Automation in the Thrombosis and Hemostasis Laboratory.

Automation continues to advance into hemostasis and thrombosis laboratories. Integration of hemostasis testing into an existing chemistry track systems and adoption of a separate hemostasis track systems are important considerations. Unique issues must be addressed to maintain quality and efficiency when automation is introduced. Among other challenges, this chapter discusses centrifugation protocols, incorporation of specimen-check modules in the workflow, and inclusion of tests amenable to automation.

Interlaboratory Performance in Measurement of Dabigatran and Rivaroxaban.

CONTEXT.­: Assessing direct oral anticoagulant (DOAC) drug levels by reliable laboratory assays is necessary in a number of clinical scenarios. OBJECTIVE.­: To evaluate the performance of DOAC-specific assays for various concentrations of dabigatran and rivaroxaban, assess the interlaboratory variability in measurement of these DOACs, and investigate the responsiveness of the routine clotting assays to various concentrations of these oral anticoagulants. DESIGN.­: College of American Pathologists proficiency testing survey data from 2013 to 2016 were summarized and analyzed. RESULTS.­: For dabigatran, the interlaboratory coefficient of variation (CV) of ecarin chromogenic assay was broad (ranging from 7.5% to 29.1%, 6.3% to 15.5%, and 6.8% to 9.0% for 100-ng/mL, 200-ng/mL, and 400-ng/mL targeted drug concentrations, respectively). The CV for diluted thrombin time for dabigatran was better overall (ranging from 11.6% to 17.2%, 9.3% to 12.3, and 7.1% to 11.2% for 100 ng/mL, 200 ng/mL, and 400 ng/mL, respectively). The rivaroxaban-calibrated anti-Xa assay CVs also showed variability (ranging from 11.5% to 22.2%, 7.2% to 10.9%, and 6.4% to 8.1% for 50-ng/mL, 200-ng/mL, and 400-ng/mL targeted drug concentrations, respectively). The prothrombin time (PT) and activated partial thromboplastin time (aPTT) showed variable dose- and reagent-dependent responsiveness to DOACs: PT was more responsive to rivaroxaban and aPTT to dabigatran. The undiluted thrombin time showed maximum prolongation across all 3 dabigatran concentrations, making it too sensitive for drug-level monitoring, but supporting its use as a qualitative screening assay. CONCLUSIONS.­: DOAC-specific assays performed reasonably well. While PT and aPTT cannot be used safely to determine DOAC degree of anticoagulation, a normal thrombin time excludes the presence of dabigatran.

Gray Platelet Syndrome Presenting With Pancytopenia, Splenomegaly, and Bone Marrow Fibrosis.

OBJECTIVES: Gray platelet syndrome (GPS) is a rare platelet storage pool disorder associated with a marked decrease or absence of platelet α-granules and their contents. It is characterized clinically by mild to moderate bleeding; moderate macrothrombocytopenia with large, agranular platelets; splenomegaly; and bone marrow fibrosis. Electron microscopy confirms markedly reduced or absent α-granules in platelets and megakaryocytes. The classic description of GPS is caused by homozygous mutations in NBEAL2 (neurobeachinlike 2). METHODS: A 28-year-old Hispanic man with a history of easy bruising and occasional episodes of epistaxis sought treatment for pancytopenia and splenomegaly. Peripheral blood smear and bone marrow analysis, electron microscopy, and next-generation sequencing were performed. RESULTS: Large and agranular platelets were present in the peripheral blood. There was bone marrow fibrosis. Electron microscopy of the platelets showed absence of α-granules. Next-generation sequencing revealed a germline apparently homozygous nonsense variant in the NBEAL2 gene: c.5674C>T, p.Gln1892X (p.Q1829X). CONCLUSIONS: The differential diagnosis of GPS includes a myeloid neoplasm such as myelodysplastic syndrome with bone marrow fibrosis. The availability of diagnostic genetic panels for hereditable platelet disorders can assist in the recognition of GPS and other platelet disorders. We also describe a previously unreported pathogenic germline homozygous nonsense variant in the NBEAL2 gene: c.5674C>T, p.Gln1892X (p.Q1829X) in a patient with GPS.

Peripheral Blood Examination Findings in SARS-CoV-2 Infection.

OBJECTIVES: Peripheral blood abnormalities in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have not been fully elucidated. We report qualitative and quantitative peripheral blood findings in coronavirus disease 2019 (COVID-19) patients and compare them with a control group. METHODS: We reviewed electronic medical records, complete blood counts, peripheral blood smears, and flow cytometry data in 12 patients with SARS-CoV-2. These were compared with 10 control patients with symptoms suspicious for SARS-CoV-2 but who tested negative. RESULTS: No significant differences were noted in blood counts, except that absolute lymphopenia was present frequently in the control group (P < .05). Acquired Pelger-Huët anomaly (APHA) was noted in all COVID-19 cases, in most cases affecting over 5% of granulocytes. This contrasted with APHA in only 50% of control cases, affecting fewer than 5% of granulocytes in all cases (P < .05). Monolobate neutrophils were exclusive to COVID-19 cases. COVID-19 patients had greater frequency of plasmacytoid lymphocytes (P < .05). Flow cytometry data revealed absolute CD3+ T-cell count reduction in 6 of 7 patients; all of them required mechanical ventilation. CONCLUSIONS: Lymphopenia was infrequent in our COVID-19 cohort; however, flow cytometric analysis revealed absolute T-cell count reduction in most cases. COVID-19 cases had significant APHA with monolobate neutrophils and plasmacytoid lymphocytes as compared to controls.

External Quality Assurance of Platelet Function Assays: Results of the College of American Pathologists Proficiency Testing Program.

CONTEXT.­: The College of American Pathologists (CAP) developed proficiency testing for platelet function assays by using blood collected by the participant added to challenge tubes containing either saline (normal) or tirofiban (abnormal). OBJECTIVE.­: To analyze platelet function proficiency testing for Platelet Function Analyzer PFA-100, platelet aggregation, PlateletWorks, and PlateletMapping. DESIGN.­: Proficiency testing data from 2012-2016 were analyzed. RESULTS.­: For PFA-100, a total of 1200 laboratories participated; the coefficient variation (CV) of cartridge closure times was 22% (saline); 44,952 of 45,616 survey responses (99%) provided an interpretation, and 42,934 of 44,952 (96%) were correct. For optical platelet aggregation, 190 laboratories participated; the CV was 17% (saline), 7444 of 7813 survey responses (95%) provided an interpretation, and 7015 of 7444 (94%) were correct. For PlateletWorks, 60 laboratories participated; the CV was 3% to 11% (saline); 2412 of 2454 survey responses (98%) provided an interpretation, and 1207 of 1276 (95%) were correct for adenosine diphosphate (ADP) and 936 of 1136 (82%) for collagen. For PlateletMapping, 200 laboratories participated. For ADP, 1128 of 2697 survey responses (42%) provided an interpretation, but only 927 of 1128 (82%) were correct. For arachidonic acid, 1139 of 2604 survey responses (44%) provided an interpretation and 964 of 1139 (85%) were correct. CONCLUSIONS.­: CAP is the first to provide proficiency testing for platelet aggregation, PlateletWorks, and PlateletMapping. Platelet aggregation, PFA-100, and PlateletWorks using ADP as an agonist performed well with more than 90% of laboratories providing an interpretation and a similar number providing correct results. PlateletWorks using collagen and PlateletMapping showed worse interpretive accuracy than the other methods.

Automated assays for von Willebrand factor activity.

von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) by platelet aggregometry has been considered the gold standard for evaluating the ability of VWF to bind platelets for over 40 years. Many automated systems no longer require platelets and rather rely on agglutination of latex particles. Automated methods of measuring VWF activity have improved performance characteristics and are performed on the same coagulation instruments used for routine testing via immunoturbidimetric methodology. Alternatively, a newer chemiluminescence assay system for measuring VWF activity demonstrates excellent performance characteristics. As these methods are becoming widely used, it is important to assess their performance in diagnosing and monitoring different types of von Willebrand disease. We review the automated methodologies and the published performance of these VWF assays. Advantages and limitations of these automated methods are discussed.

Lack of grading agreement among international hemostasis external quality assessment programs.

: Laboratory quality programs rely on internal quality control and external quality assessment (EQA). EQA programs provide unknown specimens for the laboratory to test. The laboratory's result is compared with other (peer) laboratories performing the same test. EQA programs assign target values using a variety of methods statistical tools and performance assessment of 'pass' or 'fail' is made. EQA provider members of the international organization, external quality assurance in thrombosis and hemostasis, took part in a study to compare outcome of performance analysis using the same data set of laboratory results. Eleven EQA organizations using eight different analytical approaches participated. Data for a normal and prolonged activated partial thromboplastin time (aPTT) and a normal and reduced factor VIII (FVIII) from 218 laboratories were sent to the EQA providers who analyzed the data set using their method of evaluation for aPTT and FVIII, determining the performance for each laboratory record in the data set. Providers also summarized their statistical approach to assignment of target values and laboratory performance. Each laboratory record in the data set was graded pass/fail by all EQA providers for each of the four analytes. There was a lack of agreement of pass/fail grading among EQA programs. Discordance in the grading was 17.9 and 11% of normal and prolonged aPTT results, respectively, and 20.2 and 17.4% of normal and reduced FVIII results, respectively. All EQA programs in this study employed statistical methods compliant with the International Standardization Organization (ISO), ISO 13528, yet the evaluation of laboratory results for all four analytes showed remarkable grading discordance.

Dysplastic follicular dendritic cells in hyaline-vascular Castleman disease: a rare occurrence creating diagnostic difficulty.

Follicular dendritic cell (FDC) proliferations and dysplastic FDCs can be seen in Hyaline-vascular Castleman disease (HVCD). The association between HVCD and FDC sarcoma is well-documented; dysplastic FDCs may be precursors to FDC sarcoma. Herein, we describe a case of HVCD with strikingly large and dysplastic FDCs, which raised the differential of Hodgkin lymphoma and other neoplasms. Scattered dysplastic FDCs were predominantly in germinal centers and mantle zones, and rarely in interfollicular areas. Although occasional germinal centers contained increased FDCs, no mass forming proliferations were present to suggest FDC sarcoma. Immunostaining demonstrated that the atypical FDCs expressed CD21, clusterin and CXCL13, but not CD23, S100, pankeratin or CD30; they aberrantly expressed epidermal growth factor receptor (EGFR). The present case demonstrates that dysplastic FDCs may be present as isolated cells that require immunophenotyping to distinguish them from malignant entities with similar morphologic features. A variety of FDC markers is required to confirm their origin as the expression of any single marker is not assured, as occurred in this case. Pathologists need be aware of FDC proliferations in HVCD because of their association with FDC sarcoma. Aberrant EGFR expression by dysplastic FDCs may indicate that they are pre-neoplastic and necessitate long-term patient follow-up.

Primary Central Nervous System Histiocytic Sarcoma Arising After Precursor B-Cell Acute Lymphoblastic Leukemia.

Histiocytic sarcomas (HSs) are rare malignant neoplasms derived from histiocytes that may be associated with other hematolymphoid neoplasms. Histiocytic sarcomas rarely occur in the CNS and have not previously been reported in conjunction with prior B-cell lymphoblastic leukemia. We report the case of a 23-year-old man who presented with primary CNS HS 7 years after achieving remission for precursor B-cell acute lymphoblastic leukemia (B-ALL). Molecular studies revealed clonal immunoglobulin heavy-chain (IGH) gene rearrangement within the HS, suggesting linkage to his previous B-ALL. Previously reported post-ALL HSs show a strong predilection for young males (male-to-female ratio, 20:1), whereas cases of primary CNS HS without previous ALL affected older adults with balanced sex predilection. The patient's survival at 60 months exceeds expectations when compared with that of other reported cases of de novo primary CNS HS (n = 18) and post-ALL HS at all sites (n = 19). In addition, we discuss the potential relationship between B-ALL and HS posed by other authors.

An experimental model of hemothorax autotransfusion: impact on coagulation.

BACKGROUND: Traumatic hemothorax (HTX) has been demonstrated to predictably contain low fibrinogen, low hematocrit, and low platelet counts. When analyzed on its own, shed HTX demonstrates coagulopathy. However, when mixed with normal pooled plasma (NPP) at physiologically relevant dilutions, HTX demonstrates accelerated coagulation. We hypothesize that when HTX is mixed with a patient's own plasma, the mixture will demonstrate hypercoagulability. The accelerated coagulation of this mixture would have important implications for the autotransfusion of HTX as a method of resuscitating a trauma patient. METHODS: Adult trauma patients from whom greater than 140 mL of HTX was evacuated within 1 hour of tube thoracostomy were included. HTX was sampled at 1 hour after evacuation, and a portion of the sample was centrifuged and stored as frozen plasma for later analysis. The remainder of the sample was analyzed (coagulation, hematology, electrolytes), and values were compared with concurrent venous values extracted via chart review. A citrate tube containing the patient's venous blood was additionally spun down and frozen for subsequent mixing study analysis. Coagulation was further evaluated by mixing serial dilutions of the previously frozen HTX with NPP. Additionally, the previously frozen HTX was mixed in serial dilutions with the previously frozen sample of patient plasma (PTP). RESULTS: Subjects (10) were enrolled based on inclusion criteria and collection of a discarded venous sample. In HTX samples analyzed alone, no thrombus was formed in any coagulation test (activated partial thromboplastin time [aPTT] > 180). The median aPTT value of PTP alone was 25.5. In 1-hour specimens mixed at a clinically relevant dilution of 1:4, HTX mixed with NPP had a mediana PTT value of 26.0, whereas HTX mixed with PTP had a median aPTT value of 21.7. Thus, the mixture of HTX + PTP demonstrated a statistically significantly lower aPTT than the mixture of HTX + NPP (P = 0.01). Additionally, the mixture of HTX and PTP shows a statistically significantly lower aPTT value than PTP alone (P = 0.03), indicating a hypercoagulable state. CONCLUSIONS: HTX demonstrates coagulopathy when analyzed independently, but is hypercoagulable when mixed with NPP or PTP. Furthermore, mixing studies show a statistically significantly lower aPTT when HTX is mixed with PTP versus HTX mixed with NPP. Thus, autotransfusion of HTX would likely produce a hypercoagulable state in vivo, and should not be used in place of other blood products to resuscitate a trauma patient. The autotransfusion of HTX may, however, be of use in a resource-limited environment where other blood products are not available.

PML-RARA fusion resulting from a cryptic insertion of RARA gene into PML gene without the reciprocal RARA-PML fusion: clinical, cytogenetic, and molecular characterization and prognosis.

We describe a case of acute promyelocytic leukemia in a 61-yr-old woman with a cryptic insertion of RARA gene into PML gene. Using a combination of cytogenetic and molecular methods, we confirmed the insertion and presence of the PML-RARA transcript and lack of the reciprocal RARA-PML transcript. Although such cryptic insertions leading to a PML-RARA fusion have been previously reported, we show that such variant insertions, based on our case, appear to have the same prognostic significance as the classical t(15;17)(q22;q21).

A small amount can make a difference: a prospective human study of the paradoxical coagulation characteristics of hemothorax.

BACKGROUND: The evacuated hemothorax has been poorly described because it varies with time, it has been found to be incoagulable, and its potential effect on the coagulation cascade during autotransfusion is largely unknown. METHODS: This is a prospective descriptive study of adult patients with traumatic chest injury necessitating tube thoracostomy. Pleural and venous samples were analyzed for coagulation, hematology, and electrolytes at 1 to 4 hours after drainage. Pleural samples were also analyzed for their effect on the coagulation cascade via mixing studies. RESULTS: Thirty-four subjects were enrolled with a traumatic hemothorax. The following measured coagulation factors were significantly depleted compared with venous blood: international normalized ratio (>9 vs 1.1) (P < .001) and activated partial thromboplastin time (aPTT) (>180 vs 24.5 seconds) (P < .001). Mixing studies showed a dose-dependent increase in coagulation dilutions through 1:8 (P < .05). CONCLUSIONS: An evacuated hemothorax does not vary in composition significantly with time and is incoagulable alone. Mixing studies with hemothorax plasma increased coagulation, raising safety concerns.

D-dimer: simple test, tough problems.

CONTEXT: D-dimer is widely used for exclusion, or as an aid in diagnosis, of venous thromboembolism (VTE); however, the D-dimer assay methods available from manufacturers and the laboratory application of those methods vary widely. OBJECTIVES: To describe the current laboratory practice regarding the assay and reporting of D-dimer. DESIGN: Laboratories' D-dimer proficiency testing data were analyzed and laboratory practices regarding the performance and reporting of D-dimer were surveyed. RESULTS: Initial grading of D-dimer proficiency testing demonstrated high variability within and among methods. This variability continued to be present for several years after attempts to intervene. The number of laboratories using D-dimer to exclude VTE grew from 1500 in 2004 to more than 3500 in 2012. Survey and proficiency testing data demonstrated that 33% of laboratories changed the type or magnitude of units from that recommended by the manufacturer, a practice associated with as much as a 20-fold increase in the failure of proficiency testing. Many laboratories used a threshold for the exclusion of VTE that is higher than that recommended by the manufacturer. Many laboratories continue to use qualitative assays with insufficient sensitivity for exclusion of VTE. CONCLUSIONS: There is considerable variability both within and among quantitative methods used to assay D-dimer by laboratories. Laboratory practice continues to vary widely regarding the type and magnitude of units reported and the setting of the threshold for the exclusion of VTE. Although improved, the variability continues despite initial efforts to intervene.

Cytogenetic abnormalities precede morphological abnormalities in developing malignant conditions: report of 2 cases.

We previously hypothesized that cytogenetic abnormalities precede morphological abnormalities in developing malignant conditions. In this context we evaluated additional cases to further confirm that hypothesis. We report on 2 additional cases in which clonal cytogenetic abnormalities were observed in otherwise morphologically normal samples. Case 1 is a bone marrow from a 73 year old male with transformed follicular lymphoma (FL), while case 2 is a lymph node from a 53-year-old with lymphadenopathy, both referred to the cytogenetics laboratory for evaluation. A 73-year-old male presented with an enlarging left inguinal mass surrounding and obliterating the left iliac vein. A tissue core biopsy of the mass revealed recurrent high grade FL with diffuse large B-cell lymphoma (DLBCL). Examination of a random bone marrow biopsy of the adjacent left posterior iliac crest showed only mild hypercellularity (50%) and no evidence of malignancy, and the results were confirmed by flow cytometry. Cytogenetic evaluation revealed an interstitial deletion, del (9)(q13q32). In case 2, morphologically the lymph node showed extensive paracortical hyperplasia with numerous eosinophils and no clear indication of a neoplastic process with no abnormal lymphoid population observed by flow. PCR studies for TCR gamma and IgH gene rearrangements were negative for clonality. Chromosome analysis demonstrated 47,XY,+add(1)(p22),t(3;14)(q27;q11.2)[13]/46,XY[7]. FISH studies showed a BCL6 gene rearrangement but no TCRAD rearrangement. A subsequent inguinal lymph node biopsy showed DLBCL. These cases along with the other cases in the literature provide further evidence of genetic abnormalities preceding morphological abnormalities in developing malignant conditions.

Isolated vaginal myeloid sarcoma in a 16-year-old girl.

Involvement of the female genital tract by myeloid sarcoma as the initial presentation is extremely uncommon, especially in the vagina. The lack of specific histologic features and the unusual location can be a diagnostic challenge to both the surgical pathologist and the clinician. The very few reported cases of myeloid sarcoma occurring in the vagina have been exclusively seen in adults. We report a 16-year-old girl who presented with a vaginal mass of 4 weeks duration. The initial clinical impression was a Bartholin cyst vs an abscess. However, because of persistence of the vaginal mass after a full course of antibiotic treatment, a biopsy was performed. Immunohistochemistry supported the diagnosis of myeloid sarcoma. Peripheral blood and bone marrow studies were normal. The patient received 4 cycles of chemotherapy and remained disease free 5 months from therapy completion. The clinical course, diagnostic workup, and differential diagnosis of our patient are discussed. Reported cases of myeloid sarcoma occurring in the vagina are reviewed and summarized.

A prospective randomised study of a rotary powered device (OnControl) for bone marrow aspiration and biopsy.

INTRODUCTION: Bone marrow aspiration and biopsy is an invasive procedure associated with morbidity and mortality risk. We compared a powered bone marrow aspiration and biopsy device to the traditional method by relatively assessing pain scores, procedure times, biopsy capture rates, quality of material retrieved, and safety and operator satisfaction. METHODS: Two large academic medical centres participated in this trial. Patients were randomised to have procedures carried out using the powered system or the manual technique. A visual analogue scale pain score was recorded immediately following skin puncture and once again at the end of the procedure for each patient. Procedure time was measured from skin puncture to core specimen acquisition. Pathologic assessment of 30 randomised samples was carried out. Operator satisfaction with devices was measured on a scale of 0-10, with 10 as the highest rating. RESULTS: Five operators from two sites enrolled 50 patients (powered, n=25; manual, n=25). Groups were evenly matched, with no significant differences in the means for age, weight and height. The powered system was superior to the manual system with respect to patient perceived pain from needle insertion (2.6±2.0 vs 4.1±2.5, p=0.022) and procedural time (100.0±72.8 s vs 224.1±79.0 s, p<0.001). Overall pain scores at the end of both procedures were comparable (3.2±2.2 vs 3.8±3.0, p=0.438). No complications were observed in either arm of the study. Blinded pathologic analysis of the specimens retrieved revealed that cores obtained using the powered system were longer and wider than those obtained using the manual technique (25.4±12.3 mm² vs 11.9±5.6 mm², p=0.001). For marrow aspiration, no difference was seen between groups for clot/particle spicules or smear spicules. Operator assessment favoured the use of the powered device. CONCLUSIONS: Results of this trial suggest that the use of a powered bone marrow biopsy device significantly reduces needle insertion pain and procedural time when compared to a manual technique. The superior size and overall quality of core specimens retrieved by the powered device provides more material for pathologic evaluation, thereby increasing diagnostic yield and reducing the need for repeat procedures.

Anaplastic large cell lymphoma: twenty-five years of discovery.

CONTEXT: The year 2010 commemorates the 25th year since the seminal publication by Karl Lennert and Harald Stein and others in Kiel, West Germany, describing an unusual large cell lymphoma now known as anaplastic large cell lymphoma (ALCL). Investigators at many universities and hospitals worldwide have contributed to our current in-depth understanding of this unique peripheral T-cell lymphoma, which in its systemic form, principally occurs in children and young adults. OBJECTIVE: To summarize our current knowledge of the clinical and pathologic features of systemic and primary cutaneous ALCL. Particular emphasis is given to the biology and pathogenesis of ALCL. DATA SOURCES: Search of the medical literature (Ovid MEDLINE In-Process & Other Non-Indexed Citations and Ovid MEDLINE: 1950 to Present [National Library of Medicine]) and more than 20 years of diagnostic experience were used as the source of data for review. CONCLUSIONS: Based on immunostaining for activation antigen CD30 and the presence of dysregulation of the anaplastic lymphoma kinase gene (2p23), the diagnosis of ALCL has become relatively straightforward for most patients. Major strides have been made during the last decade in our understanding of the complex pathogenesis of ALCL. Constitutive NPM-ALK signaling has been shown to drive oncogenesis via an intricate network of redundant and interacting pathways that regulate cell proliferation, cell fate, and cytoskeletal modeling. Nevertheless, pathomechanistic, therapeutic, and diagnostic challenges remain that should be resolved as we embark on the next generation of discovery.

Clinical application of array-based comparative genomic hybridization for the identification of prognostically important genetic alterations in chronic lymphocytic leukemia.

Genomic aberrations have increasingly gained attention as prognostic markers in B-cell chronic lymphocytic leukemia (CLL). Fluorescence in situ hybridization (FISH) has improved the detection rate of genomic alterations in CLL from approximately 50% using conventional cytogenetics to greater than 80%. More recently, array comparative genomic hybridization (CGH) has gained popularity as a clinical tool that can be applied to detect genomic gains and losses of prognostic importance in CLL. Array CGH and FISH are particularly useful in CLL because genomic gains and losses are key events with both biologic and prognostic significance, while balanced translocations have limited prognostic value. Although FISH has a higher technical sensitivity, it requires separate, targeted hybridizations for the detection of alterations at genomic loci of interest. Array CGH, on the other hand, has the ability to provide a genome-wide survey of genomic aberrations with a single hybridization reaction. Array CGH is expanding the known genomic regions of importance in CLL and allows these regions to be evaluated in the context of a genome-wide perspective. Ongoing clinical trials are evaluating the use of genomic aberrations as tools for risk-stratifying patients for therapy, thus increasing the need for reliable and high-yield methods to detect these genomic changes. In this review, we consider the use of array CGH as a clinical tool for the identification of genomic alterations with prognostic significance in CLL, and suggest ways to integrate this test into the clinical molecular diagnostic laboratory work flow.