The majic of hema analyzer new hematological parameter

NAZAR AHMED MAHAMED ABD-ALLA (SANGOOR) SEP 2014

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THE MAJIC OF HEMA-ANALYZERNEW HEMATOLOGICAL PARAMETER

NAZAR AHMED MOHAMED ABD-ALLABSC - OMDURMAN AHLIA

HIGH DOPLOMA DGREE - ELZAEM EL-AZHARYFORMER HEAD OF HEMATOLOGY & BLOOD BANK

MINISTRY OF HEALTH – LABORATORY ADMINISTRATIONKHARTOUM STATE

MARKETING MANAGER-LAB EQP –DIVISIONALGAM 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 electronicimpedance variation principle. This means that anelectronic field is generated around the micro aperturewithin 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 in1- 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.

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 greatlyincreased 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.

Transfusion Assessment

Low Retic + Low IRF • No Production• Transfuse

Low Retic+ High IRF • Production• Do Not Transfuse


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*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 PredictingBacteremia and or Sepsis especially if IG% is >2%.


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Immature granulocytes

*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.

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


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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 Outcome1- 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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Summary1- 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

The majic of hema analyzer new hematological parameter

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