Pruthi, Rajiv K.

Abstract Background Heparin-induced thrombocytopenia (HIT) is an immune-mediated drug reaction in which heparin-dependant IgG antibodies (HIT-IgG) to heparin-platelet factor 4 complexes form with the potential for platelet activation with resultant thrombocytopenia (HIT) and thrombosis (HITT). The frequency of HIT-IgG formation and clinically apparent HIT or HITT varies considerably, according to the patient population (surgical versus medical) and type of heparin (unfractionated [UFH] vs low molecular weight [LMWH]). One large prospective study showed a frequency of HIT-IgG formation of 7.5% with clinical HITT in 34.5% of these orthopedic patients receiving prophylactic LMWH and corresponding figures in cardiac surgery patients receiving UFH were 50% and 2%, respectively [Warkentin et al. Blood2000;96: 1703]. Due to daily exposure to heparin flushes (UFH) and prophylactic LMWH (to prevent venoocclusive disease) hematopoietic stem cell transplant (HSCT) recipients may be at risk for HIT/HITT. However, due to anticipated post-transplant thrombocytopenia, clinical cases of HIT/HITT may be overlooked. We retrospectively determined the incidence of HIT-IgG formation and its clinical significance in this population. Methods: HIT-IgG was measured retrospectively using a commercially available ELISA kit in samples collected from patients prior to conditioning therapy and on day 14 post-HSCT. Medical records were reviewed to in order to determine the clinical significance (development of clinical HIT/HITT) in those with detectable HIT-IgG. Patients Samples from a total of 103 HSCT recipients with hematologic malignancy (including 25 myeloma, 17 lymphoma, 16 AML, 14 amyloid, 10 MDS, 10 ALL, 8 CML and 3 CLL) were tested, of which 52 and 49 underwent allogeneic and autologous HSCT, respectively. All patients were exposed to UFH flushes during line care. In addition, all allogeneic recipients received prophylactic subcutaneous dalteparin 5000U daily beginning with conditioning until engraftment. Conditioning regimens included high dose melphalan (n=34), cyclophosphamide/TBI (n=30), carmustine, etoposide, cytarabine, melphalan (n=15), busulfan/cyclophosphamide (n=9), fludarabine/melphalan (n=10) and fludarabine/TBI (n=4). The median age was 51.5 years (20–72) and 68 were male. Results: The median platelet count prior to initiation of conditioning therapy and at day 14 was 157 × 109/L (range: 10–672) and 40 × 109/L (range: 9–289), respectively. Prior to conditioning, two of 103 (1.9%) patients had a positive HIT-IgG assay, that became negative in one and equivocal in the other by day 14 post-HSCT, and one patient had an equivocal HIT assay that became negative by day 14. All 3 were autologous recipients. None of the previously negative patients had a positive HIT-IgG assay at day 14, although 3 patients (2 autologous and one allogeneic) had an equivocal result of uncertain significance. No patients developed clinical or symptomatic thrombosis (HITT). Conclusion: In spite of frequent exposure to heparin, the frequency of seroconversion is low, and in those patients with preexisting HIT-IgG, no patient developed symptomatic thrombosis, including the 2 patients with positive HIT IgG assays prior to conditioning. The low frequency of HIT-IgG formation may reflect the profound immunosuppression of the conditioning regimens employed. Nevertheless, in this population, a high index of clinical suspicion must be maintained.

Abstract Factor VIII (FVIII) inhibitors have been classified as Type I, linear inactivation kinetics and complete inhibition of FVIII at saturation, or Type II, non-linear kinetics and residual FVIII activity. To investigate the hypothesis that "inhibitor-blocking antibodies" contribute to the characteristics of Type II inhibitors, mixing studies were performed between fractionated Type II antiserum and various Type I inhibitor preparations. Type II IgG fractions were tested for inhibitory capacity and ability to prevent inhibition mediated by inhibitors from other hemophiliacs. The IgG from Type II plasma (II) (29 BU, 8% FVIII) and several Type I plasmas ((I) plasmas) were fractionated with DEAE resin. Late-eluting Type II fractions (II):6 and (II):7, showed slight protection of FVIII from the inhibiting activity of several Type I plasmas and additive inhibition with others. When tested against Type I IgG fractions instead of type I plasmas, (II):6 and (II):7 provided significant protection of FVIII from the inhibitory action of some immunoglobulins and additive inhibition with others. When (II):6, which contained slightly less than 1 BU of inhibitory activity, was preincubated with normal plasma, it blocked about 3 BU of inhibitory activity in an equal volume of fraction (I):1; residual FVIII increased from 11.4% to 27.2%. IgG fraction (II):6 provided protection from DEAE fraction 2, (I):2, from another type I patient (residual FVIII went from 15.1% to 31.7%) but was additive with (I):4 from the same patient (residual FVIII went from 8.0% to 3.2%). Preincubation with (II):7 increased residual FVIII activity in normal plasma incubated with (I):1 from 10.1% to 20.7%. Fraction (II):7 was processed further by hydrophobic interaction chromatography on phenyl sepharose and two fractions, Phen(II):5 and Phen(II):6 provided significant protection of FVIII from inhibition by (I):1; residual FVIII activity increased from 11.5% to 34.1% and from 7.9% to 28.2%, respectively. DEAE fractions (II):6 and (II):7 were shown to contain anti-FVIII IgG. Depletion of mixtures of plasma or rFVIII and (II):6 with immobilized Protein A, reduced FVIII activity by 42.8% and 68.8%, respectively. In addition, adsorption of (II):6 with immobilized Protein A, eliminated its ability to block FVIII inhibition by (I):1. The co-existence of these antibodies in "acquired hemophiliac" plasma would contribute to the complex kinetics seen in these patients. If a sufficiently diverse panel of such blocking IgGs occurred naturally, or could be produced in the laboratory, and their interaction with the target antigen did not adversely affect the half-life, then these blocking antibodies might prove useful to alleviate the effects of inhibitory antibodies in hemophilia and other diseases.

Abstract Background: Coagulation factor V (FV) is an essential cofactor of the prothrombinase complex, catalyzing conversion of prothrombin to thrombin. Severe FV deficiency is a rare coagulation defect with a prevalence in the general population of approximately 1 in 1,000,000 and a variable bleeding risk (Asselta R et al. J Thromb Haemostas 2006). Genetic mutations underlying a FV-deficient hemorrhagic diathesis have been described in a limited number of cases, display considerable allelic heterogeneity, and have been due to point mutations in the FV gene located at chromosome 1q23. Here, we report a 15 year-old girl with psychomotor delay, CNS abnormalities, and a solitary left kidney - features consistent with a congenital 1q deletion including band 1q23 - who was found to have severely reduced plasma FV activity after a routine preoperative laboratory evaluation prior to an elective spinal stabilization surgery revealed a mildly prolonged prothrombin (PT, 13.2 seconds; normal 8.4 – 12 seconds) and activated partial thromboplastin (aPTT, and 38 seconds; normal 21 – 33 seconds) time. She had undergone minor surgical procedures in the past without reports of significant bleeding, and her family members were also clinically unaffected. Methods: FV procoagulant activity level was measured in the patient with an MDA-180 automated coagulation analyser (Trinity Biotech, Ireland). The patient’s peripheral blood leukocytes were separated by density centrifugation and fixed in methanol:glacial acetic acid (2:1). We then performed interphase fluorescent in-situ (FISH) analysis using probes covering the FV gene and the more telomeric HPC1 gene as a control. Genomic DNA was also isolated from the patient, and the coding region (25 exons) of the FV gene amplified by polymerase chain reaction (PCR) and sequenced. Results: FV activity assays revealed a severely reduced level of 9% (normal range: 60–130%). Factor V activity levels in the patient’s family members were not assessed. Peripheral blood karyotyping revealed an interstitial deletion of 1q: 46,XX,del(1)(q24.2q25.3 or q23.3q25.1). Interphase FISH analysis was performed. Red signals corresponded to the FV probe and green signals corresponded to HPC1, which is approximately 13 MB telomeric to FV. In contrast to healthy controls, the patient had a signal pattern of only 1 green HPC1 signal and 1 red FV signal, corresponding to heterozygous loss of the FV gene and adjacent chromosomal material. Sequencing of the retained allele revealed a novel hemizygous S234W mutation (encoded by exon 6). The lost serine is part of a conserved “SGP” peptide segment predicted to be a solvent-exposed region of the heavy chain of the FV protein. Conclusion: Most mutations causing FV deficiency are autosomal recessive and introduce frame shifts resulting in the generation of a premature termination codon. Karyotypic and FISH analysis of this patient revealed a novel mechanism for FV deficiency: loss of an entire FV allele as part of a chromosomal deletion, coupled with a point mutation in the other allele. Structural modeling of the consequences of the S234W point mutation, which is predicted to alter the hydrophobicity of the protein, is ongoing. Syndromes involving an interstitial deletion of 1q have been described in association with ATIII deficiency (Pallotta, Dalpra et al. 2001), but to our knowledge, this is the first description of a 1q deletion with FV deficiency.

Abstract Background AL amyloidosis can be associated with coagulopathy due to acquired factor X (FX) deficiency. Published experience, limited to case reports, suggests that infusion of fresh frozen plasma (FFP), platelets, plasma exchange, and vitamin K are of limited hemostatic efficacy for periprocedural management of coagulopathy. Therefore, we reviewed our cumulative experience of managing patients (pt) with FX deficiency due to AL amyloidosis who underwent an invasive procedure. Methods Pt with AL amyloidosis and isolated FX deficiency (≤ 50%), who underwent an invasive procedure between January 1975 and May 2007, were retrospectively identified from institutional databases. We excluded bone marrow biopsies as well as procedures >3 months prior to the diagnosis of FX deficiency. Endpoints of the study were bleeding, thrombosis, and death. Results Sixty-one pt fit the study criteria. They were arbitrarily classified as having severe (<10%; n=6), moderate (10–25%; n=16), or mild (26–50%; n=39) FX deficiency. The pt underwent a total of 115 procedures, 13 (11.3%) of which were considered major procedures and 102 (88.7%) were minor procedures. Major Procedures 13 pt underwent a major procedure; 4 had severe FX deficiency, 2 moderate, and 7 mild. Procedures included splenectomy, colon resection, renal transplant, and others. All 4 of the pt with severe deficiency were treated with hemostatic agents (FFP, plasma exchange, tranexamic acid, danazol, aminocaproic acid, and rFVIIa) in the perioperative period. Two (50%) of these patients had complications: intraabdominal hematoma and death. The death occurred in a patient with splenic rupture and uncontrolled hemorrhage refractory to rFVIIa and FFP. None of the pt with moderate deficiency experienced complications, despite not receiving hemostatic agents. Similarly, 0/7 patients with mild deficiency developed complications (2 received hemostatic agents FFP, platelets, vitamin K, and DDAVP). Minor Procedures All 61 pt underwent a minor procedure; central venous catheter placement was the most common (n=26), followed by EGD with biopsy (n=16) and renal biopsy (n=10). Six pt with severe deficiency underwent 7 minor procedures, 2 (29%) of which were associated with excess bleeding, despite rFVIIa treatment in both cases. An additional 3 procedures were performed with hemostatic agents and were uncomplicated. 2/16 (13%) pt with moderate deficiency had excess bleeding, 1 of whom was treated with rFVIIa and the other was not treated. Two others received perioperative treatment and did not have a complication. There were 7 complications in the 39 patients with mild deficiency (18%), including 1 AV fistula thrombosis after rFVIIa. Five of the patients with a bleeding complication did not receive treatment. One patient with mild deficiency received procoagulants and did not have excess bleeding. Conclusions -This is the largest series examining the perioperative management of pt with FX deficiency due to AL amyloidosis -Complications occurred in 11.3% of procedures, including one death -In this series, there were no clear predictors of bleeding -FX deficiency did not contribute to mortality, with the exception of 1 case -Bleeding risk is relatively infrequent in pt with mild or moderate FX deficiency, particularly with non-vascular procedures -Although optimal management of pt with severe FX deficiency is unclear, options include rhFVIIa +/− FFP and aminocaproic acid in those requiring procoagulant therapy.

Abstract Background: Primary systemic amyloidosis (AL) is an incurable plasma cell disorder associated with varying degree of organ infiltration with light chain derived amyloid protein and consequent organ dysfunction. Various acquired coagulopathies, predominantly Factor X (FX) deficiency have been previously described in AL. The factor deficiencies are thought to be due to the direct binding of blood coagulation proteins to amyloid fibrils. Little is known regarding the prognostic significance of abnormal coagulation parameters in AL patients (pts). Methods: After Institutional Review Board approval, we performed a retrospective review of the medical records of 625 consecutive pts who had presented within 3 months of a diagnosis of AL to be evaluated at Mayo Clinic from June 2001 to June 2006. 540 pts had coagulation studies performed within 2 months of presentation. 51 of those pts were on oral anticoagulation and were excluded from the analysis. Results: Of the 489 pts included in the analysis, the median age was 62 years and 61% were male. The median overall survival (OS) of the entire cohort was 26.8 months. Table 1 lists the prevalence of abnormal coagulation tests in the remaining 489 patients. Prothrombin time (PT) was available in the majority of pts. 61 pts (12.6%) had elevated PT, and 63 pts (20.4%) had a decreased FX. 37 of the 61 pts with elevated PT had available FX levels, of which 31 (84%) of those had a decreased FX, suggesting that the cause of elevated PT was mostly due to a low FX. Indicators of hepatic involvement such as alkaline phosphatase and bilirubin levels were higher among the patients with prolonged PT and low FX. An elevated PT (more than 12.4 seconds) was strongly predictive of an adverse outcome, with a median OS of 8.7 months compared to a median OS of 29 months (p<0.0001) in pts with normal PT (Figure 1). Similarly, pts with decreased FX (less than 60% activity), was associated with significantly worse outcomes with a median OS of 14.9 months versus a median OS of 33 months (p<0.0001) in pts with normal FX (Figure 2). In a Cox proportional hazards model containing cardiac troponin (cTnT), NT-Pro BNP, serum alkaline phosphatase, and free light chain difference (involved minus uninvolved), prolonged PT or FX deficiency were independently prognostic for overall survival. Conclusion: Abnormalities of PT and FX are seen in a sizable proportion of pts with newly diagnosed AL. An elevated PT and or FX deficiency appear to adversely affect the outcome of these patients. It is likely that the elevated PT in these patients relate to FX deficiency in the majority which in turn may reflect the amyloid ‘burden’. The effect of other organ involvement like the hepatic involvement may also have an impact on FX and PT. Table 1. Prevalence of abnormal coagulation testing in patients with newly diagnosed primary systemic amyloidosis. Coagulation study N (Test results available) N of abnormal results (%) Elevated prothrombin time 486 61 12.6 Elevated activated partial thromboplastin time 312 48 15.4 Elevated thrombin time 315 84 26.7 Elevated fibrinogen 140 118 84.3 Decreased Factor II activity 62 26 41.9 Decreased Factor V activity 63 9 14.3 Decreased Factor VII activity 63 17 27 Decreased Factor X activity 309 63 20.4 Elevated D-dimer 130 72 55.4 Figure 1. Survival grouped by normal versus elevated Prothrombin time. Figure 1. Survival grouped by normal versus elevated Prothrombin time. Figure 2. Survival grouped by normal versus low Factor X levels. Figure 2. Survival grouped by normal versus low Factor X levels.

Abstract Background & Objectives: Diagnosis and classification of VWD are currently based on integration of personal and family bleeding histories and results of protein-based diagnostic tests (VWF:Ag, VWF:RCo, FVIII:C, VWF:CB and VWF multimers) which have performance limitations. Genetic testing is emerging as a complementary diagnostic tool. We have identified mutations and correlated VWF multimer patterns in our patients (pt) with types 2 & 3 VWD, and report novel candidate mutations. Patients & Methods: Unrelated pt with type 3 (n=6) and subtypes 2 (n=22) VWD, from Mayo Comprehensive Hemophilia Center, consented to this IRB approved study. PCR amplification, from genomic DNA, of VWF gene (splice junctions, coding and promoter regions), avoiding pseudogene amplification, followed by ABI® sequencing and analysis (Mutation Surveyor: SoftGenetic®) were performed. Mutations were compared to VWD (ISTH), NCBI NR nucleotide and DV SNP databases. Comparison with available VWF sequences from other vertebrate species was performed. Selected regions not known to contain mutations remain to be sequenced. VWF multimer analysis was performed using a novel in-gel immunostaining and infrared imaging system. Results: Type 2A VWD (n=10), a novel mutation in exon 28 (E28) (V1524G: 2 pt) with characteristic VWF multimer abnormalities (decreased HMWM, increased satellite banding, suggesting enhanced VWF proteolysis). Type 2B VWD (n=6), 2 pt were compound heterozygous for 2 previously reported mutations, the first in E28 (R1306W)/E48 (T2647M) and the second in E28 (V1316M)/E20 (R854Q). Type 2M VWD (n=4): an 84 yr female with personal (spontaneous and post-surgical) and family bleeding histories, VWF:RCo 72%, VWF:Ag 118% (RCo:Ag ratio 0.61), normal FVIII and platelets, and aberrant VWF multimer banding pattern without substantive reduction of HMWM, had a novel mutation in E52 (S2775C; conserved in mouse, rat, chicken and dog); a 14 yr female with a history of bleeding, VWF:RCo 34%, VWF:Ag 68% (RCo:Ag ratio 0.5) and normal FVIII and platelets, was compound heterozygous for a known mutation (R1399C subtype not classified in VWD database) and two novel mutations: E30 (P1725S) (conserved in mouse, rat, dog, cow and chimp) & E49 (T2666M) (conserved in mouse, dog, rat not in chicken). VWF multimers demonstrated abnormal banding pattern without substantive reduction of HMWM; 2 unrelated pt and families with ultra-large and smeary multimers, previously classified as Vicenza variant, had R1374C mutations and were reclassified as Type 2M VWD. Type 2N VWD (n=1) previously reported mutation E20 (R854Q) was confirmed. Type 3 VWD, 2 pt had novel mutations, the first in E22 (E950X) and the second was a compound heterozygote for E43 (R2478Q)/E40 (E2322V) mutations. In general, patients with similar mutations had similar multimer patterns. Conclusions: We report novel candidate mutations in types 2 & 3 VWD and demonstrate novel VWF multimer patterns with our in-gel staining system. Multimer patterns correlated well with the underlying genotype. The novel mutations are likely causative, given their occurrence at highly conserved residues, but this needs to be confirmed with expression studies. Integration of genetic testing into the diagnostic workup of VWD will potentially lead to more accurate diagnosis and subtyping of VWD, and may further refine protein based testing and provide additional biological insight into VWD.

7094 Background: Obesity is associated with comorbidities that could cause negative outcome upon delivering intensive care. In pediatric AML patients (pts), obesity was associated with more toxicity and worse prognosis. Here, we study Body-Mass Index (BMI) impact on clinical outcome of adult AML pts. Methods: A total of 180 adult pts with AML between 2003-2011 were enrolled. Retrospective data included demographics, labs, cytogenetics and outcome. LeukemiaNET Standardization (LNS), complete remission (CR), overall survival (OS) and relapse free survival (RFS) were obtained (Dohner E, Blood 2010). BMI of 25-30 was defined as overweight, while >30 as obesity. Fischer’s and Wilcoxon tests were used for comparatives between groups, cox proportional hazards and logistic regression for associations for OS/RFS and CR, Kaplan-Meier test for OS and RFS estimates via JMP software V9.0. IRB approval was obtained according to Helsinki declaration. Results: The median age was 63 years, with 115 (64%) were men. Of 159 pts, karyotype was favorable, Intermediate I, II and adverse in 21 (13%), 76 (48%), 23 (%14) and 39 (25%) pts respectively. Median BMI was 28.2 (range 16.8-47.8). 48 (26%) had normal BMI, 62 (34%) were overweight, and 70 (38%) were obese. At diagnosis, BMI classes were not associated with age, sex, glucose, white blood count (WBC), platelets, blasts, ECOG status, LNS, FLT3/NPM1 status; nor treatment toxicities, CR rates, or relapse after CR. BMI classes were associated with presence of concomitant comorbidities (p=0.047) and glucose levels (p=0.044). In univariate analysis, overweight (OR=1.8, p=0.16) and obesity (OR=1.9, p=0.13) did not affect CR rates. On adjusting for age, sex, LNS, WBC and blast count at diagnosis, only overweight pts had a significant higher CR (76% vs 63%) rates (OR=2.99, p=0.043). OS and RFS were not associated with BMI in univariate (p=0.51) and multivariate (p=0.32) models. Median OS and RFS were not different across BMI subgroups (p=0.52 and 0.59). Conclusions: BMI subgroups showed no correlation with treatment toxicity, LNS, relapse rates, OS or RFS. This should encourage giving therapy to pts regardless of their BMI status. Overweight was associated with better CR rates despite increased concomitant morbidities.