Blood And Lymph- Hematology By Turnicky

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Blood and Lymph- Hematology by Turnicky

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Blood and Lymph- Hematology by Turnicky

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Where should you normally find hematopoeisis?

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in the bone marrow.

Blood elements are first produced by precursor cells in the yolk sac, then the fetal liver, and finally in the bone marrow.

Pluripotent stem cells both self renew and differentiate into all of the specialized circulating blood elements.

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What do cytokines do?

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-Produced by a variety of cells both hematopoietic and stromal.
-Aid in communication between cells
-Influence the survival, proliferation and differentiation of hematopoietic stem and progenitor cells
-Act only in the local environment

Others produced by distant organs and act in an endocrine fashion:
Thrombopoietin- liver to regulate megakaryocytes
Erythropoietin- kidney to increase red cell mass

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2 choices of Hematopoietic Regulation

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The choice between self-renewal, in which one or both daughter cells retains the properties of the parent HSC, versus differentiation, in which one or both daughter cells lose capacity to self re-new and become irreversibly committed along a differentiation pathway.
-The central question is whether self-renewal versus commitment is controlled intrinsically by a property of the HSC itself (stochastic), or whether external cues such as cytokines can directly influence HSC fate decisions (instructive)

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So what is erythropoiesis?

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The process by which a committed erythroid progenitor cells proliferate and differentiate into the mature circulating non-nucleated erythrocytes that transport oxygen.
The purpose of the heart is to pump these things to the various organs

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Contents of buhlud. How much is RBCs?

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HUMANS HAVE 5L OF BLOOD
-ONLY 2.5L OR 45% CONSISTS OF CELLS
-0.037 L LEUKOCYTES (1.6%)
-0.0065 L PLATELETS
-THE REST IS PLASMA
-93% WATER
-7% SOLID, MOSTLY PROTIENS (ALBUMIN)

*that means about 2.5L is all RBCs

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EMDEN-MEYERHOF in RBC metabolism?

How about
HEXOSE-MONOPHOSPHATE SHUNT?

How about RAPOPORT-LEUBBERING SHUNT?

How about METHEMOGLOBIN REDUCTASE?

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EMDEN-MEYERHOF- Provides ATP for regulation of intracellular cation concentration (Na, K, Ca, Mg)

HEXOSE-MONOPHOSPHATE SHUNT -NADPH & glutathione to reduce oxidants

RAPOPORT-LEUBBERING SHUNT- 2,3 DPG to facilitate 02 release to the tissues

METHEMOGLOBIN REDUCTASE- Protects Hemoglobin from oxidation

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Single most abundant intracellular protein is...

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hemoglobin.

-Each tetramer is composed of two pairs of globin chains (Dimer)
-There are two identical pairs of globins in the tetramer
-There are four types of globin chains, alpha, beta, delta and gamma.

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What's in the center of the heme ring? How many oxygens can each Hb carry?

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-Different types of Hb formed depend on the types of associated globin chains.
-Each globin contains a Heme ring each containing a ferrous iron located in the center of the ring.
-Each Heme can carry one molecule of O2
-Thus each Hb can carry 4 oxygen molecules

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What are the 3 types of adult Hb?

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A pair of alpha chains combines with a pair of beta, delta or gamma chains to form one of 3 adult Hbs:

A (97%) Alpha2/Beta2
A2 (2%) Alpha2/Delta2
F (1%) Alpha2/Gamma2 (fetal)

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What are the two causes of erythrocytosis?

(An increase in the number of circulating red cells; Not a disease, but a result of some other problem; Symptoms: Often none, an incidental finding on a CBC
headache, blurred vision, dizziness, vertigo)

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Relative Erythrocytosis (a hemoconcentration): No increase in red cell mass, but a decrease in plasma volume. Causes: Dehydration, diuretics, ethanol, Excessive perspiration

Absolute (actual increase in rbc mass);
Hypoxia: Certain hemoglobinopathies (high O2 affinity), High altitude, Pulmonary disease, Kidney disease (artery stenosis, cysts), Neoplastic conditions: Polythyemia vera, other tumors via paraneoplastic syndromes

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ANEMIA

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-Any condition resulting from a significant decrease in total body red cell mass.
-Reduction in oxygen carrying capacity of the blood leading to tissue hypoxia.
-Mass= Production X Survival
-Production may increase 10 times normal, which is the maximal capacity of the BM.
-Anemia is a symptom, not a disease
-Anemia is a dynamic process
-Natural aging is not a cause of anemia
-The diagnosis of anemia requires further clinical work up for associated disease conditions

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What are the physiological compensations for anemia?

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Increased cardiac output
-Tachycardia
Redistribution of blood flow
-Vasoconstriction and shunting
Decreasing O2 affinity to hemoglobin
-Increased production of 2-3 DPG shifting the oxygen dissociation curve to the right

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Erythrocyte Count

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# of red cells within a fixed volume (not red cell mass). Counts all the cells (including white) but since white cells are such a small proportion, it doesn’t matter too much. Automated Impedance Counter (Coulter) is the way to do this.
-more RBC is males, highest at birth, lowest at 2 months, then by puberty hits normal adult value and stays there. Increases hypoxemia (ex high altitude and smoking)

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Hematocrit

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aka PCV (packed cell volume). Total volume of red cells relative to total volume of whole blood in a sample. Expressed as percent 45%, or unit-less .45.

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Hemoglobin Conc

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Indirect measure of O2 carrying capacity. Red cells are lysed and the free Hb reacted with potassium cyanide to form a stable pigment (cyanmethemoglobin) which is measured spectophotometrically.

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Mean Corpuscular Volume-

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The mean volume of all rbcs. Expressed in volume units (femtoliters, fL) Calculated: MCV=Hct / rbc count.
Direct Measure: as individual rbcs pass in an orifice, which an elec. current is flowing, the cell produces a voltage pulse which is proportional to the cell vol.
-Measures only average cell vol.. MCV can be normal while ind. Cells may vary widely.
Used to classify cells and type of anemia:
Normocytic: 80-100fl ; Microcytic: <80 ; Macrocytic >100

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Mean Corpuscular Hemoglobin Concentration (MCHC)

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-Measures the average conc. of Hb in a given volume of packed red cells ; MCHC (g/dL) = Hb / HCT
-Just as MCV relates to cell size, MCHC relates to the color of the cells ; Defines normochromic, and hypochromic cells relating to anemia classification.
-Because there is a physical limit to the amount of Hemoglobin that can fit in a cell, there is no hyperchromic category
-Not sex dependent
-Normal Ref Range: 32-36 g/dL

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Red Cell Distribution Width. Why isn't it reliable?

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it's a mean, so its less reliable in identifying marked variation in cell size (anisocytosis)
-RDW measures the variation in size; calculated index
-RDW is a coefficient of variation of red cell volume
-RDW (%) = Std of MCVx 100 / mean MCV
-Normal RDW: 10% to 14.5%
-Not related to age, or sex; Normally red cell are a standard size
-High RDW: Increase in the heterogeneity of RBC size
-Low RDW: A more uniform RBC population, little or no variability in size.

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Where do you want to review the smear?

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feathered edge. look for RBC morphology, white cell differential and morphology, and platelet estiamte and morphology.

Normal RBC is 7um disc with central pallor 1/3 the diameter of the cell.
-POIKILOCYTOSIS: Nonspecific variation in shape.
-ANISOCYTOSIS: Nonspecific variation in size.

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Macrocytosis, size, shape, -chromic level

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>8 um diameter; MCV>100
Oval or Round
Normochromic; no polychromasia

Oval macrocytes seen in vitamin B12 and folate deficiences, aplastic anemia, myelodysplastic syndromes, chemotherapy effect

Round macrocytes seen in: LIVER DZ, ALCOHOLISM, post-splenectomy, hyper/hypo thyroidism, malaria

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Ok, now go with microcyte

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<6 um : MCV<80 fl
Hypochromic (with increased central pallor)
More than 1/3 of the cell
Associated with: Iron deficiency, Thalassemia, Lead poisoning

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Acanthocytes. key features to remember

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Smaller than normal red cell
**3 to 12 irregular membrane spikes, UNEVENLY distributed.
-narrow base, sharp tipped, some may be knobby
-No Central Pallor
Look – alikes: Echinocytes, Schistocytes, Microspherocytes

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Greater than 10% and less than 10% acanthocytes... what are we thinking?

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Usually >10% acanthocytes
-Abetalipoproteinemia
-Advanced Liver Disease: due to neonatal hepatitis, metastatic disease, alcoholism.
*see a lot...think LIVER disease

Usually <10% acanthocytes
-Post splenectomy
-Myeloproliferative disorders
-Microangiopathic hemolytic anemia
-Autoimmune hemolytic anemia

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Echinocyte features, most common cause.

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-Spiculated RBCs with 10 to 30 short, EVENLY distributed projections. Broad based.
-Same size as normal RBC
-Unlike, acanthocytes, the cells RETAIN central pallor

Most common cause is laboratory artifact of improperly prepares smears (slow drying, thick smears, aged blood) or “glass effect” causing an elevated ph in the medium surrounding the cell.
-also seen in uremia and chronic RENAL failure. immediate post-transfusion with aged or metabolically depleted blood.

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What do you call a small fragment of a red cell? What disease are you thinking about if you see them?

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Schistocyte. Size: Varies, usually microcytic or fragment. Irregularity of cell size and shape. Small fragments lack any significant central pallor.

Think microangiopathic anemia (Disseminated intravascular coagulation, Thrombotic thrombocytopenia purpura, Hemolytic Uremic Syndrome)

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How do schistocytes form?

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initiation of the coagulation system, fibrin is formed in the microvasculature. rbcs are floating along and hit a piece of fibrin and fragment.

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What does a teardrop cell look like?

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-Resembles a pear: round with a single elongated or pointed extremity. Unipolar tapered end with a blunt tip
-Maybe artifactual in improperly made smears. (tails line up in same direction)

*think something abnormal in the BONE MARROW or SPLEEN. ex replaced by fibrous tissue.

Associated Diseases: Myelofibrosis, Myelofibrosis with myleloid metaplasia (MPS), BM infiltration by hematologic or carinomatous malignancy.

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What exactly is a sickle cell?

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Drepanocyte. RBC deformed by polymerized Hb.
-Sickle shaped cells are found in homozygous (SS), but NOT the heterozygous (SA) state.
-Size: variable, usually >10um, with two pointed ends/crescent shaped.

Associated Disease States:
SS disease, SC disease, SD disease, S-beta Thalassemia

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Spiculated Red Blood Cells

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-Acanthocyte (spur cell)
-Echinocyte (burr cell)
-Teardrop cell
-Sickle Cell
-Schistocyte (horn cell, helmet cell)

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If you have greater than 10% Ovalocyte, what type of abnormality do you suspect?

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Rod shaped with nearly parallel sides
Normal finding, but <1%
If >10% suspect hereditary ovalocytosis (25% is diagnostic), an abnormality of skeletal membrane proteins.
Also seen in: Megaloblastic anemia, thalassemia, severe Fe deficiency.

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What do spherocytes look like? What are the associated diseases?

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-Round, thicker than normal, and without central pallor.
-Often smaller than normal RBC
-MCV normal to slightly reduced (dependent on reticulocyte count)
-assd with Immune hemolytic anemia and microangiopathic hemolytic anemia (clinically obvious)

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Stomatocyte

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-Central pallor appears slit like, straight, or fish mouth.

Associated Diseases:
-Hereditary Stomatocytosis (if see many)
-Rh null disease

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Target Cells. What does it look like? What are the broad causes?

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Increased surface membrane to volume ratio results in a central darker region within the area of central pallor. “Bulls eye” cell.

Broad causes: excessive membrane lipid in liver disease; less interior content in Thalassemia. Also artifact of slow drying.

Associated Disease: iron deficiency anemia, thalassemia, hemoglobinopathies, post-splenectomy, liver disease, artifact

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We're going to go through a whole bunch of Erythrocyte Inclusions.

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Basophilic Stippling
Howell-Jolly Body
Pappenheimer Body
Cabot Rings
Siderosomes
Heinz Body (denatured Hb)
Crystals (HbC)
Bacteria
Parasites (malaria, babesiosis)
Artifacts (stain contamination, platelets)

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Basophlic Stippling. Difference between coarse and fine?

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Numerous, evenly distributed blue-gray granules within the cytoplasm. Maybe fine or coarse

Coarse:
-Clinically SIGNIFICANT
-Suggests impaired Hb synthesis
-Lab should only report Coarse stippling
-Coarse stippling results from abnormal aggregates of ribosomes
-Easily seen on Wright-Giemsa staining, unlike fine stippling which is an artifact of drying and is more dust like and almost imperceptible.

Fine most likely artifact

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What are assd diseases of coarse basophilic stippling?

What causes basophilic stippling?

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Lead poisoning, Thalassemia, Refractory Anemia (MDS), Sideroblastic Anemia (MSD), Megalobastic Anemia (B12/folate), Sickle Cell Anemia

-In pathologic states RNA degradation is incomplete or the ribosomes are abnormal.
-WG stain is capable of precipitating RNA, but inefficiently, if too little RNA (normal) no visible results. If RNA increased get precipitation.
-Coarse stippling is never a normal finding.

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Howell-Jolly Body. What causes them?

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-A DNA fragment produced when nuclear extrusion takes place
-Rarely seen in the normal state since rapidly removed by spleen
-A chromosome may become separated from the mitotic spindle and remain in the cytoplasm.
-Abnormal cell division seen in Megaloblastic anemias or MPS
-Spherical intracytoplasmic inclusion that is iron stain negative.
-Round, eccentrically placed. Small; 1 um.

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Disease states of Howell-Jolly Bodies

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Disease states:
Hyposplenism
Asplenia
Megaloblastic anemia
Leukemias and MPS
Severe hemolytic anemia

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Pappenheimer Body

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-Iron containing particles visible on WG stain.
-Abnormal sidersomes composed of both iron and protein matrix.
-Normal sidersomes are composed of iron only and do not stain on WG.
-Blue granules, with irregular sharp edges.
-Usually at periphery of cell.
-Iron stain is positive and confirmatory

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Pappenheimer bodies associations

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Disease Associations:
Siderblastic Anemia (MDS)
Thalassemia
Megaloblastic Anemia
Hemolytic Anemia
Post splenectomy states

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Try to differentiate Howell-Jolly Bodies and Pappenheimer bodies

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-Howell-Jolly bodies are round and papenheimer bodies are angular.
-Both maybe single or multiple
-Pappenheimer usually form tight clusters of inclusions in pairs or tetrads.
-HJ can be multiple either grouped or dispersed, but vary in size.
-An iron stain may be necessary to confirm
Howell-Jolly: Negative
Pappenheimer: Positive

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Cabot Rings. What are they remnants of? Look like? Dz assn

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-Remnants of mitotic spindle
-Red-purple thread like rings
Disease Association:
Lead poisoning
Megaloblastic anemia
Dyserythropoiesis

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Rouleaux, look and association

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-Linear arrangement of four or more RBCs
-Simulating a “stack of Coins”
Associations:
-None if in Thick area of smear
-Hypergammaglobulinemia
-Liver Disease, chronic infections
-Multiple myeloma, lymphoma

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Anemia-Laboratory Diagnosis

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-CBC for Hemoglobin/Hematocrit, Red cell number
-CBC for MCV, MCHC ( to classify)
-Peripheral smear for red cell morphology
-Reticulocyte count (corrected) and IRF
-Lab assays pending above
-Serum Fe, TIBC, % sat., B12/folate, haptoglobin, bilirubin, LDH, test for rbc survival, ect.

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Bone Marrow exam

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(pending other results)
-Complements clinical and lab information in determining the cause of:
-anemia, leukopenia/leukocytosis
-thrombocytopenia/thrombocytosis
-diagnosis of Leukemia/lymphoma
-Staging of Lymphomas
-Obtained from the iliac crest (posterior superior), in both adults and children
-Children ,2yrs; tibia
-Direct smears prepared from fluid and biopsy obtained
Aspirate: preferred for the cytology of marrow
Biopsy: for assessment of cellularity and architecture.

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Bone Marrow-Ancillary Studies

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-Flow cytometry
-Cytogenetic analysis
-Chromosomal analysis for translocations, chromosomal alterations for diagnosis and prognosis.
-Molecular diagnostic studies
-PCR/FISH for detection of gene re-arrangements, chromosomal translocations or detection of infectious agents.

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Anemia is the result of 3 pathophysiologic mechanisms:

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-Proliferation Defect- abnormal proliferation of stem cells (ex aplastic anemia)
-Maturation Defect- a lot of cells produced, but abnormal and killed in marrow (iron/folate deficiency anemia)
-Survival Defect- cells made, but there's rapid destruction

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Which classification are we going to pay particular interest to?

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Morphology/Cytometric
-By red cell size and Hemoglobin concentration using:
-MCV (cell size)
-MCH and MCHC (cell color).
-Does not determine the cause of the anemia.

1. Practical to work from
2. Easily and less expensively measured
3. Anemia is placed in one of three categories

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Which are the NORMACHROMIC NORMACYTIC ANEMIAS?

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Normochromic Normocytic
(normal MCV, Normal MCHC)

These include:
-Anemias of chronic disease
-Immune hemolytic anemia
-Anemia of acute hemorrhage
-Aplastic anemia
-End organ failure (endocrinopathy, renal)

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how about HYPOCHROMIC, MICROCYTIC

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Hypochromic, microcytic
(low MCV, low MCHC)

These include:
-Iron deficiency anemia
-Thalassemia
-Anemia of chronic disease (infrequently)
-Long standing disease

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MACROCYTIC, NORMACHROMIC ANEMIA

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Increased MCV, Normal MCHC

These include:
-Vitamin B12/ Folate Deficiency
-Refractory Anemias/Myelodysplastic Syndrome (MDS)
-Liver disease

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Reticulocytes

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-Immature, anuclear red cells containing increased ribosomal RNA for hemoglobin synthesis
-Reticulocytes spend 2 to 3 days in the BM and an day in the PB before becoming mature red cells.
-The normal red cell life span is 120 days.
-Since it is immature it is larger than mature red cells
-The RNA content gives it a blue coloration (polychromatophilic)
-A reticulocyte count is available from the CBC.

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Reticulocyte Count

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In anemia reticulocytes are released into the blood prematurely.

Reticulocytes indicate the degree of effective BM activity, e.g is the BM compensating for anemia. (high retic--> compensating for anemia...low retic--> marrow is exhausted and cant compensate for anemia)

A cost effective and useful test
- in classifying the pathophysiology of anemia.
- monitoring marrow response to therapy.

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What is the Corrected Reticulocyte Count?

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In the anemic individual , the relative retic count appears normal (1%), when in actuality the individual is making fewer rbcs than the healthy individual
Corrected Reticulocyte count over comes this dilutional problem and is useful in evaluating the marrow response to anemia.

%observed reticulocytes x patients Hct/45

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Immature Reticulocyte fraction (IRF) measures what

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-The maturity level of the released reticulocyte can also be quantitative by its RNA concentration
-Younger more immature reticulocytes have more RNA
-Instruments now assess reticulocyte maturity level and report an index of maturity known as the IRF in addition to reticulocyte % and absolute numbers.

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What is IRF good in monitoring/evaluating?

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-Helpful in evaluating marrow compensation for anemia, and monitoring response to therapy.
-An elevated IRF indicates adequate response while normal or subnormal IRF reflects inadequate response
-There is an observable increase in IRF before there is an increase in reticulocyte count, or an increase in hemoglobin, hematocrit or RBC count
-In patients receiving rEPO or iron therapy for anemia and increased IRF predicts a successful outcome
-In BM transplant patients it is the first sign of successful engraftment.