Hematology

UCSD Murine Hematology and Coagulation Core Laboratory Dr. Dzung T. Le, M.D., Ph.D., Director

Hematology, blood serum chemistry, and coagulation services for mice can be arranged by contacting Qiongyu Chen at q2chen@ucsd.edu or (858) 534-3172.

Service prices for UCSD investigators:

Sample Processing / Analysis: The charge for hematology is $30 each sample. That includes duplicate CBC with WBC differential and platelet count and a smear for staining and viewing.

For chemistry, the charge is $60 each sample for each panel (see specific analytes below). The charge for coagulation analysis (requires citrated plasma, performed in duplicate) is $60 each sample for each assay. Coagulation assays offered are: PT, aPTT, antithrombin, protein C, plasminogen, antiplasmin, vWF, fibrinogen, and coagulation Factors II, V, VII, VIII, IX, X, XI, and XII.

Additional Services: Platelet aggregometry and additional serum chemistry analyses are also available. Inquire for prices and methods.

Methods:

Sample Processing for Hematology: Samples are kept at room temperature until analyzed (within four hours from collection). Whole EDTA blood samples are analyzed in duplicate for CBC with leukocyte differential and platelet count on a Hemavet 950FS Multi Species Hematology System (Drew Scientific, CT) programmed with mouse hematology settings. Sample processing and system maintenance are performed as described in manufacturer's operating instructions. Mouse control reference supplied by manufacturer is tested each time samples are run for calibration control. Background checks are run before samples to verify that the background counts are within acceptable limits. Individual report sheets and an excel file with data are created. A whole blood smear is prepared from each sample.s

Retro-Orbital Sinus Blood Collection (for serum for chemistry): Mice are kept under general (inhalant) anesthesia. The venous plexus in the orbit behind the eyeball is punctured with a 50µl micropipet (microhematocrit tube) with no additive. Blood is allowed to drip into a Microtainer Serum Separator Tube(s) (SST, product number 365957) with no anticoagulant. The microhematocrit tube is removed, and light pressure to the area around the eye is applied with a Kimwipe tissue to close the eye and stop any bleeding. Mice are replaced into cage for observation during recovery.

Cardiac Puncture Blood Collection (for serum for chemistry): Mice are maintained under general (inhalant) anesthesia. Either open (body cavity opened to expose heart) or closed (needle inserted through intact skin and between ribs) method is used. Up to 1ml of blood is quickly withdrawn from heart using a 1ml plastic syringe with a 25ga. needle. Needle is removed from heart and then from syringe, and blood is emptied into Microtainer Serum Separator Tube(s) (SST, product number 365967) with no anticoagulant. Mouse is euthanized by cervical dislocation.

Sample Processing for Chemistry: Blood is allowed to clot for 4 to 6 hours at room temperature, then spun in SST for 5 minutes at 7000 rcf. Serum is removed and placed into a 0.5ml mc tube. For the Comprehensive Panel, 300µl of serum is analyzed for Albumin, ALP, ALT, AST, Anion gap, Bicarbonate, BUN, Calcium, Creatinine, Chloride, Glucose, Potassium, Sodium, Total Bilirubin, and Total Protein. A Lipid Panel requires an additional 300µl of serum and includes cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol calculated.

Cardiac Puncture Blood Collection (for citrated plasma for coag.): Mice are maintained under general (inhalant) anesthesia. Either open (body cavity opened to expose heart) or closed (needle inserted through intact skin and between ribs) method is used. Up to 1ml of blood is quickly withdrawn from heart using a 1ml plastic syringe with a 25ga. needle containing 30µl buffered citrate (0.06 mole/L sodium citrate, 0.04 mole/L citric acid, pH 7.4). Needle is removed from heart and then from syringe, and blood is emptied into a plastic mc tube containing sufficient additional citrate to achieve a final ratio of nine parts whole blood to one part citrate. Blood is immediately mixed well by tapping and inverting the tube five times to ensure proper anticoagulation. Mice are euthanized by cervical dislocation. Citrated platelet poor plasma is prepared from the citrated blood by centrifugation twice at 2000 rcf for 15 min at 22°C. Plasma samples are aliquoted and frozen at -80°C within four hours of cardiac puncture.

Prothrombin Time (PT): Clotting times are determined in duplicate with an ST4 semi-automated mechanical coagulation instrument (Diagnostica Stago, NJ). 30µl of citrated plasma are incubated at 37°C for 3 min, followed by the addition of 60µl of thromboplastin reagent prewarmed to 37°C to initiate clotting. Time until clot formation is measured in seconds.

Activated Partial Thromboplastin Time (APTT): Clotting times are determined in duplicate with an ST4 semi-automated mechanical coagulation instrument (Diagnostica Stago, NJ). 30µl of citrated plasma are incubated with 30µl of APTT reagent at 37°C for 5 min followed by the addition of 30µl of 25 mM 37°C CaCl2 to initiate clotting. Time until clot formation is measured in seconds.

Antithrombin Activity Assay: 40µl of plasma samples, tested in duplicate, are diluted 1:40 and 1:80 in 25mM Hepes, pH 7.5, 150 mM NaCl and 0.1% BSA (HN/BSA) and incubated in microtiter plate wells with 40µl factor Xa/heparin reagent (1 µg/ml factor Xa (Enzyme Research Labs, IN) and 10 U/ml unfractionated heparin) for 3 min at 37°C. Then, 40µl of 1.25 mg/ml chromogenic substrate S-2765 (DiaPharma, OH) is added to each well for color development and read at 405 nm by a Versa Max microtiter plate reader (Molecular Devices, CA). Standard curves are prepared with each plate by diluting normal mouse plasma (NMP) 1:20 to 1:640 in HN/BSA, analyzed simultaneously on the sample plate.

Protein C Activity Assay: Protein C activity is measured using a chromogenic substrate specific for Activated Protein C (APC) in diluted citrated plasma samples, in duplicate, with Protein C activator using 96-well plates read by a Versa Max microtiter plate reader (Molecular Devices, CA). 10µl of sample plasma dilution [1/10 in TBS (25mM Tris, 150mM NaCl, pH 7.5) with 100mM CsCl] are incubated at 37°C for 15 min with 25µl of pProtein C activator (2 u/ml) isolated from copperhead snake venom (PROTAC, BioMedica Diagnostics, Windsor B0N2T0, NS CA). 25µl of 2.5mM chromogenic substrate specific for APC (S-2366, DiaPharma, West Chester, OH) is added, plate is covered and kept at 37°C for 1 hour. 25µl of 20% acetic acid is added, and absorbance is read at 405nm. Absorbances are converted to percent normal reference mouse plasma (NMP) protein C from a standard curve made from dilutions of NMP (1/4 to 1/64) prepared and assayed simultaneously with samples.

Protein S Antigen Assay: A 96-well microtiter plate is incubated overnight at 5°C with 100µl per well of 10µg/µl rabbit anti-human Protein S polyclonal antibody (Dako, Denmark) prepared in 50mM Na2CO3, pH 9.6. The wells are then blocked with 200µl 25mM Tris, pH 7.5, 150mM NaCl (TBS) containing 3% BSA for 3-5 hr at 37°C or overnight at 5°C. After washing with TBS containing 1% BSA (TBS/1%BSA), 100µl of plasma samples, diluted 1/100 in TBS/1%BSA, are incubated in the wells overnight at 5°C followed by washing five times with TBS containing 0.05% Tween 20 (TBS/Tween). The wells are then incubated with 100µl of horseradish peroxidase-conjugated rabbit anti-human Protein S polyclonal antibodies (Enzyme Research Laboratories, Inc.) diluted 1/1000 in TBS/1%BSA overnight at 5°C. After washing again five times with TBS/Tween, the color is developed using a TMB peroxidase substrate (Bio-Rad, CA) according to the manufacturer's instruction. After 4h, 100µl of 1N H2SO4 are added to the wells to stop color development. Absorbance at 450nm is measured using a Versa Max microplate reader (Molecular Devices, CA). Absorbances are converted to percent normal reference mouse plasma (NMP) protein S from a log-log standard curve prepared with dilutions of NMP (1/25 to 1/800) in TBS/1%BSA. Samples are tested in duplicate, and analyzed using standard curves read simultaneously on the same plate

Plasminogen Activity Assay: Urokinase activated plasminogen is measured by chromogenic substrate specific for plasmin and urokinase activated plasminogen in diluted citrated plasma samples using 96-well plates read by a Versa Max microplate reader (Molecular Devices, CA). 60µl of plasma sample dilution (1/50 in 100mM Tris pH 8.5 with 8.3mM EACA (Calbiochem, La Jolla, CA)) are incubated at 37°C for 90 sec. 20µl of 2500 Ploug u/ml urokinase (EMD Millipore) are added to each sample well and incubated for 60 sec. Next, 100µl of 1.2 mM chromogenic substrate specific for plasmin and urokinase activated plasminogen (S-2403, DiaPharma, West Chester, OH) in 25mM HEPES, 150mM NaCl, pH 7.4 (HN buffer) are added. After 10 min, 100µl of 20% acetic acid are added to the wells to stop color development. Absorbance at 405nm is measured and converted to percent normal reference mouse plasma (NMP) plasminogen from a log-log standard curve prepared with dilutions of NMP (1/10 to 1/320) in Tris buffer with EACA. Samples are tested in duplicate, and analyzed using standard curves read simultaneously on the same plate.

Alpa-2 Antiplasmin Assay: Alpha-2 antiplasmin inactivation of plasmin is measured by chromogenic substrate specific for plasmin in diluted citrated plasma samples and plasmin using 96-well plates read by a Versa Max microplate reader (Molecular Devices, CA). 50µl of plasma sample dilution (1/50 in TBS with 120mM methylamine) are incubated at 37°C for 10 min. 50µl of 10µg/ml plasmin (EMD Millipore) are added to sample in well and incubated for 90 sec. 50µl of 3mM chromogenic substrate specific for plasmin (S-2403, DiaPharma, West Chester, OH) are added. After 10 min, 50µl of 20% acetic acid are added to the wells to stop color development. Residual plasmin is measured by reading absorbance at 405nm. Absorbances are converted to percent normal reference mouse plasma (NMP) alpha-2 antiplasmin from a standard curve prepared with dilutions of NMP (1:10 to 1:640) in TBS with methylamine. Samples are tested in duplicate, and analyzed using standard curves read simultaneously on the same plate.

Von Willebrand Factor (vWF) Antigen Assay: A 96-well microtiter plate is incubated overnight at 5°C with 100µl per well of 10µg/ml rabbit anti-human vWF polyclonal antibody (Dako, Denmark) prepared in 50mM Na2CO3, pH 9.6. The wells are then blocked with 200µl 25mM Tris, pH 7.5, 150mM NaCl (TBS) containing 3% BSA for 3-5 hr at 37°C or overnight at 5°C. After washing with TBS containing 1% BSA (TBS/1%BSA), 100µl of plasma samples, diluted 1/200 in TBS/1%BSA, are incubated in the wells overnight at 5°C followed by washing five times with TBS containing 0.05% Tween 20 (TBS/Tween). The wells are then incubated with 100µl of horseradish peroxidase-conjugated rabbit anti-human vWF polyclonal antibodies (Enzyme Research Laboratories, Inc.) diluted 1/2000 in TBS/1%BSA overnight at 5°C. After washing again five times with TBS/Tween, the color is developed using a TMB peroxidase substrate (Bio-Rad, CA) according to the manufacturer's instruction and read at 655nm using a Versa Max microplate reader (Molecular Devices, CA). Absorbances are converted to percent normal reference mouse plasma (NMP) vWF from a log-log standard curve prepared with dilutions of NMP (1/25 to 1/1600) in TBS/1%BSA analyzed on the same plate.

Fibrinogen Activity Assay: Fibrinogen activity is measured by thrombin clotting time of diluted citrated test plasma. 45µl of sample plasma dilution (1/15; in Owren's Veronal Buffer) are incubated for 3 min at 37°C. Then, 22.5µl of bovine thrombin reagent (Siemens Healthcare Diagnostics, Inc) are added to activate formation of fibrin clot. Clotting times are converted to fibrinogen concentration from a log-log standard curve prepared with dilutions (1/5, 1/10, 1/20, 1/30, 1/40; in Owren's Veronal Buffer) of a standardized plasma of calibrated fibrinogen concentration (Siemens Healthcare Diagnostics, Inc). Samples were tested in duplicate, and converted using standard curves prepared the same day.

Factor VIII: Clotting times are determined in duplicate with an ST4 semi-automated mechanical coagulation instrument (Diagnostica Stago, NJ). 30µl of citrated sample plasma diluted 1/20 in HN/BSA buffer are incubated with 30µl of APTT reagent and 30µl of citrated plasma deficient of factor VIII at 37°C for 5 min, followed by the addition of 30µl of 25mM 37°C CaCl2 to initiate clotting. Time until clot formation is measured and interpolated on a standard curve of serial dilutions citrated normal (BL/6 pool) mouse plasma tested as described to give reported result in % BL/6.

Factor IX, Factor XI, and Factor XII: Same as factor VIII method, with plasma deficient of the specific factor being measured in place of factor VIII deficient plasma.

Factor VII: Clotting times are determined in duplicate with an ST4 semi-automated mechanical coagulation instrument (Diagnostica Stago, NJ). 30µl of citrated sample plasma diluted 1/200 in Owren's Veronal Buffer are incubated with 30µl of citrated plasma deficient of factor VII at 37°C for 3 min, followed by the addition of 60µl of thromboplastin reagent prewarmed to 37°C to initiate clotting. Time until clot formation is measured and interpolated on a standard curve of serial dilutions citrated normal (BL/6 pool) mouse plasma tested as described to give reported result in % BL/6.

Factor II, Factor V, and Factor X: Same as factor VII method, with plasma deficient of the specific factor being measured in place of factor VII deficient plasma.

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