Blood sample Types, Anticoagulants, Preservatives, Adverse effects of Additives

Types of the blood samples

Indications for the whole blood, plasma, and serum:

1. A whole blood sample is used for blood gases and ammonia.
   1. It may be used for glucose, urea nitrogen, and lactate estimation.
2. Serum and plasma are used for the majority of the chemical tests.
3. The disadvantage of plasma is if you store the sample, then there are chances to form fibrin clots.
   1. These microclots may block the probe of the analyzer.
   2. Plasma is not a good sample for electrophoresis.

Type of patients for the blood samples:

1. Pediatric patients: If this is the first time sample from the child, then gain his confidence.
   1. Blood for neonatal screening is collected to rule out hypothyroidism, phenylketonuria, galactosemia, and hemoglobinopathies.
   2. For phenylketonuria, take the blood at least 24 hours, and the infant has taken the feed.
2. Adult patients: Be friendly and explain the procedure.
3. Patients in the ICU are unconscious: No doubt, the patients are unconscious, but still, there may be a need to take the blood samples.
   

   Blood sample types

Types of blood samples and collection procedures:

1. 1. Capillary blood (skin puncture).
      1. This is good for a small quantity of blood.
      2. Warm the finger from where taking the blood sample.
      3. In a newborn under 3 months, the heel is the best site to get a small blood quantity.
         1. The depth should not be >2.4 mm on the heel.
         2. Avoid the central portion and back of the heel.
            

            Various sites for the capillary blood

            

            Blood sample finger prick

   2. Venous blood (venipuncture):
      1. For larger quantities, will take venous blood.
      2. The blood sample is taken from the forearm, wrist, or ankle veins.
      3. A forearm site is preferred. Blood is taken directly from the vein, called phlebotomy.
      4. The median cubital vein is usually preferred.
      5. Mostly venous blood is drawn in the fasting state.
      6. Blood collected after the meal is called a postprandial sample.
      7. There are biological variables in the blood collection like:
         1. Patient lying in bed or standing up.
         2. After the exercise.
         3. Diurnal variations.
         4. Recent food intake.
         5. Recent intake of Tea/coffee (caffeine), smoking (nicotine), alcohol ingestion, and drug administration.
      8. Can take a blood sample in vacutainers, syringes, and with the help of butterfly needles.
      9. The blood samples can also be taken for blood culture.
         

         Venous blood various sites

         Difference between the values of venous blood and the capillary blood serum:

         Capillary blood values < than venous blood	No difference in capillary and venous blood values	Capillary blood values > than venous blood.
         Bilirubin     = 5.0% Chloride      = 1.8% Calcium      = 4.6% Total proteins = 3.3% Sodium         = 2.3%	Phosphorus Urea	Glucose = 1.4% Potassium = 0.9%

1. 3. Arteria blood is needed for the blood gases.
      1. Arterial blood is usually taken from the femoral artery.
      2. Blood for gases should be processed immediately without any delay.
         

         Can take a blood sample from the  femoral artery

Types of blood samples:

Type of blood sample	Special features	Indications
Whole blood	There is anticoagulant The tube contains cells and plasma. Mix the tube well	This sample is used for hematology studies. Can get plasma from the whole blood
Clotted blood	There is no anticoagulant.	Can separate the serum Serum good for biochemistry tests
Plasma	There is anticoagulant Centrifuge and will get plasma. Plasma contains fibrinogen	Used for the coagulation studies Can do biochemistry tests
Serum	No anticoagulants Centrifuge to get serum Fibrinogen is absent	This is the best sample for biochemistry tests. It can use for serology tests. Can in blood banking.
Buffy coat	After centrifugation, this is the middle layer between plasma and RBCs It contains white cells and platelets.	It can use for hematological studies. It is used for special stains.

Summary of the various types of blood samples:

Whole blood

1. Obtain a blood sample in the test tube containing an anticoagulant.
2. This sample will contain cells (white blood cells, platelets, RBCs, proteins) and plasma.

Plasma

1. This is a pale yellow liquid that contains RBCs, white cells, and platelets.
2. Plasma forms with the help of anticoagulants, which will prevent clotting.
3. There is the presence of fibrinogen in the plasma.

Serum

1. This is a clear fluid that is separated from the clotted blood. There are no RBCs, white cells, or platelets. There is no need for anticoagulants.
2. Clotted blood is kept at 37 C for at least 20 minutes and then centrifuged.
3. The upper portion is called serum.
4. There is no fibrinogen.
   

   Serum and plasma difference

   Difference between the capillary and venous blood values:

   Characteristic features	Capillary blood/Venous blood
   No difference in values	Urea Phosphorus
   Capillary value > than venous blood	Glucose = 1.4% Potassium = 0.9%
   Capillary values < than venous blood	Bilirubin = 5% Total proteins = 3.3% Sodium = 2.3% Chloride = 1.8% Calcium = 4.6%

   Buffy Coat

   1. This is the middle layer between the plasma and RBCs.
   2. This will contains white cells and platelets.
      

      Buffy coat

Table showing the difference between the contents of  plasma and serum: 

Contents of plasma and serum	Plasma	Serum
Proteins	Contains all proteins (albumin, globulins, and fibrinogen)	Fluid remaining after coagulation
Fibrinogen	Contains fibrinogen	No fibrinogen
Water contents	90% of water (92 to 95%)	90% of water
Cellular elements	RBCs, WBCs, and platelets are suspended in plasma	No RBCs, No WBCs, No platelets
Electrolytes contents	Electrolytes same level	Electrolytes same level
Presence of prothrombin		No prothrombin
Presence of antibodies	Antibodies are present	Antibodies are present
Presence of gases	Gases (CO2, O2, and N2)
Presence of clotting factors	Fibrinogen	No clotting Factor VIII, V, XIII Contain factor XII, XI, X, IX, VII
Presence of chemicals	Glucose, amino acids, cholesterol, and fats	Contains like plasma
Presence of hormones	Hormones	Contain rest of all products like plasma
Excretory products	Excretory products like urea, uric acid, creatinine, and bile	Excretory products present
Value of chemical substances	The same value of bilirubin, cholesterol, and creatinine	The same value of bilirubin, cholesterol, and creatinine

The difference between plasma and serum:

Characteristics	Plasma	Serum
Fibrinogen	0.2 to 0.4 G/dL	Nil
Formation site	Present in the body fluid	Prepared outside the body
Outside, the body contains	Always contains anticoagulant	Never anticoagulant added

The following table elaborates on the composition between plasma and serum regarding the values of constituents of blood.

Chemical substances	Plasma values more than serum	Plasma values less than serum.	No difference in the value in serum and plasma
Calcium	0.9%
Chloride	0.2%
Total protein	4%
LDH	2.7%
Albumin		1.3%
SGOT		0.9%
Alkaline phosphatase		1.6%
glucose		5.1%
Bicarbonate		1.8%
Sodium		0.1%
Phosphate		7%
Potassium		8.4%
Urea		0.6%
Uric acid		0.2%
			Bilirubin
			Creatinine
			Cholesterol

Purpose of anticoagulants

1. To prepare the whole blood or the plasma, anticoagulants are needed.
   1. The anticoagulants are added to the container before collecting the blood sample.
   2. These are used to prepare the whole blood or plasma during the collection of blood samples.
2. Definition of the blood:
   1. Blood is a combination of formed elements (RBCs, WBCs, Platelets) in a liquid portion called plasma.
      

      Blood components

3. There is a difference in the plasma and the serum for estimating various substances in the blood.
   

   Serum and plasma formation

Specimens to be rejected are:

1. Sample with lipemia.
2. Sample showing hemolysis.
3. Specimens with contamination like not proper cleaning of the site.
4. The sample quantity is not enough with the proper ratio for the tests.

In routine used anticoagulants are:

EDTA (Ethylenediaminetetraacetic acid)

1. Indications:
   1. This is useful for the hematological examination.
   2. It is used for cell count, hematocrit, hemoglobin estimation, and the cell differential count.
2. EDTA is used as a disodium or dipotassium salt.
   1. Mostly potassium EDTA is used as an anticoagulant, recommended for hematology studies. This is more soluble.
3. Mechanism of action:
   1. This is a chelating agent that binds the calcium, which is needed for coagulation. Chelation prevents coagulation.
      

      EDTA role as an anticoagulant

   2. It is effective at a final concentration of 1 to 2 mg / mL of blood.
   3. This can be used as a powder or make the solution and then add to vials. Let it dry.
   4. It is used as disodium, or dipotassium, or tripotassium salt.  
4. Solution:
   1. EDTA solution of 0.1% can be prepared and used. Let it evaporate at room temperature.
   2. Or 1.5 mg/mL.
   3. More than 2 mg/mL causes shrinkage of the cells.
5. Advantages:
   1. EDTA preserves the morphology of the blood cell structure.
   2. This is the anticoagulant of choice for hematocrit, Hb, and differential count.
   3. This is the best anticoagulant for peripheral blood smears and studies.
   4.  It has little effect on the various tests.
   5. They produce less shrinkage of RBCs.
   6. There is less increase in the cell volume after keeping the blood.
6. Drawbacks:
   1. It inhibits alkaline phosphatase, creatine kinase, and leucine aminopeptidase activities.
   2. EDTA is not suitable for Calcium and iron estimation.

Heparin

Indications:

1. This is used in the DVT (deep vein thrombosis)
2. It is used in pulmonary embolism.
3. This is also used in unstable angina.
4. This is used as a prophylactic drug in venous thrombosis.
5. If needed in pregnancy, this is the drug of choice because it can not cross the placenta.
6. This is used in cardiopulmonary bypass surgery. This will maintain the patency of the blood vessels.
7. It can be used in DIC if there are predominantly vasoocclusive manifestations.
8. Low molecular weight heparin is given subcutaneously because this has a longer half-life than heparin.
   1. A prophylactically single dose is needed. Lastly, it is used as an anticoagulant and mostly used in hematology.

Properties of Heparin:

1. This is an anticoagulant and causes the least interference with the test.
   1. This is theoretically the best anticoagulant because it is a normal blood component and does not introduce any foreign contaminants to the blood specimen.
2. This acidic mucopolysaccharide with a molecular weight of 15,000 to 18,000 is a blood coagulation inhibitor by potentiating the antithrombin activity.
3. This is more costly than the others.
4. It is present in powder form but is hygroscopic and dissolves rapidly.
5. It is mucoitin poly sulfuric acid available as sodium, potassium, lithium, and ammonium salts.
6. Mechanism of action of heparin:
   1. The GI tract does not absorb it, so given by injection in case of therapy.
   2. Heparin accelerates antithrombin III action, which neutralizes thrombin, thus preventing the formation of fibrin from fibrinogen.
   3. It forms the complex of thrombin + antithrombin cofactor + heparin and prevents fibrin clot formation.
   4. It prevents the coagulation for 24 hours by neutralizing the thrombin, thus preventing fibrin clots’ formation from the fibrinogen.

 

7. Solution preparation of the heparin:
   1. Heparin is added 0.2 mg / mL of blood in each test tube.
      1. Or 20 units of heparin for 1 mL of blood (in another reference, 15 U/mL).
   2. Or a drop of heparin is drawn into the syringe.
   3. Or simply coating the inside of the tubes or syringe is enough for the anticoagulant effect.
   4. After collecting blood, inverts the tubes 5 to 7 times for proper mixing of the blood.
8. Advantage:
   1. 1.  This is the best anticoagulant to use dry when minimal hemolysis is desired, e.g., sodium and potassium estimation.
      2. This is the best anticoagulant used to estimate pH, blood gases, electrolytes, and ionized calcium.
9. Drawback
   1. 1. It is costly.
      2. It inhibits the acid phosphatase activity.
      3. It gives a blue background for Wright’s stain smears, so not good for peripheral blood smear interpretation.
      4. It also affects the binding of triiodothyronine and thyroxine to their carrier protein and produces a higher free concentration of these hormones.
      5. It interferes with the binding of calcium to EDTA.
      6. It is not used for coagulation and hematology studies.
      7. Ammonium heparin affects the RBCs volume.

Sodium Citrate

1. Citrate is used as trisodium citrate salt.
2. It is a white hygroscopic crystalline powder.
3. Indications:
   1. Sodium citrate is widely used for coagulation studies.
   2. For PT and PTT.
   3. The sample can be used for ESR by the Westergren method.
4. Mechanism of action:
   1. it is used in solution form.
   2. This will chelate calcium. Inactivates Ca⁺⁺ ions.
   3. This will prevent the rapid deterioration of labile coagulation factors like factor V and factor VII.
      

      Mechanism of Sodium citrate as an anticoagulant

5. Solution preparation and uses:
   5. Trisodium citrate= 3.2 to 3.8 g/dL (3.2% solution).
   6. Mix well Trisodium citrate 3.8 grams in distle water.
   7. This can be used as 0.109 mg/mL.
   8. In blood, its ratio is 1:9, where 9 parts are blood, and 1 part is sodium citrate.
      5. PT and PTT= Blood: Sodium citrate = 9: 1 part (blood 9 parts: sodium citrate 1 part)
      6. ESR = Blood: Sodium citrate = 4:1  (1.6 mL of  blood: o.4 mL Sodium citrate).
6. Drawbacks
   1. This is used in liquid form (liquid anticoagulant).
   2. This is not a good anticoagulant for a complete blood examination.
   3. This is not good for the estimation of calcium.
   4. It inhibits aminotransferase and alkaline phosphatase.
   5. This will stimulate acid phosphatase when phenyl phosphate is used as the substrate.
   6. It has little value in clinical chemistry.

Potassium Oxalate

1. Mechanism:
2. This may be sodium, potassium, ammonium, or lithium oxalic acid salt used as an anticoagulant.
   1. This forms an insoluble complex with calcium ions (precipitate with calcium as a salt).
      

      Potassium oxalate mechanism as an anticoagulant

2. This is the most popular oxalate salt used as an anticoagulant in powder form.
3. Solution:
   2. Potassium oxalate at a concentration of 1 to 2 mg/mL of blood is used.
   3. Bulk solution: when you mix 30 grams/dL in distal water.
      2. Now add a few drops to the test tube side and dry it in the oven below 100 °C.
4. The combination of ammonium/potassium oxalate does not lead to shrinkage of the RBCs.
5. While other oxalates cause shrinkage.
6. Drawbacks
   1. If the concentration is >3 mg/mL, then there are chances for hemolysis.
   2. There is a reduction of 10% hematocrit.
   3. Oxalates inhibit several enzymes like acid phosphatase, alkaline phosphatase, amylase, LDH.
   4. It may cause precipitation of calcium as oxalate salt.

Sodium Fluoride

1. 1. This is a weak anticoagulant but used an antiglycolytic agent to preserve the glucose.
      1. This inhibits the system involved in glycolysis and preserves the glucose.
      2. This can be used as a dry additive.
   2. Mechanism of action: It acts in two ways:
      1. As an anticoagulant by binding the calcium.
      2. As an enzyme inhibitor that prevents the glycolytic enzyme from destroying the glucose.
      3. Sodium fluoride acts after the enolase, so it will not be effective in the first 1 to 2 hours. It prevents glycolysis after this period.
         1. Glucose can fall during this period, around 10 mg/dL.
         2. Transport on ice and rapid separation of the serum within 30 minutes can prevent glycolysis. There is no need for the addition of sodium fluoride.
         3. Not good for clinical chemistry tests.
            

            Sodium fluoride role in stopping the glycolysis

            

            Sodium fluoride as an anticoagulant

   3. Solution:
      1. This is effective at a concentration of 2 mg/mL of blood along with another anticoagulant like potassium oxalate.
      2. When used alone, then more concentration than 2 mg/mL is needed.
      3. This can be used in combination with oxalate as a fluoride-oxalate mixture.
   4. Most specimens are preserved at 25 °C for 24 hours and at 4 °C for 48 hours.
   5. Sodium fluoride is poorly soluble, so mix blood thoroughly before effective anti-glycolysis occurs.
   6. This is mostly used for glucose estimation.
   7. The rate of decreases is faster in newborns because of the increased metabolic activity of the white cells.
   8. Drawback
      1. This is also an inhibitor of many enzymes.
      2. Also, effect urease for the estimation of urea.

Sodium Iodoacetate

1. 1. This is an effective antiglycolytic agent and substitute for sodium fluoride.
   2. This does not affect urease for glucose and blood urea levels instead of sodium fluoride and on a single sample.
   3. Solution use:
      1. It can be used at the concentration of 2 g/L and is an effective glycolytic agent.
      2. This may be substituted for sodium fluoride.
      3. This does not affect urease.
   4. Drawback:
      1. It inhibits creatine kinase but no effect on other chemistry tests.

Adverse effects of the additives:

1. The additive may contain the substance to be tested like Na⁺oxalate for the estimation of Na⁺.
2. The additive may remove the component to be tested like in oxalate, removes the calcium.
3. The additive may affect enzymes like Na⁺flouride. This may destroy many enzymes.
4. A small amount of the anticoagulant gives rise to microclots, and this will interfere with cell count.
5. The additive may distort the cells like oxalate and change cell morphology like RBCs, which will become crenated. While WBCs show vacuoles. Lymphocytes and monocytes will have distorted shapes.
6. If the excess quantity is used, that will dilute the substance to be tested.