FREQUENTLY ASKED QUESTIONS

If blood taken from vein into a tube is left on its own, the shaped elements it contains collapse together with coagulation factors, and a clot is formed; the liquid part above the clot is also serum. Coagulation of the blood can be prevented by some substances called anticoagulants, so if the anticoagulant-prevented blood is left in a tube, the shaped elements are separated by settling to the bottom, the upper liquid part is the plasma. Blood plasma is the liquid portion of blood that travels in the veins. The difference of serum from plasma is that it does not contains come coagulation factors, especially fibrinogen. Bilirubin and carotenes give the serum a yellow color.

Recommended tube sequence for blood collection:
1. Blood culture 2. Coagulation 3. Gel / Non-Gel Serum 4. Gel / Non-Gel Heparin 5. EDTA 6. Glucose 7. Others.
 

To ensure that the additive and blood are mixed thoroughly, gently invert the tubes and bring them upright again immediately after blood collection. This double motion is a complete cycle. Pay attention to the number of cycles recommended according to the tubes used.

Do not shake the tubes. Vigorous stirring action may cause foaming or hemolysis. Insufficient or delayed mixing of serum tubes may result in delayed clotting. In tubes containing anticoagulant, insufficient mixing causes platelet accumulation, coagulation and/or false test results.

The usage areas of the Vacuum Blood Collection Tubes are as follows, according to their colors.

With EDTA tubes (MOR); Hemogram, Reticulocyte, Formula leukocyte, Blood Group, Cross-match, Direct Coombs, Indirect Coombs test are performed.

With citrate tubes (MAVİ); PT, aPTT, Fibrinogen, D-Dimer, Coagulation Factors (FII, FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII), Collagen-ADP, Collagen-Epinephrine, Protein C, Protein S, ATIII, TT, Lupus Anticoagulant, VWF:Ag and VWF:RiCoF, Antifactor Xa are studied.

Sedimentation test works with black capped (Citrated) tubes.

Serum tubes (Yellow-Red); It is used for many tests such as Biochemistry-Hormone-Microbiology-Serology-Immunology-Blood Bank.

Heparin (Green) tubes; Other analyzes requiring NT-ProBNP and Plasma are used for biochemistry analysis. Sodium heparin is used for the lithium level. Sodium or lithium heparin is used for the ammonia level.

Glucose Tubes (Gray); It is used in the long-term storage of some substances such as glucose measurement and ethanol. Antiglycolytic agents retain glucose for up to 5 days.

before the tubes are used. You can use the following formulation in order to work at the recommended RCF values according to the rotor radius of the centrifuges to be used.

RPM=1000*√(RCF11.18*rcm) 

r: Centrifuge Rotor Diameter

RCF: Force of Rotation

RPM: Number of Rotation

Depending on the condition of the sample taken, it is sometimes recommended to use a higher g-force and longer centrifugation time if the gel movement is not sufficient (especially due to a hematocrit value greater than 50%).

Centrifugation should be done in a cooled centrifuge. Higher temperatures may adversely affect the physical properties of the gel. 20°C-22°C is ideal for serum and plasma efficiency.

Gel separation tubes should be centrifuged within 2 hours at the latest. Contact of blood cells with serum and plasma for a long time causes false results. Once the barrier is formed, repeating the centrifuge is not recommended. Re-centrifugation of the tubes may result in possible loss of gel barriers, thereby causing gel particles to separate and appear in serum or plasma.

Before shipping gel tubes by mail or pneumatic system, the tubes should be kept upright for approximately one hour after centrifugation at room temperature to minimize damage to the gel barriers by sudden shaking.

Considering the feature of the gel barrier used by the tube manufacturers, it is recommended to centrifuge the tubes with a certain g force. If this recommended speed and time is not taken into account, it may cause the gel barrier to fail to fulfill its function properly. Our recommended centrifuge value and time for our tubes is 1800-2000 RCF (g) and minimum 10 minutes. In addition, it is recommended to use a movable rotor centrifuge instead of a fixed rotor to ensure the separation of the gel.

Since centrifuges used in laboratories may have different diameters, it is important to enter an RCF value. Devices that cannot be entered as RCF value will have to enter RPM value. Even if the RPM value entered here is the same, the RCF values to be obtained will be more or less according to the length of this centrifuge diameter. For this, verification is recommended by the laboratory staff before the tubes are used. You can use the following formulation in order to work at the recommended RCF values according to the rotor radius of the centrifuges to be used.

RPM=1000*√(RCF11.18*rcm) 

r: Centrifuge Rotor Diameter

RCF: Force of Rotation

RPM: Number of Rotation

Depending on the condition of the sample taken, it is sometimes recommended to use a higher g-force and longer centrifugation time if the gel movement is not sufficient (especially due to a hematocrit value greater than 50%).

Centrifugation should be done in a cooled centrifuge. Higher temperatures may adversely affect the physical properties of the gel. 20°C-22°C is ideal for serum and plasma efficiency.

Gel separation tubes should be centrifuged within 2 hours at the latest. Contact of blood cells with serum and plasma for a long time causes false results. Once the barrier is formed, repeating the centrifuge is not recommended. Re-centrifugation of the tubes may result in possible loss of gel barriers, thereby causing gel particles to separate and appear in serum or plasma.

Before shipping gel tubes by mail or pneumatic system, the tubes should be kept upright for approximately one hour after centrifugation at room temperature to minimize damage to the gel barriers by sudden shaking.

The outdoor pressure is also different according to the altitude (elevation) values of the place where the tubes will be used. This will cause the standard vacuumed tube to collect less sample at high altitudes as the external pressure will be less, and at low altitudes it will cause more sample collection due to high external pressure.  

For this reason, the manufacturer should be informed with the altitude values, if possible, by considering the regions where the tubes will be used when ordering. Thus, by ensuring that the tubes are vacuumed with the correct vacuum values according to the place where the tubes will be used, incorrect sample collection and erroneous test results will be prevented.

It is not recommended to transfer a sample from syringe to tube. Pressing the syringe plunger during the transfer can cause damage to blood cells as it will move the sample by creating a positive pressure, and this causes conditions such as hemolysis to occur. On the other hand, the pressure inside the tube will cause the cap to open, causing the sample to scatter around.

In addition, the use of a syringe for blood collection may cause tubes to either underfill or overfill, resulting in incorrect blood/additive ratio and potentially inaccurate analysis results.

To ensure that the additive and blood are mixed thoroughly, gently invert the tubes and bring them upright again immediately after blood collection. This double motion is a complete cycle. Pay attention to the number of cycles recommended according to the tubes used.

Do not shake the tubes. Vigorous stirring action may cause foaming or hemolysis. Insufficient or delayed mixing of serum tubes may result in delayed clotting. In tubes containing anticoagulant, insufficient mixing causes platelet accumulation, coagulation and/or false test results.

After mixing, the tubes should be kept upright. Fibrin formed in serum tubes stored horizontally may adhere to the cap or side walls of the tube and may not be separated from the serum despite being centrifuged.

Centrifugation should be done in a cooled centrifuge. Higher temperatures may adversely affect the physical properties of the gel. 20°C-22°C is ideal for serum or plasma efficiency.

Gel separation tubes should be centrifuged within 2 hours at the latest. Contact of blood cells with serum and plasma for a long time causes false results. Once the barrier is formed, repeating the centrifuge is not recommended. Re-centrifugation of the tubes may result in possible loss of gel barriers, thereby causing gel particles to separate and appear in serum or plasma.

Before shipping gel tubes by mail or pneumatic system, the tubes should be kept upright for approximately one hour after centrifugation at room temperature to minimize damage to the gel barriers by sudden shaking.

Hemolysis: It is the breakdown of erythrocytes in the blood and the mixing of hemoglobin and other contents into the blood. Hemolysis affects the results due to mixing of erythrocyte substances in the serum, hemodilution and chemical interference in some tests and causes erroneous results. In this case, the serum is not as it should be, but a redo color that gets darker depending on the severity of hemolysis.

Some of the tests most affected by hemolysis are given below.

Iron binding capacity, Lactate dehydrogenase, Potassium, Urea, Phosphorus, Alanine aminotransferase, Bilirubin, Chloride, Sodium, Iron, Aspartate aminotransferase, Calcium, Creatine kinase, Amylase.

Lipemia: The presence of lipids above normal in serum or plasma. Lipemia can be observed in non-fasted samples or in serum or plasma of patients with hyperlipidemia. Turbidity, density and color change in lipemic serum may cause erroneous test results.

Icterus: Increased bilirubin levels in serum or plasma.

Some of the tests most affected by icterus are:

Total cholesterol, Albumin, Uric acid, HDL cholesterol, Ferritin

Apart from these parameters, erroneous results may be obtained in other tests due to turbidity in the serum.

Situations such as daily biological rhythm, exercise, hunger, diet, medication, alcohol or smoking can affect many laboratory tests. Lipemic samples can cause clogging of analyzer probes and affect some test parameters. While 10-12 hours of fasting is recommended especially for glucose, blood lipid panel, phosphorus, iron, ferritin, ALP, folate and vitamin B12 in the blood, the fasting period should not exceed 16 hours. During the hunger period, water can be drunk only if needed.

It is recommended that heavy and strenuous exercises should be avoided for 24 hours before the examinations and that the patient should rest for half an hour before blood draw. Carboxy hemoglobin, plasma catecholamines and serum cortisol increase in smokers. While changes in these hormones increase the number of white blood cells such as neutrophils and monocytes, they decrease the number of eosinophil cells. As a result of chronic smoking, hemoglobin concentration and the numbers of erythrocyte and leukocyte may increase. If there is a regular use of medication due to the existing disease, the use of medication should not be stopped except for the control and recommendation of the physician.

The obtained test results vary depending on factors such as dietary habits, nutrition style, nutritional quality, exercise habits and style, genetic structure, geographical structure of the living area among societies.

We can group the variables that affect the test results under two main sub-headings as controllable variables such as individual’s posture, bed rest, exercise, smoking and alcohol consumption, and uncontrollable variables such as age and gender.

A- Controllable variables;

1. Posture

In an adult, the blood volume decreases by approximately 10% (600-700 mL) when moving from lying to an upright position. This situation may cause an increase of approximately 8-10% in plasma protein concentration. This decrease in plasma volume can cause an increase in the levels of enzymes, protein hormones and protein-bound drugs. Changes in posture may also cause changes in aldosterone, catecholamine, and antidiuretic hormone levels.

2. Prolonged bed rest

Serum protein and albumin levels decrease during prolonged bed rest, levels of protein-bound substances other than serum calcium decrease, and blood and urine calcium levels increase as a result of bone calcium mobilization.

3. Exercise

Exercise at a moderate intensity increases blood glucose levels and stimulates insulin release. Blood lactate levels increase after low intensity exercise. This can cause pH changes of the environment and affect the test results. An increase in the levels of enzymes such as CK, AST, LDH can be observed even in very short walks. At the same time, the levels of some hormones (T4, testosterone, LH, etc.) may increase after exercise.

The serum skeletal muscle enzyme activity of athletes at rest may be higher than normal individuals. Serum urea, uric acid, creatinine and thyroxine levels may be higher in athletes.

While physical exercise causes a decrease in serum cholesterol levels up to 25%, it causes an increase in HDL levels.

4. Circadian change

Most of the components in body fluids show cyclical or circadian changes throughout the day. Factors contributing to these changes are posture, activity, nutrition, stress, daylight/darkness, sleep/wakefulness. Since this change is high in some parameters, attention should be paid to the sample collection time. For example, serum iron levels can drop by 50% towards noon. Serum cortisol peaks in the morning hours and low in the afternoon. While the release of growth hormone is at its highest during sleep, it decreases with wakefulness.

5. Travel

Travels of a few hours can change the circadian rhythm. During flight, while serum glucose and triglyceride levels increase, glucocorticoid secretion is stimulated. Fluid and sodium are retained during long flights.

6. Nutrition

In the case of giving blood when one is full, the parameters most affected (in the upward direction) are glucose, iron, total lipids and alkaline phosphatase. After a high protein diet in the evening, increases in serum urea nitrogen, phosphate and uric acid can be observed even 12 hours after feeding.

Not only nutrition, but also substances in food content can affect test results. Caffeine can disrupt glucose tolerance, causing it to be high. Two cups of coffee can increase the free fatty acid concentration in plasma by 30%. In long-term intake of caffeine, total cholesterol levels decrease slightly, while triglyceride levels may increase. While avocado impairs glucose tolerance by affecting insulin secretion, onion reduces both plasma glucose and insulin response to glucose.

Malnutrition can cause a decrease in serum total protein and albumin levels as well as decrease in enzyme levels. Serum thyroid hormones may decrease.

7. Obesity

There is a positive correlation between serum cholesterol levels and obesity. Serum uric acid levels increase in relation to the body weight in individuals over 80 kg. As the body weight increases, serum LDH levels increase in both genders. While serum AST, creatinine, hemoglobin and total protein levels increase with body weight in men, serum calcium levels increase in women. While plasma insulin levels increase in obese patients, response to glucose decreases. Serum testosterone levels decrease in obese men.

8. Smoking

The effects of smoking on test results are related to how long it has been smoked. While the number of erythrocytes in the blood is high in smokers, the amount of carboxyhemoglobin in heavy smokers may exceed 10% of the total hemoglobin. The number of blood leukocyte increases.

Smoking affects the immune response. While serum IgA, IgG and IgM levels are lower in smokers compared to non-smokers, IgE levels are higher. Slight positivity can be observed in CEA levels in smokers.

9. Intake of alcohol

Moderate intake of alcohol has little effect on laboratory tests. Moderate intake of light alcohol causes an increase in triglyceride, glucose, lactate and cortisol levels. GGT, AST and ALT levels may be higher in individuals with chronic intake of alcohol compared to the healthy population.

10. Use of medication

Many drugs can affect laboratory test results. The effects of the drugs used on the tests can be learned from the laboratory specialist. If necessary, the laboratory should be informed about the drugs the patient uses regularly.

B- Uncontrollable variables;

1. Age

The reference range of many tests varies by age group. For example, children in the adolescence period have higher plasma proteins-, muscle- and bone-related enzyme levels than adults. Serum urea, uric acid, triglyceride and cholesterol levels increase in adults, enzyme levels are lower than in adolescence. In the elderly, creatinine clearance decreases, urine protein increases. Serum testosterone decreases in men over the age of 50, FSH and LH increase and estrogen decreases in women.

2. Gender

While there is not much difference between the genders in the pre-pubertal period, such enzymes as serum ALP, ALT, AST, CK are higher in men in the post pubertal period. Serum albumin, calcium, magnesium and cholesterol levels are higher in men. Hemoglobin and iron are lower in women. ALP is high in postmenopausal women.

3. Pregnancy

Test results may vary, depending on the increase in mean blood volume. Low levels of glucose in the urine may be positive.

4. Seasonal changes, environmental factors

Some of the laboratory test results may vary between summer and winter seasons. For example, the synthesis of vitamin D (1,25 dihydroxy cholecalciferol) increases during the summer months. Hemoglobin levels are higher in individuals living in high places. The characteristics of the geographical area where the individual lives can change the body fluid components.

5. Fever

Many parameters such as glucose, lipids and uric acid, especially proteins with positive and negative acute phase reactant character, can be affected. It also affects hormone responses.

6. Transfusion

An increase in plasma protein concentrations may occur with the administration of whole blood or plasma.

The reference value is the value obtained as a result of the measurements or observations made in the defined sample individual or group. A reference distribution range is created with the statistical interpretation of these values. Results obtained in clinical laboratories are interpreted taking into account the reference range values. Thus, the health or disease state of an individual is defined.

Reference ranges are determined according to the recommendations of the National Committee for Clinical Laboratory Standards: NCCLS and the International Federation of Clinical Chemistry: IFCC. Likewise, the selection of reference individuals is standardized in NCLSS- and IFCC-related documents. NCCLS later changed its name to the Clinical and Laboratory Standards Institute: CLSI.

Critical (panic) value is the laboratory result that indicates a life-threatening situation and may require urgent intervention in order for the patient to survive. In laboratories, the terms panic value and critical value are used in terms of patient’s health, follow-up and treatment when a result is seen that is far below or above the reference values or when there is a finding suggesting a serious infection.

Below is an example table for generally determined panic values. Although the values here are average values, they can vary according to the baby, child, adult and even gender. Therefore, each laboratory should determine its own reference range.

Biochemistry Critical (Panic) Values:

Hematology Critical (Panic)  Values:

Hormone Critical (Panic)  Values: