case_images: PMC4397521_01
case_images: BMB Hypercellular marrow, megakaryo-granulocytes proliferation, clustered polymorphous atypical megakaryocytes (HE stain, ob x20)
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case_images: F1
case_images: Bone marrow biopsy (BMB) examination showed a hypercellular marrow (about 80% marrow cellularity), with pronounced proliferation of megakaryo- and granulopoiesis: very frequently polymorphous megakaryocytes, with an atypical morphology (from giant megakaryocytes with cloud-like, bulbous nuclei to small dwarf megakaryocytes with hyperchromatic nuclei); megakaryocytes were densely clustered perivascularly and paratrabecularly; sinusoids were proliferated, without intravascular hematopoiesis (Fig. 1). The immunohistochemical (IHC) reevaluation of the BMB with AE1/ AE3 pan cytokeratin and kappa and lambda light chains showed interstitial plasma cells clonal infiltration (approx. 20-22%) with lambda light chain restriction (Fig. 8); AE1/ AE3 was negative.
case_images: 2 BMB - Grade 2 fibrosis (Gomori stain, ob x20)
case_images: JMedLife-08-55-g002.jpg
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case_images: F2
case_images: Gomori silver stain for fibrosis showed grade 2 myelofibrosis (Fig. 2).
case_images: ARMS-PCR method used for detection of the V617F mutation in JAK 2 gene positive for Case 1. 1st lane - Molecular weight marker (of 100smp in 100 smp); 2nd 9th lanes - Pacient samples 2 locus for each pacient; 10th-11th lanes - Positive control; 12th-13th lanes - Negative control; 14th-15th lanes - Blank. The first lane of each patient is mutant allele (JAK2 V617F) and the 2nd is the normal allele (wildtype JAK2)
case_images: JMedLife-08-55-g003.jpg
case_images: PMC4397521_01_JMedLife-08-55-g003.jpg
case_images: F3
case_images: This mutation was identified in heterozygous form in the JAK2 gene (Fig. 3).
case_images: Aligned sequences showing mutation in JAK2 exon 14 - c1849 G>T (V617F). Prevalence of this mutation in this patient was 72%. Sequencing was performed on Miseq Illumina. For JAK2 exon 12, 13 and 14 sequencing, specific primers, which amplify exon 12, 13 and 14, were used. After amplification, amplicons were purified by using Agencourt Ampure XP magnetic beads following the manufacturer's protocol. Purified amplicons were indexed by using indexes provided by Illumina and resulting fragments sequenced on Miseq sequencer from Illumina, after another round of purification by using Agencourt Ampure XP. Sequencing was performed by using MIseq kit Nano and 2x250 sequencing protocol. Resulting sequences were aligned by using LaserGene 11.2 software and visualized and analyzed by using IGV version 2.3.40. An average coverage of 10000x was obtained for all exons.
case_images: JMedLife-08-55-g004.jpg
case_images: PMC4397521_01_JMedLife-08-55-g004.jpg
case_images: F4
case_images: This result was confirmed by JAK2 gene sequencing using next generation sequencing (Fig. 4).
case_images: Vertebral tumor - Extramedular infiltration of plasma cell myeloma (HE stain, ob 40x)
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case_images: F5
case_images: Serum protein electrophoresis, no monoclonal spike; normal values
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case_images: F6
case_images: Immunofixation electrophoresis: positive for light lambda chain
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case_images: F7
case_images: BMB IHC examinatin - interstitial lambda chain myelomatous infiltrate (IHC stain for lambda light chain, ob x20)
case_images: JMedLife-08-55-g008.jpg
case_images: PMC4397521_01_JMedLife-08-55-g008.jpg
case_images: F8
case_images: The immunohistochemical (IHC) reevaluation of the BMB with AE1/ AE3 pan cytokeratin and kappa and lambda light chains showed interstitial plasma cells clonal infiltration (approx. 20-22%) with lambda light chain restriction (Fig. 8); AE1/ AE3 was negative.
case_images: PMC4397521_02
case_images: BMB Hypercellular marrow by diffuse plasma cells infiltrate; small clusters of giant megakaryocytes (HE stain, ob x40)
case_images: JMedLife-08-55-g0010.jpg
case_images: PMC4397521_02_JMedLife-08-55-g0010.jpg
case_images: F10
case_images: The bone marrow biopsy was repeated and this showed a hyper cellular marrow with diffuse atypical plasma cells infiltration (approx. 60%); small clusters of giant megakaryocytes still remaining (Fig. 10).
case_images: BMB Plasma cells infiltrate was clonal, with lambda light chain restriction (IHC stain for lambda light chain, ob 40x)
case_images: JMedLife-08-55-g0011.jpg
case_images: PMC4397521_02_JMedLife-08-55-g0011.jpg
case_images: F11
case_images: IHC stain showed clonality of marrow plasma cell infiltrate, with lambda light chain restriction (Fig. 11).
case_images: ARMS-PCR method for the detection of V617F mutation in JAK 2 gene low level positive for Case 2. 1st lane - Molecular weight marker (of 100 samples/ s in 100s); 2nd 9th lane - Pacient samples 2 lanes for each pacient; 10th - 11th lane - Positive control; 12th-13th lane - Negative control; 14th-15th - Blank. The first lane of each patient is mutant allele (JAK2 V617F) and the 2nd is the normal allele (wildtype JAK2)
case_images: JMedLife-08-55-g0012.jpg
case_images: PMC4397521_02_JMedLife-08-55-g0012.jpg
case_images: F12
case_images: The molecular biological tests were repeated, showing the V617F somatic mutation allele in the JAK2 gene low level positive (Fig. 12) in the DNA sample analyzed by ARMS - PCR.
case_images: Aligned sequences showing mutation in JAK2 exon 14 - c1849 G>T (V617F). Prevalence of this mutation in this patient was 9%. Sequencing was performed on Miseq Illumina
case_images: JMedLife-08-55-g0013.jpg
case_images: PMC4397521_02_JMedLife-08-55-g0013.jpg
case_images: F13
case_images: This result was confirmed by JAK2 gene sequencing by using next generation sequencing (Fig. 13).