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Hematology Objectives Mod 4 and 5

Terms in this set (73)

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.
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.
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.
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.
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.
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 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