Blood drawing

Peripheral venous blood of adults is most often collected using closed collection systems. The person collecting blood is protected from contamination with the patient's blood and at the same time external contamination of the collected biological material is prevented. Mainly vacuum collection tubes are used. The disposable collection tube already contains preparation agents (e.g. anticoagulants or, conversely, coagulation accelerators). It is hermetically sealed and a vacuum is created in it, which ensures that the right amount of blood is sucked in during sampling. This also ensures the correct ratio of blood and preparation reagents.

Serum
Blood serum is the basic material for most routine clinical-biochemical determinations. It is prepared from collected whole venous blood by its precipitation and centrifugation. This will get rid of blood elements, fibrinogen and most other coagulation factors from the material. Precipitation of fibrin reduces the total protein concentration in serum by about 4 g/l compared to plasma. The concentration of calcium, which is consumed during coagulation, also decreases slightly.

The advantage of serum is the good stability of most analytes (usually hours to days when stored in the cold), it is often even possible to freeze the sample and store it for a long time. The disadvantage is the longer preparation of the material, coagulation and centrifugation takes around 45 minutes.

Blood for obtaining serum can be collected in a test tube without any reagents, coagulation occurs after contact with glass. Precipitation, however, takes quite a long time, for reliable results you need to wait at least 60 minutes before centrifugation. This increases the risk of hemolysis and release of intracellular components from blood elements. At the same time, metabolism in blood cells can continue for some time, which also affects the concentration of some substances. That is why tubes are mainly used, in which coagulation activators have been added – e.g. different forms of silicon dioxide. The addition of the activator reduces the time required for reliable coagulum precipitation to 30 minutes.

For easier separation of the serum, there may be a separation gel in the collection tube. During centrifugation, blood elements and coagulum sediment under the gel, the serum remains above it. The gel facilitates the separation of the serum and at the same time it prevents contamination of the serum with the intracellular content of blood elements, which is gradually released. The separation gel is usually made of acrylate polymers with the addition of silicon dioxide, which further accelerates coagulation.

Collection tubes for the preparation of blood serum usually have a golden yellow (with separation gel) or a red (without gel) stopper.

Plasma and heparinized whole blood
The basic material for most statistical biochemical tests is blood plasma. Lithium heparin is used as an anticoagulant for its preparation. The preparation of plasma is faster than the preparation of serum, because the time required for blood clotting is eliminated. The tubes have walls coated with dried heparin. After collection, the blood is mixed with it by repeatedly turning the test tube, and centrifugation can be started immediately afterwards. Tubes for plasma preparation may also contain a gel to facilitate the separation of plasma from blood elements.

The addition of heparin has relatively little effect on common biochemical determinations. The lithium salt of heparin is most often used because, unlike sodium or potassium heparin, it does not change the concentration of sodium or potassium, i.e. basic and commonly determined ions.

As an anticoagulant, heparin can also be used for some examinations of "whole blood", e.g. examination of acid-base balance and blood gases. In that case, heparinized capillaries or special sampling sets with a syringe for anaerobic sampling are used. However, heparinized blood is not suitable for hematological examinations (heparin interferes with the staining of blood smears) or for molecular biological methods (it inhibits the polymerases used in PCR).

Vacuum tubes with heparin are usually marked green – tubes with separation gel have a light green stopper, and tubes without the separation gel have a dark green stopper.

Whole blood with EDTA
For examination of blood elements (blood count) non-coagulable whole blood is used. One of the salts of ethylenediamine tetraacetic acid (EDTA) serves as an anticoagulant in this case. The most commonly used are well-soluble potassium salts, K2-EDTA and K3-EDTA. Whole blood with EDTA is also suitable for the examination of most analytes that are intracellular in blood cells, e.g. "glycated hemoglobin" and for "DNA analysis and molecular-biological methods".

Blood with the addition of EDTA is not suitable for many other biochemical methods. The concentration of ions changes significantly and a number of enzymes are inhibited. EDTA also cannot be used to test blood coagulation, although the mechanism of its anticoagulant effect is based on the chelation of calcium ions, similar to the effect of citrate. However, the effect of EDTA cannot be completely canceled by plasma recalcification, probably because EDTA also absorbs other metals (e.g. copper) that are necessary for the function of some coagulation factors (e.g. f. V and VIII).

Test tubes with the addition of EDTA salts are most often marked with a purple (lavender) stopper.

Citrated plasma and citrated whole blood
Examination of coagulation parameters is performed from plasma decalcified with citrate. Buffered solutions of sodium citrate are mainly used. Whole blood with citrate, possibly with other additives (theophylline, adenosine and dipyridamole) is used to determine platelet functions. Citrate is also used as an anticoagulant in measuring the rate of erythrocyte sedimentation.

Citrate changes the ion concentration and inhibits some enzymes. Therefore, citrate plasma is not suitable for most biochemical determinations.

Tubes with the addition of citrate are marked with a light blue stopper, special tubes for erythrocyte sedimentation have a black stopper.

Plasma, serum and whole blood with glycolysis inhibitor
Even after blood collection, blood cells consume glucose. The glucose concentration thus gradually decreases during sample processing, depending on the temperature and other factors, by about 0.5 mmol/l per hour. Therefore, for a more reliable blood glucose determination, sampling tubes with the addition of sodium fluoride, which inhibits glycolysis, are used. There are variants both with an anticoagulant (Na2EDTA or potassium oxalate) for obtaining plasma, and without anticoagulant for serum preparation.

Fluoride collection tubes have a grey stopper.

Other and special collection tubes
A number of other variants of sampling tubes are commercially available for various other purposes – e.g. for taking samples for microbiological cultures, for toxicological analyses, determination of trace elements, with peptidase inhibitors, etc.

Other devices for venous blood collection
The quality of the material is also affected by other aids and medical devices used for sampling. Their correct choice is therefore an important part of the pre-analytical phase of the examination.

Disinfectant
For skin disinfection, alcohol disinfectants are recommended, most often 70% 2-propanol. In addition to the requirements for antimicrobial efficiency, it is desirable from the point of view of the pre-analytical phase that the disinfectant evaporates quickly. Contamination of the collected blood with alcohol leads to hemolysis and thus to the deterioration of the sample for a number of determinations.

Iodine preparations are less suitable for blood sampling, because if they even minimally contaminate the sample, they can affect a number of determinations.

Injection needles
With closed vacuum sampling systems, special double-sided needles are most often used. When assembling the system, the cover (usually white or gray) on the side facing the tube is removed from the needle. This end of the needle is also protected by a rubber sleeve to avoid injury during handling. The needle is screwed into the holder. Just before sampling, the second cover is removed (the color corresponds to the diameter of the needle). This coloured cover protects the tip, with which the vein will be punctured. Finally, after inserting the needle into the vein, the sampling tube is inserted into the holder, while its cap is pierced with the tip, still covered by the rubber sleeve.

The blood collection needle must have a suitable diameter. Using too thin a needle with a vacuum system will cause large pressure differences, which can lead to hemolysis. At the same time, the collection period will be extended. If sampling is slow, initially there is a high concentration of reagents in the tube, which again can lead to hemolysis. On the other hand, a needle that is too thick will cause a larger wound, the collection may be more painful, and smaller veins may be damaged.

The diameter of injection needles is traditionally expressed in the so-called Birmingham gauge, and is marked with the letter G. A higher number means a thinner needle. 23 G to 18 G needles are used for blood sampling.