bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical Scoring, the PF4 ENHANCED Assay, and the 14 C-Serotinin Release Assay for the Diagnosis of Heparin Induced Thrombocytopenia (HIT) in Complex Medical and Surgical Patients.
Authors: Shaheed Gurvinder , Malkovska Vera , Horne McDonald , Mendoza Jose , Patel Mehool , Rees John , Wesley Robert , Merryman Paula
Published: April 2020

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a clinico-pathological entity that presents a diagnostic challenge, especially in medically complex patients.

PATIENTS AND METHODS: In this observational and prospective study, 34 consecutive patients with suspected HIT were enrolled. They were clinically scored using two different clinical scoring systems and underwent testing for PVS-ELISA and 14C-SRA. 14C-SRA results were also available from 21 medical and surgical patients who had previously been tested.

PURPOSE: The objective of our study was to evaluate the sensitivity and specificity of the PF4 ENHANCED (GTI Diagnostics) ELISA for HIT performed at two institutions using the serotonin-release assay (14C-SRA) and clinical scoring systems as reference methods.

RESULTS: With the 14C-SRA as the reference method, the sensitivities and specificities of the ELISA were 100% and 57% respectively at one institution and 93% and 65% at the other. There was only one false-negative ELISA with the 14C-SRA as the reference standard. When compared with the clinical scoring by Greinacher et al., the sensitivity and specificity for PVS-ELISA was 81.82% and 50% and for 14C-SRA 40% and 100% respectively. Five of eleven patients who scored ’Very Likely’ by the Greinacher scoring system were either ’Unlikely’ or ’Possible’ with the Paulpard scoring system. Seven of 11patients with ’Very likely’ HIT and 7 of 8 with ’Possible’ HIT by Greinacher clinical criteria were 14C-SRA negative.

CONCLUSIONS: Our study did confirm the high specificity of 14C-SRA, however the sensitivity was low with clinical scoring as reference standard. Clinical scoring was discordant in more than 50% of patients with ’Very Likely’ HIT by Greinacher score when compared with Paulpard scoring. Thus neither the clinical score nor the 14C-SRA appears suitable as the ’gold standard’ for diagnosis of HIT. Because of its high sensitivity with the 14C-SRA as the reference method, we believe the PF4 ENHANCED ELISA should be used to identify HIT in patients with multiple potential causes of thrombocytopenia, although false-positive results will not be uncommon.
Low Platelet Counts: Diagnosis Using Flow Cytometry and Anti Platelet Antibody.
Authors: Guerra Joao Carlos C. , Kanayama Ruth H. , Nozawa Sonia S. , Ioshida Marcia R. , Takihi Irina Y. , Lazaro Robson J. , Hamerschlak Nelson , Rosenfeld Luiz Gastao M. , Guerra Celso Carlos C. , Bacal Nydia S.
Published: April 2020

In our service of hematology 9,1 % of all consults are related to low platelet number finding in the CBC. Pseudothormbocytopenia is a rather unusual phenomenon, induced by agglutination of platelets by auto-antibodies acting on EDTA collected blood.

Objective: Classify, diagnose and analyze low platelet counts as obtained by automatic blood counters.

Methods: From January 1997 to May 2005; 16761 patients were attended in our service. Among them, 1174 cases (7,0%), 429 males and 745 females (1 to 75 years), were there because of low platelet counts alone. Platelet counts were done by Coulter T-890, with blood collected in EDTA K3 and sodium citrate. Counts were repeated after letting blood at 4o C for 24 hours; also Fonio technique and Neubauer camera counts were done. Normal value was defined as platelet count between 150,000 to 450,000. Bone marrow was obtained when necessary and stained by conventional techniques. Serological and biochemical tests were done in a Cobas Core (Roche) machine. Immuno-phenotyping was done in 115 cases, to identify anti-platelet-antibody (CD41 PE Immunotech and anti human IgG FTIC conjugate Sigma)-direct method, by flow citometry (Coulter Epics XL-MCL).

Results: Among the 1158 cases, platelets counts between 120 000 to 150 000, were found in 15.8 %; laboratory error in 6,3 % and spurious low platelet counts in 1.3 %. In addition, the follow up for four years of those patients with “lower normal” platelet count (120 000 to 150 000) did not show any morbid process. Immunologycal idiopatic purpura (ITP) was diagnosed in 49,1 % of the cases, being as expected, more frequent in females, children and young adults. Hepatitis C as cause of low platelet counts was responsible for 6,9 % and HIV infection for 1.3 % of the cases Anti-platelet antibodies were present in 76.9 % of ITP cases and was not found in 88.3 % of normal platelet values.

Conclusions: In an hematological reference service should be considered a lower range for normal platelet values (120 000 to 450 000). Additionally, low platelet counts as diagnosed by automated machines should be confirmed by slide exam and anti platelet antibody is useful for asserting ITP diagnosis and to rule out spurious thrombocytopenia or normal values.
Treatment of Immune-Mediated Thrombocytopenia Purpura with Concurrent IVIg and Platelet Transfusion: A Retrospective Review of 40 Patients.
Authors: Spahr Joseph E. , Agarwal Neeraj , Rodgers George M.
Published: April 2020

Introduction: In January 2000, two patients with severe Immune-Mediated Thrombocytopenia (ITP) at our institution were successfully treated with prolonged infusions of IVIg and platelets. The dose of IVIg was 1 g/kg given by continuous infusion over 24 hours with concurrent platelets (1 pheresis unit every 8 hours). Based on these preliminary results, we evaluated this protocol in a larger series of 40 ITP patients.

Methods: We performed an IRB-approved retrospective review of adult hospitalized patients with ITP treated with this regimen from January 2000 - December 2005. Patients with clinically significant thrombocytopenia and either active bleeding, need for anticoagulation, or requirement for a surgical procedure received the combined treatment. The subjects received IVIg and platelets as described above. Additional treatments, such as steroids, immunosupressives, or rituximab, as well as splenectomy were utilized at the discretion of the hematologist overseeing their care.

Results: The average age of patients treated was 52 years. The majority of patients ranged from 20–80 years old, but 12.5% were older than 80 years. The average pretreatment platelet count was 10,000/μl, with an increase to 55,000/μl after 24 hours, and 69,000/μl after 48 hours. By 72 hours, the average platelet count had begun to decline, although the platelet count remained at an acceptable level (58,200/μl). After 24 hours, 62.7% of patients had a platelet count > 50,000/μl. Bleeding was controlled initially in all patients, and those requiring a procedure experienced no bleeding complications. Over half of the patients (52.5%) required additional treatments for recurrent or refractory ITP, and 32.5% of the patients underwent splenectomy. Six of the 21 patients requiring later retreatment (29%) received IVIg and platelets again in a similar fashion. The average retreatment platelet counts after 24 and 48 hours were 53,000/μl and 49,000/μl respectively, with clinical improvement in bleeding in all patients. No side effects of the combined treatment were noted. The response rates for the 3 IVIg products used were similar.

Discussion: For ITP, IVIg and platelets are considered to be first line treatment for patients with very low platelet counts, active bleeding, or those requiring urgent procedures. There is limited literature on the optimal dose and schedule for administration of IVIg and platelets. Our approach for administration of IVIg and platelets concurrently was associated with minimal side effects, resolution of bleeding, ability to safely undergo procedures, and rapid restoration of adequate platelet counts. Additionally, elderly patients had equivalent benefit with no increased side effects, indicating that this regimen is appropriate and safe for patients of all age groups.
Rituximab in Refractory Idiopathic Thrombocytopenic Purpura (ITP).
Authors: Santoro Cristina , Biondo Francesca , De Angelis Gioia , De Propris Mariastefania , De Vellis Annalisa , Guarini Anna , Malandruccolo Luigi , Rago Angela , Foa Robert , Mazzucconi Mariagabriella
Published: April 2020

Background. Rituximab, a chimeric anti-CD20 monoclonal antibody effective in B-cell depletion, may be useful in autoimmune disorders by interfering with the production of auto-antibodies.

Aims. To investigate the efficacy of Rituximab in patients with resistant ITP.

Patients and Methods. Fourteen adult ITP patients (3 males, 11 females; median age 44.5 years [21.1–67.6]) were treated with Rituximab (375 mg/m2/weekly for four doses). The median time between diagnosis and start of Rituximab was 2 years (0.2–33.1 months). All patients had already received at least two lines of therapy (median 3; 2–6): prednisone, pulsed high-dose dexamethasone, azathioprine, immunoglobulins, interferon or splenectomy. At the start of Rituximab, the median platelet count was 10 x 109/L (3–20 x 109/L). Response definitions: complete response (CR), platelet count ≥150 x 109/L; partial response (PR), >50 <150 x 109/L; minimal response (MR), >20 ≤50 x 109/L; no response (NR) ≤20 x 109/L. After completing therapy, patients were evaluated for platelet count after 1 and 3 months, and thereafter every 3 months until relapse or start of a different treatment. Peripheral blood B lymphocytes were evaluated by flow-cytometry as CD19+ cells before treatment, 1 and 3 months after stopping therapy, and then every 3 months up to recovery.

Results. One month after Rituximab therapy, 7 responses (2 CR, 4 PR, 1 MR; 50%) and 7 NR (50%) were observed. Two relapses occurred 5 and 18 months after response. The median follow-up of all treated patients is 6 months (1.8–34.6), while the median follow-up of all responsive patients is 6.1 months (2–18.7).

Before starting therapy, 11/14 cases were evaluable for flow-cytometry studies. The median baseline value of peripheral blood CD19+ B cells was 137 x 106/L (58–476). One month after completing therapy, 7/9 evaluable cases showed absence of CD19+ cells and 2/9 showed a count of 9 and 4.4 x 106/L CD19+ cells, respectively. At the last available control (median follow-up of 6.8 months; 1.9–32.5), 11/14 evaluable patients had still not recovered the baseline CD19+ cell count (median value: 5.5 x 106/L; 0–287).

The following side effects were observed: 1 case of papulosquamous dermatitis, 2 cases of fever.

Conclusions. Seven out of 14 (50%) ITP patients had an early response to Rituximab (2 CR, 4 PR, 1 MR), that persisted in 5 cases. No late responses were observed. The response was independent of the post-therapy CD19+ cell numbers. No serious infections were observed during the clinical follow-up. No patient had to stop therapy because of severe side effects.
Contribution of the Heparin Neutralization Step to the Anti-PF4 ELISA.
Authors: Dlott Jeffrey S. , Amen Nadia N. , Frock Ruth A. , Chou Peter P. , Gallivan Monica V. , Sahud Mervyn A.
Published: April 2020

The combination of anti-PF4 ELISA and serotonin release assay (SRA) has a reported sensitivity approximating 100% for diagnosing heparin-induced thrombocytopenia (HIT) - 7th ACCP Chest Supplement. Current therapeutic guidelines are to initiate therapy with a direct thrombin inhibitor (DTI) based on clinical parameters and to use HIT assays for diagnostic confirmation. Consequently, timely lab data may potentially minimize unnecessary drug exposure, reduce cost, and decrease morbidity. Due to test complexity, the ELISA results consistently precede the SRAs - the “gold standard” for HIT testing. This study examines the predictive value of the optical density (OD) reading and heparin neutralization procedure (100 IU/ml) for determining positive SRA. 200 patients with orders for the anti-PF4 ELISA (GTI) were retested with and without 100 IU of unfractionated heparin. Confirmation by heparin neutralization was reported as % inhibition (>50% inhibition = positive) as calculated by manufacturer’s instructions.

Negative SRA Positive SRA Negative, OD/0.4 34 2 Positive, OD/0.4 120 44

The sensitivity and specificity of the ELISA was 95.6% and 21.6% respectively. The heparin neutralization step improved the specificity to 38.6% and did not alter the sensitivity. Figure 1 shows a single variable binary logistic regression analysis demonstrating the relationship between OD and probability for a positive SRA. An additional binary logistic regression analysis showed no statistical significance for the % neutralization with heparin. A receiver operators curve (ROC) (Figure 2)demonstrating an optimal sensitivity/specificity was generated for both the neat and the heparin neutralized samples. The curves were virtually identical with the same OD cut-off of 1.008. The specificity was nominally improved with the neutralization procedure.

Figure 1 Figure 1. Figure 2 Figure 2.

The heparin neutralization step only marginally improves the specificity of the anti-PF4 ELISA and does not correlate with a positive SRA result, and therefore, does not justify the added expense. Higher ELISA OD measurements correlate significantly with the probability of a positive SRA.
Bone Marrow Reticulin in Patients with Immune Thrombocytopenic Purpura.
Authors: Mufti Ghulam , Bagg Adam , Hasserjian Robert , Bain Barbara , Kuter David , Dreiling Lyndah , Nichol Janet
Published: April 2020

Immune thrombocytopenic purpura (ITP) is an uncommon disorder, and research that characterizes the bone marrow stroma in ITP patients is lacking. It is known that megakaryocytes are often increased in number in ITP patients. Megakaryocytes are associated with reticulin formation in other conditions, including malignant and nonmalignant diseases, such as systemic lupus erythematosus and other immune disorders. Our objective was to determine if the reactive megakaryocyte proliferations in ITP are associated with increased reticulin deposition. We identified 40 ITP patients from clinical records in a retrospective survey of bone marrow biopsy material. Patients were required to have had a diagnosis of ITP, a bone marrow biopsy with available tissue blocks, and a complete blood count at the time of the biopsy. Paraffin-embedded sections from the bone marrow biopsy specimens were stained for reticulin using standard silver-impregnation methods. Reticulin was quantified using the Bauermeister scale (0 = no reticulin fibers demonstrable; 1 = occasional fine individual fibers and foci of a fine fiber network; 2 = fine fiber network throughout most of the section but no coarse fibers; 3 = diffuse fiber network with scattered thick coarse fibers but no mature collagen; and 4 = diffuse often coarse fiber network with areas of collagen). In addition, bone marrow cellularity and megakaryocyte numbers were evaluated. Of the 40 patients, 13 (33%) were considered to have absent (grade 0) bone marrow reticulin, 1 (2%) had grade 0–1, 20 (50%) had grade 1, 5 (13%) had grade 1–2, and 1 (2%) had grade 2. Thus, reticulin was present in the bone marrow of approximately two-thirds of the patients, with 15% having greater than grade 1. Previous research on bone marrow reticulin in 100 hematologically normal subjects found that 27% had Bauermeister reticulin grade 1 and 4% had grade 2 (Arch Pathol Lab Med1990;114:1241–3). Analysis is ongoing to further characterize the deposition of reticulin in the bone marrow of patients with ITP, the relationship of reticulin deposition to megakaryocyte numbers and location, and correlations with patient clinical findings.
Recombinant Plasmin from Lemna: Purification, Characterization and In Vitro Comparison with Human Plasma-Derived Plasmin.
Authors: Brookhart Gary L. , Tomalski Michael D. , Lounes Karim C. , Meijer Piet , Kluft Cornelis , Radtke Klaus-Peter
Published: April 2020

Background: The fibrinolytic enzyme plasmin has gained renewed interest as a direct thrombolytic agent. Local delivery of plasmin via catheter into thrombi, combined with the efficient neutralization of plasmin that may escape from the clot by its inhibitors α2-antiplasmin (α2-AP) and α2-macroglobulin may provide safe and effective removal of pathologic blood clots.

Objective: To overcome the potential for pathogen contamination associated with plasmin prepared from pooled human plasma, we have generated recombinant plasmin (r-PLM) from transgenic Lemna plants and compared its biological properties with those of plasma-derived plasmin (pd-PLM).

Results: Lemna minor, an aquatic plant, was transfected with the c-DNA encoding human plasminogen, and grown in media under controlled conditions. Lemna-derived plasminogen (r-PLG) was extracted from plant-tissue, separated on lysine-Sepharose and subsequently converted into plasmin (r-PLM), using urokinase-type plasminogen activator (u-PA). r-PLM was further purified to homogeneity using affinity chromatography on benzamidine-Sepharose. r-PLG and r-PLM co-migrate with pd-PLG and pd-PLM on non-reducing SDS-PAGE with the respective molecular weights of about 90,000 kDa and 82,000 kDa. When analyzed on reducing SDS-PAGE, r-PLM, just like pd-PLM, separates into a 56,000 kDa heavy chain and a 26,000 light chain. On Western blots r-PLG and r-PLM cross-react with polyclonal antibodies that were raised against pd-PLG, indicating immunologic identity of r-PLG/PLM and pd-PLG/PLM. All three plasminogen activators (PA), tissue-type PA, u-PA and streptokinase, activate r-PLG. Activity assays using the PLM-specific chromogenic substrate S-2403 show that catalytic rates of r-PLM are indistinguishable from pd-PLM, indicating that in this assay r-PLM and pd-PLM have comparable activity. Purified r-PLM and pd-PLM have similar specific activities. Like pd-PLM, r-PLM activity is inhibited by its physiological inhibitor α2-AP and Kunitz-type inhibitors SBTI and aprotinin. Inhibition of r-PLM by α2-AP results in the formation of a high-molecular weight SDS-stable complex, which migrates on SDS-PAGE with a molecular weight of 150,000 kDa, the cumulative molecular weight of r-PLM (82,000 kDa) and α2-AP (65,000 kDa). In microtiter-based fibrinolysis assays, both pd- and r-plasmin dissolve fibrin clots at the same rate and in a concentration-dependent manner.

Conclusion: This work demonstrates that proteins of the fibrinolytic system can be expressed in Lemna. Lemna-derived r-PLM and pd-PLM have comparable biochemical properties, activity, and are equally sensitive to known plasmin inhibitors. Plant-derived r-PLM may be a valuable alternative to PLM preparations from pooled human plasma.
A Study on the Molecular Mechanism for Hereditary Type II Antithrombin Deficiency in a Chinese Pedigree.
Authors: Xu Ye , Feng Ying , Pang Ying , Jing Peipei , Wei Xie , Zhou Xuhong , Ding Qiulan , Wang Xuefeng
Published: April 2020

Objective: To identify the phenotype and study for the genetic mechanism underlying the recurrence of deep vein thrombosis formation in members of a Chinese pedigree caused by antithrombin (AT) deficiency.

Methods: AT antigen level (AT:Ag) and AT activity (AT:A) were detected with respectively immuno-nephelometry and chromogenic assay. All the seven exons and the flank regions of AT gene form the propositus were amplified by PCR. The purified PCR products were sequenced directly. After mutation was found, the corresponding gene fragments covering the mutated point from the other members of the pedigree were amplified and sequenced.

Results: The plasma level of AT:Ag of the propositus was 29.80mg/dl and those of other 3 members of the kindred ranged from 30.90 to 34.00mg/dl, all were among normal range. The AT:A of the propositus was 56%, and those of the other members were 62% to 68%, all were lower than normal. A heterozygous G13830A missense mutation in exon 6 was identified, which has led to the substitution of histamine (CAT) 393 for arginine (CGT). The sequencing results from the pedigree suggested that all the three other members of the kindred also had the same heterozygous mutation.

Conclusion: The R393H mutation in AT which is similar to the AT Glasgow and AT Avranches can cause reactive site injury in AT so that it is not able to inhibit thrombin anymore. The gene mutation found in this pedigree which was unreported in China is the molecular mechanism for recurrent thrombosis events in two members of the kindred.
Routine Measurement of Endogenous Thrombin Potential (ETP) To Assess Hemostatic Function in Patients with Liver Cirrhosis.
Authors: Gadisseur Alain P. , Schouten Jeoffrey , Franque Sven , Van der Planken Marc , Michielsen Peter , Berneman Zwi N.
Published: April 2020

The maintenance of hemostasis through the production of most of the coagulation proteins is a basic liver function. In patients with cirrhosis of the liver the decrease in these proteins is one of the contributory factors to an increased bleeding tendency. Normally the hemostatic capacity of the liver is measured through routine clotting tests as the activated Partial Thromboplastin Time (aPTT) and Prothrombin Time (PT). In the Child classification for liver cirrhosis the coagulation as expressed by the PT is one of the determinants.

Recently a test has become available which will make it possible to routinely measure the endogenous thrombin generation potential (ETP) which may be a better alternative. In this test according to the method first described by Hemker (1993) thrombin generation is continuously measured by use of a chromogenic substrate. Results are calculated as area under the curve and as a percentage of normal.

We analysed 110 patients with liver cirrhosis classified according to the Child classification, 79 patients with stage A, 19 stage B and 12 stage C, without known pre-existing coagulation abnormalities like inherited bleeding disorders, or anticoagulant drugs. In these patients ETP, APTT, PT/INR, FV, FVII and FXI were measured. We used a fully automated assay for the determination of the endogenous thrombin potential (ETP) on the BCS® System (both Dade Behring, Marburg, Germany).

The results for the mean PT were 81% (CI95 77 - 85) for Child A patients, 53% (CI95 44 - 63) for Child B, and 41% (CI95 33 - 50) for Child C. For the mean INR the results were 1.11 (CI95 1.08 - 1.14) for Child A patients, 1.65 (CI95 1.22 - 2.08) for Child B, and 1.82 (CI95 1.34 - 2.31) for Child C. The results for the mean normalized ETP were 0.87 (CI95 0.84 - 0.90) for Child A patients, 0.78 (CI95 0.68 - 0.88) for Child B, and 0.58 (CI95 0.51 - 0.64) for Child C (p<0.001). While the normalized ETP correlated well with both PT and INR for Child B (P<0.05) and C (p<0.01), there were significant differences within the child A patients where no significant correlation could be identified (Pearson correlation 0.203, R2=0.04, p=0.075.

We conclude that the fully automated assay for the determination of the endogenous thrombin potential (ETP) on the BCS® System (both Dade Behring, Marburg, Germany) is a potentially interesting test to measure the coagulation abilities in patients with liver cirrhosis which in the future may supersede the PT/INR in classification systems for hepatic disease.
Comparison between Endogenous Thrombin Potential (ETP) and INR To Assess Liver Function in Patients with Liver Cirrhosis.
Authors: Schouten Jeoffrey , Gadisseur Alain P. , Franque Sven , Van der Planken Marc , Michielsen Peter , Berneman Zwi N.
Published: April 2020

The decreased hepatic synthesis of coagulation factors is a marker of liver failure and a prognostic indicator of survival in cirrhosis. The aPTT and PT/INR are the conventional coagulation tests to measure the haemostatic capacity of the liver. The INR was developed uniquely for use in patients under oral anticoagulation and may not adequately reflect the coagulation changes in liver cirrhosis. However, prognostic models for liver disease include the INR (MELD). New coagulation assessment tests are obviously needed in liver cirrhosis. Recently a test has become available to routinely measure the endogenous thrombin generation potential (ETP). This more physiological test is currently investigated in several haemostatic disorders but not yet in liver failure.

The aim of the study is to investigate correlations between ETP performed on the BCS® system (both Dade Behring, Marburg, Germany) and liver function tests, and compared with INR. We analysed 112 patients with liver cirrhosis (73 Child A, 21 Child B, 18 Child C) without known pre-existing coagulation abnormalities. In these patients ETP, APTT, PT/INR and S-cholinesterase were measured and an C14-Aminopyrine breathing test was performed.

Both INR and ETP show good correlation with the Child score (P<0.01) as a whole. Looking at the Child score for bilirubin there is a clear correlation with the normalized ETP with 0.84 (CI95 0.81 - 0.88) for Child A, 0.74 (CI95 0.55 - 0.93) for Child B, and 0.67 (CI95 0.60 - 0.75) for Child C (p<0.01). For the presence of ascites there is a correlation with the ETP with 0.86 (CI95 0.82 - 0.89) for Child A, 0.71 (CI95 0.62 - 0.79) for Child B, and 0.69 (CI95 0.59 - 0.80) for Child C (p<0.01). Looking at the albumin level there is a clear correlation with the normalized ETP with 0.83 (CI95 0.79 - 0.86) for Child A patients, 0.72 (CI95 0.57 - 0.88) for Child B, and 0.69 (CI95 0.60 - 0.78) for Child C (p=0.02). In a linear regression model bilirubin (β= −0.319, p<0.01) and ascites (β= −0.233, p=0.06) are the main predictive factor for decreasing thrombin generation. No such clear relationships can be identified for the INR. Both ETP and INR correlate well with S-cholinesterase levels (p<0.01). ETP correlated better with the breathing test than the INR.

These results indicate that the integrity of the coagulation decreases in line with deterioration of other liver functions in patients with liver cirrhosis. The seriousness of this decrease in haemostatic capacity is better demonstrated by the ETP than by the INR (currently part of MELD). In the future the ETP may supersede the INR in prognostic classification systems for hepatic failure.
Trypsin Mediated Activation of Thrombin Activatable Fibrinolytic Inhibitor (Carboxypeptidase U): Potential Clinical Implications.
Authors: Kujawski Michelle , Cunanan Josephine , Hoppensteadt Debra A. , Bick Rodger L. , Fareed Jawed
Published: April 2020

Thrombin activatable fibrinolytic inhibitor (TAFI) (EC 3.4.17.20) is now known to mediate the fibrinolytic deficit in various pathophysiologic conditions such as thrombotic stroke, acute coronary syndrome and pancreatic disorders. While such serine proteases as plasmin are known to activate TAFI into TAFIa, no information is available on the effect of trypsin (EC 3.4.21.4) on the activation process. Pancreatic diseases including inflammatory conditions, surgical interventions and malignancies are associated with hypercoagulable state and DIC. Fibrinolytic deficit is also commonly observed in these patients. In these conditions, pancreatic enzymes are released into circulation. While the pathogenic role of pancreatic leakage mediator is known the effect of such pancreatic enzymes, as trypsin on the conversion of TAFI to TAFIa has not been addressed. To test the hypothesis that the observed fibrinolytic deficit in pancreatic disorders may be related to the conversion of TAFI to TAFIa by trypsin, the effects of this serine protease were studied on immunopurified human TAFI utilizing proteomic profiling, SDS gel electrophoresis and functional TAFI evaluation employing a specific substrate for TAFIa. Immunopurified human TAFI was incubated with human trypsin at graded concentrations in standardized assays with appropriate controls. The activation of TAFI was monitored by using a TAFIa substrate (Benzoyl-(L)-lysyl-(e-benzyloxycarbonyl)-a-(L)-(2-quanidinoethylthio)-glycine) from Pentapharm Inc. (Basel Switzerland). Thrombin thrombomodulin complex (T-TM) was also used as a positive control for TAFI to TAFIa conversion. The proteomic profile of the cleavage products generated was profiled utilizing surface enhanced laser desorption ionization (SELDI) (Ciphergen, Freemont, CA). Additionally, SDS gel electrophoresis of the cleavage products with appropriate controls were also obtained. Trypsin and TAFI alone did not produce any hydrolysis of the TAFIa substrate. However, when trypsin was added to TAFI it produced a concentration dependent hydrolysis of the substrate, which was comparable to that observed upon the activation of TAFI by T-TM. The proteomic profile of trypsin activated TAFI revealed specific peaks at 45.2, 35.6, 24.4 and 19 kDa. In addition, there were several cleavage products at < 10 kDa molecular weight range, which was not seen with the activation of TAFI by T-TM. Similarly activation products as measured by SDS gel electrophoresis showed differential molecular profiles between trypsin and T-TM. These observations clearly suggest that trypsin is capable of activating TAFI into TAFIa independent of other processes such as the T-TM and plasmin mediated conversions. The observed fibrinolytic deficit in pancreatitus/pancreatic cancer may be due to the leakage of trypsin into circulation.
Extrapolating Mixing Study Results from the STACLOT-LA.
Authors: Dlott Jeffrey S. , Tushek Michael T. , Strother Rachael S. , Gallivan Monica V.
Published: April 2020

The International Society on Thrombosis and Haemostatis (ISTH) has established criteria for the diagnosis of lupus anticoagulants (LA) to include a mixing study to establish the presence of an inhibitor. Up to 30% of LAs are time and temperature dependent inhibitors, therefore only an incubated mixing study will detect this subtype. Global assays (i.e. STACLOT-LA® Diagnostica Stago) incorporate a sensitive screening reagent, a source of normal pooled plasma (npp) for an immediate mixing study, and a step to demonstrate phospholipids dependence. It does not, however, include an incubated mixing study. 493 consecutive STACLOT-LA samples were evaluated based on outcome and compared to the clotting time for Tube 1 (sample + npp + buffer) and Tube 2 (sample + npp + buffer + hexagonal phospholipids). All samples were tested according to manufacture’s instructions with a delta > 8 seconds considered positive. Figure 1 reveals the lack of clear demarcation between negative and positive outcomes. Figure 2 identifies considerable differences between the clotting times of tube 1. Tube 2, while statistically different, has greatly overlapping confidence intervals. From this, it was identified that samples with Tube 1 clotting times between 55–75 seconds represented the population most likely to give borderline confirmatory calculations (CC). Subsequently, 45 samples with this criterion were selected for study, 17 negatives and 28 positives (CC 6.2–9.7). Immediate and 1 hour incubated mixing studies were performed with two reagents - aPTT-A and aPTT-LA® (Diagnostica Stago). The results are given in the table.

Figure 1 Scatter plot of LA Calculation Figure 1. Scatter plot of LA Calculation Figure 2 Figure 2. aPTT-A aPTT-LA Correction Reference Interval NC Reference Interval NC npp + 5 seconds Correction Reference Interval NC Reference Interval NC npp + 5 seconds NC= No correction; npp= Normal pooled plasma time; †2 of 3 LA positive by dRVVT; *1 case is a time dependent inhibitor Positive, (n=28) 22 (78) 6 (21) 13 (46) 23* (82) 5 (18) 14 (50) Negative, (n=17) 14 (82) 0 3† (18) 14 (82) 0 3† (18)

Conclusions: Borderline positive STACLOT-LA results showed an inhibitor pattern only 50% of the time under the most optimal conditions of a LA sensitive aPTT reagent with select interpretative guidelines. Therefore, a borderline or indeterminate range might be a more effective way to communicate these results if the inclination is to avoid a separate mixing study.
Successful Treatment of Intra-Cranial Hemorrhage with Repeated Doses of Activated Recombinant FVII in a Newborn with Severe Factor VII Deficiency.
Authors: Farah Roula , Hamod Dany , Bechara Elie , Melick Nancy , Hage Paul , Giansily-Blaizot Muriel , Sallah Ahmed S.
Published: April 2020

Factor VII deficiency is a rare inherited bleeding disorder. Intracranial hemorrhage can occur in approximately 1 to 10% of the cases. We report the case of a one month old girl from Lebanon who presented with vomiting, abnormal cry, lethargy and partial seizures. She was diagnosed prenatally with severe factor VII deficiency after detection of a splice-site mutation in exon 2 of the FVII gene (IVS2+1 G→C) that was described previously in her sister who died at the age of 4 months from severe intracranial hemorrhage. The parents are first degree cousins and are both heterozygous. She has a healthy heterozygous two year old sister.

An emergency head CT Scan showed intra ventricular and right hemispheric intra parenchymal hemorrhage, peri-hemorrhage edema and minimal midline shift. The patient was placed immediately on mechanical ventilation and anti-epileptics and activated recombinant factor VII (rFVIIa, NovoSeven) was administered intra-venously at 60 ug/kg for the first dose then 30 ug/kg/dose every 4 to 6 hours for 10 days then once a day for 5 days. She was treated conservatively with no surgery. Stabilization of the hematoma was observed with gradual improvement of her neurological condition. Communicating hydrocephalus developed later and a ventriculo-peritoneal shunt was placed on day 16 post-hemorrhage. Subsequently, she received weekly infusions of rFVIIa at 30 ug/kg/dose for 4 weeks after this episode and showed no bleeding symptoms. Follow-up two months later showed axial hypotonia and a mild left sided spasticity. Auditory evoked potentials were normal. However, afte prophylactic rFVIIa was dicontinued, the patient developed sub-arachnoid hemorrhage and was placed again on weekly prophylactic infusions of rFVIIa at 30 ug/kg/dose. The patient tolerated the infusions very well. No side-effects were observed.

Conclusion: rFVIIa is safe and effective in Factor VII deficient newborns with intracranial hemorrhage. Rapid treatment at the onset of hemorrhage is critical to reduce mortality and improve the outcome. Prophylactic factor VII infusions can be proposed in this setting to minimize the risk of subsequent bleeding episodes. The ideal dosing and schedule for treatment and/or prophylaxis will have to be defined. Despite the short half-life of rFVIIa, weekly infusions in our experience seemed safe and effective in reducing the incidence of further bleeding episodes.
Reduction of Anti-FIX Inhibitor Titers in a Hemophilia B Patient with Continuous Use of FEIBA.
Authors: Watanabe Hiroyoshi , Watanabe Tsutomu , Onishi Toshihiro , Okamura Kazumi , Kagami Shoji , Yamada Tadashi , Kawano Yoshifumi
Published: April 2020

Background: Inhibitor formation is a severe complication of hemophilia B associated with poor response to factor replacement and uncontrolled bleeding, although an inhibitor formation in hemophilia B is a rare event compared to hemophilia A. Currently, immune tolerance is the only proven method for inhibitor eradication. However, immune tolerance induction in patients with hemophilia B is a rare occurrence. We report a case of a hemophilia B patient who had a reduction of inhibitor titer during continual use of FEIBA.

Case report: Patient is an 11-year-old boy with severe FIX deficiency and high titer inhibitor (historical peak: 65 BU/mL) since age 3 years. Since inhibitor formation, the patient had received FEIBA or FVIIa on demand. FVIIa became therapeutically not effective in 1999 October, and bleeding frequency increased as he developed a target joint. In 1999 November, 2002 February, and 2004 September, he received high dose of FIX concentrate for neutralization of inhibitor and then cyclophosphamide for 8 weeks. We achieved only a transient effect in reducing the inhibitor titer. The inhibiter titer fluctuated between 20 and 60 BU/mL. Since 2005 January, subsequently, he had received FEIBA 1,000–2,000 IU per dose (30–50 units/kg) on demand. He needed FEIBA twice or thrice a week, and continued to receive this dose till now. The inhibitor titer was gradually getting lower after a transient rise up to 39 BU/mL on 2005 February. On 2006 February, the inhibitor titer became below 1.0 BU/mL, and finally undetectable on 2006 March, despite the longevity of the inhibitor (8 years). Factor IX recovery was normalized, and bleeding frequency dramatically decreased. Elimination of the inhibitor by the continued administration of FEIBA was observed. Anaphylactic reaction and the development of nephrotic syndrome were not seen.

Conclusion: The continuing use of FEIBA safely and effectively might decrease the inhibitor titer and the frequency of bleeding episodes in hemophilia B patients. This case may provide us the optimization of the FEIBA dose, and duration of treatment for inducing tolerance by using FEIBA.
Hepatocellular Carcinoma in Patients with Hemophilia and Chronic Hepatitis C Infection.
Authors: von Auer Charis , Heinsdorf Sandra , Krause Manuela , Miesbach Wolfgang , Scharrer Inge
Published: April 2020

Hepatitis C virus (HCV) infection is often observed in hemophilic patients who were treated with non-virus inactivated clotting factor concentrates prior to the mid 1980s. HCV infected patients exhibit an increased risk of developing liver cirrhosis and hepatocellular carcinoma (HCC). Hemophilia cohort studies until 2004 suggest a prevalence of HCC development of 0,1–0,2% in HCV infected hemophilic patients. During the last two years we observed a rapid increase in HCC development in our hemophilia treatment center. In the current study we analyzed our data to investigate the impact of chronic HCV infection in hemophilic patients today. We treated 90 hemophiliacs with chronic HCV hepatitis. Seven of these patients (6,3%) developed a HCC between 2002 and 2005, six of them only between 2004 and 2005. Five patients had severe hemophilia A, one mild hemophilia A, one severe hemophilia B, two of them had co-infections (HIV and HIV+HBV). Three had HCV genotype 1b, three 3a and one unknown. Four had been treated with interferon alpha/peginterferon alone or in combination with ribavirin, only one achieved a virologic response. Two patients refused treatment of HCV infection, one patient was not treated due to HIV co-infection. Their age at HCC diagnosis ranged from 40 to 73 years (median 60), time-course between estimated HCV infection and HCC development ranged between 11 and 35 years (median 22). All patients had developed liver cirrhosis: two Child-Pugh A, one Child-Pugh B and four Child-Pugh C. Six patients were asymptomatic; only one patient had jaundice when HCC was detected. All were regularly checked at least once a year with routine screening of AFP level and ultrasound (US) scans. Three patients had normal, four elevated AFP levels (>15ng/ml), three had US scans without pathological finding when HCC was detected. All had consistently or intermittently elevated serum ALT levels during the last five years before diagnosis. HCC was diagnosed either by elevated AFP levels plus US identification (2) or plus MRT (2), by MRT plus check of arterial hypervascularisation (1) plus liver biopsy (1). One patient was only diagnosed after liver transplantation for chronic liver disease. Orthotopic liver transplantation was done in one patient, transcatheter arterial chemoembolization (TACE) in three, two patients have died of liver failure. Our data show that at present HCC is an increasingly frequent cause of mortality in hemophilic patients with chronic HCV infection. The detection of six HCC in only 15 months is striking. Especially nowadays a close follow up on hemophilic patients with chronic HCV infection seems to be mandatory. This should result in earlier detection and consequently more easily treatable tumors and longer survival of our patients.
Liver Transplantation for Prothrombin Deficiency Causing Life-Threatening Bleeds.
Authors: Dower Nancy A. , McClure Wilma , Akabutu John J.
Published: April 2020

We report here the first case of living-related liver transplantation to correct a severe bleeding diathesis. A Caucasian infant was diagnosed at 11 weeks old with prothrombin deficiency after a spontaneous subdural hemorrhage. Even though he had a Factor II activity level of 0.08, he had severe clinical complications including umbilical bleeding, hemorrhagic shock after circumcision and bleeding per rectum.

Prophylactic treatment with prothrombin complex concentrate prevented further bleeding episodes. Unfortunately, he developed clots of the superior vena cava and of the inferior vena cava in the context of central venous catheters. On-demand therapy via peripheral IV was followed for almost a year.

He then had severe subdural and intraparenchymal hemorrhages. His clinical course did not correlate with measured Factor II activity. Investigations looking for other coagulation disorders were negative.

He underwent a living-related liver transplant from his father in order to correct the bleeding diathesis. After transplant his Factor II level rose to 0.51. He required a second liver transplant (cadaveric) and died from tranplant related complications within a month.

Management decisions must reflect the clinical severity of bleeding and not the activity level in rare bleeding disorders where no treatment guidelines exist.
Combination of Bortezomib, Melphalan, Prednisone and Thalidomide (VMPT) for Relapsed Multiple Myeloma: Results of a Phase I/II Clinical Trial.
Authors: Palumbo Antonio , Ambrosini Maria Teresa , Benevolo Giulia , Pregno Patrizia , Pescosta Norbert , Callea Vincenzo , Cangialosi Clotilde , Caravita Tommaso , Morabito Fortunato , Musto Pellegrino , Bringhen Sara , Gay Francesca , Rus Cecilia , Boccadoro Mario
Published: April 2020

BACKGROUND: In newly diagnosed patients, the addition of thalidomide or bortezomib (Velcade™) to the standard oral melphalan/prednisone combination significantly increased response rate and event-free survival. In this multicenter, open-label, non randomized, phase I/II trial, the safety/efficacy profile of the 4 drug combination, bortezomib (Velcade™), melphalan, prednisone, and thalidomide (VMPT) was evaluated in patients with relapsed/refractory myeloma.

METHODS: Bortezomib was administered by IV bolus on days 1, 4, 15, 22 at three dose levels: in the first cohort (10 patients) at 1.0 mg/m2; in the second cohort (10 patients) at 1.3 mg/m2and in the third cohort (10 patients) at 1.6 mg/m2. Oral melphalan was administered at 6 mg/m2 on days 1–5, oral prednisone at 60 mg/m2 on days 1–5. Thalidomide was delivered at 50 mg on days 1–35. Each course was repeated every 35 days for a total of 6 courses.

RESULTS: Thirty patients, median age 66 years (range 38–79), with relapsed or refractory myeloma were enrolled. Fourteen patients received VMPT as second line of therapy, 16 as third line. Twenty patients received prior autologous transplant, 10 conventional chemotherapy and 9 thalidomide-based regimens. After a median of 6 courses, 20 patients (67%) achieved a partial response (PR) including 13 patients (43%) who achieved at least a very good partial response (VGPR). Among patients who received VMPT as second line treatment, the PR rate was 79%, and the immunofixation negative complete response rate 36%. The 1-year progression-free survival was 61%, and the 1-year survival from study entry was 84%. Grade 3 non-hematological adverse events included: infections (5 patients), fatigue (1), vasculitis (1) and peripheral neuropathy (2); no grade 4 toxicities were recorded.

CONCLUSIONS: Initial results showed that VMPT is an effective salvage therapy with a high proportion of responses. Toxicities were manageable. The incidence of neurotoxicities was unexpectedly low.
Digital Analysis of Vascularity in Hemophilic Synovitis.
Authors: Lin Jonathan , Hakobyan Narine , Valentino Leonard A.
Published: April 2020

Hemophilia is a genetic disorder caused by a deficiency of blood coagulation factor VIII or IX. The most serious symptom is bleeding into joints, which over time leads to a proliferative, inflammatory disorder known as hemophilic synovitis. In time, the synovitis develops into hemophilic arthropathy, a form of incapacitating arthritis. While this sequence of events is well documented, the components responsible for the development of hemophilic synovitis are largely unknown. Recent evidence suggests that there is a relation between synovitis and vessel formation. Investigation of this cause-and-effect relationship necessitates a quantitative means of analyzing synovial and vessel hyperplasia. The goal of this experiment is to determine the utility of digital analysis of vessel formation by comparison to visual “hot-spot” scoring (Valentino and Hakobyan 2006, in press). Digital scores were expressed as percentages by dividing the number of vessel pixels in the densest area (“hot-spot”) by the total pixels in the field or separating the entire joint into 10 regions then averaging the percentages from each (“ten field”). Twenty-two histological sections from mouse joints with hemophilic synovitis were scored by visual and digital methods (Fig 1). The correlation of the two “hot-spot” methods (visual and digital) was good (r2=0.81). Next, the relationship of digital “hot-spot” and “ten field” analysis is shown in Fig.2. These data support the use of digital image analysis for evaluation of vessel formation and will allow for rapid examination of multiple samples.

Figure 1: Hot Spot Scoring Figure 1:. Hot Spot Scoring Figure 2: Digital Scoring Figure 2:. Digital Scoring
Lenalidomide (Revlimid™), Adriamycin and Dexamethasone Chemotherapy (RAD) Is Safe and Effective in Treatment of Relapsed Multiple Myeloma - First Results of a German Multicenter Phase I/II Trial.
Authors: Knop Stefan , Gerecke Christian , Topp Max S. , Liebisch Peter , Hess Georg , Kotkiewitz Sandra , Einsele Hermann , Bargou Ralf
Published: April 2020

A more widespread use of thalidomide (Thal) in patients (pts) with relapsed multiple myeloma has been prevented by its considerable neurotoxicity. The immunomodulatory drugs (IMiDs) form a new class of Thal successors aiming at reduced toxicity with reliable anti-tumour effect. Recently, lenalidomide (CC-5013; Revlimid™) was the first IMiD to be approved by the FDA for treatment of both MDS and multiple myeloma. Several trials in pts with advanced myeloma demonstrated lenalidomide (alone or in combination with dexamethasone) to induce significant and durable response. We sought to establish a protocol with superior efficacy and decided to add lenalidomide (R) to adriamycin (A)/dexamethasone (D; RAD). A dose escalation study was set up to include pts after a maximum of 3 previous treatment lines. They had to have measurable disease and adequate organ and bone marrow function. No dose limiting toxicity occurred until two cases of non-febrile neutropenia were seen with R 15 mg, day 1–21; A 9 mg/m2 for days 1–4 as a continuous infusion; and D 40 mg, days 1–4, and 17–20. The protocol was amended for G-CSF support (Pegfilgrastim [Neulasta™] 6mg, day 6) resulting in an uneventful course in all pts treated subsequently at the two highest dose levels. Surprisingly, maximum tolerated dose of lenalidomide in combination with A and D as described before was 25 mg day 1–21, thus being the daily dose from the monotherapy trials. Up to now, 41 subjects have been enrolled, 37 of whom had received autologous and five additional allogeneic stem cell transplants. 31 pts are currently evaluable for response and toxicity: 26 pts achieved reduction of paraprotein levels of at least 50% for a response rate of 84 % including one confirmed CR and 14 PRs according to the EBMT criteria (Bladé et al). An additional three pts had SD while two progressed. Notably, eight of ten pts who displayed del (13) on cytogenetic analysis responded up to now, including six confirmed PRs. One patient each experienced acute renal failure due to emesis and hypovolemia, pneumocystis pneumonitis, and catheter related infection. All subjects recovered without sequelae. Neither somnolence nor constipation, thromboembolism or neuropathy occurred. We conclude that RAD induces substantial remission with an acceptable toxicity profile and thus significantly contributes to the therapeutic armamentarium even in heavily pretreated myeloma pts.