Prepared by the Turkish Biochemical Society Preanalytical Phase Working Group.

2020-ANKARA ISBN: 978-

605-70
 sop for CBC TEST

· Structure and Cellular Components of Blood

Blood is a life-sustaining fluid. Blood, also defined as a tissue, is a crucial
tool in evaluating the health status of the individual for it circulates almost all
over the tissues. Blood has two essential components;
· Cellular components,
· Plasma.
The blood count is the most frequently used measurement method for
assessing the blood’s cellular components.
Red Blood Cells (RBC, erythrocytes): Red blood cells are akaryocytes in the
shape of a concave disc. Its diameter is 6.2 - 8.2 µm in average and its volume
is 90 fL on average .

Red Blood Cells (erythrocyte)

Their main function is to mediate gas exchange . Hemoglobin is a protein

providing this function and filling almost the whole content of the cell. Red
blood cells bond oxygen in lungs mediated through hemoglobin and transport
oxygen to the tissues and make the return trip taking carbon dioxide back to
the lungs.
Red blood cells are produced in bone marrow as all the other blood
cells. Reticulocyte is an intermediate nuclear cell which is observed during
the maturation of erythrocytes. The elasticity of the cell wall of ageing red
blood cells declines and cells are caught while filtered in the sinusoids during
their passage through the spleen. Average life-span of red blood cells in blood
cir- culation is 120 days.
Red blood cells are examined in blood count with the parameters such
as RBC, hemoglobin (HGB), hematocrit (HCT), Mean Corpuscular Volume
(MCV), Red Cell Distribution Width (RDW), Mean Corpuscular Hemoglobin
(MCH), Mean Corpuscular Hemoglobin Concentration (MCHC)
The decrease in red blood cell count is called anemia. Oxygen-carrying
capacity of blood declines as a result of anemia. The case where an increase
in red blood cell count is observed is polycytemia; polycytemia is observed in
myeloproliferative disorders.
Hemoglobin: The functional protein which is found most frequently in red
blood cells is hemoglobin. The most crucial function of hemoglobin is to medi-
ate gas exchange between lungs and tissues as well as some other functions
such as maintaining acid-base balance, transportation of nitric oxide (NO).
More than 90% of the cytoplasm of a red blood cell is filled with hemoglobin.
This protein with a molecular weight of 64.000 Dalton is a tetramer
composed by the combination of four globulin chains containing one heme
loop each. Heme is the functional part of hemoglobin composed of
protoporphyrin IX ring in the middle of which an iron atom is found.
Hemoglobin is the most important part of blood count because it is used both
alone and in the meas- urements of many red blood cell indicators .

White Blood Cells (WBC, leukocytes):

White blood cells are called so because they have achromatic vision com-
pared to red blood cells under the light microscope. They are divided into five
different types.
Neutrophils: Neutrophiles which are an important component of hered-
itary immunity, have crucial functions in defense against microbial infections.
50-70% of white blood cells in blood circulation are neutrophiles and their
count is 1.7-7.5 103/µL' on average.

They stain neutral pink in hematoxylin and eosin. Two types of granules
are specified inside the neutrophiles. Primary granules are also known as
azurophilic granules; they contain enzymes such as myeloperoxidase,
elastase, proteinase. Secondary granules (specific granules) contain enzymes
such as phosphatase, NADPH oxidase, collagenase. Nuclei of the neutrophiles
of which the diameter var- ies between 12-15 µm have 2 to 5 segments (lobed
or segmented). Therefore, they are denominated as polymorphonuclear
neutrophil cells (PMN) together with eosin- ophiles and basophiles of which the
nuclei are seen lobed under the microscope .
Eosinophiles: They make just 1-3% of white blood cells; their diameter
varies between 12-17 µm. They stain well with acidic dyes; they are observed
dark red under the light microscope. The structures that stain are granules;
they are full of enzymes such as lipase, DNAse, plasminogen. Their particu-
lar function is to defend the body against parasitic infections . Eosinophile
count also increases in allergic conditions.

Basophils: Just 0.5-1% of white blood cells is basophile in blood circu-

lation; the diameter of a basophile is 12 µm in average. They are called as
ba- sophiles due to well staining with alkaline (basic) dyes; they are blue
under the light microscope. Molecules like histamine and serotonin make the
content of the granules. Particularly in allergic conditions, the count
increases.

Lymphocytes: Lymphocytes make about 20-40% of white blood cells; its

blood count is 1.0-3.2 103/µL. They are divided into sub-typed such as T cells,
B cells, natural killer cells (NK). These cells functioning both in hereditary
immunity and acquired immunity mediate almost all defence incidents of the
immune system. Under the light microscope, the nucleus of these cells
having a 7 µm size in average fills almost the whole of its cytoplasm;
however, in the cytoplasm of some NK cells, granules can be observed .

Monocytes: Monocytes are the precursors of the tissue macrophages in

blood. They make 2-10% of white blood cells. They are the largest cells in
blood circulation with their 12-20 µm diameters. Under the light microscope,
their horseshoe-like nuclei is observed .

Thrombocytes (platelet (PLT), blood-platelets): There is no nucleus in

thrombocytes derived from megakaryocytes. The function of thrombocytes
which are 2-3 µm in diameter is to plug holes by clotting where endothelial
in- tegrity is interrupted. For this reason, when necessary, they must be
activated. Availability of two kinds of granules inside thrombocytes: While
coagulation factors such as fibrinogen are found in alpha granules, delta
granules is full of substances such as ADP, calcium, serotonin .

· PRINCIPLES OF THE MEASUREMENT IN AUTOMATIC BLOOD COUNT

DEVICES

· Calculation of Complete Blood Cell Count Parameters

The complete blood count is developed by combining techniques more
than one. At present, this development has been going on by combining new
techniques with the techniques which are in use. In complete blood count,
the initial technique used was a visual examination of the cells via
microscope. HGB levels measured spectrophotometrically by
cyanohemoglobin meth- od (Drabkin method) enabled parameters known as
red blood cell indicators (erythrocyte indices) to be calculated. At present,
photometric HGB measure- ment is an integral part of the automatic blood
count analyzers.

Red Blood Cell Count (RBC), MCV: Red blood cell count is reported as RBC.
Size of the red blood cells is also essential as their count. Mean corpuscu- lar
volume (MCV) is an auxiliary indicator, particularly in the differential diagnosis
of anemia. In measurements performed under a microscope, MCV value is
cal- culated by dividing measured hemotocrite value (HCT) by red blood cell
count (RBC).

HCT: In this context, another critical indicator (parameter) is the HCT val-
ue. HCT is the proportion of total cellular components in the blood to total
blood volume in percentage. Before the development of automatic methods,
HCT levels used to be measured in the way that blood samples were
collected in capillary tubes and these capillary tubes were resolved using
micro-centrif- ugation. However, with the development of automatic blood
count, HCT has become a value calculated by using RBC counts and sizes
instead of being a measured value ,hematocrit measurement using of micro-
centrifugation (HCT (%)). On the right; calculation of hemotocrit in automatic
blood count analyzers MCH, MCHC: Another two important red blood cell
indicators are MCH and MCHC. MCH and MCHC serve in specifying the type of
anemia. When MCHC value drops below 32, red blood cells are denominated
as hypochromic because of the faint appearance of red blood cells due to iron
deficiency, but not the other way round; in fact, under physiological
conditions, MCHC level in red blood cells do not rise over 36. Hyperchromia
definition, which is a microscopic assessment is a misattribution. In such
cases, for the structure of red blood cells changes and turns into spherocyte,
they are seen much more filled and darker in color. There- fore, they have
been specified as hyperchromic. MCHC value changes a little bit lifelong of an
individual. There are minimal factors that affect MCHC level. There- fore,
MCHC is an auxiliary indicator, particularly in evaluating preanalytical errors.

RDW: It is a quantitative measurement of differences among red blood

cell sizes. The distribution of red blood cell sizes is calculated by added on
the histogram . Results of the patients are reported in standard devia- tion
(RDW-SD), variation coefficient (RDW-CV).

*LD: Lower detection limit, UD: Upper detection limit

White Blood Cell Count (WBC): High count of red blood cells in a blood
sample conceals white blood cell count. Therefore, for blood count analyzers
to perform the measurement, red blood cells are removed by lysing red blood
cells with substances in surfactant specifications (lyse solution). Then, white
blood cells are counted directly according to the measurement method of the
manufacturer company. In the patient’s results, white blood cell count (WBC)
in total and sub-component of each white blood cell are reported both in
count and in proportion In designs of blood count analyzers launched by
different manufacturers, changes can exist in measurement methods and cal-
culations, measurement and calculation methods of the most fre- quently
used blood count analyzers are summarized.

Platelet (thrombocyte) count: Platelet count can be more difficult than

other cells due to their small dimensions. In addition, while residual pieces
of other degraded cells, micro-erythrocytes and bacteria lead to false-high
platelet counts, giant platelets or platelet aggregates lead to false-low
platelet count values. In patient’s results, platelet values are reported
together with platelet distribution width (PDW), MPV and plateletcrit (PCT)
obtained by divid- ing platelet volume by platelet count.

Reference ranges, units, measurement methods and formulas

of blood count parameters
Measurement
Parameter Reference Unit Formula
method
range
Red blood cell indicators
Female:3.8-5.2
RBC 106 /µL Count
Male: 4.2-6
Female:12-15
 Male: 13.5-18 Photometric
HGB g/dL
measurement
Female: 35-49
HCT % Calculation
Male: 40-54

MCV 80-100 fL Calculation

HCT
MCV(fL) =
RBC
MCH 26-34 pg Calculation
HGB
MCH(pg) =
RBC
HGB
MCHC 32-36 g/dL Calculation MCHC (g/dL) =
HCT
RDW 11,5-14,5 % Calculation Histogram
Platelet parameters
PLT 150-450 103/µL Count

MPV 7-12 fL Calculation

PCT
MPV(fL) =
PLT
White blood cell parameters
WBC 6.3-10.6 103/µL Count
Neutrophil 1.7-7.5 103/µL Count
Neutrophil 50-70 % Calculation
#
Lymphocyte 1-3.2 103/µL Count
Lymphocyte 18-42 % Calculation
#
Monocyte 0.1-1.3 103/µL Count
Monocyte# 2-11 % Calculation
Eosinophil 0-0.3 103/µL Count
Eosinophil# 1-3 % Calculation
Basophil 0-2 103/µL Count
Basophil# 1-2 % Calculation

· VENOUS BLOOD COLLECTION FOR COMPLETE BLOOD COUNT

· Preparing the Patient

· Posture
Switching to sitting position or upright position from prone position
causes fluid flow from the vessels into the interstitial space. It is known that,
as a result of this fact, molecules which cannot diffuse into the tissue due to
their dimen- sions such as blood cells, proteins, cholesterol and iron are
measured high. It is reported that HGB values increase by 8%, RBC values by
8% and WBC up to by 15% between supine position and sitting or prone
position of the same person (14).

· Exercise
 Exercise can affect analytes such as creatinine, creatinine kinase, myo-
globin, aspartate aminotransferase as well as the results of complete blood
count . It is reported that WBC, neutrophil and lymphocyte values increase
significantly and high neutrophil count lasts more than 2 hours after heavy
exercise .

· Circadian Rhythm
It has been known that circadian rhythm has an impact on blood count.
Although RBC, HGB and HTC display a little increase at 11:00 in the morning,
an increase in leukocytes can be observed in the evening between 21:00-
24:00
. It is reported that while lymphocyte, eosinophil and basophil counts reach
their highest value, they decrease in morning hours. This change is inversely
related to the cortisol level . It is reported that PLT count increases in the
evening and decreases in the morning . For standardization in results, it is
appropriate to perform blood sampling in the morning.

· Stress
Anxiety and particularly crying of children during blood collection can
cause increase in leukocyte count .

· Diet
Blood should be drawn for sampling after at least 8-12 hours fasting period
. Glucose and lipid content of the blood increase in individuals who eat with-
in 2 hours or less before drawing the blood sample. It can affect MCV, MCHC,
HCT results in high concentration. Excessively high lipid content can lead to
interference in tests where photometric measurement is performed such as
he- moglobin . In addition, it has been reported that when blood glucose level
in- creases over 500 mg/dL, MCV value is affected due to the osmotic impact.

· Smoking
Before blood sampling, smoking can cause an increase in leukocyte
count. The long duration of smoking causes increases in hemoglobin level.

· Blood Sampling Equipment Used in Complete Blood Count

· Blood Sampling Tubes

Plastic tubes are preferred because they are flexible, resistant to high
cen- trifugation speeds and safer for the staff; in addition, they are adjustable
so they ensure a decrease in medical waste burden and they do less harm in
the environment.
Because of these reasons, plastic tubes are more frequently chosen and
are used prevalently.
Plastic tubes are manufactured using polyesters such as polyethylene
terephthalate (PET), polyolefins such as polyethylene and polypropylene (PP),
polyacrylic, polytetrafluoroethylene, polysiloxane, polyvinyl chloride,
polyacry- lonitrile and polystyrene. However, plastic tubes have more gas
permeability compared to glass tubes. PET which is non-breakable and
ensuring longer vacuum retention is prevalently used in the production of
blood sample tubes. And PP, for it has less water permeability, is another
selected plastic material because it provides keeping the volume and
concentration of liquid anticoag- ulation agents .

· Tube Additives: Anticoagulants

The most critical point of blood collection in complete blood count is to
draw the sample into the right tube. Drawing the sample into the EDTA tubes
known to be ‘lavender cap’ in daily laboratory speech has become a stand-
ard for complete blood count analyses. That is the most basic information
that must be well known by all healthcare workers taking roles in preanalyti-
cal phase. Thus, collecting blood sample in improper tubes, therefore sample
transfer from tube to tube can be prevented.

Ethylenediaminetetraacetic acid (EDTA, C10H16N2O8); is specified as the

most appropriate anticoagulant for hematological tests because it keeps both
the morphology of blood cells and cellular content. For complete blood count,
EDTA salts are used as an anticoagulant. EDTA salts, being a chelating agent,
bind calcium in blood and inhibit coagulation cascade. As EDTA salts, K3ED-
TA (tri-potassium EDTA), K2EDTA (di-potassium EDTA), Na2EDTA (di-sodium
EDTA) can be used .

· Insufficient Sample Volume

It has been reported that the second source of preanalytical error in com-
plete blood count tests is the collection of insufficient blood sample .It has
been stated above that two types of EDTA salt additives are used in the
tubes used for drawing complete blood count sample. While K 2EDTA is
sprayed onto the inner surface of the tubes in dry form, K 3EDTA exists in the
tubes as liquid solution. Therefore, it is known that K 3EDTA tubes cause dilu-
tion in samples and that this leads to reading the results under the real
values for all of the parameters that are measured by 1-2%. However,
drawing blood in lower volumes than the volume recommended for K 3EDTA
tubes will in- crease this dilution effect. Another negative effect of drawing
blood in lower volume is the shrinkage occurring in blood cells due to
hyperosmolarity in both K2EDTA and K3EDTA tubes. As a matter of this fact, it
is reported that while a decrease in MCV and HTC values is observed, there is
an increase in MCHC values .

At present, blood sampling tubes, their holders and needles are used to-
gether providing a system. In evacuated blood sample tubes, it is specified
how much volume of blood shall be drawn following vascular access. All man-
ufacturers put a fill line indicator on the tubes in order to observe this vol-
ume . In order to provide the right blood/anticoagulant ratio which is needed
for correct test results, it should be paid attention to drawing the volume of
blood specified by the manufacturer company (fill line). The overall approach
is in the direction that the tube can be filled with the sample about
+10% of the tube’s fill line (90%-110%) . For the speed of tube fill can vary
between different brands, waiting until the end of the blood flow into the tube
(till the vacuum exhausted) is important for sufficient blood drawing.

. On the left, sample drawn in insufficient volume; in the middle, sample

drawn in appropriate volume; on the right, sample drawn in excess volume
· Blood Sampling Needle
In venous blood sampling, the size of the sampling needle should be
speci- fied according to the amount of blood to be drawn, the age of the
patient and the diameter of the vein. For drawing blood from the antecubital
vein, 19-21 G (gauge) needles are ideal; smaller needles can be used in
newborns and in adults who have thinner veins. It should be kept in mind that
large G size numbers connote needles with small diameter and small G size
numbers connote needles with large diameter.

· Venous Sampling for Complete Blood Count

· Blood sampling staff

It has been recommended that venous sampling should be done by
trained nurses and phlebotomists for all tests. The staff that will draw blood
for sampling should be trained especially about tube sequence, specifications
of the equipment to be used and tube filling volume.

· Order of Draw for EDTA tubes

In patients for whom the EDTA sample is ordered together with the other
laboratory tests, the order of draw of the tubes that have different specifica-
tions is crucial. After venipuncture, drew order stated in the Guidelines for
Venous Blood Sampling should be followed .
Order of draw and mixing to comply with for sample tubes accord- ing to the
features of the tests ordered .
Cap color Tube/Additive Mixing
In order to make
Parameter (1) Blood culture/Medium medium and blood mix,
it is inverted slightly
(3) Coagulation tube / 3-4 times
Citrated
 (4) ESR tube / Citrated 3-4 times

(5) Serum tube / Without gel 5 times

(5) 5 times
Serum tube / With gel
(5) 5 times

Serum tube / Tube with

(5) 5 times
thrombin clot activator

Plasma tube / Heparin

(6) 8-10 times
tube with or without
gel

Plasma tube / EDTA

(7) 8-10 times
tube with or without
gel
Plasma tube / Fluoride /
(8) Potassium Oxalate: 8-10 times
Fluoride
/ EDTA Fluoride /
Heparin

· Mixing the EDTA tube

In addition, all tubes independently of EDTA salt used for anticoagulation
should be inverted 8-10 times in order to ensure a complete mixture of an-
ticoagulant and blood. Doing this procedure imprecisely is one of the most
essential preanalytical error sources, imprecisely. This procedure should be
repeated 8-10 times as shown in . Tubes must not be shaken, must be
inverted gently.

. Mixing the whole blood sample collected in EDTA tube by inverting

· EDTA-dependent pseudothrombocytopenia
It is considered that autoantibodies against glycoprotein originated as a
result of an interaction between EDTA and the glycoprotein IIb-IIIa found on

the platelet membrane leads to platelet aggregation. Consequently, pseudo-

thrombocytopenia is specified. Prevalence of EDTA-dependent pseudothrom-
bocytopenia is reported to be 0.1%. If such a condition is suspected, the test
is repeated after collecting a blood sample in another tube containing an
anti- coagulant other than EDTA (such as sodium citrate, heparin). If the case
is ED- TA-dependent pseudothrombocytopenia, then platelet count is
expected to be corrected ,Besides, the platelet count can be controlled by
peripheral smear with a finger stick capillary sample.

· Effect of Blood Sampling Procedures

At every stage of:
 · Identity validation,
· Choosing and preparing the equipment that will be used for blood
collection,
· Choosing vascular access site (vein),
· Cleaning vascular access site,
· Tourniquet duration,
· Venipuncture and blood collection,
· Tube sequence
which are applied during blood collection in the patient, it should be com-
ply with related guidelines
Tourniquet application should not exceed one minute for it can locally halt
circulation system associated with hemoconcentration and infiltration of
blood to tissue. If tourniquet application exceeds this period of time, analytes
hav- ing a protein structure, cellular blood volume and other cellular element
levels are found to be erroneously high. Among the parameters affected,
along with analytes such as albumin, potassium and calcium, complete blood
count pa- rameters such as RBC, WBC, HGB, HCT take place. It is reported
that if tour- niquet duration lasts for 2 minutes, there is a significant increase
in HGB and HCT values . In order to prevent this case, it is recommended to
loosen the tourniquet and reapply after two minutes if it is applied for more
than one minute .

· Blood volume
Blood volume collected should be so as to minimize iatrogenic (related to
phlebotomy) anemia risk particularly in pediatric patients and individuals
with

any critical illness. In order to prevent iatrogenic anemia, total blood amount
drawn from the patient should be monitored and limited on the base of the
time period.
In references, blood volume for children 75-80 mL/kg and for new-
borns it is higher. And in adults, it is specified as 65-70 mL/kg. It is recom-
mended to limit so as not to exceed 1-5% of the total blood volume within 24
hours and not to exceed 10% of total blood volume within 8 weeks . Also,
EDTA tubes with lower volumes providing to draw lower volumes of blood.
Blood amount filled into the tube is also essential. If it is overfilled, blood
is not well mixed with the anticoagulant in the tube, and this can lead to erro-
neous test results .

Recommendations:
· For blood count tests, blood is drawn into the tubes with lavender cap
(EDTA additive).
· Blood is to be drawn in the proper and appropriate volume. In order to
ensure proper blood/anticoagulant ratio, the tube should be filled up to the fill
line on the tube pointed out by the manufacturer. Much or less blood should
not be collected .
 · Recommended equipment should be used; blood should not be drawn
definitely using a syringe.
· In venous sampling, phlebotomists should determine the gauge of the
blood collection needle according to the blood volume that will be drawn from
the patient, his/her age and the diameter of vein of the patient. 19-21G size
numbered blood collection needles should be used for blood count tests. For
children or and individuals with thin veins, small size numbered needles
(>21G) can be used.
· Blood transfer from tube to tube must not be done.
· After venipuncture, one should be comply with the blood sampling se-
quence stated in the Guidelines for Blood Sampling .
· The tube should be inverted 8-10 times in order to ensure blood and
anticoagulant is completely mixed .
· For all tests to be performed, it is recommended that venous blood sam-
pling should be done by trained nurses, phlebotomists. These staff should be
trained in regular intervals

· SAMPLE TRANSPORTATION
Samples should be held on the tube racks straightly; swinging and shak-
ing should be avoided as possible as it can be. Specimens should be trans-
ported in nested special primary (sample tube), secondary (in a structure
that can prevent infectious contamination even if the cap is opened and
containing sorbent material) and tertiary (can be used as transportation
bag, protect- ed against temperature changes) containers so as to control
heat. Heat con- trol can be performed using devices that record temperature
changes. If the samples will be tested distant laboratories, refrigerated
transportation is the recommended application. On the other hand, it should
be kept in mind that keeping samples in the refrigerator can stimulate
coagulation and these sam- ples should be evaluated with regards to the
presence of coagulum.

· SAMPLE STORAGE

· Time Period of Complete Blood Count and Conditions of Sample Storage

If samples of complete blood count analyses are kept waiting at room
temperature, it is recommended to perform the tests within 6 (six) hours just
after blood drawing . If samples cannot be tested within 6 hours, they can be
kept in the refrigerator at +4ºC for 24 hours. In samples kept in the refriger-
ator, it is reported that MCV, HCT, WBC values are more stable .

· CRITERIA OF SAMPLE REJECTION

Many of the preanalytical variables affecting complete blood count can be
controlled. In case of complying with the blood collection instructions stated
in the previous article, a whole blood sample which is appropriate for blood
count can be obtained. However, getting an appropriate sample, meeting the
required features, may not be always provided. Recognizing such cases, if
needed, this sample or results obtained from these samples should be
rejected.
 · Clotted sample 57%
· Insufficient sample 14%
· Improper tube 7%
· Hemolysis 2.7%
· Lipemia 2.3%

· Clotted samples
Regarding complete blood count test, the most observed preanalytical
error source is reported to be the clotted samples .
Potential factors that cause clotted samples:
· Insufficient mixing of blood and the additive just after the sample
collection,
· Difficult blood drawing,
· Storing the sample in the refrigerator,
· Collecting blood sample over or below the fill line indicated by the
manufacturer,
· Blood drawing by using a syringe,
· Blood transfer from tube to tube (especially from the tubes
containing coagulant to EDTA tubes).
Removing the clot using tools like wooden applicator from the tube is a
frequent misapplication in laboratories and it is not recommended definitely.
This application can lead to hemolysis and false low values in results of all
pa- rameters. Clot residues in the sample can cause occlusions in the probes
and tubing of analyzers, and if it escapes the attention, it can cause
interferences during measurement.

It is easy to detect large clots by the naked eye. At present, there are clot
detectors in almost all blood count analyzers found in laboratories. Even if a
clotted sample is applied to the device, it will not work, but micro-clots may
not be detected by the detector. In the presence of clots in the sample, for
blood cells are wrapped with clots, it can be observed false low results of all
parameters. In such a case, low MCHC values can be a warning.

· Hemolysis
Hemolysis is the degradation of red blood cells in vitro or in vivo.
However, most of the hemolysis events occur due to the mechanical
impairment of the the integrity of red blood cells during sample collection,
transportation, processing or storage. Hemolysis is a critical problem
particularly in blood samples coming from the emergency service and in
samples drawn from children .

Potential hemolysis causes:

 · Difficult blood drawing,
· Blood drawn using a syringe,
· Prolonged tourniquet application,
· Small needle size (larger than 23 G),
· Hematoma formation,
· Sample contaminated by ethanol or water,
· Shaking the tube after blood draw or during transportation,
· Blood transfer from tube to tube,
· Insufficient blood draws into the vacuum tube.
Degradation of red blood cells within the sample is a part of hemoglobin
measurement method (analytical phase), but the early occurrence of this
event accidentally in the preanalytical phase causes particularly red blood
cell pa- rameters be counted or calculated improperly. While hemoglobin
value is not affected, RBC value decreases due to the degradation of red
blood cells, con- sequently HCT, MCV values will decrease and MCHC value
will be calculated falsely elevated. Regarding complete blood count, it is very
difficult to detect hemolysis in the preanalytical phase. In tests performed in
serum or plasma, the presence of hemolysis can be detected visually or by
analyzers and a flag can be formed.
An increase in platelet (PLT) values can be observed depending on hemol-
ysis. The reason of this issue can be that degraded red blood cells residues
are read as platelets . But hemolysis cannot be detected visually in a whole
blood sample. Therefore, while evaluating the patient results, MCHC values
can be warning.

· Icterus (Bilirubinemia)

·
High bilirubin value is an important interference reason. Bilirubin gives
ab- sorbance between 340-500 nm. For hemoglobin measurements are done
in close spectrums, high bilirubin levels can cause interference. Depending
on the high bilirubin levels, HGB can be read as falsely elevated; MCH values
cal- culated from hemoglobin will also be found to be high.

· Lipemia

Lipemia over 300 mg/dL can be visible. Elevated triglyceride levels show
the existence of chylomicron and VLDL of which the large part of the content
is triglyceride increase in blood circulation. While the diameter of VLDLs from
lipoproteins having particulate structure reaches 200 nm, chylomicron size
reaches 1000 nm (51). In complete blood count of which measurement
method is based on counting the particles, these lipoprotein particles can be
counted as platelet, even as red blood cell or white blood cell leading to
obtaining false- ly elevated results. Nevertheless, the impact of lipemia is not
limited to this. Lipemia affects the matrix of the sample, hence causes
unwanted light scat- tering in photometric methods. By the blur it creates, in
HGB measurement wavelength, it interferes with HGB causing to read falsely
elevated hemoglobin values (52). In addition, lipoprotein particles cause
analyte condensation in the sample by narrowing liquid compartment due to
liquid excluding effect, hence lead to falsely elevated results in all of the
analytes. Reflection of this fact on the blood count is again on hemoglobin
values.

· Cold Agglutinins
Cold agglutinin disorder is an autoimmune disorder which is caused by
antibodies generally in the type of IgM and sometimes IgA or IgG formed
against polysaccharide antigens on the surface of red blood cells. In cold ag-
glutinin disorder, antibodies that are activated by cold cause impairment in
the red blood cell membrane and red blood cells are auto-agglutinated, if the
case intensifies, it results in hemolysis. Its effect on complete blood cell count
is similar to hemolysis; while RBC and HCT decrease MCHC increases. If a
whole blood sample is kept waiting in 37°C water bath, agglutination clears
up and real values can be reached in measurements performed once more
again . If such a case is specified, it should be reported in patient results .

Visual comparison of a whole blood sample to control sample in cold

agglutinin disorder

Preanalytical error Parameter affect- Cause Recommendation

ed in blood count
No reading or de-
In the presence
creases in all
Clotted sample of clot residues, Sample rejection
values in the
cells cannot be
presence of
counted
micro-clots
Red blood cells
which are early
Hemolysis RBC , HCT Sample rejection
degraded cannot
be counted.
Photometric in- Decision accord-
Icterus HGB, MCH terference due ing to the
to turbidity patient’s clinical
status
Photometric in- Decision accord-
Lipemia HGB, MCH terference due ing to the
to turbidity patient’s clinical
status
In disorders such
Red blood cells as hemoglobin S Decision accord-
resistant to WBC, and C, red blood ing to the
degra- dation cells can be patient’s clinical
counted as white status
blood cell
Sample can be
RBC, Red blood cells
Cold agglutinins incubated in 37
MCV, ag- gregated
°C and retested
MCHC together
Aggregated Can be tested
plate- lets can be with a new
Platelet PLT, WBC errone- ously sample drawn
aggregation counted as white into another
blood cells (EDTA citrated,
interference). heparin- ized,
etc. tube

ISBN: 978-605-70111-0-7
This guideline is published with the unconditional scientific support of BD.