Diagnosis of inherited platelet disorders on a blood smear: a tool to facilitate worldwide diagnosis of platelet disorders.

Essentials There are many hereditary platelet disorders (HPD) but diagnosing these is challenging. We provide a method to diagnose several HPDs using standard blood smears requiring < 100 µL blood. By this approach, the underlying cause of HPD was characterized in ~25-30% of referred individuals. The method facilitates diagnosis of HPD for patients of all ages around the world. SUMMARY: Background Many hereditary thrombocytopenias and/or platelet function disorders have been identified, but diagnosis of these conditions remains challenging. Diagnostic laboratory techniques are available only in a few specialized centers and, using fresh blood, often require the patient to travel long distances. For the same reasons, patients living in developing countries usually have limited access to diagnosis. Further, the required amount of blood is often prohibitive for pediatric patients. Objectives By a collaborative international approach of four centers, we aimed to overcome these limitations by developing a method using blood smears prepared from less than 100 µL blood, for a systematic diagnostic approach to characterize the platelet phenotype. Methods We applied immunofluorescence labelling (performed centrally) to standard air-dried peripheral blood smears (prepared locally, shipped by regular mail), using antibodies specific for proteins known to be affected in specific hereditary platelet disorders. Results By immunofluorescence labelling of blood smears we characterized the underlying cause in 877/3217 (27%) patients with suspected hereditary platelet disorders (HPD). Currently about 50 genetic causes for HPD are identified. Among those, the blood smear method was especially helpful to identify MYH9 disorders/MYH9-related disease, biallelic Bernard-Soulier syndrome, Glanzmann thrombasthenia and gray platelet syndrome. Diagnosis could be established for GATA1 macrothrombocytopenia, GFI1B macrothrombocytopenia, ß1-tubulin macrothrombocytopenia, filamin A-related thrombocytopenia and Wiskott-Aldrich syndrome. Conclusion Combining basic and widely available preanalytical methods with the immunomorphological techniques presented here, allows detailed characterization of the platelet phenotype. This supports genetic testing and facilitates diagnosis of hereditary platelet disorders for patients of all ages around the world.

Functional characterization of a novel GFI1B mutation causing congenital macrothrombocytopenia.

UNLABELLED: Essentials Two groups recently reported GFI1B as a novel causative gene for congenital macrothrombocytopenia. We performed functional analysis of a novel GFI1B mutation and previous mutations. An immunofluorescence analysis of the platelet CD34 expression can be useful as a screening test. Mutant-transduced megakaryocytes produced enlarged proplatelet tips which were reduced in number. SUMMARY: Background GFI1B is an essential transcription factor for megakaryocyte and erythrocyte development. Two groups have recently identified GFI1B as a novel causative gene for congenital macrothrombocytopenia associated with α-granule deficiency. Methods We performed whole exome sequencing and identified a novel GFI1B p.G272fsX274 mutation in a family with macrothrombocytopenia, and a decreased number of platelet α-granules and abnormally shaped red blood cells. p.G272fsX274 and the previous two mutations all predicted disruption of an essential DNA-binding domain in GFI1B. We therefore performed functional studies to characterize the biochemical and biological effects of these three patient-derived mutations. Results An immunofluorescence analysis revealed decreased thrombospondin-1 and increased CD34 expression in platelets from our patient. Consistent with the previous studies, the three patient-derived mutants were unable to repress the expression of the reporter gene and had a dominant-negative effect over wild-type GFI1B. In addition, the three mutations abolished recognition of a consensus-binding site in gel shift assays. Furthermore, transduction of mouse fetal liver-derived megakaryocytes with the three GFI1B mutants resulted in the production of abnormally large proplatelet tips, which were reduced in number. Conclusions Our study provides further proof of concept that GFI1B is an essential protein for the normal development of the megakaryocyte lineage.

A prevalent founder mutation and genotype-phenotype correlations of OTOF in Japanese patients with auditory neuropathy.

Auditory neuropathy is a hearing disorder characterized by normal outer hair cell function and abnormal neural conduction of the auditory pathway. Aetiology and clinical presentation of congenital or early-onset auditory neuropathy are heterogeneous, and their correlations are not well understood. Genetic backgrounds and associated phenotypes of congenital or early-onset auditory neuropathy were investigated by systematically screening a cohort of 23 patients from unrelated Japanese families. Of the 23 patients, 13 (56.5%) had biallelic mutations in OTOF, whereas little or no association was detected with GJB2 or PJVK, respectively. Nine different mutations of OTOF were detected, and seven of them were novel. p.R1939Q, which was previously reported in one family in the United States, was found in 13 of the 23 patients (56.5%), and a founder effect was determined for this mutation. p.R1939Q homozygotes and compound heterozygotes of p.R1939Q and truncating mutations or a putative splice site mutation presented with stable, and severe-to-profound hearing loss with a flat or gently sloping audiogram, whereas patients who had non-truncating mutations except for p.R1939Q presented with moderate hearing loss with a steeply sloping, gently sloping or flat audiogram, or temperature-sensitive auditory neuropathy. These results support the clinical significance of comprehensive mutation screening for auditory neuropathy.

In vitro characterization of missense mutations associated with quantitative protein S deficiency.

OBJECTIVE: To elucidate the molecular consequences of hereditary protein S (PS) deficiency, we investigated the in vitro synthesis of the PS missense mutants in COS-1 cells and their activated protein C (APC) cofactor activities. PATIENTS: Four patients with quantitative PS deficiency suffering from venous thrombosis were examined. RESULTS: We identified three distinct novel missense mutations, R275C, P375Q and D455Y, and two previously reported missense mutations, C80Y and R314H. The P375Q and D455Y mutations were found in one patient and observed to be in linkage on the same allele. The R314H mutant showed the lowest level of expression (32.7%), and the C80Y, P375Q + D455Y, and R275C mutants exhibited a moderate impairment of expression, that is, 43.8%, 49.5%, and 72.3% of the wild type, respectively. Furthermore, pulse-chase experiments demonstrated that all mutants showed impaired secretion and longer half-lives in the cells than the wild type PS. In the APC cofactor assays, the C80Y mutant showed no cofactor activity, and the R275C mutant showed reduced activity, 62.3% of the wild type PS, whereas the R314H and P375Q + D455Y mutants exhibited normal cofactor activity. CONCLUSION: These data indicate that the C80Y and R275C mutations affect the secretion and function of the PS molecule, and that the R314H and P375Q + D455Y mutations are responsible for only secretion defects, causing the phenotype of quantitative PS deficiency observed in the patients.

The reproducibility of independently measuring human regional hepatic arterial, portal and total hepatic blood flow using [15O]water and positron emission tomography.

The reproducibility of repeated human regional hepatic blood flow quantification using [15O]water and positron emission tomography (PET) was evaluated as a method of monitoring the effect of drug administration on hepatic blood flow. Nineteen patients underwent two measurements of hepatic blood flow by PET. Fifteen minutes after the first dynamic study using [15O]water, a second dynamic study was performed, and hepatic blood flow was calculated. The correlation between the first and second dynamic study of arterial blood flow was highly significant (P=6.31 x 10(-10), r=0.946). The regression line was y=1.08x. The mean error between studies was 0.158. The correlation between the first and second dynamic study of portal blood flow also was significant (P=1.29 x 10(-7), r=0.897). The regression line was y=1.03x. The mean error between the studies was 0.164. The correlation between total hepatic blood flow in the first and second dynamic study, too, was significant (P=2.68 x 10(-7), r=0.888). The regression line was defined by y=1.06x. The mean error between studies was 0.140. Hepatic blood flow has increased if the second measurement of hepatic arterial, portal, and total blood flow is more than 115%, 111% and 114% of baseline, and has decreased if the second measurement is less than 101%, 95% and 98% of the first measurement.

Expression of low-frequency Ala108Pro substitution in the platelet glycoprotein Ibbeta gene.

We determined the gene frequency of the glycoprotein (GP) Ibbeta Ala108Pro substitution. The Pro108 allele was not found in 208 healthy Japanese and 200 healthy Caucasians. In vitro expression studies showed surface expression of the GPIbbeta Pro108 variant, suggesting the possibility of the involvement of the substitution as an alloantigen.

Are both PIVKA-II and alpha-fetoprotein necessary in follow-up management after hepatic resection for hepatocellular carcinoma?

BACKGROUND/AIMS: The aim of this study was to clarify the need for measuring of PIVKA-II (protein induced by vitamin K absence or antagonist-II) and alpha-fetoprotein as the prognostic indicator for patients after hepatic resection for hepatocellular carcinoma, and as the monitoring modality for early detection of recurrence after hepatic resection. METHODOLOGY: One hundred and thirty-one patients who underwent planned liver resections for hepatocellular carcinoma were studied. RESULTS: The survival rates in patients positive for preoperative tumor markers were significantly lower than in those in the negative patients. The first modality leading to the diagnosis of recurrence was measurement of alpha-fetoprotein and/or PIVKA-II in 25 cases (55.6%). Almost all patients (96.6%) with positive preoperative alpha-fetoprotein and recurrence had elevated alpha-fetoprotein again when recurrence was found. CONCLUSIONS: Preoperative PIVKA-II and/or alpha-fetoprotein levels can predict postoperative prognosis. Measurement of these markers is useful in monitoring recurrence. For following up patients with alpha-fetoprotein-producing tumors, alpha-fetoprotein monitoring only is sufficient to detect recurrence.

Presence of Propionibacterium acnes in blood components.

BACKGROUND: Sterility testing, as part of the QC of blood components at the Japanese Red Cross Aichi Blood Center between April 1998 and March 2000, showed that 10 of 5568 tested blood components (0.18%), all of which were RBC concentrates, were contaminated with bacteria. Nine isolates were Propionibacterium acnes and one was Staphylococcus capitis. STUDY DESIGN AND METHODS: To investigate the molecular relatedness of eight available P. acnes isolates, 16S rRNA gene analysis and random amplified polymorphic DNA (RAPD) analysis were performed. RESULTS: DNA sequencing analysis of the 16S rRNA gene showed that five isolates were identified as distinct strains and that three had identical sequences. RAPD analysis in the latter three isolates showed that two exhibited indistinguishable banding patterns that differed from that of the third isolate. CONCLUSION: P. acnes was the most frequent contaminant of blood components, and six of eight isolates were molecularly unrelated. Further studies are necessary to investigate the precise mechanisms of contamination.

Reproducibility of repeated human regional splenic blood flow measurements using [15O] water and positron emission tomography.

The reproducibility of measurement of regional splenic blood flow by dynamic positron emission tomography (PET) using [15O]water was evaluated. In 19 patients, the correlation between the first and second of two serial dynamic measurements was significant (P=1.78 x 10(-6); r=0.858). The regression equation was y = 1.06x, and the slope of the line described had a 95% confidence interval of 0.09. The error apparent between the two measurements was 0.129 (95% confidence interval 0.059). The results demonstrated sufficiently good reproducibility for measurements of regional splenic blood flow with PET and [15O]water to suggest use of this method for serial measurements intended to detect change, including drug effects.

Safety and utility of autologous blood transfusion for resection of metastatic liver tumor.

BACKGROUND/AIMS: The purpose of this study was to evaluate the safety and efficacy of predeposit autologous blood transfusion for resection of hepatic metastases. METHODOLOGY: We examined stored blood from 25 patients with advanced colorectal or gastric cancer for carcinoembryonic antigen mRNA using reverse-transcriptase-polymerase chain reaction assay to detect cancer cell in the autologous blood. We also retrospectively evaluated no transfusion (A, n = 44), autologous transfusion (B, n = 15), and homologous transfusion groups (C, n = 26) for perioperative liver function and long-term outcome after undergoing resection of liver metastases. RESULTS: In 5 of 25 patients, carcinoembryonic antigen mRNA was detected immediately after blood donation and after 7 days of storage, but not after 14-21 days of storage. The cumulative 5-year survival rates for groups A, B, and C were not different. However, disease-free survival with colorectal liver metastases was significantly higher in group A than in group C (P = 0.019). Total bilirubin concentrations in group C on the first postoperative day were also significantly higher than group A (P = 0.025). CONCLUSIONS: Stored autologous blood may contain cancer cells, but these decrease or disappear after storage for more than 7 days. For hepatic resection of metastases, transfusion avoidance yields the optimal outcome.

Mutations in the NMMHC-A gene cause autosomal dominant macrothrombocytopenia with leukocyte inclusions (May-Hegglin anomaly/Sebastian syndrome).

Macrothrombocytopenia with leukocyte inclusions is a rare autosomal dominant platelet disorder characterized by a triad of giant platelets, thrombocytopenia, and characteristic Döhle body-like leukocyte inclusions. A previous study mapped a locus for the disease on chromosome 22q12.3-q13.2 by genome-wide linkage analysis. In addition, the complete DNA sequence of human chromosome 22 allowed a positional candidate approach, and results here indicate that the gene encoding nonmuscle myosin heavy chain-A, NMMHC-A, is mutated in this disorder. Mutations were found in 6 of 7 Japanese families studied: 3 missense mutations, a nonsense mutation, and a one-base deletion resulting in a premature termination. Immunofluorescence studies revealed that NMMHC-A distribution in neutrophils appeared to mimic the inclusion bodies. These results provide evidence for the involvement of abnormal NMMHC-A in the formation of leukocyte inclusions and also in platelet morphogenesis.

A missense mutation (Tyr88 to Cys) in the platelet membrane glycoprotein Ibbeta gene affects GPIb/IX complex expression--Bernard-Soulier syndrome in the homozygous form and giant platelets in the heterozygous form.

This study examined the molecular basis of a missense mutation of the platelet glycoprotein (GP) Ibbeta gene in two families. In the propositus with a novel form of Bernard-Soulier syndrome (BSS) from Family I, only GPIbalpha was detectable in reduced amounts on platelet surfaces by flow cytometry. There were no GPIX or GPIbbeta found by immunoblotting. DNA sequencing analysis showed a homozygous mutation in the GPIbbeta gene which changed Tyr (TAC) to Cys (TGC) at residue 88. Her parents were heterozygous for Tyr88Cys in the GPIbbeta gene. In transient transfection studies on 293T cells, both Tyr88Cys and Tyr88Ala mutations suppressed the expression of GPIb/IX complexes. In addition, Tyr88Cys GPIbbeta mutation was found to exert a dominant negative effect on the GPIbalpha expression. Five individuals from Family II, four of whom reported elsewhere as having giant platelet disorders with normal aggregation (BLOOD, 1997: 89: 2404) and one newly analyzed in this study, were heterozygous for Tyr88Cys in the GPIbbeta gene. Microsatellite analysis of chromosome 22 showed a common haplotype in 8 of the individuals with Tyr88Cys mutations in Families I and II. Tyr88 in the GPIbbeta gene plays a significant role in the GPIb/IX expression; the defect causes BSS in a homozygous form and possibly giant platelets in a heterozygous form.

Novel heterozygous missense mutation in the platelet glycoprotein Ib beta gene associated with isolated giant platelet disorder.

The glycoprotein (GP) Ib/IX/V complex plays an important role in primary hemostasis, serving as the platelet receptor for von Willebrand factor (vWF). Recent studies have shown that the phenotype caused by mutations in the subunits of the GPIb/IX complex spans a wide spectrum; from the normal phenotype, to isolated giant platelet disorders (GPD), and to the full-blown bleeding disorder, the Bernard-Soulier syndrome (BSS). We characterize here a novel missense mutation of the GPIb beta gene associated with isolated GPD. In the patient's platelets, the expression level of the GPIb/IX complex was moderately reduced compared with that of the GPIIb/IIIa complex, whereas the latter was expressed at higher levels than in a normal control. Immunoblot analysis showed normal electrophoretic mobility of GPIb alpha, GPIb beta, and GPIX. However, the amount of GPIb beta was approximately 66% of the normal value. DNA sequencing analysis revealed a novel heterozygous missense mutation in the GPIb beta gene that converts Arg (CGC) to Cys (TGC) at residue 17. Transient transfection studies demonstrated that mutant GPIb beta protein was not detected in transfected 293T cells. These findings indicated that null expression of the abnormal GPIb beta causes decreased expression of the complex and results in the GPD phenotype in the patient, and suggested that homozygosity of the mutation may lead to a BSS phenotype in vivo.

Identification of six novel MYH9 mutations and genotype-phenotype relationships in autosomal dominant macrothrombocytopenia with leukocyte inclusions.

The autosomal dominant macrothrombocytopenia with leukocyte inclusions, May-Hegglin anomaly (MHA), Sebastian syndrome (SBS), and Fechtner syndrome (FTNS), are rare platelet disorders characterized by a triad of giant platelets, thrombocytopenia, and characteristic Döhle body-like leukocyte inclusions. The locus for these disorders was previously mapped on chromosome 22q12.3-q13.2 and the disease gene was recently identified as MYH9, the gene encoding the nonmuscle myosin heavy chain-A. To elucidate the spectrum of MYH9 mutations responsible for the disorders and to investigate genotypephenotype correlation, we examined MYH9 mutations in an additional 11 families and 3 sporadic patients with the disorders from Japan. Korea, and China. All 14 patients had heterozygous MYH9 mutations, including three known mutations and six novel mutations (three missense and three deletion mutations). Two cases had Alport manifestations including deafness, nephritis, and cataracts and had R1165C and E1841K mutations, respectively. However, taken together with three previous reports, including ours, the data do not show clear phenotype-genotype relationships. Thus, MHA, SBS, and FTNS appear to represent a class of allelic disorders with variable phenotypic diversity.

Safety and advantage of perioperative autologous blood transfusion in hepatic resection for hepatocellular carcinoma.

BACKGROUND: The safety and advantages of perioperative autologous blood transfusion (ABT) were evaluated on hepatectomy for hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Blood samples were obtained and stored from 30 patients with HCC. HCC cells were investigated by the presence of AFPmRNA using RT-PCR after storage. We also reviewed postoperative liver function and the long-term outcomes of 138 patients who underwent hepatectomy receiving ABT compared with patients receiving homologous blood transfusion (HBT) and patients without blood transfusion. RESULTS: AFPmRNA was not detected in all samples stored for more than 14 days. Postoperative ALT, AST and total bilirubin in the HBT group were significantly higher than those of other groups. Patients in the HBT group had significantly lower survival rates than patients in the ABT group. CONCLUSION: ABT was safe after storage and it had advantages compared with HBT with regard to postoperative liver function and survival rate after the hepatectomy for HCC.