A Novel Amino Acid Substitution, Fibrinogen Bßp.Pro234Leu, Associated with Hypofibrinogenemia Causing Impairment of Fibrinogen Assembly and Secretion.

We identified a novel heterozygous variant, Bßp.Pro234Leu (fibrinogen Tokorozawa), which was suspected to be associated with hypofibrinogenemia. Therefore, we analyzed the assembly and secretion of this fibrinogen using Chinese hamster ovary (CHO) cells. To determine the impact on the synthesis and secretion of fibrinogen of the Bßp.P234L and γp.G242E substitutions, we established recombinant variant fibrinogen-producing CHO cell lines. Synthesis and secretion analyses were performed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting analysis with the established cell lines. In addition, we performed fibrin polymerization using purified plasma fibrinogen and in-silico analysis. Both Bßp.P234L and γp.G242E impaired the secretion and synthesis of fibrinogen. Moreover, immunoblotting analysis elucidated the mobility migration of the Bßγ complex in Bßp.P234L. On the other hand, the fibrin polymerization of fibrinogen Tokorozawa was similar to that of normal fibrinogen. In-silico analysis revealed that the Bßp.P234 residue is located in the contact region between the Bß and γ chains and contacts γp.G242 residue. The present study demonstrated that the Bßp.P234L substitution resulted in hypofibrinogenemia by decreasing the assembly and secretion of fibrinogen. Therefore, there is a possibility that substitutions in the contact region between the Bß and γ chains impact the assembly and secretion of fibrinogen.

Rapid ABO genotyping by high-speed droplet allele-specific PCR using crude samples.

BACKGROUND: ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele-specific PCR (droplet-AS-PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells. METHODS: We designed allele-specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification. RESULTS: Droplet-AS-PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%-10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet-AS-PCR method were always consistent with those obtained by direct sequencing. CONCLUSION: The droplet-AS-PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet-AS-PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care.

A Novel Mutation in the Fibrinogen Bß Chain (c.490G>A; End of Exon 3) Causes a Splicing Abnormality and Ultimately Leads to Congenital Hypofibrinogenemia.

We found a novel heterozygous mutation in the fibrinogen Bß chain (c.490G>A) of a 3-year-old girl with congenital hypofibrinogenemia. To clarify the complex genetic mechanism, we made a mini-gene including a FGB c.490G>A mutation region, transfected it into a Chinese Hamster Ovary (CHO) cell line, and analyzed reverse transcription (RT) products. The assembly process and secretion were examined using recombinant mutant fibrinogen. Direct sequencing demonstrated that the mutant RT product was 99 bp longer than the wild-type product, and an extra 99 bases were derived from intron 3. In recombinant expression, a mutant Bß-chain was weakly detected in the transfected CHO cell line, and aberrant fibrinogen was secreted into culture media; however, an aberrant Bß-chain was not detected in plasma. Since the aberrant Bß-chain was catabolized faster in cells, the aberrant Bß-chain in a small amount of secreted fibrinogen may catabolize in the bloodstream. FGB c.490G>A indicated the activation of a cryptic splice site causing the insertion of 99 bp in intron 3. This splicing abnormality led to the production of a Bß-chain possessing 33 aberrant amino acids, including two Cys residues in the coiled-coil domain. Therefore, a splicing abnormality may cause impaired fibrinogen assembly and secretion.

STAT3 gene mutations and their association with pure red cell aplasia in large granular lymphocyte leukemia.

Large granular lymphocyte leukemia (LGL L) has been morphologically characterized as a group of lymphoproliferative diseases that include T-cell large granular lymphocytic leukemia (T-LGL L) and chronic lymphoproliferative disorders of natural killer cells (CLPD-NK). We investigated mutations in the Src homology 2 (SH2) domain of the signal transducer and activator of transcription 3 (STAT3) gene in Asian cohorts of T-LGL L and CLPD-NK (n = 42 and 11, respectively). Two mutations, Y640F and D661Y, were identified using direct sequencing or allele-specific (AS) PCR. Y640F and D661Y mutations were found in seven and 18 patients, respectively. Two patients were positive for both mutations. Frequencies of STAT3 mutations in T-LGL L and CLPD-NK were 47.6% and 27.2%, respectively. Pure red cell aplasia (PRCA) was associated with the mutations (P = 0.005). The mutations were persistently found at stable levels in some patients after more than 5 years using AS-quantitative PCR. The results of the present study indicate that the SH2 domain of the STAT3 gene is frequently mutated in Asian T-LGL L and CLPD-NK, and that PRCA is closely correlated with the mutations.

Epithelial-mesenchymal transition of A549 cells is enhanced by co-cultured with THP-1 macrophages under hypoxic conditions.

Epithelial-mesenchymal transition (EMT) is associated with pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF). In this study, we investigated EMT of human pulmonary epithelial-derived cells (A549). A549 cells was either cultured by itself or co-cultured with THP-1 macrophages under normoxic (21% O2) and hypoxic (2% O2) conditions. We evaluated the presence of EMT by determining the expression of EMT markers, E-cadherin, vimentin, and fibronectin. To determine the role of TGF-ß1 and IL-1ß in EMT of the A549 cells, we analyzed the effects of blocking their activity with TGF-ß1 inhibitor or IL-1ß neutralizing antibody respectively. The A549 cells presented EMT when they were co-cultured with THP-1 macrophages. The EMT of the A549 cells co-cultured with THP-1 macrophages was exacerbated under hypoxia. In addition, the EMT were prevented by the addition of TGF-ß1 type I receptor kinase inhibitor. The hypoxic condition increased the mRNA levels of TGF-ß1 in A549 cells and THP-1 macrophages and that of IL-1ß in THP-1 macrophages when each cells were co-cultured. Anti-IL-1ß neutralizing antibody attenuated TGF-ß1 secretion in co-culture media under hypoxic conditions. Thus, the IL-1ß from THP-1 macrophages up-regulated the TGF-ß1 from A549 cells and THP-1 macrophages, and then the TGF-ß1 from both cells induced and promoted the EMT of A549 cells when they were co-cultured under hypoxia. Together, these results demonstrate that the interaction between type II pneumocytes and macrophages under hypoxia is necessary for the development of pulmonary fibrosis.

[Case with intrauterine fetus death: interphase fluorescence in situ hybridization using buccal cells is useful for examining chromosomal abnormalities when placental villus not available].

The proband was a male fetus who died at 18 weeks of gestation. The fetus had growth retardation, hydrocephalus, exophthalmos, and micrognathia. The placental villus was not available. We performed interphase fluorescence in situ hybridization (FISH) using buccal cells of the fetus. The FISH using centromere specific probes for chromosome 7, 8 and 18, and RB1 gene (13q14)-specific probe showed three signals for each chromosome. The sex chromosome composition was XXY by FISH using centromere-specific probes for X and Y chromosomes. Thus, the fetus was diagnosed with triploidy syndrome. This report suggested that interphase FISH using buccal cells is useful for examining chromosomal abnormalities in intrauterine fetal death when placental villus is not available.

[Development of multiplex short tandem repeat (STR)-PCR for chimerism analysis in patients with hematological malignancies and comparison of chimerism in different sample sources].

Polymerase chain reaction analysis of short-tandem repeat (STR) markers (STR-PCR) has been used for chimerism testing to assess engraftment following hematopoietic stem cell transplantation (HSCT). We investigated the informativity of 7 STR loci (FGA, D5S818, SE33, TH01, VWF, PentaE, and D18S51) in 82 pre-HSCT DNA samples from 41 donor/recipient pairs and developed 2 multiplex STR-PCRs using VWF, SE33, and D18S51, D5S818 and FGA, respectively. The multiplex STR-PCRs could distinguish the recipients and donors in 92.7% of the cases. Dilution experiments using mixed DNA showed that the sensitivity of the multiplex STR-PCRs for detecting the minor population was 1-5%. To compare chimerism in different samples such as peripheral blood, mononuclear cells (MNC), and CD3-positive cells (CD3+), we investigated the relationship between the chimerisms at approximately day 30 post-HSCT and the interval from the day of HSCT to achievement of complete chimerism (CC) in 70 patients undergoing HSCT. CC was found in all samples of 54 patients at day 30 post-HSCT, and these samples showed CC thereafter. Eleven patients with mixed chimerism (MC) in all samples or in MNC and CD3+ showed CC at day 60-270 post-HSCT or persistent MC. The remaining 5 patients with MC in only CD3+ showed CC at day 30-60 post-HSCT. Taken together, MNC which can be separated easily may be a useful source for detecting patients who require longer time to achieve CC and those with high risk of graft failure.

Redox index of Cys-thiol residues of serum apolipoprotein E and its diagnostic potential.

BACKGROUND: The redox modulation of Cys-thiol participates in various pathophysiological processes. We explored the proper index for estimating the redox status of Cys-thiol of serum apolipoprotein E (apoE), named "redox-IDX-apoE," which is necessary to understand the redox biology of age-related diseases. METHODS: The fractions of the reduced form (red-), reversible oxidized form (roxi-), and irreversibly oxidized form (oxi-) apoE in serum, obtained from the patients with no apparent disease (controls, n=192) and with atherosclerosis and type 2 diabetes (patients, n=16), were measured by a band-shift assay using a maleimide compound. Redox-IDX-apoE candidates were determined by calculating the values of these fractions and the total apoE concentration. RESULTS: Cys number of apoE significantly increased for the ratio of roxi-apoE to total-apoE (roxi/total) (E2/E3>E3/E3>E3/E4) but decreased for the ratios of red-apoE to roxi-apoE (red/roxi) and [red-apoE + oxi-apoE] to roxi-apoE ([red + oxi]/roxi) (E2/E3

Novel variant fibrinogen γp.C352R produced hypodysfibrinogenemia leading to a bleeding episode and failure of infertility treatment.

INTRODUCTION: We identified a patient with a novel heterozygous variant fibrinogen, γp.C352R (Niigata II; N-II), who had a bleeding episode and failed infertility treatment and was suspected to have hypodysfibrinogenemia based on low and discordant fibrinogen levels (functional assay 0.33 g/L, immunological assay 0.91 g/L). We analyzed the mechanism of this rare phenotype of a congenital fibrinogen disorder. MATERIALS AND METHODS: Patient plasma fibrinogen was purified and protein characterization and thrombin-catalyzed fibrin polymerization performed. Recombinant fibrinogen-producing Chinese hamster ovary (CHO) cells were established and the assembly and secretion of variant fibrinogen analyzed by ELISA and western blotting. RESULTS: Purified N-II plasma fibrinogen had a small lower molecular weight band below the normal γ-chain and slightly reduced fibrin polymerization. A limited proportion of p.C352R fibrinogen was secreted into the culture medium of established CHO cell lines, but the γ-chain of p.C352R was synthesized and variant fibrinogen was assembled inside the cells. CONCLUSION: We demonstrated that fibrinogen N-II, γp.C352R was associated with markedly reduced secretion of variant fibrinogen from CHO cells, that fibrin polymerization of purified plasma fibrinogen was only slightly affected, and that fibrinogen N-II produces hypodysfibrinogenemia in plasma.

Quantitative assessment of PTPN11 or RAS mutations at the neonatal period and during the clinical course in patients with juvenile myelomonocytic leukaemia.

To evaluate minimal residual disease (MRD) after chemotherapy and haematopoietic stem cell transplantation in juvenile myelomonocytic leukaemia (JMML), a locked nucleic acid-allele specific quantitative polymerase chain reaction (LNA-AS-qPCR) was developed for 13 patients (four types of PTPN11 mutation and four types of RAS mutation). The post-transplant MRD detected by LNA-AS-qPCR analysis was well correlated with chimerism assessed by short tandem repeat PCR analysis. Non-intensive chemotherapy exerted no substantial reduction of the tumour burden in three patients. There was no significant difference in the quantity of RAS mutant DNA after spontaneous haematological improvement in 4 patients with NRAS or KRAS 34G > A during a 2- to 5-year follow-up. PTPN11, NRAS, or KRAS mutant DNA was detected from Guthrie card dried blood in five of seven patients (who were aged <2 years at diagnosis) at a level of 1.0-6.5 x 10(-1) of the values at diagnosis. Accordingly, these five patients might have already reached a subclinical status at birth. Considering the negative correlation between mutant DNA level in neonatal blood spots and age at diagnosis, JMML patients with a larger tumour burden at birth appeared to show earlier onset.

Congenital fibrinogen disorder with a compound heterozygote possessing two novel FGB mutations, one qualitative and the other quantitative.

INTRODUCTION: Congenital fibrinogen disorders result from genetic mutations in FGA, FGB, or FGG resulting in quantitative fibrinogen deficiencies (afibrinogenemia or hypofibrinogenemia) or qualitative fibrinogen deficiencies (dysfibrinogenemia). Hypodysfibrinogenemia sharing features with hypo- and dysfibrinogenemia is rare. We performed genetic and functional analyses of a 31-year-old woman with suspected hypodysfibrinogenemia. MATERIALS AND METHODS: Functional and antigenic fibrinogen values of patient were 1.05 and 1.24 g/L, respectively. DNA sequence and western blotting analyses for plasma fibrinogen were performed. A minigene incorporating the mutational region was transfected into a Chinese hamster ovary cell line (CHO), and reverse transcription products were analyzed. Assembly and secretion were examined using the recombinant variant fibrinogen. We purified the patient's plasma fibrinogen and analyzed thrombin-catalyzed fibrin polymerization (TCFP). RESULTS AND CONCLUSIONS: DNA sequencing revealed compound heterozygous nucleotide mutations with FGB 35 bp c.1245-17_1262 or -16_1263 del and FGB c.510T>A (resulting in Bßp.N170K substitution) on different alleles. We did not detect shortened Bß-chain peptides in the plasma using western blotting analysis. A minigene incorporating the deletion DNA showed two aberrant mRNA products. The secretion of Bßp.N170K-fibrinogen-CHO was almost same as normal Bß-fibrinogen-CHO. TCFP of plasma Bßp.N170K fibrinogen was slightly lower than that of normal plasma fibrinogen. Aberrant splicing products derived from the 35 bp deletion caused hypofibrinogenemia due to nonsense-mediated mRNA decay and suggested the presence of only Bßp.N170K fibrinogen in patient's plasma. Bßp.N170K caused dysfibrinogenemia due to a delay in lateral aggregation. These findings demonstrated that these mutations respectively affected the fibrinogen quality and quantity, resulting in hypodysfibrinogenemia.

Heterozygous variant fibrinogen γA289V (Kanazawa III) was confirmed as hypodysfibrinogenemia by plasma and recombinant fibrinogens.

INTRODUCTION: Congenital fibrinogen disorders are classified as afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia. However, difficulties are associated with discriminating between dysfibrinogenemia, hypofibrinogenemia, and hypodysfibrinogenemia using routine analyses. We previously reported a heterozygous variant fibrinogen (γA289V; Kanazawa III) as hypodysfibrinogenemia; however, the same variant had previously been described as hypofibrinogenemia. To clarify the production of γA289V fibrinogen, we expressed recombinant γA289V (r-γA289V) fibrinogen and compared it with wild-type (WT) and adjacent recombinant variant fibrinogens. METHODS: Target mutations were introduced into a fibrinogen γ-chain expression vector by site-directed mutagenesis, and the vector was then transfected into Chinese hamster ovary cells to produce recombinant fibrinogen. Fibrinogen was purified from the plasma of the proposita, and culture media and fibrinogen functions were analyzed using fibrin polymerization, plasmin protection, and FXIIIa-catalyzed fibrinogen cross-linking. RESULTS: The fibrinogen concentration ratio of the culture media to cell lysates was markedly lower for r-γA289V fibrinogen than for WT. Because the secretion of recombinant γF290L (r-γF290L) fibrinogen was similar to WT, we compared r-γF290L fibrinogen functions with WT. The fibrin polymerization of Kanazawa III plasma (K-III) fibrinogen was significantly weaker than normal plasma fibrinogen. Moreover, K-III fibrinogen showed a markedly reduced "D:D" interaction. However, all functions of r-γF290L fibrinogen were similar to WT. An in silico analysis confirmed the above results. CONCLUSION: The present results demonstrated that γA289 is crucial for the γ-module structure, and the γA289V substitution markedly reduced fibrinogen secretion. Moreover, K-III fibrinogen showed markedly reduced fibrin polymerization and "D:D" interactions. γA289V fibrinogen was confirmed as hypodysfibrinogenemia.

Comparison of molecular structure and fibrin polymerization between two Bß-chain N-terminal region fibrinogen variants, Bßp.G45C and Bßp.R74C.

We identified two heterozygous dysfibrinogenemias, Bßp.Gly45Cys (Kyoto VII; K-VII) and Bßp.Arg74Cys (Iida II; I-II). The impairment of polymerization of Bßp.G45C has been well analyzed; however, that of Bßp.R74C has not. Thus, we compared fibrin polymerization between these variants. To determine the structural and functional characterization of purified fibrinogens, we performed immunoblotting analysis, kinetic analyses of fibrinopeptide A and B release, and thrombin- or batroxobin-catalyzed fibrin or fibrin monomer polymerization. Immunoblotting analysis showed that both variant fibrinogens had variant fibrinogen-albumin complexes and variant fibrinogen multimers, and the amounts of fibrinogen-albumin complexes with fibrinogen K-VII was more than with fibrinogen I-II. Moreover, fibrinopeptide B release from fibrinogen K-VII was about 50% of the control, whereas the others were normal. The maximum slopes of polymerization for variant fibrinogens were reduced, but fibrinogen K-VII was reduced more than fibrinogen I-II. The present study demonstrated that both Bßp.G45C and Bßp.R74C variants showed the presence of variant fibrinogen-albumin complexes and variant fibrinogen multimers, and polymerization of Bßp.G45C was impaired more than Bßp.R74C. Our study and several previous reports concerning the clinical phenotype of both variants suggested the risks of bleeding for patients with Bßp.G45C and thrombosis for patients with Bßp.R74C.

γD318Y fibrinogen shows no fibrin polymerization due to defective "A-a" and "B-b" interactions, whereas that of γK321E fibrinogen is nearly normal.

BACKGROUND: The fibrinogen γ-module has several functional sites and plays a role in dysfibrinogenemia, which is characterized by impaired fibrin polymerization. Variants, including γD318Y and γΔN319D320, have been reported at the high affinity Ca2+-binding site, and analyses using recombinant fibrinogen revealed the importance of this site for fibrinogen functions and secretion. We examined the polymerization abilities of the recombinant fibrinogen variants, γD318Y and γK321E. MATERIALS AND METHODS: γD318Y and γK321E were produced using CHO cells and fibrinogen functions were examined using thrombin- or batroxobin-catalyzed polymerization, gel chromatography, protection against plasmin degradation, and factor XIIIa cross-linking. RESULTS: γD318Y did not show any polymerization by thrombin or batroxobin, similar to γΔN319D320, whereas γK321E had slightly impaired polymerization. The functions of Ca2+ binding, hole 'a', and the "D-D" interaction were markedly reduced in γD318Y, and gel chromatography suggested altered protofibril formation. In silico analyses revealed that structural changes in the γ-module of these variants were inconsistent with polymerization results. The degree of structural changes in γD318Y was moderate relative to those in γD318A and γD320A, which had markedly impaired polymerization, and γK321E, which showed slightly impaired polymerization. CONCLUSION: Our results suggest that no polymerization of γD318Y or γΔN319D320 was due to the loss of both "A-a" and "B-b" interactions. Previous studies demonstrated that "B-b" interaction alone causes polymerization of neighboring γD318A and γD320A fibrinogen, which is subsequently decreased. Marked changes in the tertiary structure of the γD318Y γ-module influenced the location and/or orientation of the adjacent ß-module, which led to impaired "B-b" interactions.

Development of a rapid and sensitive one-step reverse transcription-nested polymerase chain reaction in a single tube using the droplet-polymerase chain reaction machine.

BACKGROUND: Reverse transcription (RT)-nested polymerase chain reaction (PCR) is a time-consuming procedure because it has several handling steps and is associated with the risk of cross-contamination during each step. Therefore, a rapid and sensitive one-step RT-nested PCR was developed that could be performed in a single tube using a droplet-PCR machine. METHODS: The K562 BCR-ABL mRNA-positive cell line as well as bone marrow aspirates from 5 patients with chronic myelogenous leukemia (CML) and 5 controls without CML were used. We evaluated one-step RT-nested PCR using the droplet-PCR machine. RESULTS: One-step RT-nested PCR performed in a single tube using the droplet-PCR machine enabled the detection of BCR-ABL mRNA within 40min, which was 10(3)-fold superior to conventional RT nested PCR using three steps in separate tubes. The sensitivity of the one-step RT-nested PCR was 0.001%, with sample reactivity comparable to that of the conventional assay. CONCLUSIONS: One-step RT-nested PCR was developed using the droplet-PCR machine, which enabled all reactions to be performed in a single tube accurately and rapidly and with high sensitivity. This one-step RT-nested PCR may be applicable to a wide spectrum of genetic tests in clinical laboratories.

Rapid single nucleotide polymorphism based method for hematopoietic chimerism analysis and monitoring using high-speed droplet allele-specific PCR and allele-specific quantitative PCR.

BACKGROUND: Chimerism analysis is important for the evaluation of engraftment and predicting relapse following hematopoietic stem cell transplantation (HSCT). We developed a chimerism analysis for single nucleotide polymorphisms (SNPs), including rapid screening of the discriminable donor/recipient alleles using droplet allele-specific PCR (droplet-AS-PCR) pre-HSCT and quantitation of recipient DNA using AS-quantitative PCR (AS-qPCR) following HSCT. METHODS: SNP genotyping of 20 donor/recipient pairs via droplet-AS-PCR and the evaluation of the informativity of 5 SNP markers for chimerism analysis were performed. Samples from six follow-up patients were analyzed to assess the chimerism via AS-qPCR. These results were compared with that determined by short tandem repeat PCR (STR-PCR). RESULTS: Droplet-AS-PCR could determine genotypes within 8min. The total informativity using all 5 loci was 95% (19/20). AS-qPCR provided the percentage of recipient DNA in all 6 follow-up patients without influence of the stutter peak or the amplification efficacy, which affected the STR-PCR results. CONCLUSION: The droplet-AS-PCR had an advantage over STR-PCR in terms of rapidity and simplicity for screening before HSCT. Furthermore, AS-qPCR had better accuracy than STR-PCR for quantification of recipient DNA following HSCT. The present chimerism assay compensates for the disadvantages of STR-PCR and is readily performable in clinical laboratories.

Rapid detection of PML-RARA fusion gene by novel high-speed droplet-reverse transcriptase-polymerase chain reaction: possibility for molecular diagnosis without lagging behind the morphological analyses.

BACKGROUND: Acute promyelocytic leukemia (APL) is an aggressive disease requiring prompt diagnosis and treatment. Rapid detection of the PML-RARA fusion gene provides the molecular basis for a highly effective therapy with all-trans retinoic acid. We developed a rapid assay by novel droplet-reverse transcriptase-polymerase chain reaction (droplet-RT-PCR) for the detection of the PML-RARA fusion gene in APL patients. METHODS: RNA was extracted from 7 samples obtained from 5 APL patients with the PML-RARA fusion gene confirmed by nested RT-PCR and fluorescence in situ hybridization. Using these 7 samples, we evaluated the reaction time and amplification efficiency of the droplet-RT-PCR. RESULTS: Using the droplet-RT-PCR, we could detect the PML-RARA fusion gene in all 7 samples. The reaction time for 50 cycles of droplet-RT-PCR was 27 min. The amplification by the droplet-RT-PCR assay was considered positive for the PML-RARA fusion gene in less than 22 min, at the point when the fluorescence exceeded the threshold level. CONCLUSIONS: Our novel droplet-RT-PCR assay is specific for the detection of the PML-RARA fusion gene and has a markedly reduced reaction time. Thus, the novel droplet-RT-PCR assay contributes to the rapid diagnosis of APL without lagging behind the morphological assessment.