Cardiac Puncture Blood Collection in Rats

University Animal Care Committee Standard Operating Procedure

Document No: 10.10.3

Subject: Cardiac Puncture Blood Collection in Rats

Date Issued: July 7, 2011

Location: Queen’s University

Responsibility: Principal Investigators, Research Staff, Veterinary Staff

Purpose: The purpose of this Standard Operating Procedure (SOP) is to describe how to properly collect blood via cardiac puncture in a rat.

Abbreviations:

Animal Care Services ACS, Principal Investigator PI, subcutaneous SC, intravenous IV, intraperitoneal IP, intramuscular IM, per os PO, per rectum PR

1. Introduction and Definitions:

Use the following table to ascertain the most appropriate site for blood collection based on the volume required.

Site	Tail Vein	Saphenous	Cardiac Puncture	Jugular
Multiple sampling	Yes	Yes	No	No
Volume	0.05 - 0.1 ml/site	0.1-0.3 ml	1.0-3.0 ml	1.0 ml
Gauge (maximum)	23	25 (23)	23	25 (23)

The following are “good practice” guidelines recommended for blood collection volumes Collection sites and needle gauges.

The Circulating Blood Volume (CBV) of an adult rat is ~64mL/kg (0.064mL/g).
1% (maximum) of the CBV can be collected every 24 hours.
7.5% (maximum) of the CBV can be collected in a single collection, once a week.
10% (maximum) of the CBV can be collected in a single collection every 2 weeks.
15% (maximum) of the CBV can be collected in a single collection every 4 weeks.

To calculate blood collection volumes:

Body weight x Circulating Blood Volume = Total Blood Volume (TBV)
TBV x % blood sample required = acceptable volume to be collected (i.e. 100g x 0.064mL/g = 6.4mL/g then 6.4 x 0.075 = 0.5mL is the max accepted volume)

Body Weight (g)	Total Circulating Blood Volume (ml/g)	Acceptable volume for collection μl (mL)
Body Weight (g)	Total Circulating Blood Volume (ml/g)	7.5% Single collection/ 1 week	10% single collection/ 2 weeks	15% single collection/ 4 weeks
100	6.4	500 (0.5 ml)	600 (0.6 ml)	900 (0.9 ml)
150	9.6	700 (0.7 ml)	900 (0.9 ml)	1400 (1.4 ml)
200	12.8	900 (0.9 ml)	1200 (1.2 ml)	1900 (1.9 ml)
250	16	1200 (1.2 ml)	1600 (1.6 ml)	2400 (2.4 ml)
300	19.2	1400 (1.4 ml)	1900 (1.9 ml)	2800 (2.8 ml)
350	22.4	1600 (1.6 ml)	2200 (2.2 ml)	3300 (3.3 ml)
400	25.6	1900 (1.9 ml)	2500 (2.5 ml)	3800 (3.8 ml)
450	28.8	2100 (2.1 ml)	2800 (2.8 ml)	4300 (4.3 ml)
500	32	2400 (2.4 ml)	3200 (3.2 ml)	4800 (4.8 ml)

When collecting blood it is very important that the handler is able to recognize signs of shock and anemia. The combined effect of sample volume and sample frequency without appropriate fluid replacement can cause an animal to go into hypovolaemic shock or anemia.

Signs of hypovolemic shock include a fast and thready pulse, pale dry mucous membranes, cold skin and extremities, restlessness, hyperventilation, and a sub-normal body temperature.
Signs of anemia include pale mucous membranes of the conjunctiva or inside the mouth, pale tongue, gums, ears or footpads (non-pigmented animals), intolerance to exercise and increased respiratory rate at rest with severe anemia.
Packed cell volume, haemoglobin level, red blood cell and reticulocyte counts should be monitored throughout the series of bleeds using the results from the first sample from each animal as the baseline for the animal.
If volumes larger than10% are collected, replace volumes by 3-4 times the blood volume collected with warmed (30-39 degrees) isotonic fluids.

2. Materials:

Sterile syringes (1 – 3 ml)
Sterile needles (multiple sizes ranging from 23-30g)
Collection tubes
Anaesthetics

3. Procedures:

Only University Animal Care Committee (UACC) approved blood collection techniques can be performed.
The least volume required should be collected at all times.
All collections should be performed by trained and competent individuals.
The smallest needle size that complements collection location without causing hemolysis should be used.
Each and every animal requires a new sterile syringe and a new sterile needle/lancet.
Only three attempts per site should be practiced. If unsuccessful, allow another (trained and competent) person to collect the sample.
Apply pressure with gauze until hemostasis occurs.