Good CBC Morphological AssessmentPDF

NAZAR AHMED MOHAMED ABD-ALLA
BSC - OMDURMAN AHLIA
HIGH DOPLOMA DGREE - ELZAEM EL-AZHARY
FORMER HEAD OF HEMATOLOGY & BLOOD BANK
MINISTRY OF HEALTH – LABORATORY ADMINISTRATION
KHARTOUM STATE
MARKETING MANAGER-LAB EQP –DIVISION
ALGAM COMPANY FOR DRUGS & CHEMICAL LTD
NAZAR AHMED SANGOOR 2008 1
Good CBC Morphological
Assessment

Learning Objective
*1- TO KNOW Red Blood Cell Analysis .
*2- TO KNOW THE IMPORTANCE OF INDICES
CALCULATIONS IN BLOOD TESTING.
*3- TO Know THE Classification Of Anemia.
*4- TO KNOW How To Check Leishman Stain.
*5- TO KNOW The differential white cell
count.

Learning Objective
*6- TO KNOW The Value of Good Morphological
Assessment.
*7- TO KNOW Microscopic Examination of Blood
Films.
*8- TO KNOW PROCEDURE FOR EXAMINATION OF
STAINED BLOOD SMEAR.
*9- TO KNOW Sources of error OF STAINED
BLOOD SMEAR.

Red Blood Cell Analysis
*Red Blood Cell Are Define :
* 1-Three Quantative Value:
1- Volume of packed red cell (VPRC) or
hematocrite.
2- Hb concentration .
3- The red cell concentration per unite of
volume.

Red Blood Cell Analysis
• *2- Three Qualative Characteristic Of The
Red Cell Population.
• 1- MCV (Mean Cell Volume)
• 2- MCH (Mean Cell Hemoglobin)
• 3-MCHC (Mean Cell Hemoglobin
Concentration)

CONTENUE
• *1- volume of the red cell hematocrite :
• *It is the proportion of the volume of blood sample
that is occupied by red blood cell.
• *determine manually by centrifugation of blood at
agiving speed and time in standardized glass tube
with uniform bore.
• *The height of the column of red cell compared with
that of the total column of blood following
centrifugation yields the hematocrite .

Source Of Inherent Error
• *Hematocrite measure the red cell
concentration not red cell mass there for:
• *1-Pateiont in shock or with volume
depletion may have normal or high
hematocrite measurement due to
hemoconcentration despite a decreased red
cell mass.

Source Of Inherent Error
• *2- Trapping of plasma in the red cell column
• *The plasma may trapped in:
• *Sickle cell –microcytic cell- macrocytic cell-
spherocyte cell at a high level because of
increase cellular rigidity.

Technical Error
• *1- poor sample mixing
• *2- Inappropriate anticoagulant
concentration
• *3-Insufficient centrifugation

THE IMPORTANCE OF INDICES CALCULATIONS
IN BLOOD TESTING
• *The RBC indices is a mathematical calculation
to yield three clinical parameters useful in
diagnosing anemia.
• *1- Mean Corpuscular Volume (MCV):
• average RBC volume is calculated by dividing
the hematocrit value by the RBC count value.
• *MCV = Hematocrit (L/L) x 1000
Red cell count 10/L)

* Normal values :are 76 to 96 femtoliters (fL).
*value is below 76 fL, then this indicates
microcytic anemia.
value greater than 96 would suggest
macrocytic anemia.
* falsely elevated:
*1-Agglutination of RBC as in cold agglutinin
disease .
*2- Sever hyperglassemia may cause somatic
swelling of RBC during analysis

• *2-Mean Corpuscular Hemoglobin (MCH). This
describes the hemoglobin content of each RBC .
• *obtained by dividing the hemoglobin value by
the RBC count value.
• *The normal value ranges :from 27 to 32
picograms (pg).
• *Values below 27 pg are suggestive of
hypochromic anemia.
• * falsely elevated by hyperlibidemia or
leukocytosis.

• *3-Mean Corpuscular Hemoglobin
Concentration (MCHC):
• *describing the hemoglobin concentration in
RBC’s.
• *MCHC := Hb (g/dl)/ hematocrit(L/L)
• *Normal values range : 31 to 36 g/dL.
• * Low value suggest hypochromic anemias.
• * MCHC affected by factor effect the PCV &Hb
g/dl
• *Note: Indices normal values may vary slightly
from region to region or lab to lab.

Red Cell Distribution Width
• *Reflects the range of red cell sizes measured
within a sample.
• *RDW has been proposed to be useful in early
classification of anemia
• *1- become abnormal earlier in nutritional
deficiency anemia than any of the other red cell
parameter especially in case of IDA .

Red Cell Distribution Width
• *2- useful in characterizing microcytic anemia
particularly distinguishing between IDA (high
RDW low MCV )and uncomplicated
heterozygous thalassemia (normal RDW low
MCV).
• *3-useful to identified the following (red cell
fragmentation –agglutination –dimorphic cell
populations).
• *4-Cold agglutinin disease increase MCV
&RDW volume .

ANEMIAS MAY BE CLASSIFIED BY RDW AND
MCV
 RDW = N MCV = N RDW = I MCV = N
Normocytic: acute hemorrhage sideroblastic
chronic liver disease early IDA
 RDW = N MCV = D RDW = I MCV = D
Microcytic: heterozygous thalassemia iron deficiency anemia
anemia of chronic disease homozygous thalassemia
 RDW = N MCV = I RDW = I MCV = I
Macrocytic: myelodysplastic syndromes B12 deficiency
aplastic anemia immune hemolytic with
marked reticulocytosis

ANEMIA
• *Anemia is simply a reduction in the number
of circulating RBC’s and/or hemoglobin
concentration and or PCV below the normal
range according to age & sex.
• *This means that the hemoglobin value will
drop below 13 g/dL in the adult male and
below 11 g/dL in the female.

Classification Of Anemia
• *Anemia’s may be classified in more than one
way.
• *1- morphologically in which there are three
general types of anemia:
*a. macrocytic
*b. microcytic
*c. normocytic
.

• *2- pathophysiological or etiological classification
*two general groups of anemias appear.
* A-Those anemias that are caused by a reduction
in the number of RBC’s due to a maturation defect.
*B-The other group of anemias are those due to
increased RBC destruction

• *Morphological Classification Of Anemia
• *Groups anemia by red blood cell size.
• *1- Macrocytic Normochromic Red Blood Cell
• *A- vitamin B12 deficiency. Folic acid deficiency:
• 1- pernicious anemia 2- sprue
• 3- following gastrectomy 4- dietary
• 5- abnormal intrinsic factor 6- tape worm
infection
• *B- disease of the liver:

• *2- Normocytic Normochromic Red Blood Cell:
• *A- defective formation of the blood cell or the
presence of tumor cell in the bone marrow
• 1- a plastic anemia . 2- leukemia .
• 3- Hodgkin's disease . 4- multiple myeloma.

• 5-leukoerythroblastosis. 6- metstatic cancer
• 7- anemia associated with renal and endocrine
disease.
• 8- anemia associated with inflammatory disease
(chronic disorder).

CONTENUE
• *B- Abnormal hemoglobin increased
destruction of the red blood cell
• 1-certain acquired hemolytic anemia.
• 2- paroxysmal nocturnal hemoglobinuria.
• 3- sickle cell anemia.
• 4- hemolytic disease of the new born.
• 5- anemia of chronic renal insufficiency.

*3-Microcytic Anemia
• A-iron deficiency anemia
• B-thalassemia
• C- sidroblastic anemia
• D- chronic blood loss

*Classification Of Anemia According To
Causes (kinetic )
• *Involves evaluating production, destruction and loss
• *1- decrease or impair production of red blood
cell:
• A- bone marrow damage, Infiltration, Atrophy.
• 1- leukemia . 2- leukoerythroblastosis.
• 3-aplastic anemia. 4- lymphoma .
• 5- multiple myLoma. 6- myelofibrosis.
• 7- pure red cell aplasia.

*B- Decrease Erythropoietin
• 1- inflammatory process.
• 2 – anemia of chronic disorder.
• 3- chronic renal disease .
• 4- hypothyroidism.
• *C- Vitamin And Mineral Deficiencies
• 1-iron deficiency anemia.
• 2- vitamin B12 and folic acid deficiency.
• *D- Defect In Globin Synthesis
• 1- ά & β thalassemia. 2- μ thalassemia trait.

• * E- iron over load
1- sidroblastic anemia.
2-hemochromatosis.
*F- ineffective erythropoiesis :
• 1- congenital dyserythropoietic anemia.
• 2- increased RBC destruction.

*2-Destruction And Loss
1-Intrinsic Defect With In RBC:
• 1- Hereditary Membrane Defect:
• A- spherocytosis
• B- Elleptocytosis
• C- Abetalipoproteinemia
• D- stomatocytosis
• E- Rh null disease

*1-Intrinsic Defect With In RBC:
• *2- Hereditary Enzyme Defect:
• A- Glucose -6- phosphate dehydrogenase.
• B- Pyruvate Kinas.
• *3- Hereditary Hemoglobin Defect
(Hemoglobinopathy):
• 1- Sickled cell (disease & trait ) .
• 2- HbC.
• 3- HbE .
• 4- HbD.

• *4- Hereditary Defective Globin Synthesis
• A- α or β Thalassemia (Major or minor).
• B- Sickle – β thalassemia.
• C- ε β thalassemia.
• D- HbH Disease.

*2- Extra Corpuscular Causes:
• * A-Non Acquired Hemolytic Anemia
• 1- chemical, toxin, venom .
• 2- physical trauma disorder causing fragmentation
• a- Burns. b- Cardiac Replacement Valve.
• c- Microangiopathic Hemolytic Anemia.
• d- Hemolytic Uremic Syndrome.
• 3- Infection ( parasitic).

*2-Extra Corpuscular Causes: (immune
hemolytic anemia)
*B- Acquired Hemolytic Anemia
• 1- Isoimmune Anemia.
• a- Incompatible Blood Transfusion
• b- HDN
• 2- Autoimmune Antibodies.
• a- warm
• b- cold (CHAD)
• 3- Drugs.

• 4-Miscellaneous.
1- Anemia Of Liver Disease
• 2- Sulfhemoglobinemia
• 3- Porphyries
• 4- Methemoglobinemia
• 5- Acute Blood Loss (e.g. car accident)
• 6-Proximal Nocturnal Hemoglobinurea

*How To Check Leishman Stain
• *Check the nuclose of wbc & the cytoplasm
• *Check if there is eosinophil to see the pinkish
color of granules.
• *Check the reddish brown color of RBC.
• *To obtain good staining film avoid the air
which well evaporate the glycerol.
• *Buffer the stain by use buffered DW
• PH 7.2-7.4.

Staining characteristics of a correctly stained
normal film
PurpleNuclei
Cytoplasm
Deep pinkErythrocytes
Grey-blueReticulocytes
Orange-pinkNeutrophils
Blue ; some small
lymphocytes deep blue
Lymphocytes
Grey-blueMonocytes
NAZAR AHMED SANGOOR 2008
36

*Staining characteristics of a correctly
stained normal film
BlueBasophils
Granules
Fine purpleNeutrophils
Red-orangeEosinophils
Purple-blackBasophils
Fine reddish (azurophil)Monocytes
PurplePlatelets

Staining faults
Smear too thickToo blue
Inadequate time in buffer
Buffer pH too high
Staining time too long
Diluted stain overused,
requires replenishment
Stock MGG solution
incorrectly made or made
from impure dye
Stock solution left
exposed to bright daylight

Staining faults
Excessive time in bufferToo pink
Buffer pH too low
Stock MGG solution
incorrectly made or made
from impure dye
Cover slip mounted before
film is completely dry
Staining time too shortToo faint
Excessive washing after
staining

Staining faults
Stain solution left in
uncovered jar or tray
Stain deposit
Stain solution not filtered
Dirty slides
Inadequate fixationBlue background
Prolonged storage before
fixation
Blood
anticoagulated with
heparin

*Making a blood film
*A blood film may be made from non-
anticoagulated (native) blood, obtained either
from a vein or capillary.
* from EDTA-anticoagulated blood Chelation of
calcium by EDTA hinders platelets aggregation so
that platelets are evenly spread and their numbers
can be assessed more easily.

*Films prepared from capillary blood usually show
prominent platelet aggregation .
*films from native venous blood often show small
Aggregates .
*Films prepared from native venous or capillary
blood are free of artifacts due to storage or the
effects of the anticoagulant.

*Good laboratory practice includes:
*recording the date and time the specimen is
received in the laboratory.
*making a film shortly after receipt of the specimen.
*In this way the length of any delay in transit is
known and attribution of morphological changes to
prolonged storage of EDTA-anticoagulated blood.

*A drop of blood (either native or
anticoagulated) is placed near one end of the
slide.
*Anticoagulated blood from screw-top
containers can be applied to the slide using a
capillary tube, which is then discarded.

*A drop of blood from specimen containers with
penetrable lids can be applied to the slide by means
of a special device that perforates the lid.
*The spreader is applied at an angle of 25–30°, in
front of the drop of blood, and is drawn back into it.
*Once the blood has run along its back edge, the
spreader is advanced with a smooth steady motion
so that a thin film of blood is spread over the slide.

*If the angle of the spreader is too obtuse or the
speed of spreading is too fast, the film
will be too short.
*An experienced operator learns to recognize
blood with a higher than normal haematocrit
(Hct), which is more viscous and requires a more
acute angle to make a satisfactory film and,
conversely, blood with a lower than normal Hct,
which requires a more obtuse angle.

*The differential white cell count
*A differential white cell count is the assigning of
leucocytes to their individual categories, this
categorization being expressed as a percentage or,
when the WBC is available, as an absolute count.
*The ICSH recommends that the differential
leucocyte count be expressed in absolute
numbers.

*Cells that are normally present in the peripheral
blood can be assigned to five or six categories,
depending on whether non-segmented or band
forms of neutrophils are separated from segmented
neutrophils or are counted with them.
*The differential count also includes any abnormal
cells that may be present.
*NRBC can be included as separate category in the
differential count or, alternatively,their number can
be expressed per 100 white cells.

The Value of Good Morphological
Assessment
*1. Many blood diseases can confidently be
diagnosed, or at least strongly suspected, on the
morphology.
*This can save a great deal of time, money, and even
discomfort for the patient .
*2. There are some diseases where the FBC can be
completely normal and only the morphology will
show abnormalities, such as lead poisoning.

*3. It has been pointed out that the absolute amount
of plasma water affects the counts and many of the
measurements in the FBC, which may produce
spuriously abnormal results.
* Plasma water does not affect the morphology, and
this may be of assistance in such cases.
*4. The presence in a report of an obviously careful
morphological assessment indicates that the FBC as a
whole was looked at by a professional.

*Blood films is essential in cases of
1. Leukopenia. 2. Leukocytosis.
3. Thrombocytosis. 4. Thrombocytopenia.
5. Anemia of unknown etiology.
6. A moderately to severely raised RDW even in an
otherwise normal count.
7. A moderate to severe left shift with a normal WCC.
8. The presence of nucleated RBCs.

*Microscopic Examination of Blood Films
 *1-Survey the film at x10 magnification to get a general
impression of its quality.
 *2- Find an area where the red cells are evenly
distributed, just touching but not overlapping, and
study their morphology at x 40.
 *3-Scan the film to get an impression whether the
leucocytes are increased or decreased.

*Microscopic Examination of Blood Films
*4- Identify any unusual or abnormal
cells.
*5-Estimate the relative proportion of
platelets and note the presence of
abnormally large platelets.
*6-Use the x100 lens for studying the
fine details of the cell morphology.

*PROCEDURE FOR EXAMINATION OF STAINED BLOOD
SMEAR
 **1- Check the blood smear to ensure that it is well
made .
 *The tail of the film should be smooth
 **2- Examine the blood smear using the low power
(10x) objective.
 *A- the red cell and white blood cell and platelets
must be correctly stained

The Smear

PROCEDURE FOR EXAMINATION OF STAINED
BLOOD SMEAR
• *B- check that there is even distribution of the
WBC on the smear.
• *C- Estimate the WBC count by noting the number
of WBC / field and the number of WBC in relation
to the RBC count it should agree with the test
result obtained

CONTENUE
• *D- when scanning the blood smear it is
important to note any thing unusual or irregular
such as large abnormal looking cell or rouleaux
formation of the RBC.
• *E- choose the area of the blood smear where
the differential count is to begin place a drop of
oil on the slid and carefully change the oil
immersion to objective (100x ).
• **3- perform the differential cell count and at the
same time examine the morphology of the WBC.

* type of examination can be choose
*1- using the cross – sectional or crenellation
technique .
*The wbc are counted in consecutive fields as
the blood film moved from side to side .
*Counting should begin in the thin area of the
smear where the red blood cell are slightly
over lapping and procedure into the thicker
area.

*2-longitudinal method
• *The wbc are counted in consecutive field from tail
toward the head of the smear.
• *This is the ideal method if the smear is thin enough
so that the wbc may be identified all the way to the
beginning of the smear.

*3- The battlement field
 *uses a pattern of consecutive field beginning near
the tail on a horizontal edge count three
consecutive horizontal edge field ( moving away
from the tail count two fields toward the center of
the smear count this fields horizontal moving away
from the tail count two fields vertically to the edge.

Recommended Method For Differential

CONTENUE
• **4- Identify each wbc seen and record on
differential cell counted until 100 wbc have been
counted.
• *If nucleated red cell have been counted enumerate
them on a separate counter these cell are not to be
included in the 100 cell differential count but are
reported as # of NRBC /100 WBC

CONTENUE
• *If any megakaryocytic cell or fragments
smudge cell or epithelial cell are seen these
cell should also be enumerated in the same
manner as the NRBC and reported as the
• # /100 wbc

CONTENUE
 *2- Examine the RBC morphology in a thin area
of the slide where only a few of the RBC slight
over lap not any variation from normal and
classify these irregularities as slight, moderate,
marked ,or sever

ERYTHROCYTE MORPHOLOGY EVALUATION
CRITERIA
 Morphology Normal SL MO MAR SEV
Characteristics Limits
Acanthocyte none 1-5 6-10 11-20 >20
Anisocytosis 0-2 3-10 11-20 21-50 >50
Basophilic Stippling 0-1 1-5 6-10 11-20 >20
Burr 0-2 3-10 11-20 21-50 >50
Elliptocytes 0-2 3-10 11-20 21-50 >50
Helmet (Bite) none 1-5 6-10 11-20 >20
Howell-Jolly none 1-2 3-5 6-10 >10
Hypochromia 0-2 3-10 11-20 21-50 >50
Macrocytes 0-5 6-10 11-20 21-50 >50
Microcytes 0-5 6-10 11-20 21-50 >50
Pappenheimer bodies None 1-2 3-5 6-10 >10

ERYTHROCYTE MORPHOLOGY EVALUATION
CRITERIA
 Morphology Normal SL MO MAR SEV
Characteristics Limits
 Poikilocytosis 0-2 3-10 11-20 21-50 >50
Polychromatophilia (adult) 0-1 2-5 6-10 11-20 >50
Polychromatophilia (infant) 1-6 7-15 16-20 21-50 >50
Schistocytes None 1-5 6-10 11-20 >20
Sickle Cells None (if present in any number, report as positive)
Spherocytes 0-2 3-10 11-20 21-50 >50
Stomatocytes 0-2 3-10 11-20 21-50 >50
Target Cells (Codocytes) 0-2 3-10 11-20 21-50 >50
Tear Drop Cells 0-2 2-5 6-10 11-20 >20
Rouleaux Formation
Aggregates of 3 to 4 RBC’s = 1+
Aggregates of 5 to 10 RBC’s = 2+
Aggregates of >10 RBC’s = 3+ (only a few free RBC’s noted)

CONTENUE
 *3- Examine the platelets on the smear for
morphology and number present.
 * Determine the approximate number of platelets
per field a normal blood smear (normal RBC &
normal platelets count )should show approximately
8-20 platelets per field .
 One method for reporting platelets estimates is to
determine the average number of platelets per
field 5 to 10 different field and multiply this result
by 20.000 .

Platelet Estimation Report Platelet
Estimation
• 0-49.000 /ml (marked decrease)
• 50.000-99.000/ml (moderate decrease)
• 100.000-149.000/ml (slight decrease)
• 150.000-199.000/ml (low normal )
• 200.000-400.000/ml (normal )
• 401.000-599.000 /ml (slight increase)
• 600.000-800.000/ml (moderate increase)
• Above 800.000/ml (marked increase )

DISCUSSION
 *1- when reporting manual differential Wbcc the result are
reported as a percentage of each cell type present.
 *The most accurate and also the most preferable method of
reporting is the absolute count because the result expressed
in percent is relative number and can be misleading to the
clinician.
 *The absolute count is calculated as:
 Absolute# of cell/L=% of cell type in deferential X Wbc/L

• *2- when counting a deferential the
following outline may be followed
• *A- WBC :
• *- check for even distribution and estimate the
number present also look for any gross
abnormalities present on the smear
• *-perform the differential count
• *-examine for morphology abnormality

*Relative And Absolute Normal Adult Values
 Normal Range: WBC Count = 5000 µL to 10,000/µL
 Relative Values Absolute Values Bands = 0 to 6% Bands = 0 to
600/µL
 Neutrophils = 55 to 68% Neutrophils = 2,000 to 6800/µL
Lymphocytes = 20 to 40% Lymphocytes = 1000 to 4000/µ
 Monocytes = 2 to 8% Monocytes = 200 to 800/µL
Eosinophils = 0 to 3% Eosinophils = 0 to 300/µL
Basophils = 0 to 1% Basophils = 0 to 100/µL

CONTENUE
• *B- RBC : Examine for
• -size and shape
• -Relative Hb content
• -polychromatophilia & NRBC
• -inclusion
• -rouleaux formation and agglutination
• -parasite.

• *C- Platelets
• - Estimate number present
• -Examine for morphologic abnormalities
• 3-When studding stain smear do not examine
in the thick area always look for the cell where
it well distributed

 *4- when the white count below 1.000 it may be
difficult to find many wbc on stained smear so
differential may be perform by counting 50 wbc
annotation on the report must be made that only
50 wbc were counted (alternatively Buffy coat
smear may be perform

*5- when the differential show an abnormal
distribution of cell type as
a- over 10%eosinophils b- over 2%
basophile
• c- over 11%monocytes d- more lymphocyte
than neutrophil (except for children)
• *A 200 cell differential may performed then
the result are average (divided by 2)and
notation is giving that 200 cell where counted

• *6- before reporting the platelets is decrease
• *Scan the film for clump on low power especially
the feathered edge also recheck the tube for clot
• *7- if the differential count show the present of
immature granulocyte cell this is termed a shift to
left (leukemia, bacterial infection)
• A shift to right refer to an increase number of
hyper segmented neutrophils.

*Sources of error
• *Irregular spread with ridges and long tail:
Edge of spreader dirty or chipped; dusty slide
• *Holes in film: Slide contaminated with fat or
grease
• *Irregular leucocyte and platelet distribution,
especially in tail: poor film-making technique
• *Film too short and too thick: spreader held at
incorrect angle

*Sources of error
• *Film extending to end of slide: blood drop
too large
• *Short thin film: blood drop too small
• *Film extends to edge of slide: spreader too
wide or not positioned correctly
• *Cellular degenerative changes: delay in
fixing, inadequate fixing time or methanol
contaminated with water

ANY QUESTION ?

Learning Outcome
*1- KNOWING Red Blood Cell Analysis .
*2- KNOWING THE IMPORTANCE OF INDICES
CALCULATIONS IN BLOOD TESTING.
*3- KNOWING THE Classification Of Anemia.
*4- KNOWING How To Check Leishman Stain.
*5- KNOWING The differential white cell
count.

Learning Outcome
*6- KNOWING The Value of Good
Morphological Assessment.
*7- KNOWING Microscopic Examination of
Blood Films.
*8- KNOWING PROCEDURE FOR
EXAMINATION OF STAINED BLOOD SMEAR.
*9- KNOWING Sources of error OF STAINED
BLOOD SMEAR.

Summary
*1- CBC morphological assessment is very
important diagnostic tool need to be done
professionally by expert person who
followed good laboratory practices and get
enough knowledge to examine the smear.

Summary
*2- person who examine the smear
should have background knowledge
about normal and abnormal blood cell
morphology.
*3- interpretation of result obtain is very
important.

THANK YOU FOR GOOD ATTENTION

Good CBC Morphological AssessmentPDF

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