant Spring (HBA2: c.427T > C) by an Automated High Performance Liquid Chromatography Method

Abstract

Laboratory investigation of hemoglobinopathies includes complete blood count (CBC), hemoglobin (Hb) typing by high performance liquid chromatography (HPLC) and DNA analysis. DNA analysis is the most reliable method but requires a manually laborious procedure and is time consuming. A more practical method of detecting abnormal Hbs is the HPLC technique, because it is more rapid and easier to interpret. Hb Constant Spring (Hb CS; HBA2: c.427T?>?C) is an abnormal variant that is labile and difficult to detect using conventional methods. To evaluate the efficiency of Hb CS determination by HPLC, blood samples from 578 subjects were analyzed using an automated cell analyzer for hematological parameters, automated HPLC for Hb identification, and polymerase chain reaction (PCR) for α-thalassemia (α-thal) and Hb CS confirmation. These included 169 normal, 119 heterozygous α-thal-2, 30 homozygous α-thal-2, 177 heterozygous α-thal-1, 59 heterozygous Hb CS, seven homozygous Hb CS and 17 compound heterozygous α-thal-2 and Hb CS subjects. The results showed that sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of Hb CS by HPLC were 93.78, 99.80, 98.73 and 99.00%, respectively. The mean of misdiagnosis value of the three groups of Hb CS subjects (total 83) was 6.02% (n?=?5), with percentages for heterozygous Hb CS, homozygous Hb CS, and compound heterozygous α-thal-2 and Hb CS being 6.8, 0.0 and 5.9%, respectively. The HPLC method yielded good results, although it may also lead to misdiagnosis of Hb CS due to the relatively small amount and lability.     

Hb Ullevaal [β78(EF2)Leu→Val; HBB: c.235C>G], a New Hemoglobin Variant Interfering with Hb A1c Measurement Using a Cation Exchange High Performance Liquid Chromatography Method

A new hemoglobin (Hb) variant was detected during Hb A_1c measurement. DNA sequencing showed heterozygosity for the single nucleotide substitution (C?>?G) on the β-globin gene causing an amino acid change [β78(EF2)Leu→Val; HBB: c.235C?>?G]. The new Hb variant was designated Hb Ullevaal after the hospital at which it was discovered. Heterozygosity for Hb Ullevaal appears to have no clinical significance. However, it interferes with Hb A_1c measurement by cation exchange high performance liquid chromatography (HPLC), causing falsely low Hb A_1c concentration when using the Tosoh G7 apparatus in variant mode.     

Inaccuracy of high-performance liquid chromatography estimation of haemoglobin F in the presence of increased haemoglobin A1c

Increasingly high-performance liquid chromatography is being used for identification and quantification of normal and variant haemoglobins. In many laboratories, the Beta Thal Short programme of the Bio-Rad Variant II instrument is used for this purpose. We noted that a factitious elevation of haemoglobin F was sometimes observed in diabetic patients and therefore carried out a systematic study of this phenomenon. We found discrepant results in 41% of samples from diabetic patients but in no normal volunteers. This factitious elevation could be predicted from a retention time for haemoglobin F of more than 1.15 min, the normal retention time being 1.08-1.15 min. Haemoglobinopathy laboratories need to be alert to the possibility of this erroneous result.     

Detection of Hb A2 and Hb Constant Spring (HBA2: c.427T>C) by Capillary Electrophoresis in a Patient with Hb H-Hb CS Disease

Abstract

Hb A₂ (α2δ2) is one of the key components looked for in hemoglobinopathies screening programs. Therefore, quantitative and accurate method for Hb A₂ value determination is essential for routine screening. Here, we report a case of Hb A₂ and Hb Constant Spring (Hb CS, HBA2: c.427T>C) with Hb H-Hb CS disease that was not detected by high performance liquid chromatography (HPLC), while Hb A₂ and Hb CS were clearly quantified by capillary electrophoresis (CE).     

Hb Agenogi [β90(F6)Glu→Lys (GAG>AAG) HBB: c.271G>A)] in a Pregnant Thai Woman

Hb Agenogi [β90(F6)Glu→Lys (GAG>AAG) HBB: c.271G>A)] is a very rare β-globin chain variant. We report for the first time this hemoglobinopathy in a pregnant 20-year-old Thai woman. She was seen by an obstetrician at her 14th week of gestation. She was pale and had an inflammatory lesion of her lower left leg. The hemoglobin (Hb) analysis by high performance liquid chromatography (HPLC) and low pressure liquid chromatography (LPLC) showed a peak of abnormal Hb at the C window. On capillary electrophoresis (CE), the abnormal Hb peak was observed at electrophoretic zone 4 that corresponded to the Hb E (HBB: c.79G>A) peak. Direct DNA sequencing revealed a GAG>AAG mutation at codon 90 of the β-globin gene. Thus, even though Hb Agenogi is very rare, it can be found in Thai people. The knowledge and understanding of this hemoglobinopathy will be used to assist in diagnosis, management and counseling for patients.     

Neonatal Cyanosis Due to a Novel Fetal Hemoglobin: Hb F-Circleville [Gγ63(E7)His→Leu,CAT>CTT]

Neonatal cyanosis can result from a multitude of acquired and inherited causes. Cyanosis resulting from fetal M hemoglobin (Hb) variants is very rare. Only two ^Gγ variants causing methemoglobinemia and cyanosis in the newborn have been reported to date. Here we describe a novel fetal Hb variant, Hb F-Circleville [^Gγ63(E7)His→Leu], associated with methemoglobinemia and cyanosis in the newborn. The patient's sister also had neonatal cyanosis at birth.     

Complex Interaction of Hb Q-Thailand (HBA1: c.223G>C) with β-Thalassemia/Hb E (HBB: c.79G>A) Disease

Hb Q-Thailand [α74(EF3)Asp→His (α1), GAC>CAC, HBA1: c.223G>C] is an abnormal hemoglobin (Hb) frequently found in Thailand and Southeast Asian countries. The association of the α^Q-Thailand allele with other globin gene disorders has important implications in diagnosis. Here, we report how to diagnose the coinheritance of Hb Q-Thailand with β-thalassemia (β-thal)/Hb E disease in four Thai samples from high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) testing results. Understanding of the HPLC chromatogram and CE electropherogram patterns of this complex mutation is important for interpretation of testing results and providing genetic counseling.     

Diagnosis of Thalassemia on Dried Blood Spot Samples by High Performance Liquid Chromatography

High performance liquid chromatography (HPLC) on fresh lysates is the standard test for identification of thalassemia. Samples in the form of dried blood spot(s) (DBS) mailed to reference laboratories where HPLC is available could be an alternative. Hemoglobin (Hb) on DBS at day 1, 7, 15 and 30 were analyzed by HPLC and compared to those analyzed from fresh liquid whole blood at day 0. A 100% consistent interpretation of β-thalassemia (β-thal) trait and β-thal/Hb E disease between liquid whole blood and DBS was observed when analyzing Hb A₂ on DBS at a level of 2.7–9.9% in conjunction with a lower MCV (<80 fL) and MCH (<27 pg) and analyzing β-thal/Hb E by using Hb E and Hb F at a level of 30–48% and >10%, respectively. Therefore, our results show that detection of thalassemia carriers using DBS is possible and is the alternative of choice in a low resource setting.     

Hb Bart's in Cord Blood: An Accurate Indicator of α-Thalassemia

We quantified Hb Bart's (γ₄) levels by high performance liquid chromatography (HPLC) in 103 fresh cord blood samples from Homerton Hospital, East London, UK. The α-globin gene arrangement was determined by Southern blot hybridization and genomic sequence analysis of the α-globin genes. The cord blood Hb Bart's levels ranged from 0.5 to 11.9% of total hemoglobin (Hb) and were arranged into three categories: i) levels below 1.5%; ii) levels between 1.5 and 5.7%; iii) levels above 6.1%. These corresponded to a normal α-globin genotype, a single deleted/inactivated α-globin gene and two deleted/inactivated α-globin genes, respectively. The study identified the 3.7 kb and 20.5 kb α-thalassemia (thal) deletions, three non deletional α-thal mutations and a novel α-globin gene rearrangement. Hb Bart's screening of fresh umbilical cord blood is an effective method to evaluate globin chain imbalance. This strategy could be utilized to screen populations for the incidence of α-thal and also to identify rare or new molecular lesions that reduce α-globin gene expression.