Hematology Objectives Mod 4 and 5

For each of the six Genotypes of Beta Thalassemia describe the: A. Individuals Affected B. Basic Pathophysiology C. Symptoms D. Laboratory results including cell morphology and hgb electrophoresis

Beta Thalassemia Major (B0/B0, B0/B+, B+/B+) A. Frequently found in people from Italy, Greece, Algeria, Saudi Arabia and Southeast Asia. B. The dramatic reduction or absence of B-chain synthesis affects the production of HbA. The symptoms result from B-thalassemia major begin to manifest in infants at 6 months of age. Other non-Beta-contain hemoglobins, HbA2 and HbF are increased in parieal compensation for the decreased HbA levels. Ineffective erythropoiesis with hemolysis and erythroid hyperplasia. - The dramatic reduction of HbA compromises the blodd's oxygen carrying capacity. HbF andHbA2 is increased. HbF has a higher affinity for oxygen than HbA. C. In infants include irratability, pallor, and a failure to thrive and gain weight at about 6 months of age. Diarrhea, fever and an enlarged abdomen are also common findings. D. Hemoglobin can be as low as 2 or 3 g/dL in more severe forms. Anemia is microcytic and hypochromic with an MCV of <67gL. Reduced MCH and MCHC. Blood smear shows anisocytosis and poikilocytosis. Precipitates of alpha chains can be visualized with methyl violet stain. Variable basophilic stippling and polychromasia re also noted. NRBCs are almost always found, and the RDW can be normal to increased. - Hemoglobin eleictrophoresis performed on cord blood provides evidence of deficient B-chain production at birth. Although normal cord blood contains about 20% HbA, cord blood from infants with B-thalassiema major has <2% HbA. In adults electrophoresis shows variable results depending on the thalassemia alleles inherited. Absence of HbA, 90% HbF and low, normal or increased HbA2 is characteristic of B0/B0 thalassemia. Beta Thalassemia Minor (B0/B or B+/B) A. About 1% of african americans are heterozygous. B. The normal B-gene directs synthesis of sufficient amounts of Beta-Chains to synthesize enough HbA for nearly normal oxygen delivery and erythrocyte survival. In the case of heterozygous B+ patients, the thalassemic gene will also contribute to B-chain production. C. Asymptomatic except in periods of stress that can occur during pregnancy and with infections. Under such conditions a moderate microcytic anemia can develop. Concomitant folate deficiency can produce macrocytic anemia. D. Hemoglobin values are in the rage of 9-14g/dL, with the mean value for women being 10.9g/dL and mean 12.9g/dL. The RBC count is increased to >5 x10^12/L. RBCs are microcytic (MCV = 55-70 fL) and hypochromic )MCHC = 29-33 g/DL) or sometimes normochromic with an MCH that is usually <22 pg. - Although the anemia is mild, the blood smear shows variable anisocytosis and poikilocytosis with target cells and basophilic sippling. NRBCs are usually NOT found, but anemic patients can have a slightly elevated reiculocyte count. Bone marrow shows slight erythroid hyperplasia and erythroblasts poorly filled with hemoglobin. Beta Thalassemia Intermedia (B+/B+, B0/B+, B0/B) A. All three patterns of inheritance - homozygous, double heteroxygous and heterozygous can produce B-Thalassemia intermedia. The homozygous and double heterozygous forms reprecent a mutation in both B-alleles, resulting in moderate reduction in B-chain synthesis. B. The B0/B+ genogype produces the greatest reduction in Beta-chain synthesis but has a variable clinical presentation. C. The CBC reflects a moderate microcytic hypochromic anemia with a hemoglobin value range of 7-10g/dL. In milder cases, patients express only a slight reduction in hemoglobin values. The RBC count is disproprtionately higher than the hemoglobin. Target cells are the predominante poikilocytes observed. Basophilic stippling and nucleated rbcs are also present. Bone marrow shows hypochromic erythroblasts in the context of erythroid hyperplasia. Beta Thalassemia Minima (Bsc/B) A. Asymtomatic, no major laboratory findings and is only defined by a mildly imbalanced alpha to non--alpha globin chain synthesis ratio. DeltaBeta-Thalassemia A. Rare thalassemia observed primarily in patients of Greek, African, Italian and Arabian ancestry whose production of both B- and Delta chains is affected. The DeltaBeta-mutation can be categorized into two genotypes. DeltaBeta0 and DeltaBeta+. GammaDeltaBeta - Thalassemia A. This rare form of thalassemia has several variants and is characterized by deletion or inactivation of the entire Beta-gene complex. Deletion of the gamma, delta and beta genes would result in the absence of all normal adult hemoglobin production from that chromosome.

Metamyelocyte

VAries in size 10-18 mcM in diameter and is not capable of cell division. Nuclear indentation that gives the nucleus a kidney bean shape can be a characteristic of a metamyelocyte from a myelocyte but nuclear shape is variable and is not the most reliable identifying feature. Care should be taken to review other cellular features such as the degree of the chromatin clumping, color of the cytoplasm, predominant granules present and the cell size. Nuclear chromatin is coarse and clumped and stains dark purple. Nucleoli are not visible. The cytoplasm has a predominance of secondary and secretory granules. The raio of secondary to primary granules is 2:1. Tertiary or gelatinase-containing granules are synthesized mainly during the metamyelocyte and band neutrophil stages.

Myelocyte

Varies in size from 12-18 mcM. The nucleus is reduced in size due to Nuclear chromatin condensation and appearsmore darkly stained than the chromatin of the promyelocyte. Nucleoli can be seen in the early myelocyte but are usually indistict. The myelocyte nucleus can be round, oval, slightly flattened on one side or slightly indented. The clear light area next to the nucleus, represents the Golgi apparatus. The myelocyte goes through 3 cell divisions; this is the last stage of the maturation process in whihc the cell is capable of mitosis. The early myelocyte has rather basophilic cytoplasm where as the later, more mature myelocyte has a more tan to pink cytoplasm as the cell beings to lose cytoplasmic RNA. The hallmark of the myelocyte is the appearance of primary or secondary granules. Synthesis of peroxidase-positive primary granules is halted and the cell switches to synthesis of peroxidase - negative secondary granules. Secondary granules are detected first near the nucleaus in the Golgi apparatus. SEcondary granules are small, sandlike with a pinkish-red to pinkish lavender tint.

Screening tests for Hemochromatosis

Screening tests usually include percent saturation of transferrin and serum ferritin. The criterion for a diagnosis is ususally > or = 45% transferrin saturation. Saturation often approaches 100%. Adding serum ferritin analysis increases specificity for HH. Iron overload is suggested when the serum ferritin is elevated.

Define Hemosiderosis

(AmE) or haemosiderosis (BrE) is a form of iron overload disorder resulting in the accumulation of hemosiderin. Types include: Transfusion hemosiderosis. Idiopathic pulmonary hemosiderosis.

Identify poikiliocytes and anisocytes on a blood smear and describe their mechanism of formation and correlate them with pathological conditions

Acanthocyte - Spur Cell Spiky shape. Red cells with spicules of varying length irregularly distributed over the surface; no area of pallor. Abetalipoproteinemia; alcoloic liver disease; disorders of lipid metabolism; post splenectomy; fal malabsorption; reinitis pigmentosa. Codocyte - Target Cell Bell shaped, thing with increased surface to volume ratio; on stained blood smears, appears as a target with a central bull's eye, surrounded by achromic zone and outer ring of hemoglobin. Hemoglobinopathies; thalassemias; obstructive liver disease; iron deficiency anmemia; splenectomy; renal disease; LCAT deficiency Dacrocyte - Tear Drop Round cell with a single elcongated or pointed extremity; may be microcytic and/or hypochromic Seen in Myelophthsic anemias; primary myelofibrosis (PMF); thalassemias Drepanocyte (Sickle Cell) Contain polymerized hemoglobin showing various shapes: Sickle, cresent or boat shaped. Echinocyte, Burr Cell, Crenated cell Sea urchin shape, spiculated red cells with short equally spaced projections over the entire surfice. Seen in liver diease, uremia, pyruvate kinase deficiency, peptic ulcers, caner of stomach, haparin therapy

Describe the relationship of the anemias associated with alcoholism and malignant disease to sideroblastic anemia

Alcoholism - Megaloblastic and Sideroblastic anemia occur to the majority, but is also often coupled with ACD, IDA, acute blood loss and chronic hemolysis. Malignancy - Ring sideroblasts can be found. Some investigators believe that the presence of the ring sideroblast in these disorders suggest that the malignancy can result from abnormal clone of pluripotential stem cells that affects the erythroid as well as other lineages

Correlate clinical severities of both alpha and beta thalassemia with their respective genotypes

Alpha (--/--) a0/a0 - Hydrops fetalis, Fatal. - Marked anemia, microcytic/hypochromic RBCs, Marked increase in anisopoiklocytosis, increase in NRBCs. - Bart's Hb (80-90%). Hb Portland (10-20%) - Homozygous alpha-thal-1 (--/-a) a0/a+ - Hemoglobin H disease - Moderate to marked anemia, microcytic/hypochromic rbcs, target cells, basophilic stippling and poikilocytosis. - Chronic, moderately severe hemolytic anemia - Birth = Hb Bart's. Adult = HbH - Heterozygous alpha-thal-1/alpha-thal-2 (--/aa) a0/a+ - a-Thalassemia-minor - Mild to moderate severity - Slight anemia, microcytic/hypochromic rbcs. Target cells, basophilic stippling - Birth = Hb Bart's. Adult = Normal -Heterozygous alpha-thal-1 (-a/-a) a+/a+ - Alpha-thalassemia-minor - Mild severity - Slight anemia, target cells, basophilic stippling. - birth = Barts. adult= normal - Homozygous alpha-thal-2 (-a/aa) a+/a -Silent Carrier -Normal severity - Heterozygous alpha-thal-2 - normal hemoglobins - normocytic or slightly microcytic RBCs (aa/aa) - All normal Beta look at picture!

Compare and contrast the etiology of Alpha and Beta Thalassemia

Alpha - Human genome has two Alpha genes that are located on each of the two #16 chromosomes, totaling four alpha=genes, resulting 4 mutations. A patient in whom all 4 Alpha-genes are deleted produce no Alpha-genes, a condition referred to as Hydrops fetalis. - when only 3 alpha genes are deleted its a disorder called Hemoglobin H (HbH) disease. - The deletion of 2 alpha genes is known as Alpha- Thalassemia minor - The deletion of 1 alpha genes is known as a silent carrier. Beta - There are only 2 beta-globin genes, one located on each chromosome 11. IF the prominent type of mutation found in Beta-Thalassemia were also deletional, one would expect two severities, the severe homozygote and the mild heterozygote. Most mutaions are nondeletional resulting in a near continuu, of clinical severities. There are two classiciation systems are currently used for the group of diseases. - The genotypic system, all B-gene mutations are cateforized into two groups based on the impact of the mutation on B-globin production. The two gene varieties are termed B+ and B0. The B+ gene mutation causes a partial block in B-chain synthesis, and the B-0 gene mutation results in a complete absence of B-chain synthesis from that allele. A minimally affected B-allele called silent carrier (Bsc) has been identified and is only found in the most benign version of B-Thalassemia. - In a heterozygous state, hematological features are normal; it is clinically benign.

For each of the four Genotypes of Alpha Thalassemia, describe the: A. number of affected alleles B. Individuals affected C. Basic Pathophysiology D. Symptoms E. Laboratory Findings

Alpha Thalassemia Major (a0/a0 or a-thal-1/a-thal-1: Hydrops Fetalis) A. All four allels are deleted B. Asians this is the Major ethnic group carrying the a-thal-1 allele C. Because all four of the Alpha genes are deleted in hydrops fetalis, no physiologically useful hemoglobins can be synthesized beyond the embryonic state. This disorder is imcompatitble with life and infants are either stillborn or die within hours of birth. In the absence of alpha chains, erythrocytes assemble hemoglobin using the gamma/delta and Beta chains available. Therefore abnormal hemoglobin tetramers involving gamma-chains (Hb Bart's is gamma4) are produced. - Barts has a bery high oxygen affinity and no Bohr effect, therefore this hemoglobin cannot supply tissues with sufficient oxytgen to sustain life. D. Infants who survive until birth exhibit significant physical abnormalities upon routine exam. The babies are underweight and edematous with a distended abdomen. The liver and often the spleen are enlarged due to extramedullary hematopoiesis. There is massive bone marrow hyperplasia. Hemolysis in the fetus is severe and there is extensive deposition of hemosiderin. E. Lab results confrim the clinical obersvation of severe anemia. Hemoglobin vales range from 3-10h/dL and RBCs markedly microcytic and hypochromic. Hemoblobin electrophoresis on cellulose acetate or agarose alkaline pH shows 80-90% bart's and 10-20% Hb Portland;HbH is sometimes also detectable. HbA and HbF and HbA2 are absent due to the lack of alpha chain production. Hemoglobin H disease (a0/a+ or a-thal-1/a-thal-2) A. 3 of 4 alleles affected B. African Americans seldom present with HbH disease because they rarely express a deletion of two alpha genes on the same chromosome. C. The dramatic reduction in alpha-chain (25-30% of normal) results in a decrease in the assembly of HbA, HbA2, and HbF. In addition, a decrease in Alpha-chains creates a relative excess of Beta-chains, which assemble to form Beta-chain tetramers called HbH. Gamma chains are also produced in excess of alpha-chains, especially at birth and combine to form Gamma-chain tetramers or Hb Bart's. Triggers chronic hemolytic anemia. It's oxygen affinity is 10 times that of HbA, reducing oxygen delivery to the tissues, this affinity is due to the lack of heme-heme interaction and the absence of the Bohr effect. D. Symptoms are related to anemia and chronic hemolysis. Hemoglobin H disease shows a wide variation from mild to severe anemia, which worsens during pregnancy, in infectious states and during administration of oxidant drugs. E. Characterized by Microcytic, hypochromic anemia with hemoglobin levels usually ranging from 8-10g/dL. Recticulocytes are maderatly increased to 5-10% and nrbcs are also seen. Electrophoresis of affected neonates shows about 25% Hb Barts's with decreased levels of HbA, HbA2, and HbF. Shortly before birth, Beta Chains begin to replace gamma-chains, and HbH eventually replaces Hb Bart's. HbH is a fast migrating hemoglobin at alkaline pH. and constitutes 2-40% of the hemoglobin in adults with HbH disease. Alpha Thalassemia Minor (a-thal-2/a-thal-2, [a+/a+] or a-thal-1/normal [a0/a] A. two of four alleles are affected B. Found in all geographic locations, in african americans the homozygous form (a-thal-2) is the most common presentation. In people from Southeast Asia or Mediterranean, genetic testing has identified at least nine haplotypes. C. Measurable decrease in the production of alpha-containing hemoglobins occurs, the unaffected alpha-globins are able to direct synthesis of alpha-globin chains to a greater than normal degree and therefore partially compensate for the deleted genes. Only minor changes occur in the RBC count, indices, hemoglobin electrophoresis patterns and RBC morphology. D. Asymptomatic with mild anemia and are often diagnosed incidentally or when being evaluated for family studies. E. Most abnormalities are observed in the newborn. The precense of 5-6% Bart's in neonates can be helpfor for diagnosing this condition. Silent Carrier (a-thal-2/normal a+/a) A. 1 of 4 alleles are affected. B. More than 25% of african americans have shown to express a deletion of one alpha gene. C. Asymptomatic and benign. But Adults often present with borderline normal MCV of around 78-80fL. In infants 1-2% barts may be found at birth but cannot be detected after three months of age. The only difinitive diagnostic test for thalassemias in adults with one or two gene deleteions is globin gene analysis.

Define Sideropenic as it relates to Anemia

Anemia characterized by deficient iron for hemoglobin synthesis is know as sideropenic anemia. More commonly referred to as Iron deficiency anemia (IDA) Iron deficiency Primarily affects the erythrocyte and devloping central nervous system. Sideropenic anemia is caused by inadequate iron intake or absorption or increased bloos loss responds to iron therapy given either orally. or less commonly parenterally.

Cabot Rings

Appear as a figure 8, ring or incomplete ring. Thought to be composed of microtubules of the mitotic spindle. Stain reddish violet with wrights stain. Seen in severe anemias, dyserythropoiesis

Reticulofilamentous substance (reticulocyte)

Artifactual aggregation of ribosomes. Not visible on Wright's stained smears; supravital stain (e.g. new methylene blue) must be used. Appears as deep blue reticular network

Heinz Bodies

Bodies composed of denatured or precipitated hemoglobin. Not visible on Wright's stained blood smears. With supravital stain appear as purple. round shaped bodies of varying size, usually close to the cell membrane. Can also be observed with phase microscopy on wet preparations. Seen in G6PD deficiency; unstable hemoglobin disorders; oxidizing drugs or toxins; post splenectomy.

Neutrophils Bone Marrow, Peripheral Blood and Tissues

Bone Marrow:The largest compartment of neutrophils is found within the bone marrow and is referred to as the mature neutrophil reserve. The number of neutrophils circulating in the peripheral blood, the blood compartment is about one-third the size of the bone marrow compartment. Production and Release of neutrophils from the Bone Marrow is likely a feedback loop between circulating neutrophils and the bone marrow. In normal conditions the mediator is likely G-CSF, produced by marrow stromal cells and macrophages. Inflammatory cytokines such as IL-1 and Tumor necrosis factor (TNF) are impoartent in causing an increase in neutrophilic concentration in pathologic conditions by inducing the macrophage to increase the release of G-CSF and GM-CSF. Peripheral Blood: Neutrophils are released from the BM to the PB but not all neutrophils are circulating freely at the same time. About half of the total blood neutrophil pool is temporarily marginated along the vessel walls and is called the marginating pool (MP), whereas the other half is freely circulating and is referred to as the circulating pool (CP). The average neutrophil circulates 7.5 hours in the blood before diapedesing to the tissues. Tissues: Neutrophils constitute the majority of the circulating leukocytes. The absolute concentration varies between 1.8 and 7.0 x10^9/L Adherence: Neutrophils flow freeling along the vascular endothelium when neither the neutrophil nor the VEC is activated.

Assess bone marrow response to anemia given CBC and reticulocyte results

Bone marrow evaluation can provide supplemental diagnostic information in anemic patients when other lab tests are not conclusive. For example; bone marrow evaluation in hypoproliferative anemias can reveal myelodysplasia or infliltration of the marrow with malignant cells or granulomas. Erythroid hyperplasia of the bone marrow with decreased amopunts of fat is pronounced in hemolytic anemia than in any other of the nonhemolytic anemias. Consequently, the myeloid-to-erythroid ratio (M:E) is decreased. The IRF can be helpful in evaluating bone marrow erythropoietic response to anemia. Monitoring anemia and evaluating response to therapy. In anemia an increased IRF gnerally indicates an adequate eyrthropoietic response, whereas a normal or subnormal IRF shows an inadequate or no response. - The bone marrow increases production of RBCs, an observable increasse in the IRF occurs before an increase in the reticulocyte count or an increase in hemoglobin, hematocrit, or RBC count.

Correlate polychromatophilia on a blood smear with other laboratory results of erythrocyte production and destruction

Poluchromatophilic RBCs (reticulocytes) are usually larger than normal cells with a bluish tinge on romanowsky stains. The bluish tinge is caused by risidual RNA in the cytoplasm, large numbers of the cells are associated with decreased RBC survival or hemorrhage and an erythroid hyperplastic marrow.

Explain the molecular control of total body iron and cellular iron

Cellular level - The control of iron balance at the cellular level occurs by regulation of transcription and translation proteins involved in iron metabolism.

Define Hemoglobinopathy

Clinical diseases that result from a genetically determined abnormality of the structure or synthesis of the hemoglobin molecule are called hemoglobinopathies.

Pappenheimer Bodies

Clusters of granules containing iron that are usually found at the periphery of the cell. Visible with Prussian Blue stain and Wright's stain Seen in Sideroblastic anemia, thalassemia; other severe anemias

Define Thalassemia

Constitutes a family of inherited disorders in which mutations in one or more of the globin genes of hemoglobin cause decreased or absent synthesis of the corresponding globin chains.

5 Genetic Defects in Thalassemia

Deletion (Large) - Predominantly alpha-thalassemia, some Beta- Thalassemia. - Loss of gene - Absence of production for globin chains Promoter - Predominatnly Beta-Thalassemia - Reduced or absent globen chain production Nonsense - Beta-Thalassemia - Imparied transcription, In frame substitution, frame shift. - Effect on globin chain - Amino acid change, amino acid changes distal to shift. Stop Codon - Beta-Thalassemia - Convert stop codon to amino acid codon - Slightly lengthened globin chains (retained), significantly lengthened globin chain (degraded) Splice site - Beta-thalassemia - Creates now splice sites - Slightly shortened globin chain (retained) - Significantly shortened globin chain (degraded) - Loss of splice sites - Slightly lengthened globin chain (retained) Significantly lengthened globin chain (degraded) -Unalterd globin chain

Define Hemolysis and reconcile a normal hemoglobin concentration in compensated hemolytic disease

Destruction of the RBC resulting in the release of hemoglobin. In hemolytic anemia, the premature destruction of RBCs. Compensated Hemolytic Disease - Disorder in which the RBC life span is decreased but the bone marrow is able to increase erythropoiesis enough to compensate for the decrease; anemia does not develop. - Which can readily develop into anemia if 1. erythrocyte destruction accelerates beyond the compensatory capacity of the BM (hemolytic crisis) or 2. the marrow suddenly stops producing RBCs (aplastic crisis)

Identify poikiliocytes and anisocytes on a blood smear and describe their mechanism of formation and correlate them with pathological conditions

Elliptocyte (oval) Ovalcyte, pencil cell; cigar cell Oval to elongated ellipsoid cell with central area of pallor and hemoglobin at both ends. Seen in hereditary elliptocytosis; iron deficiency anemia, thalassemia;l anemia accodiated with leukemia Keratocyte (horn) helmet cell Red cells with one or several notches with projections that look like hons on either end Seen in microangiopathic hemolytic anemias, heart-valve hemolysis; heinz-body hemolytic anemia, glomerulonephritis; cavernous hermgiomas Knizocyte RBC with more than two concavitites; on stained blood smears has a dark band of hemoglobin across the center with a pale area on either side

Describe the basic defect of porphyria.

Porphyria is characterized by a block in porphyrin synthesis. As a result of these enzyme deficiencies the porphyrin precursors behind the enzyme defect accumluate in the tissues, and large amounts are excreted in the urine and feces.

Hemoglobin Chain Formations

Embryonic - Gower 1 = Zeta2Epsilon2 - Gower 2 = Apla2Epsilon2 - Portland = Zeta2Gamma2 Fetal - HbF = Alpha2Gamma2 (90-95% before birth) and (50-85% at birth) - HbA = Alpha2Beta2 (10-40% at birth) - HbA2 = Alpha2Delta2 (<1% at birth) >1 Year Old - HbF = Alpha2Gamma2 (<2%) -HbA = Alpha2Beta2 (>95%) - HbA2 = Alpha2Delta2 (<3.5%) Adult -HbA = Alpha2Beta2 (>95%) - HbA2 = ALpha2Delta2 (1.5-3.7%) - HbF = Alpha2Gamma2 (<2%) Note: Hb Bart's = Gamma4

Identify Laboratory Tests used to evaluate erythrocyte destruction and production.

Erythrocyte survival studies A sample of the patients blood is removed and labeled in vitro with trace amounts of readionuclide. The most common label for erythrocytes and that recommended by the international committee for standardization in Hematology, is radioactive chromium (51^Cr). The cromium penetrates the RBCs and remains trapped there. This labeled sample is injected intravanously into the patient. To determin the RBC survival pattern, small samples of the patients blood are assayed at specific time intervals for radioactivity levels. The life span is expressed as the time it take for blood radioactivity to decrease by one-half, starting 24 hours after injection. The expected life spance is 100-120 days. Increased RBC Destruction Lab findings Anemia, Presence of spherocytes, schistocytes and/or other poikilocytes, Positive DAT, Decreased Haptoglobin and hemopexin, Decreased glycosylated hemoglobin, increased fecal and urine urobilinogen, increased bilirubin (unconjugated), hemoglinemia, hemoglibinuria, hemosiderinuria, methemoglinemia, increased serum LD, increased expired CO. Increased Bone Marrow Production of RBCs - Reticulocytosis (>100 C 10^9/L; RPI >2) - Increased IRF - Leukocytosis - NRBCs in peripheral blood - Polychromasia of RBCs on romanowsky stain - Normoblastic erythroid hyperplasia in the BM Decreased BM production of RBC - Anemia - Decreased retics (<25 X 10^9/L; RPI <2). Corrected Retic count <2%; Decreased IRF - Erythroid hypoplasia in the bone marrow; increased M:E ratio.

Identify the different therapies for SCA (sickle cell anemia)

Frequent moniroting of the patient status using laboratory assays is needed. Treatmnet includes both immediate and prophalyactic or long-term approaches. Immediate transfusion therapy may be ordered for complications. Transfusion from normal donors dilutes the amount HbS present, restores oxygenatation of tissues and semporarily suppresses erythropoiesis of new cells containing HbS. Hemopoietic stem cell transplantation, affords the potential for the cure of sickle cell disease. Results of multicenter case series show disease-free survival rates. Additional treaments include prophylactic penicillin, folic acid, immunization, hydration and analgesics for pain.

Identify the globin chain defects causing SCA, hemoglobin C (HbC) disease and hemoglobin E (HbE) disease

HbS - Normocytic, Normochromic anemia; reticulocytosis; poikilocytosis with sickled and boat-shaped cells. Homozygous: HbS, F, A2 Heterozygous: HbA, S, F and A2 Geographical Distribution: Tropical Africa and Mediterranean areas, Middle East, India and Nepal Mutation Lys: B6(A3) Glu--> Val HbE: Microcytic, Hypochromic anemia; Target Cells Homozygous: HbE, F and A2 Heterozygous: HbA, E, F, A2 Geographical Distribution: Burma, Thailand, Cambodia, Malaysia, Indonesia HbC: Normocytic, normochromic anemia; reticulocytosis, poikilocytosis with folded, irregularly contracted cells; target cells. Homozygous: HbC, F, A2 Heterozygous: HbA, C, F, A2

Recognize and identify abnormal laboratory test results, including peripheral blood findings and screening and confirmatory tests, typically associated with homozygous and heterozygous conditions involving HbS HbC, HbD and HbE and other variant hemoglobins. Also List major clinical findings typically associated with the hemoglobinopathies listed.

HbS - The first clinical signs of sickle cell anemia appear at about 6 months of age when the concentration of HbS predominates over HbF. Clinical manifestations result from chronic hemolytic anemia, vaso-occlusion of the microvasculature, overwhelming infections and acute splenic sequestration. - Normocytic, Normochromic anemia is characteristic of sick cell anemia; however with marked reticulocytosis, the anemia can appear macrocytic. Retic count of 10-20% is common. The hemoglobin ranges from 6-10g/dL and the hematocrit from 18-30%. - The blood smear shows variable anisocytosis with polychromatophilic macrocytes and variable poikilocytosis with a presence of sickle cells and target cells. NRBCs can also be found. Increased RDW. HbC - Mild to moderate normocytic, normochromic anemia is present. Hematocrit is usually >25% and the hemoglobin concentration is between 10 - 14 g/dL. The higher hemoglobin concetration does not necessarily mean that hemolysis is less severe than sickle cell anemia. Blood smears will show large numbers of target cells but rarely sickled forms. Typical HbC crystals are rarely found.

Compare and contrast other thalassemia and thalassemia like conditions including: Hemoglobin Constant Spring Hereditary persistence of fetal hemoglobin (HPFH) Hemoglobin Lepore Thalassemia/Hemoglobinopathy combination disorders.

Hemoglobin Constant Spring - is a hemoglobin tetramer formed from the combination of two structurally abnormal alpha-chains, each elongated by 31 amino acids at the carboxy-terminal end and two normal Beta-chains. This genetic mutation is common in Thailand. - The homozygous state is phenotypically similar to alpha-thalassemia minor. A slight anemia accompanied by mild jaundice and splenomegaly is typical. Heterozygotes show no clinical abnormalities. - Small amounts of HbCS (0.2 to 1.7%) can be found on electrophoresis. HPFH - characterized by either deletion or inactivation of the Beta and delta structural gene complex. Mutations in the gamma globin gene promoter region affecting the binding of transcription factors or mutation of gene inhibitor proteins. Hemoglobin Lepore - Dispersed throughout the world but primarily in the middle east and eastern europe - The non-alpha-chain is a deltabeta-globin hybrid in which the N-terminal end of the delta chain is fused to the C-terminal end of a B-chain. Combination disorders Occasionally an individual is doubly heterozygous for a structural hemoglobin variant and thalassemia, inheriting one of each of the two abnormalities from each parent. The most common structural hemoglobin varants involved in combination disorders are HbS, HbC and HbE. When a structural variant is inherited with a Beta-Thalassemia gene, the severity of the combination disorder dpends on the type of B-gene mutation. In the case of homozygous BsBs individual with an alpha-thalassemia syndrome, the clinical severity of sickling is often reduced because of net decrease in MCH which reduces the tendency of HbS to produce cell sickling.

Compare the prevalence of Hemoglobins S,C,D, and E

Hemoglobin D - Found in Indians, and african americans Hemoglobin E - Second most prevalent Hemoglobinopathy worldwide. MOst common in people from south-east asia. Trait reached frequencies of almost 50% in areas of Thailand. Estimated 15-30% of immigrants from southeast asia living in north americaa have HbE. HbS - Worldwide this is the most common symptomatic hemoglobinopathy. Greatest prevalence in tropical Africa. Additional testing for HbS - Solubility test - is a rapid test for detecting HbS in the heterozyous or homozygous state. In severe anemia, the amount of HbS can be too low to be accurately detected and the procedure may need to be altered. Should not be used on newborns due to the high level of HbF and low concentration of HbS in this age group. Heinz bodies can cause false positives along with HbC, Harlem and HbI and elevated plasma proteins and lipds. Sickling test - Another confirmatory test that is performed less often is the sodium metabisulfite slide test. Tests positive for both Sickle Cell anemia and trait. HbC - the second hemoglobinopathy to be recognized, and the third most prevalent hemoglobinopathy variant world wide. Found predominatly in west african blacks whom the incidence of the trait can react 17-28% of the population. From 2-3% of African Americans carry the trait and 0.02% have the disease.

Explain the basis of hemoglobin electrophoresis in identifying variant hemoglobins

Hemoglobin carries an electrical charge resulting from the presence of ionized carbcyl (COO-) and protonated (H+) amino (NH3+) groups. The type (net positive, net negative) and Strength of the charge depend on both the amino acid sequence of globin chains in the hemoglobin molecule and the pH of the surrounding medium.

Explain the physiologic abnormality resulting in unstable hemoglobins, the methemoglobinemia, and hemoglobin variants with increased or decreased oxygen affinity.

Hemoglobin variants with Increased oxygen affinity - High-affinity hemoglobins are inherited as autosomal dominant traits. All such hemoglobins discovered have been in the heterzygous state. These bind to oxygen more readily than normal and retain more oxygen at lower PO2 levels, which results in a shift to the left of the oxygen dissociation curve. RBC counts and Hct are increased. and hemoglobin increases to about 20g/dL. Hemoglobin Variants with Decreased oxygen affinity - Resutls from mutations that stabilize or favor the deoxygenated (T) conformation of the hemoglobin molecule or destabilize the oxygenated (R) form. These mutations impair oxygen binding to reduce heme-heme subunit interactions. These are often associated with Alpha1Beta2 contact points, which are involved in intramolecular movement as hemoglobin goes from the oxy (R) to deoxy (T) state. These show a right shift on the oxygen dissociation curve. Methemoglobinemias - Is a hemoglobin with iron oxidized to the ferric state, which cannot carry oxygen. This is a clinical condition that occurs when methemoglobin encompasses >1% of the hemoglobin. It is either acquired or inherited in the methemoglobin reductase system. or as a result of the presence of a structurally abnormal globin chain.

Differentiate thalassemias from hemoglobinopathies based on definition and basic pathophysiology.

Hemoglobinopathy RBC - Decreased Indices - Normocytic, Normochromic Erythrocyte Morphology - Target Cells, Sickle cells, HbC crystals Abnormal Hb - HbS, HbC, HbE etc.. Hb Solubility test - + in HbS, Hb Bart's and HbCharlem. Ancestry - Afraican, Mediterranean, middle eastern, southeast asian. Reticulocyte count - Double increase Thalassemia RBC - Increased (compared to what is expected for the Hb level) Indices - Microcytic, Hypochromic Erythrocyte Morphology - Target cells, basophilic stippling Abnormal Hb - HbH (Beta4), Hb Bart's (Gamma4) Hb Solubility test - Negative Ancestry - Mediterranean, South east asian, african. Reticulocyte count - increased.

Describe the function of the proteins invlolved in iron metabolism including hepcidin, HFE, transferrin receptor, hemojuvelin, divalent metal transporter 1, duodenal cytochrome-B reductase, hephaestin, and ferroportin.

Hepcidin - Master iron-regulating protein; regulates iron recycling/balance via interaction with ferroportin 1; negative regulator of intestinal iron absorption; additional proteins influence synthesis HFE - Interacts with Transferrin receptor1 and Transferrin receptor 2 to control hepcidin expression. Found primarily in hepatocytes. Can bind to both Transferrin receptors. In the basal state of iron metabolism, HFE and TfR1 exist as a complex on the cell membrane. Diferric Transferrin and HFE compete for binding to TfR1. Transferrin Receptors - Receptor 1 (TfR1) binds diferric-transferrin to the cell for internalization of iron; can form molecular complex with HFE. Receptor 2 (TfR2) Forms molecular complex with HFE to regulate hepatic hepcidin expression; can bind to transferrin Hemojuvelin (HJV) - Regulates hepcidin expression, is a glycosylphosphatidylinositol- anchored membrane bound BMP coreceptor that enhances hepcidin gene expression. HJV mutations are associated with decreased hepcidin synthesis and severe iron overload. Divalent metal Transporter 1 (DMT1)- Transports iron across the enterocyte apical plasma membrane; transports iron across endosome into cytoplasm of cells. Duodenal Cytochrome-B reductase (DCytB) - Reduces Fe+++ iron to Fe++ at enterocyte apical border Hephaestin - FAcilitates cellular export of iron by ferroportin; oxidizes Fe++ iron to Fe+++ for binding to apotransferrin Ferroportin - Exports cellular iron from enterocytes, macrophages and hepatocytes

Describe the role of molecular diagnostic in hereditary sideroblastic anemia

If hereditary sex-linked SA is suspected based on erythrocyte morphology and iron studies, the patient should be tested for the pesence of ALAS2 gene mutations. This can be done by studying enomoic DNA or cDND (from RNA) from reticulocytes. If a mutation is found, other family members should be tested because even if anemia is not present, there is a risk of iron overload. Molecular studies also allow a distinction between hereditary SA and RARS

Differentiate primary (hereditary) and secondary hemochromatosis and summarize typical results of iron studies in these conditions.

Primary Hemochromatosis - Characterized by increased iron absorption in the guy and progressive iron overload. It is a genetic disorder with a prevalence of 1 in 200-250 persons. Secondary Hemochromatosis - Associated with a number of conditions including aneamis that have an ineffective eyrthropoiesis component and increased iron absorption. These include Beta-Thalassemia.

List the laboratory tests that can be used to help identify the pathophysiological mechanisms of anemia and provide expected results.

Initial screening test is the CBC, which includes the RBC count, hemoglobin, hematocrit, indices, WBC count, platelet count, and depending on the instrumentation, the differential count. - Depending on the test results additional tests such as reticulocyte count, bilirubin and microscopic review of the blood for abnormal cell morphology can be suggested. In addition, the urine and stool can be examined for the presence of blood.

Assess laboratory results in Intravascular and Extravascular hemolysis.

Intravascular Hemolysis - Caused by 1. the activation of complement on the RBC membrane. 2. Physical or mechanical trauma to the RBC or 3. the presence of soluble toxic substances in the erythrocytes environment. - Free hemoglobin is release into the plasma and binds to haptoglobin and the haptoglobin-hemoglobin complex is taken to the liver to be catabolized. - Lab findings include hemoglobinema and hemoglobinuria, hemosiderinuria, methemoglobinemia, decreased haptoglobin and decreased hemopexin. Serum lactic Dehydrogenase (LD) can increase as much as 800 IU/L. LD can be an early and sensitive indicator of intravascular hemolysis because the RBCs releases LD into the plasma. And LD leaves the plasma slower than the Hemoglobin does. Extravascular Hemolysis- Is more common than intravascular hemolysis. Results in premature RBC death when phagocytes in the tissues remove RBCs from circulation. Hemoglobin is not released directly to the plasma so there no hemoglobinemia, uria, or siderinuria. Hemoglobin is degraded within the phagocyte to heme. -Lab Findings in extravascular hemolysis are the measurements of products of heme catabolism. These findings include increases in expired carbon monoxide, carboxyhemoglobin, serum bilirubin (especially unconjugated) and both urine and fecal urobilinogen. In severe or chronic extravacular hemolysis, haptoglobin and hemopexin levels also can be decreased.

Explain the difference between Intrinsic and Extrinsic RBC defects

Intrinsic (Intracorpuscular) Mode of onset - Inherited Site of hemolysis - Extravascular Predominant Poikilocyte - Spherocyte Extrinsic (Extracorpuscular) Mode of Onset - Acquired Site of Hemolysis - Extravascular or intravascular depending on the extent of cell damage Predominant Poikilocyte - Schistocyte

Treatment for Iron deficiency, Anemia of Chronic disease and Sideroblastic anemia

Iron Deficiency - Oral treatment by oral administration of ferrous sulfate Anemia of chronic disease - Can be treated by successful treatment of the underlying disease, transfusion is rarely required except in older patients with vascular disease and circulatory insufficiency. Sideroblastic Anemia - Pyridoxine therapy is generally tried on patients with hereditary SA: <50% expereince a return to normal hemoglobin levels. Iron overload decreases responsiveness to pyridoxine, but when the iron load is reduced by phlebotomy, hemoglobin concentration increases.

Explain the etiology and pathophysiology of iron-deficiency anemia, anemia of chronic disease and sideroblastic anemia

Iron Deficiency Anemia - Etiology - Can occur because of normal or pathologic conditions that result in an increased demand for iron, malabsorption, or poor diet. In malabsorption or with an iron-deficient diet, iron stores can become depleted over a period of years. Pathophysiology - defined as a diminished total body iron content, develops in sequential stages during a period of negative iron balance. Anemia of chronic disease - Block in release of iron from macrophages due to increased hepcidin. Cytokine ihibition of EPO production, Direct cytokine inhibition of erythropoiesis, Shortened erythrocyte survival. Sideroblastic anemia - Mutations that affect the first enzymatic step in heme synthesis, the formation of ALA, result in sideroblastic anemia. This is the result of diverse clinical biochemical manifestations that reflect multiple underlying hereditary, congenital or acquired pathogenic mechanisms.

Compare and Contrast the typical blood features and iron studies associated with Iron Deficiency Anemia (IDA), Anemia of Chronic Disease (ACD), Lead poisoning, and sideroblastic anemia

Iron Deficiency Anemia - Microcytic (MCV 55-74fL) Low Hypochromic (MCHC 22-21 g/DL, MCH 14-26 pg) Ferritin - Decreased (single best way to detect Iron deficiency) Iron Saturation - Low Serum Iron - Low Serum Transferrin Receptor - Increased Bone Marrow Iron Stores - Decreased Anemia of Chronic Disease - MCV - Low/Normal Serum Iron - Low Saturation % - Low to normal Ferritin - Normal to Increased Serum Transferrin Receptor - Normal to Increased Bone Marrow Iron Stores - Increased Lead Poisoning - Basophilic Stippling Serum Ferritin - Normal Serum Iron - Variable TIBC - Normal Transferrin Saturation - Increased FEP/ZPP - Increased (Marked) Bone Marrow - Normal Sideroblastic Anemia- Serum Iron - Increased TIBC - Normal or Decreased Ferritin - Increased Bone Marrow Stores - Increased Saturation - Increased

Sideroblast

Iron Granules found in erythroblasts. Stains blue with Perl's Prussian blue stain but not with romanowsky stains

Correlate the following laboratory features with iron deficiency and sideroblastic anemia: RBC morphology Iron studies Bone Marrow

Iron deficiency RBC Morphology - Microcytic and Hypochromic (MCV 55-74fL) and (MCHC 22-31 g/L, MCH 14-28pg) Iron Studies - serum iron is decreased usually less than 30ug/dL, TIBC is increased and Transferrin saturation is decreased to less than 15%, serum ferritin is decreased. Bone Marrow - Total Cellularity is often moderately increased. mild to moderate erythroid hyperplasia with decreased M:E ratio. Sideroblastic Anemia RBC morphology - Dimorphic red cells, normochromic and hypochromic cells. Bone Marrow - Erythroid hyperplasia often accompanied by various degrees of megalobastosis. Erythroblasts appear poorly hemoglobinized with scanty, irregular cytoplasm. Ring sideroblasts constitute more than 40% of the erythroblasts.

Siderocyte

Iron granules found in erythrocytes. Stains blue with Perl;s Prussian blue stain not stain with romanowsky stains.

Monocyte

Is produced in the bone marrow from a bipotential progenitor cell, the GMP. Which is capable of producing either mature monocytes or neutrophils. The differentiation of the monocyte or neutrophil is dependent on the action of CM-CSF, IL-3 and M-CSF. The primary function is host defense. Monocytes and macrophages can be stimulated by T lymphs and endotoxin to liberate endogenous M-CSF, which can be one mechanism for the monocytosis assiated with some infections. M-CSF also activates the secretory and phagocytic activity of monocytes and macrophages. Monocytes and Macrophages make up the monocyte-macrophage syste, (Mononuclear phagocyte (MNP) system.)

State Principles of light scatter used in cell counting and identify the instruments that use this technology

Light scattering principle of blood cell counting is based on light-scattering measurements obtained as a single blood cell passes through a beam of light (optical or laser). Blood cells create forward scatter and side scatter that photodetectors detect. The degree of forward scatter is a measurement of cell size and the degree of side scatter is a measurement of cell complexity or granularity. The siemens healthcare instruments use this principle for all blood cell counts and newer models manufactured by sysmex corporation and abbot diagnostics use this principle for leukocyte enumeration and differentials.

Explain the significance of Microcytic anemia in the presence of lead poisoning. What kind reflex testing should be ordered?

Microcytic, hypochromic anemia is not characteristic of elevated lead levels. Evidence suggests that the precence of microcytic anemia is most likely due to complications of Iron Deficiency or to the coexistance of thalassemia trait. ZPP should NOT be used for reflex testing in this case. Direct lead measurement SHOULD be used.

Explain the Morphologic and Functional Classification of Anemia

Morphologic Anemia can be classified by size, and hemoglobin concentration of the RBCs as indicated by the Indices. The morphologic classification is helpful because MCV, MCH, and MCHC are determined when anemia is diagnosed and certain causes of anemia are characteristally associated with specific RBCs size, and hemoglobin content. Lab Findings: Microcytic, Hypochromic: Anemias are associated with defective hemoglobin synthesis. Serum Iron studies and occasionally hemoglobin electrophoresis are usually adequate to differentiate the causes of these anemias. - Macrocytic anemia: Are associated with hemolytic anemias (RPI >2) nuclear maturation defects (megaloblastic anemia (RPI <2) or nonmegaloblastic anemia (RPI <2). Diagnostic features include hypersegmented granulocytes can be found on peripheral blood smears and supported by low Vit B12 or Folic acid levels. Hmolytic anemias with an increased MCV can be due to reticulocytosis can be diagnosed by reviewing the blood smear and other tests. - Normocytic, normochromic anemia: Many anemias have normal RBC morphology, these include hypoproliferative anemias characterized by an RPI <2 and survival defects with a RPI >2. The hypoproliferative anemias are chacterized by a hypocellular bone marrow with normal or increased M:E ratio. Hemolytic anemias have a hypercellular bone marrow with decreased M:E ratios. Functional Classification Because the normal bone marrow compensatory response to decreased peripheral blood hemoglobin levels is an increase in erythrocyte production, persistent anemia can be expected as the result of three pathophysiologic mechanisms. 1. Proliferation defect (decreased production), 2. a maturation defect or 3 a survival defect. Proliferation Defects: Characterized by decreased proliferation, maturation and release rates of RBCs in response to anemia. The most characteristic lab finding iof proliferation defects are nomrocytic, normochromic RBCs, decreased absolute Retic count and decreased corrected retic count and IRF and RPI <2. Decreased proliferation can be caused by inappropriate erythropoietin production or production of cytockines that inhibit erythropoiesis. Maturation Defects: disrupt the orderly process of either nuclear or cytoplasmic development producing qualitatively abnormal cells. The RBCs are macrocytic in nuclear defects and microcytic in cytoplasmic defects. Survival defects: are the result of premature loss of circulating erythrocytes either by hemorrhage or hemolysis.

Neutrophils Differentiation, Maturation and Morphology

Neutrophils are the most numerous leukocyte in the peripheral blood. Leukocytes deleop from HSC in the bone marrow. The common myeloid progenitor (CMP) cell gives rise to the committed precursor cells for the neutrophilic, eosinophilic and basophilic and monocytic lineages. The life span of the neutrophil is spent in three compartments, the bone marrow, peripheral blood (where they stay for a few hours) and the tissues (where they perform host defense). -Neutrophilic production is primarily regulated by three cytokines. Interleukin-3 (IL-3), granulocyte monocyte-colony-stimulating factor (GM-CSF), and the granulocyte-colony-stimulating factor (G-CSF). GM-CSF and G-CSF regulate the survival and functional activity of mature neutrophils. The phases of life for the neutrophil is 1. Myeloblast 2. Promyelocyte 3. Myelocyte 4. Metamyelocyte 5. Band 6. Segmented Neutrophil Granule Contents Primary Granules - Myeloperoxidase -Lysozyme - Cathepsin G, B, D - Defensins (group of cationic proteins) - Bactericidal permeability increasing protein (BPI) - Esterase N _ Esterase Secondary Granules - Lactoferrin - Lysozyme - Histaminase - Collagenase - Gelatinase - Heparinase Secretory Vesicles - Alkaline phosphatase - Complement Receptor 1 - Cytochrom B558 Teritiary Granules - Gelatinase - Lysozyme Function: Neutrophils must move ot the site of the foreign agent, engulf it and destroy it. Thus neutrophils function primarily in the tissues where microbial innvasion typyically occurs. Monocytes-macrophages help in this process but are slower to arrive at the site. When blood is drawn from a vein only half of the neutrophils are actually sampled. Why? half of the neutrophils are marginated in the vessels

Hemoglobin Concentrations

Normal Adult: A - 97% A2 - 3% F - <1% S - 0 C - 0 Normal Neonate A - 20-25% A2 - <1 F - 75-80% S - 0 C - 0 Sickle Cell Disease (SS) A - 0 A2 - N F - 5-20% S - 80-95% C - 0 S trait (SA) A - 50-65% A2 - N F - N S - 35-45% C - 0 C disease A - 0 A2 - N F - <7% S - 0 C - >90 C trait (CA) A - 60-70% A2 - N F - N S - 0 C - 30-40% SC disease A - 0 A2 - ? F - ? S - >50% C - <50%

Identify poikiliocytes and anisocytes on a blood smear and describe their mechanism of formation and correlate them with pathological conditions

Picture Leptocyte - Thin Cell Thin, flat cell with hemoglobin at peripher; usually cup-shaped, MCV is decreased but cell diameter is normal Seen in Thalassemia, iron deficiency anemia. hemoglobinopathies, liver disease Schistocyte - Fragmented Cell Fragments of red cells; variety of shapes including triangles, commas; and microcytic. Microangiopathic hemolytic anemias, heart-valve hemolysis, disseminated intravascular coagulation, severe burns, uremia Sphereocyte - Spherocytic red cells with dense hemoglobin content (hyperchromatic ) lack an are of central pallor Stomatocyte Uniconcave red cells with the shape of a very thick cup; on stained blood smears cells have an oval or slitlike area of central pallor. Seen in hereditary stomatocytosis, shereocytosis, alcoholic cirrhosis, anemia associated with Rh null disease, lead intoxication, neoplasms

Explain the basis of defects resulting in the production of variant hemoglobins

Qualitiative defect or Quantitative defect. Qualitiativly abnormal hemoglobin molecules arise from genetic mutations in the coding region of a globin gene. Resulting in amino acid deletions or substitutions in the globin protein chains. Quantitative globin disorders result from various genetic defects that reduce synthesis of structurally normal globin chains.

Caclulate the reticulocyte production index from reticulocyte results, hematocrit, and RBC count

Retic count calculation Patient Hct \ Normal Hematocrit x Retic % = Corrected Retic count Retic Production Index Patient's Hct \ 0.45 (normal hct) X retic count % \ Reticulocyte maturation time (days) = RPI Maturation time >= to 0.35 - 1.0 days >= to 0.25-0.35 - 1.5 days >= to 0.15-0.25 - 2.0 days <= 0.15 - 2.5 days

RBC Inclusions Basophilic Stippling

Round or irregularly shaped granules of variable number and size, distributed throughout the RBC. Composed of aggregates of ribosomes (RNA) Stain blueish black with wrights stain Seen in Lead poisoning, anemias associated with abnormal hemoglobin synthesis and thalassemia.

Segmented Neutrophil

Same size as the band. Observing 3 or more 5 lobed neutrophils (heypersegmented) is considered pathogenic. Neutrophilic granules contain protein, lipids and carbohydrates.

Describe the typical periperal blood morphology associated with thalassemia

Thalassemia is characterized by microcytic, hypochromic anemia with a decrease in MCV, MCH, and usually MCHC. The RBC count is often normal or slightly decreased but increased relative to the hemoblobin and hematocrit levels.

Compare and contrast the pathophysiology of common hemoglobin variants in terms of altered solubility, function and stability.

Sickle Cell (HbS) Altered Solubility - Hemoglobin S and C are examples of a nonpolar amino acid is substituted for a polar residue near the molecule's surface, the solubility for the hemoglobin molecule can be affected. In deoxygenated state, the HbS molecule polymerizes into insoluble, rigid aggregates. The majority of surface substitutions , however do not affect the teriary structure, heme funtion or subunit interactions and are therefore innocuous. Altered Function - Some amino acid substitutions can affect the oxygen affinity of hemoglobin by stabilizing hene iron in the ferric state, producing methemoglin which cannot combine with oxygen. Mutations within the subunit interface, Alpha1Beta2 can affect the allosteric properties of the molecule. Altered Stability - Amino acid substitutions that reduce the stability of the hemoglobin tetramer result in unstable hemoglobins. The mutations usually disrupt hydrogen bonding or hydrophobic interactions that retain the heme component within the heme-binding pocket of the globin chain or that hold the tetramer together.

Howell-Jolly Bodies

Small round bodies composed of DNA usually locatged eccentrically in the red cell. USually occurs singly, rarely more than two per cell. Stains Dark purple with Wright's stain. Seen in post-splenectomy; megaloblastic anemias; some hemolytic anemias; functional asplenia; severe anemia.

Band Neutrophil

Smaller diameter than the metamyelocyte. The meta myelocyte becomes a band when the indentation of the nucleus is more than half the diameter of the hypothetical round nucleus. All 4 of the granules types (primary, secondary, secretory and tertiary) can be found at this stage.

List the three stages of iron deficiency and define characteristic RBC morphology of each stage AND Compare and Contrast iron stores, hemoglobin, serum iron, TIBC saturation, serum ferritin, and RBC morphology in the three stages of iron deficiency.

Stage 1 During iron depletion, iron stores are exhausted as indicated by a decrease in serum ferritin. There is no anemia and erythrocyte morphology is normal. but the RDW is frequently elevated. The RDW can be the first hematologic indicator for developing Iron Deficiency. Ferritin - Decreased Hemoglobin - Normal Serum iron - Normal TIBC - Normal or Slight increase Saturation % - Normal RBC morphology - Normocytic, Normochromic Stage 2 Characterized by iron=deficient erythropoiesis. There is insufficient iron to insert into the protoporphyrin ring to form heme. As a result the protoporphyrin accumulates in the cell and complexes with zinc to form ZPP. Bone marrow sideroblasts are absent; macrophage iron is not seen. - Anemia and hypochromia are still not detectable. But the erythrocytes can become slightly microcytic. The CHr which measures the functional availability of iron during hemoglobin synthesis can decrease even when the anemia is absent. Ferritin - Decreased Hemoglobin - Normal Serum iron - Decreased TIBC - Increased Saturation % - Decreased RBC morphology - Normocytic, Normochromic Stage 3 A long standing negative iron flow eventually leads to the last stage. Blood loss can significantly shorten the time for this stage to develop. Most significant finding is classic microcytic hypochromic anemia. Ferritin - Decreased Hemoglobin - Decreased Serum iron - Decreased TIBC - Increased Saturation % - Increased RBC morphology - Microcytic, Hypochromic

Calculate Transferring saturation and Unsaturated binding capacity (UIBC)

The formula for Transferrin saturation is TIBC X 100 = % Transferrin Saturation. The formula for Unsaturated Iron-binding capacity is (UIBC) = TIBC - serium iron.

Cite the electrical impedance principle of cell counting and identify the instruments that use this technology.

The impedance prinicple of blood cell counting is based on the increased resisance that occurs when a blood cell with poor conductivity passes through an electrical field. The number of pulses indecates the blood cell count and amplitude of each pulse is proportional to the volume of the cell. Examples include: Beckman Coulter, Inc. Sysmex Corporation and Abbot Diagnostics. Coulter LH series, Beckman-Coulter Unicel DxH 800, Sysmex XE-Series, Sysmex XN-series, Abbot Cell-Dyn Sapphire, Siemens Healthcare ADVIA 120.

Monoblast

The monoblast nucleaus is most often ovoid or round but can be folded or indented. Monoblasts are large (12-20 mcM) The pale blue-purple nuclear chromatin is finely dispersed (lacy) and several nucleoli are easily identified. The monoblast has abundant agranular blue-gray cytoplasm.

Myeloblast

The myeloblast is the earliest morphologically recognizable precursor of the myeloid lineage. The myeloblast size varies from 14-20 um. in diameter. Has a high Nuclear to cytoplasmic ratio (N:C). Nucleus is usually round or oval and contains a delicate, lacy, evenly stained chromatin. One to five nucleoli are visable. The small amount of cytoplasm is agranular, staining from deep blue to a lighter blue. A distinct unstained area adjacent to the nucleus representing the golgi apparatus can be seen. - Myeloblasts can stain faintly posive for peroxidase and esterase enzymes and for lipids (sudan black B) although granules are not evident by light microscopy. Staining reactions with peroxidase and esterase help differentiate myeloblasts from monoblasts and lymphoblasts. - CD markers include CD33, CD13, CD38 and CD34.

Promyelocyte

The promyelocyte/progranulocyte varies in size 15-21 mcM. The nucleaus is still quite large and the N:C ratio is high. Nuclear chromatin structure, although coarser than that of the myeloblast. It is still open and rather lacy, staining purple to dark blue. Several nucleoli can be seen. The basophilic cytoplasm is similar to that of the myeloblast but is DIFFERENTIATED by the presence of prominent, reddish-purple primary granules also called nonspecific or azurophilic granules, which are synthesized during this stage. The primary granules are surrounded by a phospholipid membrane and contains peroxidase and a nmber of animicrobial compounds.

Define the following terms and explain their role in iron metabolism: Transferrin Hemosiderin Ferritin TIBC

Transferrin - Plasma iron transport protein that mediates iron exchange between tissues. It is NOT lost in delering iron to the cells but returns to the plasma to be reused. - Transferrin with no iron attached is called apotransferrin. One iron attached is Monoferric transferrin and if both binding sites are used its called Diferric transferrin Hemosiderin - Is a hertogeneous aggregate of carbohydrate, lipid, protein and iron. Up to 50% of it's weight is iron. Found primarily in macrophages and is formed by the partial degradation of ferritin. At lower celluar iron concentrations ferritin predominates, but at higher cellular concentrations The majority of storage iron exists as hemosiderin. - Hemosiderin can be estimated on bone marrow tissue sections. Bone marrow macrophages contain hemosidering if body iron stores are normal or increased. Hemosiderin appears yellow to brown refractile pigment on unstained marrow or liver specimens. On prussian blue-stained specimens, the iron appears as blue intracellular particles. Normally from 20=60% of the erythroblasts contain stainable iron deposites. Ferritin - Consists of a spherical protein shell that can store up to 4500 molecules of Fe+++ iron. Ferritin is 17-33% iron by weight; without iron it is called apoferritin. Acts as the primary storage compound for the body's iron and is readily abailable for erythropoiesis also increased in inflammation. It controls the amound of iron released for cellular activity and by binding the iron, protects the cellular constituents from oxidative damage catalyzed by free ferrous ions. = Ferritin is found in the bone marrow, liver, and spleen, usually within membrane-bound vesicles called siderosomes. Mature erythrocytes generally do not contain iron particles because any excess iron in the cell after hemoglobin synthesis is complete is removed by splenic macrophages. (Reference Values 20-300 ug in males 12-200 in females) Total Iron Binding Capacity (TIBC) - Each gram of transferrin binds 1.4 mg of iron. Enough transferrin is generally present in the plasma to bind 250 - 450 ug of iron per deciliter of plasma. This is referred to as the TIBC. - The formula for Transferrin saturation is TIBC X 100 = % Transferrin Saturation. - The formula for Unsaturated Iron-binding capacity is (UIBC) = TIBC - serium iron.