HEMATOLOGY | WEEK 1 | VIRTUAL INTERNSHIP

st nd rd
Sysmex Processing – 1 : CBC analyzer, 2 : Slide making, 3 : microscopy
CBC PARAMETERS
INTRODUCTION TO THE CBC
• Blood = plasma (albumin, clotting factors) + cells
o Venous Whole blood: used for complete blood count testing ▪ Oxygen carrying cells
RBC
• CBC = complete blood count, quantifies 3 types of blood cells ▪ Anucleate (unless immature)
o Red blood cells (RBCs)
o White blood cells (WBCs) – (NLMEB)
o Platelets WBC ▪ Immune function
• Gives additional info, e.g.,
o % of blood composed of RBCs – “hematocrit”
o Different types of WBCs present – “differential” ▪ Clotting
o Volume or the amount of hemoglobin (important for PLATELETS
▪ Anucleate cell “fragments”
transportation of oxygen in the body)

COLLECTION MINIMIZING ERROR

▪ 3 to 10 ml of whole blood drawn into tube containing anticoagulant
▪ All laboratory processes are subject to error
o EDTA – Chelates Ca+ ion
▪ Examples of sources of error in the CBC will be discussed under each cell type
o Heparin
▪ Automated analyzer computers have multiple programs to detect possible
o Citrate error
▪ Most common: PURPLE-TOP TUBE with EDTA to chelate Ca2+
o Tell what the problem is
CELL COUNT ANALYSIS ▪ If data meets possible error criteria, data is “flagged” for operator review
▪ Sample dilution and even distribution of cells essential to accurate counts: prior to release
Dilution, and distribution of all cell types o To validate the reading of analyzer, perform Peripheral blood smear
▪ Different solution for different cells and check in microscope
o RBC counts need isotonic solution o Check for blood clots
o WBC and platelet count use RBC-lysis solution (Acetic Acid, Hayem, o Make sure you do quality control
Gower solution) – remove RBC for WBC counting
o Acid hematin depends on visual
o Manual reagents are used as backups (hemoglobin blocks, acid RBC PARAMETERS
hematin) • QUANTITATIVE
▪ Performed by manual counts or automated analyzers o Hemoglobin (Hgb, g/dL)
o Hematology analyzer – the machine will get sample 0.05ml-0.10ml o Hematocrit (Hct, %)
o Methodology for using hematocrit analyzer – Mathematically o RBC count (per μL)
derivation • QUALITATIVE (averages) – read through mathematical derivation
MANUAL COUNTS o Mean corpuscular volume (MCV, fL)
▪ Rarely used for absolute count o Mean corpuscular hemoglobin (MCH, pg)
▪ May use hemocytometer for platelet or low WBC counts o Mean corpuscular hemoglobin concentration (MCHC, g/dL)
o Counting chamber with specific volume o Red cell distribution width (RDW, %)
o Viewed with microscope • Sometimes – reticulocyte count
▪ Peripheral blood smear can be used for estimation for platelets and WBC
AUTOMATED ANALYZERS Hemoglobin ▪ Colored protein, measured by absorbance at 540nm
▪ Increased accuracy and speed over manual count (Hgb, g/dL)
▪ Based on electrical impedance, light scattering, radiofrequency conductivity, ▪ Proportion of volume occupied by RBCs
Hematocrit
or cytochemical reactions ▪ Manual – height of column after centrifugation
(Hct, %)
▪ Electrical impedance ▪ Automated – RBC number/RBC volume
o Change of voltage when cells pass through aperture disrupting a flow ▪ Obtained via electrical impedance and/or light scatter
RBC count
current ▪ RBCs and WBCs counted together by analyzer
(cells per μL)
o Don’t need to enter patient name they have their ▪ RBC outnumber WBC ~500:1, negligible error
own barcode
o Beckman Coulter uses electrical impedance
o Electrical impedance came from the principle of
Beckman coulter Manual hematocrit tube
o Forward scatter
o Cytochemical reactions for identification of the cell RBC INDICES FORMULA
o The aperture will conduct MCV Mean Cell Volume
signal/wave which will then be 𝑯𝑪𝑻(%) × 𝟏𝟎
80- Average volume of red cell; indicator of the
measured 𝑹𝑩𝑪 𝒄𝒐𝒖𝒏𝒕 (× 𝟏𝟎𝟏𝟐 /𝑳)
100fL red cell size
▪ Flow cytometry and light scatter MCH Mean Cell Hemoglobin
o Capable of reading blast, 28- Average hemoglobin content per red 𝑯𝑮𝑩(𝒈/𝒅𝑳) × 𝟏𝟎
immature cells 32pg blood cell – individual (or in an average 𝑹𝑩𝑪 𝒄𝒐𝒖𝒏𝒕 (× 𝟏𝟎𝟏𝟐/𝑳)
▪ Laser hits single stream of cells, light RBC)
scatter interpreted into info on size, MCHC Mean Cell Hemoglobin Concentration
structure, granularity 32- Average concentration of hemoglobin in a 𝑯𝑮𝑩(𝒈/𝒅𝑳) × 𝟏𝟎𝟎
o Cell passes through the tube 36% given volume of blood – indicator of 𝑯𝑪𝑻 (%)
and then the single stream will pallor/color of RBC
lead cells into the laser beam These three are calculated averages – may not accurately describe mixed
forming scattered light population of cells

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Red Cell Distribution ▪ Range of RBC sizes WBC DIFFERENTIAL
Width (RDW, %) ▪ Assess the distribution of RBC (Anisocytosis)
• Automated
Reticulocytes ▪ Immature, anucleated cells containing RNA
o Individual cells analyzed by flow cytometry
▪ Reflect bone marrow’s ability to make new RBCs
o Light scatter
▪ Manual – “supravital” stain of ppt RNA inside the
▪ Forward (cell size)
cell membrane ▪ Side (complexity, granularity)
▪ Automated – fluorescent dye stains RNA o Cells identified based on expected profile

RBC PARAMETERS – CLINICAL SCENARIOS

Anemia Decreased Hgb, Hct, or RBC count E.g., neutrophils – larger than lymphocytes with
• 1° - iron deficiency anemia granular complexity, monocytes (First in X axis
• 2° - acute blood loss (normal mcv, mchc) because of larger number) – fewer granules than
Polycythemia Increased Hgb, Hct, or RBC count neutrophils and therefore, less SSC (At top of Y axis
• 1° – bone marrow proliferative disease because of big size)
• 2° – compensation due to chronically low oxygen
from smoking, sleep apnea, high altitude
Due to oxygen issue in loading and unloading (problem in • Abnormal cells that do not identify as WBCs are
lungs, heart, and kidneys) tagged for manual review
o E.g., atypical lymphocytes, immature blasts
RBC PARAMETERS – EXAMPLES OF ERROR • Manual review
PARAMETER FALSELY INCREASED FALSELY DECREASED o Drop of blood smeared on glass slide
Total RBC count High WBC count Hemolysis (in vitro) clotting o Dyes
Hct Giant platelets Hemolysis (in vitro), clotting ▪ Basic (nuclei, basophilic)
MCV Cell clumping Giant platelets ▪ Acidic (eosinophilic)

If abnormality is seen perform Peripheral blood smear and to validate results

from automated analyzers

WBC PARAMETERS – CLINICAL SCENARIOS

GIANT PLATELETS Hemolyzed sample Non-hemolyzed sample
WBC PARAMETERS
• Quantitative cell count (cells per μL)
• Differential (cells per μL and % of total WBCs)
How to compute absolute count: WBC – 7.5
➢ Neutrophil – 69% = 0.69 x 7.5 = 5.175
➢ Lymphocyte – 15% = 0.15x 7.5 = 1.125
➢ Monocyte – 10% = 0.10 x 7.5 = 0.750
➢ Eosinophil – 5% = 0.05 x 7.5 = 0.375
➢ Basophil – 1% = 0.01 x 7.5 = 0.075
Total = 7.5
QUANTITATIVE CELL COUNT (CELLS PER UL)
▪ Dilution of blood in RBC lysis buffer, usually acid or detergent (lyse RBC)
▪ Total count of nucleated cells obtained by electrical impedance or flow
cytometry (automated) or hemocytometer (manual)
DIFFERENTIAL (CELLS PER UL AND % OF TOTAL WBCS)
Leukopenia Decreased total WBC count
▪ 1° - HIV, bone marrow disease, e.g., aplastic anemia
▪ 2° - immunosuppressants
Leukocytosis Increased total WBC count
▪ 1° - acute leukemia
▪ 2° - neutrophilia and lymphocytosis from infection (Due
to infection, bacterial or viral infection)
WBC PARAMETERS – EXAMPLES OF ERROR
▪ Total WBC count – falsely increased
o Nucleated RBCs (counted as WBCs)
o Antibodies that cause RBC clumping
o To correct WBC, check through microscope. >5 nRBC, you need to
correct. <5nRBC declare the result of <5nRBC
▪ Differential
o Automated –Abnormal cells incorrectly identified
o Manual – Poor staining leading to cell recognition errors and larger
cells pushed to edge, missed in count (Read in thicker part)
PLATELET PARAMETERS
• Platelet count
• Obtained by electrical impedance or light scatter (automated) or
hemocytometer (manual)

PLT PARAMETERS – CLINICAL SCENARIOS

Immature neutrophil
“Band form” – can be
seen in circulation
Thrombocytopenia Decreased platelet count
▪ 1° - decreased bone marrow production
▪ 2° - immune-mediated destruction/sequestration
(Idiopathic thrombocytopenia)
Thrombocytosis Increased platelet count
Neutrophil Basophil ▪ 1° - proliferative bone marrow disease (>750
platelet count)
▪ 2° - acute phase reactant, e.g., inflammation

PLATELET PARAMETERS – EXAMPLES OF ERROR

Parameter FALSELY INCREASED FALSELY DECREASED
Platelet count Hemolysis (in vitro), Giant platelets,
microcytic (small) red cells platelet clumping

Eosinophil Lymphocyte Monocyte

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 MCH Impedance Fluorescence Impedance Light scatter
Flow Cytometry
MCHC (HGB/HCT)x100 (HGB/HCT)x100 (HGB/HCT)x100 (HGB/HCT)x100
Reticulocyte Supravital Fluorescence Supravital Fluorescence
staining light Flow Cytometry staining light Flow Cytometry
scatter scatter
Platelet Light Scatter Hydrodynamically Light Scatter Light scatter
impedance in focused impedance in
common impedance common
Microcytes Giant platelets
VENOUS BLOOD COLLECTION FOR COMPLETE BLOOD COUNT
Summary:
• CBC is an important screening and diagnostic tool
• Majority of specimens run on automated analyzers which PREPARING THE PATIENT
• use impedance and flow cytometry for cell identification and Posture Switching to sitting position or upright position from prone
enumeration position causes fluid flow from the vessels into the interstitial
• Manual review and count reserved for abnormal specimens or patients space. It is known that, as a result of this fact, molecules which
with clinical history cannot diffuse into the tissue due to their dimensions such as
• Knowledge of error sources essential to accurate interpretation (Need blood cells, proteins, cholesterol and iron are measured high. It is
to use flow cytometry) 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
CBC – ACTIVITY 1 – ADDITIONAL NOTES Exercise Exercise can affect analytes such as creatinine, creatinine kinase,
myoglobin, aspartate aminotransferase as well as the results of
HEMOGLOBIN complete blood count It is reported that WBC, neutrophil and
The functional protein which is found most frequently in red blood cells is lymphocyte values increase significantly and high neutrophil count
hemoglobin. The most crucial function of hemoglobin is to mediate gas exchange lasts more than 2 hours after heavy exercise.
between lungs and tissues as well as some other functions such as maintaining
Circadian It has been known that circadian rhythm has an impact on blood
acid-base balance, transportation of nitric oxide (NO). More than 90% of the
Rhythm count. Although RBC, HGB and HTC display a little increase at 11:00
cytoplasm of a red blood cell is filled with hemoglobin. in the morning, an increase in leukocytes can be observed in the
evening between 21:00-24:00. It is reported that while
WHITE BLOOD CELLS (WBC, LEUKOCYTES) lymphocyte, eosinophil and basophil counts reach their highest
Neutrophils Neutrophiles which are an important component of hereditary value, they decrease in morning hours. This change is inversely
immunity, have crucial functions in defense against microbial related to the cortisol level. It is reported that PLT count increases
infections. 50-70% of white blood cells in blood circulation are in the evening and decreases in the morning. For standardization
neutrophiles and their count is 1.7-7.5 103/µL' on average in results, it is appropriate to perform blood sampling in the
Eosinophiles They make just 1-3% of white blood cells. The structures that morning.
stain are granules; they are full of enzymes such as lipase, Stress Anxiety and particularly crying of children during blood collection
DNAse, plasminogen. Their particular function is to defend the can cause increase in leukocyte count
body against parasitic infections Diet Blood should be drawn for sampling after at least 8-12 hours
Basophils Just 0.5-1% of white blood cells is basophile in blood fasting period. Glucose and lipid content of the blood increase in
circulation; the diameter of a basophile is 12 µm in average. individuals who eat within 2 hours or less before drawing the
Molecules like histamine and serotonin make the content of blood sample. It can affect MCV, MCHC, HCT results in high
the granules. Particularly in allergic conditions, the count concentration. Excessively high lipid content can lead to
increases interference in tests where photometric measurement is
Lymphocytes Lymphocytes make about 20-40% of white blood cells; its performed such as hemoglobin. In addition, it has been reported
blood count is 1.0-3.2 103/µL. They are divided into sub-typed that when blood glucose level increases over 500 mg/dL, MCV
such as T cells, B cells, natural killer cells (NK). These cells value is affected due to the osmotic impact
functioning both in hereditary immunity and acquired Smoking Before blood sampling, smoking can cause an increase in
immunity mediate almost all defense incidents of the immune leukocyte count. The long duration of smoking causes increases in
system. hemoglobin level
Monocytes Monocytes are the precursors of the tissue macrophages in
blood. They make 2-10% of white blood cells. They are the Recommendations:
largest cells in blood circulation. Under the light microscope, • 8-12 hours fasting before blood sampling is recommended.
their horseshoe-like nuclei are observed • Heavy physical exercise should be avoided within 24 hours prior to
blood sampling.
THROMBOCYTES (PLATELET (PLT), BLOOD-PLATELETS) • It is recommended not to smoke at least 2 hours before blood sampling
There is no nucleus in thrombocytes derived from megakaryocytes. The function for blood count.
of thrombocytes which are 2-3 µm in diameter is to plug holes by clotting where CAP COLOR TUBE/ADDITIVE MIXING
endothelial integrity is interrupted. Parameter Blood culture/Medium In order to make medium
and blood mix, it is inverted
Parameter Beckman Coulter Sysmex XN Series Abbott CELL-DYN Siemens ADVIA slightly
UniCel DxH 800 Sapphire 2120i
WBC Impedance Fluorescence Light scatter Light scatter
Coagulation tube / Citrated 3-4 times
Flow Cytometry ESR tube / Citrated 3-4 times
RBC Impedance Hydrodynamically Impedance Laser light Serum tube / Without gel 5 times
focused scatter Serum tube / With gel 5 times
impedance
Hemoglobin Cyanohemoglobin Cyanide- free Cyanohemoglobin Cyanohemoglobin Serum tube / Tube with 5 times
525 nm Sodium lauryl 540 nm 546 nm thrombin clot activator
sulfate Plasma tube / Heparin tube 8-10 times
555nm
with or without gel
Hematocrit (RBCxMCV)/10 Total RBC peak (RBCxMCV)/10 RBCxMCV)/10
magnitude Plasma tube / EDTA tube 8-10 times
MCV Average Hct/RBC)/10 Average (Hct/RBC)/10 with or without gel
obtained from obtained from Plasma tube / Fluoride / 8-10 times
RBC diameter RBC diameter
Potassium Oxalate: Fluoride
distribution distribution
histogram histogram / EDTA Fluoride / Heparin
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 SAMPLE TRANSPORTATION COLD AGGLUTININS
Samples should be held on the tube racks straightly; swinging and shaking Autoimmune disorder which is caused by antibodies generally in the type of
should be avoided as possible as it can be. Specimens should be transported in IgM and sometimes IgA or IgG formed against polysaccharide antigens on the
nested special primary (sample tube), secondary (in a structure that can surface of red blood cells. In cold agglutinin disorder, antibodies that are
prevent infectious contamination even if the cap is opened and containing activated by cold cause impairment in the red blood cell membrane and red
sorbent material) and tertiary (can be used as transportation bag, protected blood cells are auto-agglutinated, if the case intensifies, it results in hemolysis.
against temperature changes) containers so as to control heat. Its effect on complete blood cell count is similar to hemolysis; while RBC and
SAMPLE STORAGE HCT decrease MCHC increases.
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 FREQUENTLY SEEN PREANALYTICAL ERRORS, POTENTIAL CAUSES AND
after blood drawing. If samples cannot be tested within 6 hours, they can be SOLUTION RECOMMENDATIONS
kept in the refrigerator at +4ºC for 24 hours. In samples kept in the refrigerator, Preanalytical Parameter Cause Recommendation
it is reported that MCV, HCT, WBC values are more stable error affected in
HEMOLYSIS blood count
Hemolysis is the degradation of red blood cells in vitro or in vivo. However, Clotted No reading or In the presence Sample rejection
most of the hemolysis events occur due to the mechanical impairment of the sample decreases in of clot residues,
the integrity of red blood cells during sample collection, transportation, all values in cells cannot be
processing or storage. the presence counted
of micro-clots
Hemolysis RBC ↓, Red blood cells Sample rejection
HCT↓ which are early
degraded cannot
Potential hemolysis causes:
be counted.
• Difficult blood drawing,
Icterus HGB↑, Photometric Decision according to
• Blood drawn using a syringe,
MCH↑ interference due the patient’s clinical
• Prolonged tourniquet application, to turbidity status
• Small needle size (larger than 23 G), 36 Lipemia HGB↑, Photometric Decision according to
• Hematoma formation, MCH↑ interference due the patient’s clinical
• Sample contaminated by ethanol or water, to turbidity status
• Shaking the tube after blood draw or during transportation, Red blood WBC↑, In disorders such Decision according to
• Blood transfer from tube to tube, cells resistant as hemoglobin S the patient’s clinical
• Insufficient blood draws into the vacuum tube. to and C, red blood status
An increase in platelet (PLT) values can be observed depending on hemolysis. degradation cells can be
The reason of this issue can be that degraded red blood cells residues are read counted as white
as platelets (48). But hemolysis cannot be detected visually in a whole blood blood cell
sample. Therefore, while evaluating the patient results, MCHC values can be Cold RBC↓, Red blood cells Sample can be
warning. agglutinins MCV↑, aggregated incubated in 37 °C
ICTERUS (BILIRUBINEMIA) MCHC↑ together and retested
High bilirubin value is an important interference reason. Bilirubin gives Platelet PLT↓, Aggregated Can be tested with a
absorbance between 340-500 nm. For hemoglobin measurements are done in aggregation WBC↑ platelets can be new sample drawn
close spectrums, high bilirubin levels can cause interference. Depending on the erroneously into another citrated,
high bilirubin levels, HGB can be read as falsely elevated; MCH values calculated counted as white heparinized, etc.
from hemoglobin will also be found to be high blood cells (EDTA tube
interference).

LIPEMIA
Elevated triglyceride levels show the existence of chylomicron and VLDL of
which the large part of the content is triglyceride increase in blood circulation.
. 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 falsely elevated results.

The absorption spectrum of lipid, bilirubin and hemoglobin

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