Application of indirect linkage analysis and direct genotyping to hemophilia A carrier detection in Sichuan, China.

Hemophilia A (HA) is an inherited X-linked bleeding disorder caused by mutations in the factor VIII gene. Prenatal detection in female carriers from families with HA is important to reduce the number of HA patients. The purpose of this study was to detect carriers in families with HA from Sichuan, China, using linkage analysis and a direct genotyping method. A total of 18 HA families were studied. Using a combination of intron 22 inversion, intron 1 inversion, the BclI polymorphic site in intron 18, the HindIII polymorphic site in intron 19, and dinucleotide CA-repeat markers in introns 1, 13, 22, and 24, we were able to detect HA in 88.9% (16/18) of the families studied. HA was detected in the remaining two families by direct genotyping. This study gave the participants a good understanding of their genetic condition and gave us a preliminary understanding of the prevalence of each mutation in Sichuan HA patients.

Cohn process influences the functional anticoagulant activity of human protein C.

Cohn fraction IV (CFIV) is a byproduct of a plasma fractionation process known as the Cohn process. It is an inexpensive source of protein C, retaining about 90% of protein C (PC) in human plasma. We investigated whether PC is affected during the Cohn process and evaluated correlations among coagulant activity, amidolytic activity and PC antigen during the Cohn process. CFIV was redissolved with citrate-buffered saline for 5 h at 4°C, and then centrifuged at 3500 g for 40 min at 4°C. Functional anticoagulant activity was measured with a one-stage coagulation method based on activated partial thromboplastin time. The functional amidolytic activity of PC was determined using chromogenic substrate assay, and measurement of PC antigen was performed by ELISA. In CFIV, anticoagulant activity declined significantly, with a loss of >80%, while amidolytic activity was not significantly altered, compared to PC antigen. Prior to the Cohn process, high-rank correlations were observed in cryosupernatant, with rs = 0.921 for anticoagulant and amidolytic activities (P = 0.009), 0.896 for anticoagulant activity and antigen (P = 0.014) and 0.832 for amidolytic activity and antigen (P = 0.031). After the Cohn process in CFIV, there was also a high correlation between amidolytic activity and antigen (rs = 0.782, P = 0.038). There were no significant correlations between anticoagulant activity and antigen (rs = 0.223, P = 0.653), or anticoagulant and amidolytic activity (rs = 0.236, P = 0.675). We conclude that the Cohn process significantly influences the anticoagulant activity of PC. Compared to the antigen, PC lost greater than 80% of its anticoagulant activity, but retained its amidolytic activity, during the Cohn process.

Prevalence of hepatitis A viral RNA and antibodies among Chinese blood donors.

Like other developing countries, China was reported to have a relatively high seroprevalence of anti-hepatitis A antibodies (anti-HAV). However, no studies have evaluated the prevalence of anti-HAV and HAV RNA among voluntary blood donors with or without elevated serum alanine transaminase (ALT) levels. Anti-HAV antibodies were detected using an enzyme-linked immunosorbent assay, and reverse transcription quantitative polymerase chain reaction was carried out for detection of HAV RNA. In the current study, we analyzed a total of 450 serum samples with elevated ALT levels (≥40 U/L) and 278 serum samples with non-elevated ALT levels. Seroprevalence rates of anti-HAV were 51.6% in donors with elevated ALT and 41.4% in donors with non-elevated ALT; however, none of the samples was positive for HAV RNA. The results of our study showed lower seroprevalence rates of anti-HAV in blood donors (irrespective of ALT levels) than those in published data on Chinese populations. Although donors with elevated ALT had statistically higher prevalence rates of anti- HAV than did those with non-elevated ALT, none of the serum samples had detectable levels of the active virus. In conclusion, our results demonstrate that the transmission of hepatitis A by blood transfusion will occur rarely.

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells.

Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 µg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 µg/mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.

Quantitative determination of activated coagulation factor XI as an impurity in therapeutic immunoglobulins from Chinese blood fractionation companies.

Residual activated coagulation factor XI (FXIa) has been suggested to play an important role in thromboembolic events associated with the use of intravenous immunoglobulin (IVIG) lots. This study investigated the predominant plasma proteases in 42 IVIG lots from 4 Chinese manufacturers. In one-stage clotting assays, the procoagulant activities of factors II, VII, IX, X, XI, and XII were quantified. Non-activated partial thromboplastin time and a modified thrombin generation test served as global and FXIa-specific clotting assays, respectively. We found that coagulation factor clotting activities of the 42 IVIG lots were below the detection limit of the assays, except for the products of manufacturer B (lots of 2010), in which 0.030 to 0.032 IU/mL FXI:C were detected. The peak time of thrombin using a thrombin generation test was greater than 35 min, the relevant amount of FXIa was below 0.37 nM, and non-activated partial thromboplastin time was greater than 200 s. Consequently, the 42 IVIG lots showed non-significant procoagulant potential. Further study is required to determine whether a program for FXIa determination in IVIG products should be launched in China.

Spontaneous dissection of internal carotid arteries. Spontaneous resolution documented by serial ocular pneumoplethysmography and angiography.

Spontaneous dissection of the internal carotid artery can appear on an angiogram as a total occlusion of this vessel. This appearance differs from that typical of atherothrombotic occlusion. In four patients with spontaneous dissections of internal carotid arteries, serial ocular pneumoplethysmography demonstrated spontaneous restoration to functional patency. Repeated angiography confirmed the noninvasive testing in all four patients.

Ocular pneumoplethysmography in carotid-cavernous sinus fistulas.

Pulsatile exophthalmos in association with carotid-cavernous sinus fistulas has been well defined anatomically, by angiography. This paper presents the physiological assessment of this entity, as measured with ocular pneumoplethysmography (OPG-Gee). The abnormal arteriovenous communication lowers resistance to arterial flow. This is characterized by a lowered ophthalmic systolic pressure and an increased ocular blood flow. The OPG readily documents the physiological result of therapeutic intervention.

Ocular pneumoplethysmography in head-injured patients.

Severe head injury is frequently associated with multiple trauma. In the comatose patient, endotracheal intubation and ventilator support are often required, if there is associated dyssynchronous spontaneous effort. The latter is managed with therapeutic (drug) paralysis. An elaborate life-support and monitoring system coupled with controlled paralysis limits the mobility of the patient for diagnostic procedures, and a continuing reevaluation of neurological status is difficult. Under these circumstances the ocular pneumoplethysmograph provides a simple rapid noninvasive assessment of ocular blood flow, and this reflects cerebral blood flow and alterations in brain compliance. Alterations in the therapeutic regimen can be based on these observations.

[A study of ITS sequence as a molecular marker in the phylogeny of Drosophila nasuta subgroup].

A total of 1.1 kb including whole ITS (intertranscribed spacer), part of 5.8S rDNA and 2S rDNA were sequenced. The results reveal that D. pallidifrons, Taxon I and Taxon J share the same sequence, and D. albomicans and D. s. neonasuta have the other same one. Among the sequences, there were a handful of insertions, deletions and substitutions. Insertions and deletions occur mainly between outgroup and ingroups; yet only 1 insertion and 12 deletions were detected in D. niveifrons, and 1 deletion was found in D. s. sulfurigaster. Among all the substitutions in outgroup and ingroups, totally 55 transitions and 65 transvertions were detected. The value of transition transvertion is quite different to that of mitochondrial genome. We applied parsimony and NJ methods to reconstruct the phylogenetic relationships of the 7 taxa. They show that D. niveifrons is on the basis of the trees, which suggests that it be less related to the rest; D. albomicans, D. s. neonasuta, D. pallidifrons, Taxon I and Taxon J are more related. We suggested that more related topology of D. s. sulfurigaster and D. s. neonasuta due to the specific evolution of ITS, yet not meaning their real relationships. Sequence variations and phylogeny analysis reveal that the ITS may be not an informative marker to nasuta subgroup. However, secondary structure analysis by using PCFOLD 4.0 reveals that the structure of the ITS is quite conservative; the stem-loop of ITS 3'-end may be an important structure of rDNA splicing.