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COLLECTION OF BLOOD SPECIMENS

Blood and separated serum are the most common specimens taken to investigate the etiology of communicable diseases. Venous blood can be used for isolation and identification of pathogens using sub-culture and inoculation techniques. Blood is also separated into serum for the detection of genetic material (e.g. using the polymerase chain reaction), specific antibodies, antigens, or toxins (e.g. by ELISA).

For the processing of most specimens for diagnosis of viral pathogens, serum is preferable to un-separated blood except where otherwise directed. When specific antibodies are being assayed, it is often useful to collect paired sera, i.e. an acute sample taken at the onset of illness and a convalescent sample collected one to four weeks later. Blood can also be collected by finger prick for the preparation of slides for microscopy or for absorption onto special filter paper discs for analysis. Whenever possible, blood specimens for culture should be taken before antibiotics are administered to the patient.

NOTE: Collect acute and convalescent blood for serology with at least 2 weeks between acute and convalescent specimens



A.1.0  VENOUS BLOOD SAMPLES


MATERIALS

SUPPLIES REAGENTS

Disposable latex or vinyl gloves

Tourniquet, Vacutainer, Monovette, or similar vacuum blood collection devices, or disposable syringes and needles

Vacutainer, blood culture bottles (50ml for adults, 25ml for children) with appropriate media

Swabs, gauze pads, band-aid

Labels and indelible marker pen

Skin disinfection: 70% alcohol (isopropyl alcohol, ethanol) or 10% povidone, iodine



PROCEDURE

STEP ACTION
1

Place a tourniquet above the venepuncture site.

2

Palpate and locate the vein. It is critical to disinfect the venepuncture site meticulously with 10% povidone iodine or 70% isopropyl alcohol by swabbing the skin concentrically from the centre of the venepuncture site outwards.

Let the disinfectant evaporate. Do not revalidate the vein again.

Perform venepuncture.

If withdrawing with conventional disposable syringes withdraw the following volumes

ADULTS:	5-10ml
CHILDREN:	2-5ml
INFANTS:	0.5-2ml

If withdrawing using vacuum systems, withdraw the desired amount of blood directly into each transport tube and culture bottle.

3

Remove the tourniquet.

Apply pressure to site until bleeding stops, and apply sticking plaster (if desired).

4

Using aseptic technique, transfer the specimen to the relevant cap transport tubes and culture bottles.

Secure caps tightly.
Be sure to follow manufacturer’s instructions on the correct amount and method for inoculation of blood culture bottles.

5

Label the tube, including the unique patient identification number using indelible marker pen.

6

Do not recap used sharps.

Discard directly into the sharps disposal container

7

Complete the case investigation and the laboratory request forms using the same identification number



HANDLING AND TRANSPORT

  • Blood culture bottles and blood sample tubes should be transported upright and secured in a screw cap container or in a rack in a transport box.

  • Cushion or suspend bottles during transport over rough terrain to prevent lysis of red cells. There should be sufficient absorbent paper around blood containers to soak up all liquid in case of a spill.

  • If the specimen reaches the laboratory within 24 hours, most bacterial pathogens can be recovered from blood cultures transported at ambient temperature.

NOTE: Collect acute and convalescent blood for serology with at least 2 weeks between acute and convalescent specimens




A.2.0  COLLECTION AND SEPARATION OF SERUM FROM BLOOD


MATERIALS

EQUIPMENT SUPPLIES REAGENTS

Centrifuge

Disposable latex or vinyl gloves

Tourniquet, Vacutainer, Monovette, or similar vacuum blood collection devices, or disposable syringes and needles

Vacutainer, blood culture bottles (50ml for adults, 25ml for children) with appropriate media

Swabs, gauze pads, band-aid

Labels and indelible marker pen.

Sterile Pasteur pipettes and bulb, or soft, disposable transfer pipettes (pastettes). The latter are easy to handle and dispose of in the field laboratory

Sterile screw-cap vials

Skin disinfection: 70% alcohol (isopropyl alcohol, ethanol) or 10% povidone, iodine



PROCEDURE

STEP ACTION
1  
2

Allow the blood specimen to clot for 30 minutes at ambient temperature

The specimen should be centrifuged at the laboratory at low speed (1000g for 10 minutes) to remove residual blood cells.

When serum separation is performed in a field laboratory, proper safety precautions should be taken.

Ensure that the centrifuge is in good condition and that the tubes are properly closed and balanced to avoid breakage and spilling.

If viral haemorrhagic fever is strongly suspected, samples should only be processed in properly equipped, specialized laboratories.

Discuss with the laboratory personnel whether a separation gel blood tube (see Note) would be acceptable in this case.

3

Separate the serum aseptically from the clot using a sterile Pasteur pipette and bulb or soft, disposable transfer pipette.

Transfer to a screw cap vial. Secure the cap tightly.

4

If a centrifuge is not available and there will be a delay before samples can be transported to a laboratory, serum may still be separated carefully from the retracted clot using a disposable transfer pipette.

Allow 4-6 hours to elapse after taking the blood sample to ensure adequate clot retraction.

Using the transfer pipette, remove the clear yellow serum whilst taking care to keep the tip as far as possible from the clot, and avoid agitating the blood tube during the removal process. (This may be easier if a separation gel collection tube has been used).

Transfer to plastic screw-cap vial and secure cap tightly.

5

Label the vial with the same patient details that appear on the blood sample tube.


NOTE: In some instances it may be acceptable to use a special blood tube containing a separation gel, which encourages separation of serum from clot. In this case, the centrifugation step is eliminated. This has the advantage of ease and safety of specimen processing under field conditions. However it is important to check with the laboratory in advance to ensure that these devices are appropriate for your particular investigation.



HANDLING AND TRANSPORT

  • If serum will be required for testing, separation from blood should take place as soon as possible, preferably within 24 hours at ambient temperature.

  • If the specimen will not reach a laboratory for processing within 24 hours, serum should, if at all possible, be separated from blood prior to transportation.

  • Sera may be stored at 4-8o for up to 10 days. If serological testing is to be delayed for a longer period, serum samples may be frozen.

  • If separation on site is not possible, or is inadvisable for safety reasons, the blood sample should be stored at 4-8oC.

  • Protect such un-separated samples from excessive vibrations while transporting.

  • Un-separated blood samples should not be frozen.



A.3.0  Capillary Blood Samples


MATERIALS

EQUIPMENT SUPPLIES REAGENTS
 

Disposable sterile lancets

Glass slides, cover slips, slide box

Filter paper

Fixatives such as methanol



PROCEDURE

STEP ACTION
1

Disinfect finger or thumb for adults, thumbs for children or side of heel for infants, by swabbing with 70% isopropyl alcohol, and prick with a sterile lancet.

Wipe away the first drop of blood.

2

Discard used lancets directly into the sharps disposal container.

3

Collect blood directly onto labelled glass microscope slides and/or filter paper.




Method of Preparation of Blood Films

Blood films should be made by trained personnel.


Thick films for microscopy

STEP ACTION
1

Label the slide with patient identification number and name

2

Disinfect and prick the site with a lancet as described above

3

Touch one or more large drops of blood onto the centre of the slide making sure that the slide does not touch the skin.

4

Spread the blood in a circle about 1cm in diameter using the corner of another glass slide.

5

Air-dry the film in a horizontal position.

Do not dry the film by heating over a flame or other heat source.


Thin films for microscopy

STEP ACTION
1

Label the slide with patient identification number and name/p>

2

Touch another drop of blood to the glass slide about 2 cm from one end making sure that the slide does not touch the skin.

3

Place the slide horizontally on a flat surface

4

Hold the slide of a second clean glass slide (the spreader) on to the center of the specimen slide and move it back until it touches the drop and the blood spreads along its base

5

At an angle of about 45o, move the spreader firmly and steadily across the specimen slide and air dry the film quickly.

DO NOT dry over a flame or other heat source.

6

Fix the dried film by dipping the glass slide in methanol or other fixative for a few seconds and air dry.



HANDLING AND TRANSPORT

  • Air dried and /or fixed films are transported at ambient temperature preferably within 24 hours of specimen collection. They must not be refrigerated. Thick and thin films are usually kept in separate slide boxes

Caribbean Public Health Agency © 2014