HEMATOLOGY

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

To arrange for hematology, blood serum chemistry, and coagulation services, contact the lab via email to set an appointment and receive instructions for the shipping and handling of samples.

To schedule services: Qiongyu Chen at q2chen@ucsd.edu and Michelle Abueg at mabueg@ucsd.edu

Lab phone: (858) 534-3172

Service prices for UCSD investigators:

Hematology special consideration: This service is only available locally (San Diego County), as samples are only viable 4 hours after collection. Overnight shipping of samples will not be accepted.

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

Processing Samples for Hematology (CBC)

• Whole blood samples should be collected into tubes with an anticoagulant, preferably EDTA (lavender top).
• Samples must be tested within 4 hours of collection to avoid coagulation and to ensure accurate results.
• Analysis is done using a Hemavet 950FS Multi-Species Hematology System (Drew Scientific, CT) programmed with species-specific (e.g., mouse, monkey, dog, human) settings.
• Background checks are run before sample analysis to verify that background counts are within acceptable limits.
• A mouse control reference is supplied by the manufacturer and is tested each time samples are run for calibration control.
• Control reference for other species are not kept in stock; contact our lab to discuss ordering.
• All samples are tested in duplicate (unless otherwise specified) for a Complete Blood Count (CBC) with leukocyte differential, and a Platelet count.
• Results may be given as individual sample report sheets and/or a cumulative excel file.
• A whole blood smear is prepared for each sample and is available for reference upon request.

SERUM CHEMISTRY

Retro-Orbital Sinus Blood Collection (for Serum Chemistry)

1. Mice are kept under general (inhalant) anesthesia
2. The venous plexus of the orbit is accessible directly behind the eyeball
3. The scruff of the animal is pulled back and the orbit is punctured behind the eyeball with a microhematocrit tube containing no additive
4. Blood drops are collected into a Microtainer Serum Separator Tube (SST, P/N: 365957) with no anticoagulant
5. Light pressure is applied to the area around the eye with a Kimwipe tissue to close the eye and stop the bleeding
6. Mice are returned to their cage and observed during recovery

Cardiac Puncture Blood Collection (for Serum Chemistry)

Materials

1. Inhalant anesthetic
2. 1mL plastic syringe with a 25ga. needle
3. Microtainer Serum Separator Tube(s) (SST, product number 365967)

Procedure

1. Mice are kept under general (inhalant) anesthesia
2. One of two methods may be used
1. Open - body cavity is opened to expose the heart
2. Closed - a needle is inserted through the intact skin, between the ribs, to reach the heart
3. The needle is inserted to the heart and up to 1mL of blood is withdrawn
4. Blood is emptied into a plastic mc tube
5. Mice are euthanized by cervical dislocation

Processing Samples for Chemistry (Comprehensive Metabolic Panel, or Lipid Panel)
300ul of serum required for each panel (e.g., if both Metabolic & Lipid panel requested, 300ul + 300ul = 600ul required)

1. Collected blood is allowed to clot over 4 hours at room temperature
2. Blood is then spun in the SST for 5 minutes at 7000 rcf
3. Serum supernatant is removed and placed into a 1.5ml mc tube

COAGULATION STUDIES

Cardiac Puncture Blood Collection (for Coagulation Studies - Citrated Plasma)

Materials

1. Inhalant anesthetic
2. 1mL plastic syringe with a 25ga. needle
3. Buffered citrate (0.06 mole/L sodium citrate, 0.04 mole/L citric acid, pH 7.4)
4. Microtubes

Procedure

1. Mice are kept under general (inhalant) anesthesia
2. One of two methods may be used
1. Open - body cavity is opened to expose the heart
2. Closed - a needle is inserted through the intact skin, between the ribs, to reach the heart
3. The syringe is loaded with 30uL buffered citrate and capped with the needle
4. The needle is inserted to the heart and up to 1mL of blood is withdrawn. Note the volume.
5. 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 (9:1)
6. Blood is immediately mixed well by tapping and inverting the tube five times to ensure proper anticoagulation. (if the sample is not mixed well, blood will clot and cannot be analyzed)
7. Mice are euthanized by cervical dislocation
8. Sample is centrifuged twice at 2000 rcf for 15 min at 22℃
9. Plasma is drawn off the top and aliquoted and frozen at -80℃ within four hours of puncture

Materials for All Clotting Time Studies*

1. ST4 semi-automated mechanical coagulation instrument (Diagnostica Stago, NJ)
2. 4-well cuvettes
3. Magnetic mixing balls
4. Citrated plasma samples

Prothrombin Time (PT)

Measures the time to clot formation, indicating the activity of the extrinsic and common coagulation pathways and their involved clotting factors.

Additional Materials*

1. Thromboplastin reagent

Procedure

1. Instrument, cuvettes, mixing balls, and thromboplastin reagent are pre-warmed to 37℃
2. 30uL of citrated plasma samples, in duplicate (2x), are incubated within cuvette wells at 37℃ for 3 minutes
3. 60uL of thromboplastin reagent is added to each well to initiate clotting
4. Time until clot formation is measured in seconds

Activated Partial Thromboplastin Time (aPTT)

Measures the time to clot formation, indicating the activity of the intrinsic and common coagulation pathways and their involved clotting factors.

Additional Materials*

1. APTT reagent
2. 25mM CaCl2

Procedure

1. Instrument, cuvettes, mixing balls, and CaCl2 are pre-warmed to 37℃
2. 30uL of citrated plasma samples, in duplicate (2x), are added to each well, followed by 30uL of aPTT reagent, and then incubated at 37℃ for 5 minutes
3. 30uL of CaCl2 is added to each well to initiate clotting
4. Time until clot formation is measured in seconds

Factor VIII

Key coagulation factor of the intrinsic pathway; activity levels are based on correction of clotting time for plasma deficient of the factor of interest and is reported as a percent.

Additional Materials*

1. HN/BSA
2. aPTT reagent
3. 25mM CaCl2
4. Citrated plasma deficient of factor VIII
5. Normal mouse plasma (NMP) BL/6 pool

Procedure

1. Instrument, cuvettes, and mixing balls are pre-warmed to 37℃
2. Citrated plasma samples are diluted 1/20 in HN/BSA
3. 30uL of sample dilutions, in duplicate, are added to each well, followed by 30uL of aPTT reagent, and then 30uL of citrated plasma deficient of factor VIII, and incubated at 37℃ for 5 minutes
4. 30uL of CaCl2 is added to each well to initiate clotting
5. Time until clot formation is measured
6. Time is interpolated on a standard curve based on NMP serial dilutions and reported as %BL/6

Factor IX, Factor XI, and Factor XII

Follow the factor VIII method, using plasma deficient of the specific factor being measured in place of factor VIII deficient plasma.

Factor VII

Key coagulation factor of the extrinsic and common pathway; activity levels are based on correction of clotting time for plasma deficient of the factor of interest and is reported as a percent.

Additional Materials*

1. Owren's Veronal Buffer
2. Thromboplastin reagent
3. Citrated plasma deficient of factor VII
4. Normal mouse plasma (NMP) BL/6 pool

Procedure

1. Instrument, cuvettes, and mixing balls are pre-warmed to 37℃
2. Citrated plasma samples are diluted 1/200 in Owren's Veronal Buffer
3. 30uL of sample dilutions, in duplicate, are added to each well, and then 30uL of citrated plasma deficient of factor VII§ , and incubated at 37℃ for 3 minutes
4. 60uL of thromboplastin reagent is added to each well to initiate clotting
5. Time until clot formation is measured
6. Time is interpolated on a standard curve based on NMP serial dilutions and reported as %BL/6

Factor II, Factor V, and Factor X

Follow factor VII method, using plasma deficient of the specific factor being measured in place of factor VII§ deficient plasma.

Fibrinogen Activity Assay

Activity of fibrinogen, activated by thrombin, is measured in time to clot formation.

Additional Materials*

1. Owren's Veronal Buffer
2. Bovine thrombin reagent

Procedure

1. Citrated plasma samples are diluted 1:15 in Owren's Veronal Buffer
2. 45uL of sample dilutions, in duplicate, are incubated at 37℃ for 3min
3. A log-log standard curve is prepared by diluting NMP 1:5 to 1:40 in Owren's Veronal Buffer
4. 22.5uL of bovine thrombin reagent are added to activate fibrin clot formation
5. Measured clotting times are converted to fibrinogen concentration using the standard curve

Materials for all Immunologic and Chromogenic Assays†

1. Versa Max microtiter plate reader (Molecular Devices, CA)
2. 96-well microtiter plate
3. Citrated plasma samples
4. Normal mouse plasma (NMP)

AntiThrombin Assay

Antithrombin activity is measured by level of inhibition of Factor Xa.

Additional Materials✝

1. HN/BSA
2. Factor Xa/Heparin reagent
3. 1.25 mg/ml chromogenic substrate S-2765

Procedure

1. Plasma samples are diluted to 1:40 and 1:80 in HN/BSA
2. Plate wells are loaded as follows:

   Sample dilutions, in duplicate	40uL	37℃	3min
   Factor Xa/Heparin reagent	40uL
   Chromogenic substrate	40uL			Color development

3. Plate is read at 405nm
4. Standard curve is prepared with each plate by diluting NMP 1:20 to 1:640 in HN/BSA, analyzed simultaneously on the sample plate

Protein C Assay

Activity is measured by the level of chromogenic substrate cleaved by activated protein C.

Additional Materials✝

1. TBS with 100mM CsCl
2. 2 u/mL Protein C activator
3. 2.5mM chromogenic substrate specific for activated Protein C (APC)
4. 20% acetic acid

Procedure

1. Plasma samples are diluted to 1:10 in TBS/CsCl
2. Plate wells are loaded as follows:

   Sample dilutions, in duplicate	10uL	37℃	15min
   Protein C activator	25uL	37℃	15min
   Chromogenic substrate	25uL	37℃	1hr	Color development; plate must be covered
   Acetic acid	25uL	37℃	1hr	Stop reaction

3. Plate is read at 405nm
4. Standard curve is prepared by diluting NMP 1:4 to 1:64 in TBS, analyzed simultaneously on the sample plate
5. Absorbance are converted to %NMP protein C using the standard curve

Protein S Assay

Concentration of protein S antigen is determined by binding to an antibody, then secondary binding by a conjugated antibody that will produce a detectable color change with the addition of a substrate.

Additional Materials✝

1. 10 ug/uL rabbit anti-human protein S polyclonal antibody prepared in 50mM Na2CO3, pH 9.6
2. TBS
3. 3% BSA in TBS
4. 1% BSA in TBS
5. 0.05% Tween 20 in TBS
6. Horseradish peroxidase (HRP)-conjugated rabbit anti-human Protein S polyclonal antibodies diluted 1/1000 in 1%BSA in TBS
7. TMB peroxidase substrate
8. 1N H2SO4

Procedure

1. Plasma samples are diluted to 1:100 in TBS/1%BSA
2. Plate wells are loaded as follows:

   Rabbit anti-human protein S Ab	100uL	5℃	Overnight
   3%BSA in TBS	200uL	37℃ or 5℃	3-5hr or Overnight	Block
   1%BSA in TBS				Wash 1x
   Sample dilutions, in duplicate	100uL	5℃	Overnight
   0.05% Tween 20 in TBS				Wash 5x
   Diluted HRP-conjugated rabbit Ab in TBS/1%BSA	100uL	5℃	Overnight
   0.05% Tween 20 in TBS				Wash 5x
   TMB peroxidase substrate			4hr	Color development
   H2SO4	100uL			Stop reaction

3. Plate is read at 450nm
4. Log-log standard curve is prepared by diluting NMP 1:25 to 1:800 in TBS/1%BSA, analyzed simultaneously on the sample plate
5. Absorbance are converted to %NMP Protein S using the standard curve

Plasminogen Activity Assay

Activity is measured by the level of chromogenic substrate cleaved by plasmin, activated by urokinase.

Additional Materials✝

1. 100mM Tris, pH 8.5 with 8.3mM EACA (Tris/EACA)
2. 2500 Ploug U/mL urokinase
3. 1.2mM chromogenic substrate specific for plasmin and urokinase-activated plasminogen in HN buffer
4. 20% acetic acid

Procedure

1. Plasma samples are diluted to 1:50 in Tris/EACA
2. Plate wells are loaded as follows:

   Sample dilutions, in duplicate	60uL	37℃	90sec
   Urokinase	20uL		60sec
   Chromogenic substrate in HN	100uL		10min	Color development
   Acetic acid	100uL			Stop reaction

3. 100uL of is added to each well for color development
4. Plate is read at 405nm
5. Log-log standard curve is prepared by diluting normal mouse plasma (NMP) 1:10 to 1:320 in Tris/EACA, analyzed simultaneously on the sample plate
6. Absorbance are converted to %NMP plasminogen using the standard curve

Alpha-2 AntiPlasmin Assay

Antiplasmin activity is measured by level of inhibition of plasmin.

Additional Materials✝

1. TBS with 120mM methylamine
2. 10ug/mL plasmin
3. 3mM chromogenic substrate specific for plasmin
4. 20% acetic acid

Procedure

1. Plasma samples are diluted to 1:50 in TBS with methylamine
2. Plate wells are loaded as follows:

   Sample dilutions, in duplicate	50uL	37℃	10min
   Plasmin	50uL		90sec
   Chromogenic substrate	50uL		10min	Color development
   Acetic acid	50uL			Stop reaction

3. Plate is read at 405nm
4. Standard curve is prepared by diluting NMP 1:10 to 1:640 in TBS with methylamine, analyzed simultaneously on the sample plate
5. Absorbance are converted to %NMP alpha-2 antiplasmin using the standard curve

vonWillebrand Factor (vWF) Antigen Assay

Concentration of vonWillebrand Factor antigen is determined by binding to an antibody, then secondary binding by a conjugated antibody that will produce a detectable color change with the addition of a substrate.

Additional Materials✝

1. 10ug/ml rabbit anti-human vWF polyclonal antibody prepared in 50mM Na2CO3, pH 9.6
2. 3% BSA in TBS
3. 1% BSA in TBS
4. 0.05% Tween 20 in TBS
5. Horseradish peroxidase(HRP)-conjugated rabbit anti-human vWF polyclonal antibodies diluted 1/2000 in 1% BSA in TBS
6. TMB peroxidase substrate

Procedure

1. Plasma samples are diluted to 1:200 in TBS/1%BSA
2. Plate wells are loaded as follows:

   Block	100uL	Rabbit anti-human vWF Ab	5℃	Overnight
   Block	200uL	3% BSA in TBS	5℃ or 37℃	Overnight Or 3-5hr
   	100uL	Sample dilutions, in duplicate	5℃	Overnight
   Wash 5x		TBS/Tween
   	100uL	HRP-conjugated rabbit anti-human vWF Ab	5℃	Overnight
   Wash 5x		TBS/Tween
   Color development		TMB peroxidase substrate

3. Plate is read at 655nm
4. Log-log standard curve is prepared by diluting NMP 1:25 to 1:160 in 1% BSA in TBS, analyzed simultaneously on the sample plate
5. Absorbance are converted to %NMP vWF using the standard curve