Unexpected Detection of HbH-CS in a Pregnant Woman by Assessing HbA1c Using Capillary Electrophoresis.

BACKGROUND: Testing for hemoglobin A1C (HbA1C) is useful for following up on glycemic control in diabetic patients as well as in pregnant women. METHODS: A pregnant Chinese woman appeared for regular pregnancy check-ups. While monitoring blood glucose, an HbA1c test was initially performed in the pregnant Chinese woman using a CE-HPLC assay and capillary electrophoresis. Then acid gel Hb electrophoresis, a reverse dot-blot (RDB) assay, gap-PCR and DNA sequencing were applied to further evaluate the Hb variants. RESULTS: The CE-HPLC system has shown that the percentage of blood HbA1c was 5.4%, while hemoglobin electrophoresis of HbA2 and HbA0 were 1.6% and 88.7%, respectively. The capillary electrophoresis measurement results were 6.4% HbA1c, 0.4% HbA2, and 84.1% HbA0. HbA2 and HbA were 0.8% and 89.3%, respectively. Further gap-PCR, the reverse dot-blot (RDB) assay, and DNA sequencing showed that the pregnant woman had HbH-CS (αα CS/--) (1800 bp and 1300 bp) and ß/ß. CONCLUSIONS: Our study demonstrates that hemoglobin variants, such as HbH-CS can affect capillary electrophoresis results. An HbH-CS diagnosis in a pregnant woman is clinically significant. Laboratories should be cautious in using the CE-HPLC assay to evaluate HbA1c results in the presence of hemoglobin variants. Our findings highlight the strong discriminatory power of capillary electrophoresis for various Hb variants.

[Analysis of hematological phenotype and genotype of 23 patients from Guangdong with co-inherited hemoglobin Hb Westmead and ß-thalassemia].

OBJECTIVE: To analyze the genotype-phenotype correlation among carriers from Guangdong with co-inherited hemoglobin Hb Westmead (HbWS) and ß-thalassemia. METHODS: Twenty three patients (including 9 males and 14 females, aged 1-53 year old) were diagnosed by hematological analysis and genetic testing. Complete blood cell count and hemoglobin electrophoresis analysis were performed on a XE4000i automatic hemocyte analyzer. Hb, HbF and HbA2 were tested by high performance liquid chromatography (HPLC). Gap-PCR was adopted to detect three common thalassemia deletions. Reverse dot-blotting (RDB) assay was applied for detecting three common non-deletional α2 gene mutations and ß-thalassemia. RESULTS: Among the 23 patients, 12 showed anemia, among whom 9 had mild anemia and 3 had moderate anemia. The lowest Hb was 68 g/L, and both mean corpuscular volume and mean corpuscular hemoglobin were lower than average, while HbA2 was higher than 3.5%. Genetic analysis confirmed that 5 cases had αWS-α/α-α, ß CD654/ß N (21.7%), 4 had α WS-α/α-α, ß CD41-42/ß N (17.4%), 5 had α WS-α/α-α, ß CD17/ß N (21.7%), 4 had α WS-α/α-α, ß CD28/ß N (17.4%), 1 had α WS-α/α-α, ß CD71-72/ß N (4.3%), 1 had αWS-α/α-α, ß CD27-28/ß N (4.3%), 1 had α WS-α/α-α, ß CD41-42/ß CD17 (4.3%), 2 had a concomitant ß-thalassemia heterozygosity and -α 3.7 deletion. CONCLUSION: Patients with co-existing Hb WS and other ß-thalassemia trait may show variable clinical features. Such compound heterozygotes are usually misdiagnosed during screening by hemoglobin electrophoresis, accurate diagnose should be attained by molecular diagnosis.

Discovery and Correction of Spurious Low Platelet Counts due to EDTA-Dependent Pseudothrombocytopenia.

BACKGROUND: Ethylene diamine tetraacetic acid dependent pseudothrombocytopenia (EDTA-PTCP) is a laboratory artifact that may lead to unnecessary evaluation and treatment of patients. The purpose of this article is to discuss how to identify EDTA-PTCP and correct spurious low platelet counts in clinical laboratories. METHODS: We use two criteria to screen for platelet aggregation: (1) an abnormal platelet count in EDTA-treated blood from a patient lacking clinical signs of a platelet disorder, and (2) an instrument flag for platelet clumps. EDTA-PTCP was confirmed by microscopic examination for platelet agglutination and by platelet counts that corrected with citrate sample. In addition, the time course of EDTA-PTCP was investigated in samples from 26 patients anticoagulated with EDTA-K2 and sodium citrate. Amikacin (5 mg/ml) was added to tubes with EDTA-K2 or sodium citrate from seven additional cases in order to confirm its dissociative effect on platelet aggregation. RESULTS: In our laboratory, the overall incidence of EDTA-PTCP was approximately 0.09%; and the duration was between 2 weeks and 6 months. EDTA-PTCP was time-dependent and occurred as early as 10 min after sample collection. Weaker agglutination could also occur in most corresponding citrate-treated samples. The dissociative effect of amikacin on platelet agglutination was case-specific and not concentration-dependent. CONCLUSIONS: The method of screening for platelet clumping with the help of XE5000 images is convenient. The decline in the platelet count is related to the length of time and the intensity of chelation. Amikacin supplement is not always effective for correcting platelet counts in vitro.

Fucoidan elevates surface organic cation transporter 2 expression via upregulation of protein kinase A in uric acid nephropathy.

Uric acid nephropathy (UAN) is caused by excessive uric acid, and is a key risk factor for uric acid nephrolithiasis, gouty arthritis, renal diseases and cardiovascular diseases. The present study aimed to evaluate the protective effect of fucoidan, a sulfated polysaccharide component of brown algae, on UAN and to elucidate the underlying molecular mechanism. A rat model of UAN was induced by adenine treatment, and rats were then randomly assigned to control, model or fucoidan treatment groups. Hematoxylin and eosin staining of the kidney tissues of rats with UAN was subjected to conventional morphological evaluation. Cellular infiltrate in the tubules, atrophic glomeruli, tubular ectasia, granuloma hyperplasia focal fibrosis and accumulated urate crystals in the tubules of UAN rat renal tissues were observed. These symptoms of kidney damage were reduced in the fucoidan treatment group. Periodic acid methenamine silver-Masson staining was performed and the results indicated that renal interstitial fibrosis was reduced among renal tissues from the fucoidan treatment group compared with the model group. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling staining revealed a lower proportion of apoptotic nuclei in the kidneys of the fucoidan treatment group compared with the model group. Protein kinase A (PKA) 2ß and phosphorylated PKA 2ß protein levels were significantly elevated in renal tissues of the fucoidan treatment group compared with the model group (P<0.05 and P<0.01, respectively), suggesting that PKA expression was upregulated by fucoidan. Immunohistochemistry staining of PKA in rat renal tissues demonstrated increased expression of PKA. The surface organic cation transporter 2 (OCT2) level was significantly increased by fucoidan treatment compared with the model group (P<0.01), with no significant change in total OCT2 level. COS-7 cells ectopically expressing OCT2 were established. It was indicated that fucoidan was able to activate PKA and upregulate surface OCT2 in OCT2-expressing COS-7 cells. This further demonstrated that upregulation of surface OCT2 expression in OCT2-expressing cells was induced by PKA upregulation. In conclusion, fucoidan upregulated surface OCT2 expression in renal tissues to alleviate the symptoms of UAN via upregulated expression of PKA.

Folic acid reverses uric acid crystal-induced surface OAT1 internalization by inhibiting RhoA activity in uric acid nephropathy.

To investigate how organic anion transporter (OAT)-1 is involved in uric acid nephropathy (UAN), a rat model for UAN was established and the serum uric acid, blood urea nitrogen and serum creatinine levels were all measured, and observed to be increased. It was additionally identified that in UAN rats the surface OAT1 expression levels were reduced. By treating HEK cells with monosodium urate (MSU) crystals, it was observed that the cells exhibited a reduction in OAT1 levels. Furthermore, MSU crystals were observed to recruit Ras homolog family member A (RhoA), a small guanosine triphosphatase, to the membrane and activate it. Following RhoA activation, the OAT1 internalization rate was identified to be increased. The dominant­negative RhoA N19 mutation was able to block MSU­induced OAT1 internalization, indicating that the process was RhoA­dependent. Finally, the results indicated that folic acid, a daily nutritional supplement, was capable of rescuing MSU­induced nephropathy and OAT1 internalization. These observations indicated that uric acid crystals were able to reduce the OAT1 membrane distribution through activating RhoA, and that folic acid was capable of preventing MSU-induced OAT1 relocation by inhibiting the RhoA signaling pathway.

High serum haptoglobin level is associated with tumor progression and predicts poor prognosis in non-small cell lung cancer.

The overall survival time of non-small cell lung cancer (NSCLC) has not improved dramatically in recent decades. An important reason is the lacking of valuable biomarkers. Haptoglobin was reported to have activities of anti-inflammatory, anti-oxidant, autoimmune and tumor angiogenesis. However its potential role as a tumor biomarker was not well recognized. We used an immunoturbidimetry method to measure serum haptoglobin levels in 205 NSCLC patients, and 210 normal healthy controls. We found that serum haptoglobin levels were significantly elevated in NSCLC patients compared with normal healthy controls (1.985±1.039 mg/mLvs. 0.922 ± 0.495 mg/mL, respectively, P < 0.0001). Higher serum haptoglobin levels were associated with advanced TNM stage, lymph node metastasis, and distant metastasis. Area under receiver operating characteristic curve (ROC) for serum haptoglobin was 0.809 (95% CI: 0.767-0.852) at a specificity of 0.881 and sensitivity of 0.639. The optimal cut-off value of haptoglobin was 1.495 mg/mL for discriminating NSCLC from normal healthy controls. Kaplan-Meier log rank analysis revealed that the higher serum haptoglobin levels group had a poorer overall survival compared with lower haptoglobin group (the median survival was 12.0 weeks , 26.0 weeks, respectively, P < 0.01). Further univariate and multivariate Cox regression analysis showed that serum haptoglobin was an independent risk factor of prognosis of NSCLC patients (P < 0.01, P = 0.01, respectively). In conclusion, our study suggests that serum haptoglobin may act as useful clinical serological biomarkers in progression and prognostic evaluation in NSCLC.

Uric acid crystal could inhibit Numb-induced URAT1 lysosome degradation in uric acid nephropathy.

To investigate whether uric acid could regulate urate transporter 1 (URAT1) protein and activity level, we established uric acid nephropathy (UAN) rat model and detected their serum uric acid and URAT1 level with or without the treatment of allopurinol. Results here showed that allopurinol could reduce serum uric acid level in UAN rat model. We further found that in UAN rats, the total and surface URAT1 expression level were both increased while this increase could be blocked by allopurinol treatment. By treating URAT1 stable expressed HEK cell with monosodium urate (MSU) crystals, we found that URAT1 level showed an increase in both total and cell surface level, and it would colocalize more with Rab11 instead of Rab7. Consistently, we also found that the total URAT1 protein level will show an increase in the presence of lysosome inhibitors but not ubiquitin-proteasome inhibitors. Furthermore, we also found that MSU crystal could drive Numb, a clathrin-coated adaptor protein which performs a key function in cell division, out of cell surface and disassociated it from URAT1. Finally, we found that Numb short hairpin RNA (shRNA)-transfected showed a phenocopy as MSU treatment, while Numb-2A mutation over-expression could resist crystal-induced phenotypes. These findings indicated that uric acid crystal could increase URAT1 membrane distribution through inhibiting Numb-induced URAT1 lysosome degradation.

5-HT7 receptors are involved in neurogenic dural vasodilatation in an experimental model of migraine.

Neurogenic dural vasodilation has been demonstrated to play an important role in migraine. 5-HT(7) receptors have been found on trigeminal nerve endings and middle meningeal arteries and demonstrated involved in the dilatation of meningeal arteries. The aim of the present study was to demonstrate whether 5-HT(7) receptors are involved in neurogenic dural vasodilation in migraine. The neurogenic dural vasodilation model of migraine was used in this study. Unilateral electrical stimulation of dura mater was performed in anesthetized male Sprague-Dawley rats. Animals were pretreated with selective 5-HT(7) receptor agonist AS19, 5-HT(7) receptor antagonist SB269970, 5-HT1B/1D receptor agonist sumatriptan, or vehicles. Blood flow of the middle meningeal artery (MMA) was measured by a laser Doppler flowmetry. AS19 significantly increased the basal and stimulated blood flows of the middle meningeal artery following electrical stimulation of dura mater, and its effect was dose dependent at the early stage. SB269970 and sumatriptan significantly reduced the basal and stimulated blood flows of middle meningeal artery. The present study demonstrates for the first time that 5-HT(7) receptors are involved in neurogenic dural vasodilation evoked by electrical stimulation of dura mater and maybe of relevance in the pathophysiology and treatment of migraine.

[Clinical significance of abnormal protein bands in multiple myeloma treated with bortezomib-based induction regimen and autologous stem cell transplantation].

OBJECTIVE: To study the clinical significance of abnormal protein bands (APB) in multiple myeloma (MM) patients treated with bortezomib-based induction regimen and autologous stem cell transplantation (ASCT). METHODS: Sixty-eight MM patients submitted to bortezomib-based induction therapy and ASCT from January 2007 to July 2012 were retrospectively studied. Monoclonal protein was detected by immunofixation electrophoresis (IFE). RESULTS: Of all 68 patients, 33 (48.5%) patients had APB. At the first emergence of an APB, two patients with light chain type achieved CR and before transplantation, and thirty-one patients were after transplantation with median time of 104 (ranged 33-404) days. The median duration of APB appearance was 105 (ranged 35-801) days. Patients who developed APB compared with those without APB, had a significantly higher CR plus very good partial response (VGPR) rates (100.0% vs 85.7%%, P=0.017) and CR rates (87.9% vs 62.9%) (P=0.03). There were no significant differences in gender, age, HGB, ALB, ß2-microglobulin, M protein type, Durie-Salmon and ISS stages, the case number of first line or second line treatment, induction courses of bortezomib-based regimen, and the mode of ASCT. With a median follow-up of 33.4 (ranged 7.0-71.7) months, patients with APB tended to have a longer overall survival (OS) versus non-APB patients, although no significant difference obtained (P＞0.05). Among APB patients, OS was longer in patients whose appearance of APB occurred ＜6 months after transplantation than those ≥ 6 months, but the significant difference was not obtained yet (P＞0.05). CONCLUSIONS: Patients who developed APB had a significantly better response to bortezomib-based induction regimen followed ASCT. APB emergence has a good prognostic significance.

Uric acid crystal could inhibit Numb-induced URAT1 lysosome degradation in uric acid nephropathy

To investigate whether uric acid could regulate urate transporter 1 (URAT1) protein and activity level, we established uric acid nephropathy (UAN) rat model and detected their serum uric acid and URAT1 level with or without the treatment of allopurinol. Results here showed that allopurinol could reduce serum uric acid level in UAN rat model. We further found that in UAN rats, the total and surface URAT1 expression level were both increased while this increase could be blocked by allopurinol treatment. By treating URAT1 stable expressed HEK cell with monosodium urate (MSU) crystals, we found that URAT1 level showed an increase in both total and cell surface level, and it would colocalize more with Rab11 instead of Rab7. Consistently, we also found that the total URAT1 protein level will show an increase in the presence of lysosome inhibitors but not ubiquitin-proteasome inhibitors. Furthermore, we also found that MSU crystal could drive Numb, a clathrin-coated adaptor protein which performs a key function in cell division, out of cell surface and disassociated it from URAT1. Finally, we found that Numb short hairpin RNA (shRNA)-transfected showed a phenocopy as MSU treatment, while Numb-2A mutation over-expression could resist crystal-induced phenotypes. These findings indicated that uric acid crystal could increase URAT1 membrane distribution through inhibiting Numb-induced URAT1 lysosome degradation