Application of Thrombolytic Drugs on Clotted Blood and Bone Marrow Specimens to Generate Usable Cells for Cytogenetic Analyses

Transcription

1 Application of Thrombolytic Drugs on Blood and Bone Marrow Specimens to Generate Usable Cells for Cytogenetic Analyses Angelique St Antoine, BS; Morgan N. Ketterling, BA; William R. Sukov, MD; Josh Lowman, BS; Ryan A. Knudson, BS; Jason P. Sinnwell, MS; Anne E. Wiktor, BS; Rhett P. Ketterling, MD N Context. blood and bone marrow specimens account for a large proportion of failed cytogenetic studies. There are no published protocols describing salvage of clotted specimens such that conventional chromosome or fluorescence in situ hybridization (FISH) studies can be performed. Objective. To evaluate the utility of thrombolytic drugs on clotted blood samples to yield intact cells suitable for cytogenetic analysis. Design. Five commercially available thrombolytic drugs (alteplase, urokinase, streptokinase, tenecteplase, reteplase) were evaluated in a series of blinded experiments to identify the best drug for lysing clots to produce samples suitable for chromosome and FISH studies. After the selection of alteplase as the drug yielding the most promising results, a comparative study between alteplase (0.75 mg/ml) and a commercially available anticlotting reagent (ACR) was performed. For each sample, mitotic index, chromosome length, and quality of slides prepared for conventional chromosome and FISH analyses were evaluated. Results. Alteplase-treated samples produced a higher mitotic index than those treated with ACR while showing equivalent quality in conventional chromosome and FISH studies. We have demonstrated the utility of treating clotted blood samples with alteplase before cell culture to yield cells suitable for cytogenetic analysis. Since clinical implementation, this technique has been applied to more than 250 bone marrow samples, with a 93% success rate. Conclusions. We believe the routine use of alteplase on clotted blood and bone marrow specimens should become standard for cytogenetics laboratories and may have similar utility in salvaging clotted specimens for other clinical assays requiring intact cells for analysis. (Arch Pathol Lab Med. 2011;135: ) Conventional chromosome studies continue to be a critically important technique for visualization of the genome in both neoplastic and nonneoplastic settings. For many hematologic malignancies, cytogenetic results are essential for providing patients and physicians with diagnostic, prognostic, and predictive information. Similarly, chromosome studies are critical for identifying both balanced and unbalanced genetic rearrangements in children with developmental delays and dysmorphic features and in couples with recurrent miscarriages. Chromosome and fluorescence in situ hybridization (FISH) studies are typically performed on peripheral blood or bone marrow samples and require dissociated cells for interphase FISH and viable cells capable of division to generate metaphases for chromosome analysis. These specimens can be difficult to acquire, particularly Accepted for publication September 15, From the Division of Laboratory Genetics, Department of Laboratory Medicine and Pathology (Mses St Antoine, Ketterling, and Wiktor, Drs Sukov and Ketterling, and Messrs Lowman and Knudson) and the Division of Biomedical Statistics and Informatics (Mr Sinnwell), Mayo Clinic, Rochester, Minnesota. The authors have no relevant financial interest in the products or companies described in this article. Reprints: Rhett P. Ketterling, MD, Division of Laboratory Genetics, Mayo Clinic, 200 First St SW, Rochester, Minnesota ( Ketterling.rhett@mayo.edu). bone marrow specimens or specimens obtained from children and infants. Clotting of blood and bone marrow specimens results in sequestration of dividing cells within the thrombus and therefore frequently results in culture failure, precluding chromosome analysis. Owing to the importance of acquiring diagnostic information from blood or bone marrow specimens, the ability of a cytogenetics laboratory to salvage clotted samples should significantly reduce the need for obtaining a new sample, will minimize delays in providing results and consequently, the time required for making appropriate treatment decisions. The application of a thrombolytic agent to a clotted blood or bone marrow specimen is a possible resolution to this recurrent problem. Thrombolytic drugs are used clinically to lyse blood clots in vivo and are administered to patients with venous or arterial thrombi. Tissue plasminogen activators are able to lyse clots by converting plasminogen to plasmin, thus activating a potent thrombolytic cascade. 1,2 Several tissue plasminogen activators are commercially available and have the potential to be used in vitro to salvage clotted blood and bone marrow samples for cytogenetic analyses. Herein, we describe the application of 5 commercially available thrombolytic drugs and 1 commercially available anticlotting reagent to a series of clotted peripheral Arch Pathol Lab Med Vol 135, July 2011 Thrombolytic Drug Treatment of Specimens St Antoine et al 915

2 Table 1. Average Number of Metaphases Observed per Field of View (3400) From PB Max Cultures Control a Control b Alteplase Urokinase Metaphases per field of view a Sodium heparinized sample. b Untreated clotted blood sample blood samples to determine if dissociated interphase cells for interphase FISH, and intact cells capable of mitotic activity, can be isolated for subsequent cytogenetic analyses. MATERIALS AND METHODS After institutional review board approval, a series of experiments were performed to identify the most effective reagent, optimum concentration, and protocol to treat clots before cell culture initiation. Five thrombolytic drugs and a commercial anticlotting reagent were evaluated using 2 clot manipulation techniques before initiating cell cultures. The thrombolytic drugs tested included: alteplase (Cathflo Activase, Genentech, Inc, San Francisco, California), urokinase (Abbokinase, Abbott Laboratories, Abbott Park, Illinois), streptokinase (Streptase, Aventis, Paris, France), tenecteplase (TNKase, Genentech, Inc), and reteplase (Retavase, Boehringer Mannheim, Mannheim, Germany). We also evaluated a commercially available laboratory solution, Anti-Clotting Reagent (ACR), available from Genial Genetic Solutions Ltd (Upton, Wirral, United Kingdom). Statistical analysis was performed with a paired t test. Evaluation of 5 Thrombolytic Drugs on Peripheral Blood Specimens The initial exploratory experiment was conducted with peripheral blood samples collected from 6 volunteers. From each volunteer, 6 clot tubes containing no anticoagulant and 1 sodium heparinized tube were collected. The samples collected into tubes without anticoagulant were allowed to clot for 2 hours. The clotted samples were then treated with alteplase, urokinase, streptokinase, tenectoplase, or reteplase. These samples, as well as the normal control (sodium heparinized sample) and the clotted control (untreated clotted sample), were concurrently processed. The 5 thrombolytic drugs were diluted in sterile water according to manufacturer instructions to the following concentrations: alteplase, 1.0 ; urokinase, 100 ; streptokinase, I; tenecteplase, 5 ; reteplase, 1. For each drug tested, the entire contents of 1 vial were added to each clotted sample. specimens were manipulated with a sterile pipette to break up the clot and incubated for approximately 60 minutes at 37uC. When the clots were sufficiently dissociated, 1.0 ml was used to establish each culture. Two cell cultures, standard and high resolution, were established for each of the 7 samples per volunteer (84 cultures total). 3 Standard cultures were established with PB Max Karyotyping Medium (Gibco, Invitrogen, Carlsbad, California) and harvested after 72 hours. High-resolution cultures were initiated in RPMI media with GlutaMAX (Gibco) and treated with 5-fluoro-29-deoxyuridine 48 hours after culture initiation and ethidium bromide and colcemid at the time of harvest. Two slides were prepared per culture for Geimsa-trypsin-Leishman banded chromosome analysis. All slides were evaluated in a blinded fashion and assessed for metaphase quality and quantity. Fluorescence in situ hybridization slides were also prepared from each patient culture (84 samples) according to standard laboratory protocols. Each specimen was hybridized with X (DXZ1) and Y (DYZ1) chromosome probes (Abbott Molecular, Inc) to assess for signal quality in interphase cells. Testing of the 2 most successful drugs, alteplase and urokinase, was repeated as described above, to determine the optimal thrombolytic concentration for effectively lysing the clot and maximizing metaphase yield. The concentrations evaluated for alteplase included 0.25 mg/ml, 0.5 mg/ml, 0.75 mg/ml, and 1.0 mg/ml, and for urokinase, 25, 50, 75, and 100. Results were quantified in terms of average analyzable metaphases per field at 3400 magnification (Table 1). Comparison Between Alteplase and ACR on Peripheral Blood Specimens After determining the alteplase concentration for optimum metaphase yield in the initial exploratory experiment, a more thorough comparative study between alteplase (0.75 mg/ml) and ACR was performed on peripheral blood specimens from 10 volunteers. Blood samples for 4 tubes were drawn from each volunteer: 1 sodium heparin tube and 3 tubes without an anticoagulant, which were allowed to clot for 24 hours before addition of the lysing reagent. The 24-hour clot time simulated the average length of time between specimen collection and receipt in the cytogenetics laboratory. The sodium heparin tube and 1 clotted tube from each donor served as controls. Two clotted samples from each volunteer were transferred to individual sterile centrifuge tubes, centrifuged for 8 minutes at 1200 rpm, and the supernatant was aspirated and discarded. Five milliliters of alteplase or ACR was added to the remaining clots, the clots were manually manipulated with a sterile glass pipette, and incubated at 37uC for 40 minutes. The treated samples were centrifuged for 8 minutes at 1200 rpm and the supernatants were aspirated and discarded. Five milliliters of RPMI medium was added to each tube, which was inverted for mixing. The samples were centrifuged for 8 minutes at 1200 rpm and the supernatant was aspirated and discarded. For each sample, 2 cultures were established (80 cultures total) and slides were prepared for chromosome and FISH analysis as previously described. Geimsa-trypsin-Leishman banded slides were prepared from both standard and high-resolution cultures established from the alteplase, ACR, sodium heparin, and untreated clotted samples for each of the 10 specimens. The mitotic index (number of metaphase cells per 1000 cells) was calculated for all samples, and chromosome length was evaluated. All FISH slides were hybridized with X and Y probes and evaluated for signal intensity and integrity. Analysis of all Geimsa-trypsin-Leishman banded and FISH slides was performed in blinded fashion to pretreatment and culture conditions. RESULTS Evaluation of 5 Thrombolytic Drugs on Peripheral Blood Specimens Assessment of metaphase cells from the clotted peripheral blood specimens treated with the 5 thrombolytic drugs indicated that 2 drugs, alteplase and urokinase, yielded metaphase cells of a quality and quantity (at least 20 metaphase cells) sufficient for routine chromosome analysis. Samples treated with streptokinase, tenecteplase, and reteplase did not yield a sufficient number of quality metaphases (less than 20 metaphases) for routine clinical 916 Arch Pathol Lab Med Vol 135, July 2011 Thrombolytic Drug Treatment of Specimens St Antoine et al

3 Sample Control a Table 2. Mitotic Index (Number of Metaphase Cells per 1000 Cells) for 10 Samples Standard Culture Control b Alteplase ACR Control High-Resolution Culture Control Alteplase ACR Median Mean Abbreviation: ACR, Anti-Clotting Reagent. a Sodium heparinized sample. b Untreated clotted blood sample. analysis. For the samples treated with the 5 thrombolytic drugs, no deleterious effects on chromosome quality were observed. Control samples performed as anticipated: all clotted samples failed to produce metaphase cells and all specimens collected in sodium heparin produced metaphase cells sufficient for analysis. Analysis of urokinase and alteplase at varying concentrations showed that clotted samples treated with alteplase at a concentration of 0.75 yielded the highest average number of analyzable metaphases (Table 1). This concentration was used for all subsequent studies. Comparison Between Alteplase and ACR on Peripheral Blood Specimens As part of a blinded comparison of alteplase and ACR on clotted specimens, the mitotic index (MI) was calculated for a series of 40 standard and 40 highresolution cultured specimens, including 20 each treated with alteplase or ACR, 20 sodium heparin and 20 clot-tube specimens (Table 2). Once unblinded, the data for standard cultures indicated that the average MI of the 10 alteplase-treated samples was comparable to that of the heparin control and significantly higher than that of the ACR-treated samples (P 5.001, paired t test; Figure 1). The MI range for the alteplase-treated samples was 13 to 38 (mean, 27.6), paralleling the MI range for the heparin control (12 to 40; mean, 28.8). The MI for ACR-treated specimens ranged from 3 to 25 (mean, 14.2) and for the untreated clotted specimens, the MI ranged from 1 to 33 (mean, 7.6). The mitotic index of the high-resolution cultures was lower than that of the standard cultures for all groups. Samples treated with ACR produced slightly more metaphases (average MI, 10.8) relative to samples treated with alteplase (average MI, 8.9), although this difference was not significant (P 5.57, paired t test). On the basis of the banding quality and chromosome length (550 band stage), the metaphase cells observed from the alteplase or ACR-treated samples were equivalent in quality to those of the heparin and clotted controls in both standard and highresolution cultures (Figures 2, A and B, and 3, A and B). All FISH studies were successful, regardless of whether the interphase cells were isolated from the clotted, sodium heparin, or thrombolytic-treated specimens. No differences were identified in the signal quality among the samples (data not shown). COMMENT Conventional chromosome analysis requires viable cells capable of mitosis. blood and bone marrow specimens result in entrapment of these cells within the thrombus, eliminating their availability for culture. In Figure 1. The mitotic index (MI) calculated for each standard culture sample (x-axis) treated with either alteplase (triangle) or Anti-Clotting Reagent (ACR) (square) is depicted in a scatter plot relative to the average MI of sodium heparin (normal control) and clot-tube (abnormal control) specimens. Arch Pathol Lab Med Vol 135, July 2011 Thrombolytic Drug Treatment of Specimens St Antoine et al 917

4 Figure 2. Representative metaphase images from standard cultures. Chromosome banding quality of standard culture metaphases obtained from clotted specimens treated with alteplase (A) was equivalent to that obtained from heparin control specimens (B) (original magnifications 31000). Figure 3. Representative metaphase images from high-resolution cultures. Chromosome banding quality of high-resolution culture metaphases obtained from clotted specimens treated with alteplase (A) was equivalent to that obtained from heparin control specimens (B) (original magnifications 31000). many instances, cytogenetic results are essential for providing patients and physicians with diagnostic and prognostic information. The ability of a laboratory to salvage clotted peripheral blood and bone marrow aspirate samples should significantly decrease the need for obtaining a new sample and prevent delays in providing critical results for time-sensitive treatment decisions. Thrombolytic drugs have been shown to be effective in salvaging clotted specimens for laboratory testing requiring functional nucleated cells from blood or bone marrow specimens. 4 Streptokinase has been shown to be effective in salvaging clotted blood and bone marrow specimens for flow cytometric immunophenotyping and has been successfully used to treat clotted samples before proceeding with cellular immune assays. 5 Herein, we explored the application of thrombolytic drugs to clotted peripheral blood specimens and demonstrated the ability of thrombolytic drugs to yield viable cells capable of mitotic activity. In the present study, our initial comparison between commercially available thrombolytic drugs indicated that only 2 drugs, urokinase and alteplase, were able to salvage clotted specimens such that the yield of metaphase cells was sufficient for routine clinical analysis (Table 1). Importantly, there was no detectable deleterious effect on chromosome quality. Similarly, interphase FISH studies were not affected by use of either drug. Further blinded comparisons between alteplase and ACR on 10 samples indicated that both were successful in producing quality metaphase cells suitable for chromosome analysis. In standard cultures, alteplase-treated samples had a significantly higher MI (average, 27.6) than samples treated with ACR (average MI, 14.2) (P 5.001) (Figure 1). Interestingly, these samples yielded not only a higher mitotic index but also superior banding quality and chromosome length (Figures 2, A, and 3, A). In highresolution culture, no difference was noted between alteplase- and ACR-treated samples; however, both yielded a quantity of metaphases at least equivalent to 918 Arch Pathol Lab Med Vol 135, July 2011 Thrombolytic Drug Treatment of Specimens St Antoine et al

5 that of the unclotted heparin control. Fluorescence in situ hybridization analysis was successful on all samples tested. This validation process demonstrated the utility of alteplase in salvaging clotted blood specimens for routine chromosome and FISH analyses. We have since instituted the use of alteplase on clotted bone marrow aspirate specimens in clinical practice. After the implementation of alteplase treatment for all clotted specimens, 232 of 250 clotted bone marrow aspirate samples (93%) successfully yielded an adequate number of metaphase cells suitable for chromosome analysis. Of these specimens, 43 (18%) demonstrated an abnormal karyotype, indicating that intact neoplastic cells are also released from the thrombus by this technique. We conclude that the treatment of clotted peripheral blood and bone marrow aspirate samples with alteplase is an effective means of salvaging samples for chromosome and FISH analysis. samples, which were previously unusable, can be salvaged successfully to provide important diagnostic and prognostic cytogenetic information. The application of alteplase to clotted blood and bone marrow specimens should decrease the requirement for patients to undergo repeated specimen collection procedures, which can be costly and invasive. We believe the routine use of a thrombolytic agent on clotted blood and bone marrow specimens should become standard for all cytogenetics laboratories. References 1. Meretoja A, Tatlisumak T. Thrombolytic therapy in acute ischemic stroke basic concepts. Curr Vasc Pharmacol. 2006;4(1): Tanswell P, Tebbe U, Neuhaus KL, Glasle-Schwarz L, Wojcik J, Seifried E. Pharmacokinetics and fibrin specificity of alteplase during accelerated infusions in acute myocardial infarction. J Am Coll Cardiol. 1992;19(5): Brown MG, Lawce HJ. Peripheral blood cytogenetics methods. In: Barch M, Knutsen T, Spurbeck J, eds. AGT Cytogenetics Laboratory Manual. 3rd ed. Philadelphia, PA: Lippincott-Raven; 1997: Niku SD, Hoon DS, Cochran AJ, Morton DL. Isolation of lymphocytes from clotted blood. J Immunol Methods. 1987;105(1): De Vis J, Renmans W, Segers E, Jochmans K, De Waele M. Flow cytometric immunophenotyping of leukemia cells in clotted blood and bone marrow. J Immunol Methods. 1991;137(2): Arch Pathol Lab Med Vol 135, July 2011 Thrombolytic Drug Treatment of Specimens St Antoine et al 919