The majic of hema analyzer new hematological parameter

NAZAR AHMED MAHAMED ABD-ALLA
(SANGOOR) SEP 2014
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THE MAJIC OF HEMA-ANALYZER
NEW HEMATOLOGICAL PARAMETER
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
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Learning Objective
1- Introduction to hematology analyzer.
2- To know the general principle of
hematology Analyzers.
3- To known the new parameter
measure by hematology analyzer
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Introduction
*Hematology analyzer procedure are
usually depend on the following general
principle witch separate the type of
analyzer to:
*1- three part differential.
*2- five part differential.
*3- seven part differential .
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General principle
The general principle is:
*1- The RBC’s and PLT’s are measured by an electronic
impedance variation principle. This means that an
electronic field is generated around the micro aperture
within the chamber in which the blood cells are pulled
through.
*2- the hemoglobin: The hemoglobin freed by the lysis of the
red blood cells combines with potassium cyanide to form a
cyanmethemoglobin compound. Absorbance is then
measured by spectrophotometry, at a wave length of 550 nm.
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Electrical impedance
principle
*The sample is diluted with an electrolytic
Diluent (electronic current conducting fluid),
mixed then pulled through a calibrated
micro-aperture. Two electrodes are placed
on either side of the aperture and electric
current continuously passes between the
two electrodes.
*As the blood cells pass through the
aperture, they create resistance (Impedance)
in the electronic field between the two
electrodes. The voltage, which measures the
cells, is proportional to the size of the cell.
Since the current is constant and remains
unchanged, the larger the cell is, the «more»
resistance it has. The smaller the cell is, the
«less» resistance it has.
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Impulse generation
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This give rise to three part
differential
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General principle
*3- white blood cell :measure using DOUBLE Matrix &
electronic impedance variation principle at the same
time
*The Double matrix is based on 3 essential principles:
1- The double hydrodynamic sleeving «DHSS» (HORIBA
Medical patent).
2- Volume measurement: impedance changes.
3- Cytochemical staining and optical absorbance
measurement.
4- fluorescent dye absorbance and light emission.
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Monocytes
Eosinophils
Neutrophils
Large immature cells
Atypical Lymphocytes
Lymphocytes
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General principle
*These principles give us the opportunity to
measure and calculated variety of hematological
parameter witch contribute to highly effective
diagnosis of several complicated hematological
disease.
This parameter have to be understood and
correctly interpret to reach the exact and valuable
diagnosis .
*histogram and scatrograme should also
understood and correctly interpret
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This give rise to five & seven
part differential
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What parameter to be
understood
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PLATELETS
*Thromboembolic diseases are among the
major cause of mortality in developed
countries. Early diagnosis of progressive
activation of coagulation can help manage
these diseases successfully.
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PLATELETS
*A significant list of reliable markers have been
investigated recently, concerning :
*1-activation of coagulation: such as prothrombin
fragment 1+2, thrombin-antithrombin complex
(TAT).
*2- platelet activation:such as β-thromboglobulin
(β-TG) or soluble platelet P-selectin.
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PLATELETS
*However, laboratory measurement of
these indices is laborious and expensive.
Additionally, the above mentioned indices
cannot be included in routine laboratory
tests
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PLATELETS
*Automated hematological analyzers have
contributed to more precise and faster
results. They also make it possible to
measure several blood cell parameters
automatically.
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PLATELETS
*Among the parameters provided, platelet
indices are probably the most ignored by
clinical laboratories due to the difficulty of
standardization, as well as being affected by a
range of methodological problems.
*It has been suggested that each laboratory
determines its own reference intervals with
the equipment used.
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PDW
*Automated counters provide platelet counts and
generate the MPV and a measure of their size
variability (PDW).
The great dispersion of platelet volumes (log-normal
distribution) depends on the process of platelet
production, by fragmentation of cytoplasm of
megakaryocytes and proplatelet formation.
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PDW
*In healthy populations, there is a direct
relationship between MPV and PDW; this
relationship is maintained in idiopathic
thrombocytopenic purpura and chronic myeloid
leukemia, in which both are increased
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Platelets
* This does not occur in hypo plastic anemia's or
megaloblastic anemia or during chemotherapy, in
which the MPV decreases with an increasing PDW.
*The PDW can also be useful in differentiating reactive
thrombocytosis from the essential type, especially
when it is combined mathematically with the MPV and
platelet count to obtain a discriminant function.
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PDW
*Determination of the PDW reference range is
fundamental, and the association of this parameter with
the platelet number and mean platelet volume may be
used for the diagnosis and differentiation of several
pathologies.
*MPV and PDW are simple platelet indices, which increase
during platelet activation.
*The combined use of MPV and PDW could predict activation
of coagulation more efficiently.
* PDW is a more specific marker of platelet activation.
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Mean platelets volume(MPV)
*Mean platelets volume (MPV):
*test measures the sizes of platelets in a blood sample to
determine the average.
*a patient’s MPV may enable a doctor to detect a
problem before it shows up in a Plt test.
*The two tests in combination are used to detect a variety
of conditions, many of them serious.
*A high MPV indicates increased production of platelets,
* a low MPV, decreased production.
*Normal measurement for MPV is typically in the range of
5.0 – 15.0 femtoliters
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Conditions Associated with High
MPV
*Elevated mean platelet volume is common in
patients with certain forms of diabetes.
*If combined with a low Plt result, this indicates a
condition that results in destruction of platelets.
These include:
*1- immune thrombocytopenia: where the
patient’s immune system destroys platelets.
*2- pre- eclampsia: a complication during
pregnancy that elevates blood pressure.
*3- sepsis: an inflammatory response to an
infection; or various hereditary conditions.
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Conditions Associated with High
MPV
High MPV with a Normal Plt test result may be a
sign of:
*1- chronic myeloid leukemia: a condition in
which too many of a type of white blood cell are
produced.
*2- hyperthyroidism: over-production of thyroid
hormones.
*Accompanied by a High Plt result, it may
indicate a Bone marrow disorder that causes
excessive cell production.
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Conditions Associated with Low MPV
*association with a low Plt value, this can indicate:
*1- a form of anemia.
2- may result from therapy involving treatment with
drugs that are toxic to cells, for example, in
chemotherapy used to treat cancer.
*combined with a normal Plt, it may indicate:
* chronic kidney failure.
Accompanied by a high Plt, it can indicate :
*1-an infection.
*2- inflammation .
*3-some form of cancer.
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Problems Caused by Abnormal MPV
*Elevated MPV means that the blood has a
greater tendency to clot, which can increase the
risk of:
*thrombosis.
*stroke.
*cardiovascular disease.
*Aspirin is sometimes prescribed for this
condition, as it makes it more difficult for
platelets to clump together and form clots.
*A person with low MPV may bleed more easily.
Aspirin should be avoided in this case.
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Immature Platelet Fraction (IPF)
*Reticulated platelets are a measure of Immature Platelets.
*The Immature Platelet Fraction is an index of thrombopoiesis
and can help to determine the mechanism of thrombocytopenia.
* increased IPF in the presence of thrombocytopenia is indicative
of a platelet destruction or consumption.
*Values at or below the range in combination with
thrombocytopenia are indicative of decreased marrow
production.
*Normal range : The reference intervals of IPF were 0.5-3.2% in
males and 0.4-3.0% in females
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Immature Platelet Fraction (IPF)
*immature platelet fraction (IPF): useful to evaluate patients with
thrombocytopenia.
*IPF is the platelet equivalent of the red blood cells reticulocyte count
and is typically elevated in :
*1- disorders of platelet destruction –
1- idiopathic thrombocytopenic purpura (ITP),
2- thrombotic thrombocytopenic purpura (TTP)
3- disseminated intravascular coagulation (DIC).
2- early indicator of marrow recovery in post-chemotherapy and
stem cell transplant patients. IPF is normal or minimally elevated in
1- marrow suppression disorders such as aplastic anemia and in liver
failure.
2- IPF can be elevated in a subset (24%) of myelodysplasia patients.
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IPF
*Use in conjunction with patient diagnosis and
platelet count.
*May assist in determining cause/differential
diagnosis of thrombocytopenia.
*Provides a direct cellular measurement of
thrombopoietic activity.
*May help in determining need for prophylactic
transfusions.
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Transfusion Assessment
Low PLT + Low IPF
• No Production
• Transfuse
Low PLT+ High IPF
• Production
• Do Not Transfuse

Clinical Utility: Transfusion Management
• IPF should allow a more controlled prophylactic
platelet transfusion policy to be implemented at
specified threshold count, particularly when
platelet recovery is imminent.
• Predicting platelet recovery would permit more
reasoned use of prophylactic platelet transfusion
and provide the potential to reduce the use of
platelet concentrates, minimizing possible
transfusion-transmitted infections.
Briggs, C. (2003) Transfusion Management 16:101 - 109

Solution: Immature Platelet Fraction
C
• Use in conjunction with patient diagnosis and platelet count
• May assist in determining cause/differential diagnosis of thrombocytopenia
• Provides a direct cellular measurement of thrombopoietic activity
• May help in determining need for prophylactic transfusions
O
• Automated, rapid, inexpensive, speeds information to clinicians
• Direct measurement of immature cell production for faster indication of response
to changes in therapy
F
• Could reduce time and cost of diagnosis of thrombocytopenia
• Could reduce time to assess response to changes in therapy, saving cost
• Potential savings from better transfusion management

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Red Cell Distribution Width (RDW)
*Red Cell Distribution Width (RDW):
* measure of RBC size variation.
*now reported as the standard deviation rather than as a coefficient of
variation (CV).
*The conventional method of reporting variation of cell size by CV
resulted in underestimating variation as mean cell size (MCV) increased
(i.e., CV inversely proportional to MCV).
*Normal ranges RDW: 37 – 46%.
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Reticulocyte Hemoglobin (RET-He)
*The measurement of reticulocyte hemoglobin
content:
*Direct assessment of the incorporation of iron into
erythrocyte hemoglobin and thus a direct estimate of
the functional availability of iron into the erythron.
*RET-He is a reliable marker of cellular hemoglobin
content.
* A value below the range is indicative of a decreased
amount of iron in the RBC or iron deficiency.
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Reticulocyte Hemoglobin
*Reduction of this fraction indicates iron-
deficient erythropoiesis, even in conditions in
which traditional biochemical markers such as
ferritin and transferrin are inadequate, e.g. In
cases of inflammation or anemia from chronic
disease.
*Useful for monitoring early response to
intravenous iron therapy because RET-Hb
increases significantly after only 48 hours.
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Reticulocyte Hemoglobin
*Low values are indicative of iron-deficient
erythropoiesis in patients undergoing dialysis
and even in functional deficits, which appear in
patients treated with erythropoietin.
*RET-Hb of <28 pg accurately predicts functional
anemia when compared with Ferritin and
Transferrin saturation.
*Mean Value for both adult men and women is
30.8 pg.
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Reticulocytes(RBC)
*Automated counting of reticulocytes has greatly
increased the precision and accuracy of retics assay
compared with traditional manual counts.
*Reticulocyte maturity can now be assessed based on
the staining intensity of the reticulocytes, which is
proportional to their RNA content.
*immature reticulocytes may be defined by a
relatively high degree of RNA staining, whereas more
mature forms show less staining.
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Reticulocytes(RBC)
*Reticulocytes: immature non-nucleated RBCs that
have not completed production of hemoglobin and
consequently contain residual hemoglobin synthetic
machinery, mRNA and rRNA, commonly referred to
as “reticulin.”
*Peripheral blood reticulocyte count is a measure of
erythropoeitic activity.
Rising counts :in the face of anemia are regarded as
an indicator of appropriate bone marrow response.
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Immature Reticulocyte Fraction (IRF)
*The IRF is a fractional :
*percentage of all reticulocytes that have an intermediate
and high content of RNA.
IRF normal levels : reported to ranges from 5 – 22%.
*IRF is a direct cellular measurement of erythropoiesis
that can be used to monitor erythropoietic activity.
*IRF is a useful aid in diagnosis & therapeutic
management of anemia and in monitoring erythropoietic
stimulating agent.
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Transfusion Assessment
Low Retic + Low IRF
• No Production
• Transfuse
Low Retic+ High IRF
• Production
• Do Not Transfuse

*There are a number of published studies
that suggest that:
*IRF is a more sensitive and specific indicator
than the reticulocyte count alone, of the
following:
*1. Adequacy of marrow response to anemia
in patients with a variety of chronic diseases
including chronic renal failure.
*2. Adequacy of marrow response in neonates
with anemia.
3. Response to anemia therapy
including erythropoietin, iron, B12, and folate.
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*4. Signal of successful renal transplant
engraftment and erythropoietin production.
*5. Bone marrow recovery following
myelosuppression.
*6. Bone marrow engraftment in transplanted
patients.
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*7. Measure of chronic hypoxia and resultant
increased erythropoetic activity in conditions
such as chronic lung and cyanotic cardiac
diseases.
*8. Evaluation of normochromic anemias for
erythropoeitin activity.
*Reference range is 0.11 – 0.38%
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Clinical Condition IRF Absolute Retics
Aplastic marrow Decrease Decrease
Early erythropoietic response
after anemia or engraftment
after BMT
Decrease or No
Change
Decrease
Response to EPO Rx or early
acute hemorrhage
Increase Increase
Hemolytic anemia or
hemorrhage
Increase Increase
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Immature granulocytes
*Usually immature granulocytes are classified on the basis of
cell morphology by the microscopic exam of a stained blood
film.
*However, the manual differential count is imprecise
because of the small number of cells counted and
interobserver variability.
All IG% quantitations, 1% or over, are flagged for manual
review. The presence of IG above 2% can be significant
whether or not anything abnormal is detected in the manual
review, because the automated instrument has screened
thousands of cells in contrast to 100 cells reviewed manually.
*The Immature Granulocyte can be an Aid in Predicting
Bacteremia and or Sepsis especially if IG% is >2%.
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*The IGs, normally absent from peripheral blood, are
increased also in other conditions such as bacterial
infections, acute inflammatory diseases, cancer
(particularly with marrow metastasis), tissue necrosis,
acute transplant rejection, surgical and orthopedic
trauma, myeloproliferative diseases, steroid use, and
pregnancy (mainly during the third trimester).
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*The increase in IGs is accompanied by an increase in
neutrophils, which are freed from the marginal pool and
bone marrow. In some subjects, especially elderly
people, neonates, and myelosuppressed patients, the
increase in neutrophils may be absent, and, in other
conditions, such as sepsis, there can even be
neutropenia.
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*In these situations, the increase in
IGs (>2%), even if isolated, can be
useful for identifying an acute
infection, even when not suspected.
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Clinical Utility: IG
• More sensitive/specific for infection than WBC
comparable to ANC
• IGs >3% predicted positive blood cultures with
92% specificity
• Improve predictive value of infection by adding
the IG into an algorithm with other lab tests to
target a careful workup on patients with a IG >3%
• IG count can ID potential acute infection or
inflammatory response at early state … when
other parameters are within normal range and
nonspecific indicator

Solution: Automated Immature
Granulocyte Count
C
• Useful to complement to current infection surveillance program even
when other tests are negative
• Help physicians identify patients with infection sooner
O
•Automated, rapid, accurate, decreases manual reviews
•Speeds information to physicians
F
• Decreased labor needed for manual slide reviews
• Reduce cost of care when part of comprehensive infection surveillance

Large immature cell (LIC)
*LIC :cell appear as result of present
of blast cell or atypical lymphocyte
there present should investigate
carefully to the present of leukemia
or other associated disorder.
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ANY QUESTION ?
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Learning Outcome
1- Introduction to hematology
analyzer.
2-know the general principle of
hematology Analyzers.
3-known the new parameter
measure by hematology analyzer
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Summary
1- The seven part differential analyzer
gives the opportunity to identify more
hematological parameter that gives
more detail about the patient
abnormality witch help in the diagnosis
and monitoring of the patient without
more complicated investigation .
2- It’s the time to shift from the three
part to seven part differential.
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THANK YOU FOR GOOD
ATTENTION
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The majic of hema analyzer new hematological parameter

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