Anemias and RBC Dyscrasias

Kay Case PA-CNSU Nova SWF PA Program

Fall 2010

1

Objectives1. Understand the regulation of the bone

marrow in a steady state. What are the triggers, the stimulators of each cell line, and which stem cell progenitors produce each cell line.

2. Understand hypocellular and aplastic states of the bone marrow and their causes.

3. Understand the trigger for formation of erythropoetin by the kidney.

2

Objectives

4. Identify causes of increased production of red blood cells by the bone marrow as indicated by an increased reticulocyte count – Acute blood loss– Chronic blood loss– Hemolytic anemia– Secondary polycythemia– Hemochromatosis

3

Objectives

5. Understand how the CBC is essential for the detection of RBC dyscrasias

6. Be able to utilize the CBC to determine the cause of the RBC dyscrasias

7. Understand the RBC Indices and their use.8. Know what additional tests are needed

based on patient symptoms and CBC results to help in the determination and confirmation of RBC dyscrasias.

4

Objectives.

5

9. Describe etiologies and mechanisms of the following anemias associated with decreased production of red blood cells (hypoproliferative anemias):

Iron deficiency anemia; anemia of chronic disease; thalassemias; anemia of renal failure; aplastic anemia; anemia due to replacement of bone marrow; megaloblastic anemias ( Vitamin B12 and Folate deficiency), non-megaloblastic (Liver disorders etc), sideroblastic anemia.10. Describe the laboratory findings associated with

the hypoproliferative anemias described above.

Objectives

6

11. Know the pathophysiology of hemolytic anemias. 12. List laboratory findings associated with hemolytic

anemias 13. Diagnose hemolytic anemias utilizing clinical history and CBC data interpretation:

–sickle cell anemia; disseminated intravascular coagulation ( DIC); prosthetic heart valves; immune mediated hemolytic anemia (associated with drugs and autoimmune disease ); thalassemias; G-6-PD.

14. What are the mechanisms and when do hemolytic crises occur?

Objectives

7

15. Describe how iron studies ( serum iron; serum ferritin; total iron binding capacity; transferrin saturation) can be used to help diagnose and differentiate the microcytic/normocytic anemias ( iron deficiency anemia; anemia of chronic disease; thalassemia minor)16. Describe studies for evaluation and confirmation of megaloblastic anemias (ie. B12 and folate deficiency)

Overview

8

Erythrocyte Derivation

9

RBC PRODUCTION

• Red blood cells (RBC): – Made in the bone marrow (embryo in liver)

• 2 million /sec made; formation from a stem cell to a rbc is 5-6 days.

• RBC purpose is to transport oxygen to the body’s tissues in exchange for CO2, which is then carried back and eliminated in the lung.

• Structure is biconcave for more surface area for this exchange and for flexibility to pass through small capillaries single file.

10

The Players in RBC Production

11

Iron and RBC Production• Iron is critical to the

formation of Heme.• It is recycled by the body.

(normally 1mg taken in and 1mg lost daily); rest is recycled.

• Dietary sources include:– Red meat (primarily and most

absorbable)– Some green vegetables

(spinach- less absorbable do to presence of oxalate which chelates the iron)

– Whole grains- less absorbable– Iron additives or supplements

12

Iron Metabolism• Food sources contain Fe++ +(ferric state)• In the stomach acid environment, Fe+++ is converted to Fe ++

(ferrous state) in order to pass through enteric and plasma membranes; (ascorbic acid also assists with this).

• In the duodenum, bicarbonate changes the environment to an alkaline state where the Fe++ is converted back to Fe+++ for easier dissociation within the cells and subsequent attachment to transferrin.

• Fe++ (ferrous- dietary source)• Transferrin (carries Fe+++ from intestinal wall to storage (to

marrow, liver, muscle)13

The Players in RBC Production

14

Iron Metabolism (con’t)

• Transferrin:– There is an excess amount of Transferrin available to

attach to any Fe+++ around. That way there is no unbound iron circulating which would be toxic to the body.

– Transferrin is normally only 1/3 saturated with iron and therefore an unexpected influx of iron can be handled easily.

– Transferrin is not measured directly and is therefore the test used is called Transferrin Saturation.

15

Transferrin Saturation (%)

• Transferrin Saturation = serum iron/TIBC X 100• Total Iron Binding Capacity (TIBC) is the

maximum amount of iron that can be bound.• Therefore TIBC is a measurement of how much

protein is available. Since Transferrin makes up most of this protein, the Transferrin Saturation value would be low in the case of low iron but high in the case of liver failure (not as much protein made).

16

Expected Transferrin Saturation levels

17

Disease Iron TIBC/Transferrin UIBC %Transferrin Saturation Ferritin

Iron Deficiency Low High High Low Low

Hemochromatosis High Low Low High High

Chronic Illness Low Low Low/Normal Low Normal/High

Hemolytic Anemia High Normal/Low Low/Normal High High

Sideroblastic Anemia

Normal/High Normal/Low Low/Normal High High

Iron Poisoning High Normal Low High Normal

18

Storage of Iron: FERRITIN

• Transferrin transport the iron to storage sites where it is released to apoferritin in bone marrow, liver, and muscles.

• The liver makes only enough apoferritin to equal the amount of Fe+++ being transported.

• Therefore the levels of ferritin normally equals the amount of iron levels.

19

Ferritin in Anemia of Chronic Disease (ACD)

• Exception is in anemia of chronic disease (ACD) or other inflammatory disorders where ferritin levels will be high with normal or low iron levels.

• This is because ferritin is an inflammatory protein and will be increased in ACD and pregnancy.

• This can be used as a diagnositic criteria.

20

REQUIREMENTS FOR RBC PRODUCTION

• Heme is synthesized in nucleated red cells in the bone marrow.– Four pyrrole units are arranged in a ringed

structure (1 heme and 4 porphyrin units). – When heme is catabolized, it forms bilirubin.

• Enzymes are required for this.• Lack of or defect of enzymes causes a condition called porphyria

– Porphyria patients have accummulation of porphyrinogens in the skin causing photosensitivity and neurologic symptoms.

21

RBC Destruction and Iron Metabolism

• When heme is disassembled, the pyrole units are converted to bilirubin.

• The iron is engulfed by macrophages and carried in the form of hemosiderin and carried back to the bone marrow or liver for reuse.

• In cases of excess iron intake or absorption (ie. Hemochromatosis, Beta thalasemia), hemosiderin is also stored in tissues such as brain and heart, causing death.

22

The Players in RBC Production

23

Stimulation of RBC Production

HYPOXIA• Blood Loss

– Sensors in Brain (blood pressure, blood volume, and O2 sensors)

– Kidney (02 sensors, Hgb conc, blood volume, cardiopulmonary function, and O2 affinity)

24

Erythropoietin

Erythropoietin is a hormone that stimulates the bone marrow stem cells to shift to erythroid progenitor cell production.

During fetal life, erythropoietin is produced in the liver.

After birth, it is formed in the renal peritubular by fibroblasts and interstitial cells.

Regulation is by Oxygen Feedback Mechanism. It is mainly made in response to low OXYGEN levels, low blood flow, poor oxygen exchange, and impaired oxygen release from hemoglobin.

25

Erythropoietin Function

• Accelerates RBC production by:– Increase rate of cell division– Speeds up incorporation of iron into the

developing cells– Shortens the time of cell maturation– Hastens entry of immature red cells into the

circulation.– The reticulocyte level is an indication of the

erythropoietin level.

26

Erythropoietin Levels

• Enzyme-linked assay: method of choice• Useful in distinquishing primary polycythemia

(uncontrolled bone marrow production of RBCs) from secondary polycythemia.– Erythropoietin levels are low/normal in Primary

polycythemia (Polycythemia Vera).– Erythropoietin levels are high in secondary

polycythemia in which tissue hypoxia is driving the increased production of RBCs.

27

Hemoglobin• Heme portion consists of a porphyrin ring with Fe+

suspended within the middle. Synthesized in mitochondria of the cytoplasm.

• Globin portion is composed of 2 amino acid chains (alpha and either gamma, beta, or delta)

• Most (97%) adult hemoglobin consists of 2 alpha and 2 beta chains and 4 heme molecules (Type A)

• One hemoglobin molecule can bind 4 molecules of oxygen.

• 2,3 DPG (diphosphoglycerate) binds to the two beta chains to stabilize the molecule and shift oxygen out to tissues. (enhances oxygen displacement)

28

2,3 DPG MOLECULE

HEMOGLOBIN A MOLECULEWITH 2,3 DPG FOR STABILIZATION after oxygen displacement

29

HEMOGLOBIN FORMS

• Hemoglobin Type A – 2 alpha and 2 beta (97%)

• Hemoglobin Type A2 - 2 alpha and 2 delta (<2.5%)

– Note that beta and delta are derived from the same gene, chromosome 11; alpha is from chromosome 16

• Hemoglobin Type F (fetal) – 2 alpha and 2 gamma (1%) (Normally, after 4-6 months of age, most of the gamma production shifts to beta (or delta); (1% may normally remain as gamma).

30

Normal Globin synthesis

31

Globin Synthesis• Occurs mainly in the early or basophilic

erythroblast. Small amount can occur in the reticulocyte (especially if rbc maturation is accelerated).

• Genes for globin synthesis is location on chromosome 11 (gamma, delta, beta) and on Chromosome 16 for alpha.

Anemias can occur if there is some problem at the DNA level; defects in interpreting the RNA template or nonsense messenger RNA occurs.

32

RBC MATURATION

33

Polychromatophilic normablast

34

Hemoglobin Disorders

• Switch from Fetal hemoglobin to Type A occurs 3-6 months of age. Mechanism is not known.

• Defects in chromosomes 11 and 16 can cause abnormal globin synthesis and therefore oxygen carrying capacity is decreased.

• 2,3 DPG also has less affinity to Fetal hgb

35

Normal productions of Globin Chains

36

Bone Marrow Function

1. Production of erythrocytes capable of transporting oxygen to tissues.

2.Concentration of RBC needs to be maintained.3.Red cell membranes must allow deformability

to transport through microcirculation.4.An elevated reticulocyte count indicates an

increase in bone marrow activity.

37

Reticulocytes• After the nucleus is extruded from the

nucleated red cell, residual cytoplasmic ribosomal RNA, fragments of mitochondria and organelles remain for several days.

• These cells are slightly larger and appear as polychromatic (slightly blue) and basophilic stippling.

• Most of the maturation occurs in the bone marrow, however 0.5-2.5% are in the peripheral blood normally.

38

DEFINITION OF ANEMIA

• Decrease in the concentration of hemoglobin.–Maintenance of several factors keep this

concentration within normal limits.• Also known as decreased red cell mass:

–Occurs with decrease in production, increased loss, or increase in destruction

39

Classification of Anemias

• Increased red cell destruction or loss– Excess loss (hemorrhagic anemia)– Hemolytic anemias (hereditary and extrinsic)

• Impaired production of red cells– Deficiency states– Bone marrow failure (hypoproliferative states)– Refractory anemias (ineffective erythropoiesis)

40

Anemias of Impaired Production

• Fe Deficiency• Sideroblastic• Megaloblastic (B12, folate)• Aplastic

Unknown cause• Anemia of Chronic Disease

41

Impaired Production

Decreased Hgb Synthesis• Microcytic and hypochromia implies decreased

Hgb synthesis.– Iron Deficiency– Thalassemia– Siderblastic anemia– Anemia of Chronic Disease ???

• Generally the Reticulocyte count will be normal or low.

42

Anemias of Increased Destruction

• Hemolytic Anemias– Mechanical– Autoimmune (Direct Coombs +)

• Generally the Reticulocyte count will be elevated.

43

Reticulocyte Count

Corrected retic count = observed retic count X Hct 45 Mean normal Hct

44

Reticulocyte Production Index (RPI)• 1. Calculate the Corrected Retic based on 45 as the normal HCT.

See previous slide.• 2.The next step is to correct for the longer life span of

prematurely released reticulocytes in the blood--a phenomenon of increased red blood cell production. This relies on a table:

Hematocrit% Retic Survival (days)

36-45 1.0

26-35 1.5

16-25 2.0

15 and below 2.5

45

RPI Calculation and Interpretation

• So, in a person whose uncorrected reticulocyte count is 5%, hemoglobin 7.5 g/dL, hematocrit 25%, the RPI would be:

• [Corrrected retic]/[maturation correction] = [5 x (25/45)] /2 = 1.4• Interpretation• The reticulocyte index (RI) should be between 1.0 and 2.0 for a

healthy individual.• RI < 2 with anemia indicates decreased production of

reticulocytes and therefore red blood cells.• RI > 2 with anemia indicates loss of red blood cells (destruction,

bleeding, etc) leading to increased compensatory production of reticulocytes to replace the lost red blood cells.

46

Clinical PresentationCase #1

• A 44 yr old female presents with complaints of fatigue for 6 months. She occasionally feels faint, especially with exertion. She has noticed heavy periods lasting 2 wks and are irregular. She likes to chew on ice.

• On exam, her gums and nail beds are pale. Her heart rate is 100 bpm and regular. You order a CBC as you expect an anemia.

• What do you predict her CBC results would be? Describe.

47

SO, Lets Apply Our Knowledge To Our Patient!

• . • What Laboratory results would you expect?

WBC: 5.5RBC: 2.88 Hgb: 8.0Hct: 27.0MCV: 79MCHC: 29.8RDW: 18PLT: 160,000MPV: 9.0Retic: low/normal

48

How would you classify these results?

WBC: 5.5RBC: 2.88 Hgb: 8.0Hct: 27.0MCV: 79MCHC: 29.8RDW: 18PLT: 160,000MPV: 9.0Retic: low/normal

49

What would you expect her RBCs to look like?

• Hypochromic,• Microcytic

50

What is your most likely diagnosis?

Microcytic hypochromic

What is your next step for confirming the diagnosis, short of a bone marrow biopsy? 51

Chronology of Fe Depletion

52

IRON Studies Review

• Ferritin is a ferric oxide surrounded by a hollow sphere of apoferritin (half-life is 5-10 minutes and is water soluble). Ferritin is an iron buffer as free Fe+ is toxic to the tissues.

• Transferrin transports Fe+ to the cells. It also functions to bind up any free Fe+.

• Total Iron Binding Capacity (TIBC) is the maxium capacity for iron binding.

• Total serum Fe+ concentration or 60-150 ug/dL largely reflects iron bound to transferrin.

• The percent of transferrin saturated with ferric iron is the best indicator of storage iron as serum iron is nonspecific. Serum iron/TIBC X 100 = transferrin saturation.

53

Iron Studies to CONFIRM Diagnosis

• Fe Deficiency anemia from chronic blood loss

• Fe+ level decr; TIBC incr; Ferritin decr

54

Severe Fe loss

• RBC 2.17• Hgb 3.9• Hct 14.4• MCV 66.6• MCH 18.1• MCHC 27.1• RDW 17

55

Iron Deficiency• Iron Deficiency is the Most common cause of anemia• Initially the cells change size (RDW elevated)• Then they become hypochromic.• Cell become microcytic over time: (MCV will be low; RDW may

eventually normalize if chronic)– because of the lack of iron and continued production of rbcs.

– The resulting excess of porphyrin rings stimulates heme

synthetase (rate limiting enzyme) to switch off heme synthesis because of the lack of iron and continued production of rbcs.

56

Iron requirements

• Normal levels of iron are usually maintained through intake of diet and recycling of iron.

• In normal menstrating women with 60 ml of blood loss (30 mg of iron), an extra mg of iron per day is required to maintain a steady state.

• Pregnancy depletes 600-700 mg of iron. • The body stores 500-1500 mg of iron; so one

pregnancy can deplete these stores.

57

Causes of Microcytic Anemia

– T: Thalassemia syndromes (genetic defect in Hgb)

– I: Iron Deficiency– C: Anemia of Chronic inflammation

– Slow blood loss (may also be normocytic)– Long standing infections– Lupus, RA etc.

– S: Sideroblastic anemias – Lead Poisoning– Anti-tuberculosis medications

58

Anemia of Chronic Disease (ACD)

• In ACD, you usually are unable to determine the etiology of the anemia.– It is the most common reason for anemia in hospitalized patients. – What follow-up test is most commonly ordered?

• Most get a colonoscopy.

• Primary mechanism for pathogenesis of ACD is decreased RBC production. Reason is unclear but infections and some inflammation release factors ( IL, tumor necrosis factor) that suppress erythropoiesis.

59

ACD (Con’t)

• In most cases, treatment of underlying disease corrects the anemia.

• Usually slow loss and is normocytic/normochromic because the body has adjusted to it but may be microcytic if also Fe deficient.

• Hallmark: Ferritin is usually high or normal unless accompanied by Fe def.

60

Iron Studies to CONFIRM Diagnosis

• Fe Deficiency anemia from chronic blood loss

• Fe+ level decr; TIBC incr; Ferritin decr

But in ACD the Ferritin in HIGH because ferritin is also an inflammatory protein.

61

CASE #2

• 11 yr old male with fatigue and not able to participate in sports. Mom states he has always been pale and short of breath.

• He has not been seen by a physician since early childhood. Lives in a poor neighborhood.

• What else do you want to know?

62

???? CASE #2• Hgb 9.0• Hct 30.6%• MCV 79• MCHC 29• RDW high• LOW RETIC Ct.• Serum Fe/Ferritin increased• TIBC Normal to decr; TRANSFERRIN saturation

normal to incr63

Case # 2

• BASOPHILIC STIPPLING • BONE MARROW CELL (ERYTHROBLAST) WITH Prussion Blue FE+ STAINING

64

Case # 2Differential Diagnosis

• Sideroblastic AnemiaThree etiologies:– Hereditary – X-linked recessive

– Acquired – exposure to toxins (alcohol,lead)

– Idiopathic myelodysplastic syndrome (aplastic bone marrow)

65

Who Is This?

66

Beethoven

• Tests on the hair and skull fragments of Ludwig van Beethoven show the legendary 19th century German composer died from lead poisoning, scientists say.

67

Sideroblastic Anemia

• Abnormal heme metabolism• Iron stores are adequate in the bone marrow

but rbc maturation is deficient.• Unable to incorporate the iron into the heme

molecule.• It accumulates in the

perinuclear mitochondria of nucleated rbcs.

68

Case #3

• A 35 yr old male patient presents with fatigue, shortness of breath with exercise, and weakness. Some numbness and tingling in his hands and feet. He has noticed a beefy red tongue which is tender. He is a strict vegetarian but drinks alcohol.

69

SO, Lets Apply Our Knowledge To Our Patient! (Case #3)

• What Laboratory results would you expect?

WBC: 5.5RBC: 3.40 Hgb: 10.8Hct: 32.4MCV: 103MCHC: 29RDW: 16.0PLT: 260,000MPV: 9.0Retic: Low

70

What would expect the scan and cells to look like?

71

And your diagnosis based onCLINICAL Evaluation and confirmed by Lab results

is? (Case #3)• Macrocytic Anemia

– Most likely B-12 deficiency based on his diet.• Confirmation can be made by a B-12 level

Other causes:Folic Acid deficiency (usually dietary)Pernicious Anemia: Immune disorder of Parietal cells

(Lack Intrinsic factor)Gastric CancerHistory of Small bowel resection (ileum)Blind loop syndrome (bacterial overgrowth)Parasites (tapeworm)

72

Pernicious Anemia

• Megaloblastic anemia• Gastric atrophy and

subsequent lack of intrinsic factor

• Antibodies produced to intrinsic factor

73

Pernicious Anemia

• Seven P’s of Pernicious anemia– Pancytopenia– Peripheral neuropathy– Papillary (tongue) atrophy– Psychosis (megaloblastic madness)– Posterior spinal column neuropathy– Pyramidal tract signs– pH elevation of gastric fluid

74

Pernicious Anemia• B12 deficiency occurs due to lack of intrinsic

factor which is normally produced by parietal cells of the stomach. Immune antibodies are formed against intrinsic factor.

• Intrinsic factor is essential for presentation and absorption of B12 across the intestinal muscosa.

• Unable to differentiate in bone marrow between causes of anemia due to deficiency of B12, folic acid, or that of pernicious anemia. 75

A Word about B12 deficiency symptoms

• Weakness• Fatigue• Dyspnea• Paresthesias• Mental clouding

• Edema, pallor, jaundice, smooth tongue, decreased vibratory and position sense, peripheral neuropathy

This can sometimes be mistaken for a PA student on exam day

76

LABORATORY TESTS FOR B-12 Deficiency • Macrocytic anemia exhibiting hypersegmented neutrophils. • Hemolysis occurs when these sluggish cells try to traverse the

sinuses of the spleen causing a chronic anemia.• May present itself with signs of dementia or other neurologic

defects without macrocytic anemia. This is especially true if Fe Deficiency is also present.

• Most clinicians do not rely on the CBC to confirm B-12 deficiency. Ordering a direct B-12 level is more accurate but not sensitive in early disease, where homocystiene and methylmalonic acid levels are beneficial.

• B12 def: high homocystiene and methylmalonic acid• Folic acid def: high homocystiene and low methylmalonic acid• True differentiation for pernicious anemia is anti-parietal cell

test or more specific anti-intrinsic factor test.77

B-12 Deficiency

Associated with B-12 deficiency acquired in 3 ways:– Autoimmune: Lack of Intrinsic factor in the parietal cells of the stomach.

Autoantibodies against parietal cells destroys production of intrinsic factor which is required for B-12 absorptionin the terminal ileum. This is Pernicious Anemia.

– Removal of the Terminal small intestine, where most B-12 absorption occurs.

– Dietary deficiency of B-12. Red meat , dairy, or supplements are the only source of B-12. Elderly and alchoholics are at risk. Strict vegans are also at risk.

– B-12 deficiency produces hyperhomocysteinemia, which is an independent risk factor for artherosclerotic disease and increased methylmalonic acid. These are good tests to detect early B-12 deficiency in the presence of normal B-12 levels and lack of anemia.

78

FOLIC ACID DEFICIENCY

• Also causes macrocytic anemia.• Has high levels of homocysteine also and is

commonly given to alzheimer’s patients and their family members as prevention. Prevention of arthrosclerosis is also an indication.

• Methymalonic acid levels are not elevated.• Check both B-12 and folic acid levels before treating

with folic acid, as giving folic acid will mask a B-12 deficiency.

79

Megaloblastic AnemiaPernicious anemia and/or Folic Acid deficiency

• Macrocytes on left. • Hyper segmented neutrophil (Pernicious anemia)

80

81

Howell-Jolly Bodies

• RBC Inclusion bodies• Remanents of Immature RBC nucleus; indicates

high RBC turnover.– RBC Inclusion bodies– Hemolytic anemia– Megaloblastic anemia– Splenectomy (could be incidental here)

82

Macrocytic AnemiaDifferential Diagnosis

BIG FAT RED CELLSB: B12 deficiencyI: inherited disordersG: GI disease or surgery (nontropical

sprue,enteritis,ileal resection, fish tapeworm)

F: folic acid deficiencyA: alcoholismT: thiamine-responsive anemia

83

BIG FAT RED CELLS, cont

R: reticulocytes in lg. #s may inflate the MCVE: endocrine disturbances (hypothyroidism)D: dietary deficiencies (folate, B12, protein, lipids)

C: chemotherapeutic drugsE: erythroleukemiaL: liver disease (chronic hepatitis, cirrhosis…)L: Lesch-Nyhan syndromeS: splenectomy

84

Normocytic Anemias

• SI: systemic inflammation• Z: zero production ;aplastic anemia usually

presents with pancytopenia (Deficiency of all cell elements of the blood)

• E: endocrine disorders (hypothyroidism, hyperthyroidism, adrenal insufficiency, hypogonadism)

85

Hemolytic Anemias• Hemolytic anemias are a result of premature

destruction of RBCs. • Anemia is the end result when the bone marrow

cannot keep up with production to match the destruction

• Causes are genetic or acquired• On a peripheral smear, the cells appear injured and

are called shistocytes or helmet cells. Genetic forms may appear as sickle cells, or crystalized bars within a red cell (Hgb C).

• Reticulocyte count is elevated.86

Hemolysis

87

Classification of Hemolytic Anemias

• Intrinsic defects:– Hereditary defects

• Abnormalities of the red cell membrane– Hereditary spherocytosis– Hereditary elliptocytosis – Hereditary pyropoikilocytosis– Hereditary stomatocytosis– Hereditary xerocytosis

• Enzyme deficiency disorders– G-6 –phosphate dehydrogenase (G-6-PD)– Pyruvate kinase deficiency (PK)

88

Anemia of increase destructionExtrinsic Hemolytic

anemia• Non-immune

– Mechanical destruction (heart valves)

– Microangiopathic (DIC)– Burns, toxic chemical

exposure• Immune

– Primary (ABO incompatibility)

– Secondary to leukemias or lymphomas

– Drug induced or infectious 89

Red cell membrane defects• Hereditary spherocytosis: autosomal dominant ;

deficiency in spectrin which is part of the cell membrane. RBCs lose their biconcave ability causing less surface area. Patients often present with gallbladder bilirubin stones. Spleen removes them via macrophages and recollects heme. Splenectomy is treatment if not controlled.

90

Hereditary Spherocytosis

• CBC shows normal to abnormal Hgb. MCV may be sl lowered or normal and MCHC is elevated. Why? Retic is elevated. Bili elevated

• Osmotic fragility test: cells subjected to hypotonic saline. Hereditary Spherocytes will not hold up to the shearing forces of the hypotonicity.

• Hypersplenomegaly common and a useful indicator to predict a crisis.

91

Abnormal Heme Synthesis and Hemolytic sydrome

• Thalassemia syndromes– Thalassa (sea) emia (blood) = Mediterranean

anemia– Abnormal globin synthesis leads to decreased

production of heme molecules.– Alpha thalassemia is a decrease production of

alpha chains leaving excess beta chains– Beta thalassemia is a decrease production of beta

chains leaving excess alpha chains.

92

Thalassemiamicrocytic/hypochromic with

basophilic stippling.

Normal heme globin structure: In thalassemias, the production of alpha and/or beta chains are out of wack. 93

B-thalassemia• The reverse is present in B-thalassemia where

there is substitution of alpha Hgb for beta Hgb.• Rather than forming tetramers of normal

hemoglobin, these α attach to the cell membrane causing unstable cell walls.

• Decrease in beta production results in replacement with delta chains producing more hemoglobin A2

• B-Thalassemia major (Cooley’s anemia) make none or very few B chains. They have severe hepatosplenomegaly, Hgb 2-6 g/dl . (why?)

Alpha subst

94

Thalassemias• Divided into α-thalassemia and B-thalassemia.• Both the α and B chains are structurally normal. It is

the combination frequency that is off. If excess chains of one type are over produced, the combination of the chains to form hemoglobin is defective.

• α-thalassemias are common in Southeast Asia.• B-thalassemias are most common in Mediterranean

region.• In both, increased destruction causes inadequate

production of hgb to keep up with the loss. This causes microcytic/hypochromic RBCs. What is the RDW? 95

α Thalassemia• In α-thalassemia, a substitution of α with B

leaves excess available B chains for the production of hemoglobin. If 4 substitutions are made, severe oxygen dissociation and apoxia results.

• If the substitution is by gamma chains, the condition called Bart’s hemoglobin is fatal in infants. (hydrops fetalis syndrome)

• Hgb H (4 betas) is still serious but not as fatal. 1 subst. is usually assymptomatic.

96

Thalassemia

Homozygous: HeterozygousHgb 2-6 g/dl 9-11 g/dl

RBC morph: microcytic hypochromicpoik, baso stip,target cells, nrbcsHeinz bodies

Retic: > 15% normal

Bone marrow: erythroid hyperplasia

Marked mild-modStorage iron: increased normal/sl incr 97

Case #

• A 3 year old male patient presents to the ER with symptoms of SOB, intermittent fever, pain in his extremetries and abdomen.

• CBC reveals the following:

98

Hemolytic Anemia

99

100

Sickle Cell Disorder

• Inheritance pattern • Amino acid replacement

101

Sickle Cell Anemia • Occurs 1 in 500 African Americans and 1 in 1000-

1400 Hispanic Americans (mainly South American) and Mediteranean descent.

• Autosomal recessive pattern where 2 copies of the B-hemoglobin is altered by a simple replacement of substitution of Glu with Valine (HbSS). This is caused by a mutation in the HBB gene which is located on Chromosome 11.

• HgSC is also common and cause the same severity of disease.

102

Sickle Cell

• Sickle cells die prematurely as they are inflexible and get stuck in small vessels. Fe is lost in the vascular system and out into the urine causing an anemia. In addition, painful episodes can occur depriving oxygen to the affected tissues.

• Sx usually start at early age. Anemia is the cause of most sx. They are often jaundiced from the breakdown of the Rbcs.

• Organ damage to lungs, kidneys, spleen, and brain can be serious and life threatening. Pulmonary HTN occurs in 1/3 of adults and can lead to heart failure.

• Many have no sx, however, the actual sickling of the cells depends on degree of oxygenation of the Hgb in the cell, and concentration of the HgbS by dehydration.

103

Sickle cell

• Avoid high altitudes, keep hydrated, • PCN prophalaxis at 2 months until age 6 to

protect from pneumococus. • Leg ulcers should be treated aggressively.• Strokes in children due to cerebral vascular

injury by sickled cells.• If three sickle crises/yr, median death is at age

35 because of organ damage.• Most states now screen newborns with risks.

104

Tissue Damage from Sickle cell crises

105

Solubility Test for Sickle Cells

A drop of blood is put in a chemical medium; normal cells will hemolyze.Sickle cells with crystalized hemoglobin stay insoluble and therefore is cloudy. (Not accurate in newborns; subjective read; false positives are common in polycythemic states.) 106

Hgb Electrophoresis

107

Hemoglobin C• Glutamic acid is replaced with lysine on the sixth position on

the beta chain.• Less soluble than normal Hgb due to the excess positive

charge of lysine.• Occurs in 2-3% of American blacks; heterozygous C trait has

no clinical symptoms, however, the peripheral smear may show target cells.

• 1% of Am blks are homozygous and have C crystals evident.

Hgb C trait Hgb C disease Hgb SC 108

Glucose-6-Phosphate Dehydrogenase Deficiency (G-6-PD)

• Inherited deficiency of erythrocyte enzyme G-6-PD. (X chromosome) Males inherit it; females

unaffected.G-6-PD is required for conversion of g-6-

phosphate to ultimately NADPH. This is required to reduce oxidative stress on the rbc.

Deficiency of G-6-PD causes the inability to neutralize oxidative stress leading to fragile rbcs and subsequent hemolysis.

109

Variants A and B

• There are 2 variants: A and B – 99% of white population has B variant;

20% black females have heterozygous A variant; one amino acid subst.There is a relationship to those with A and resistence to malaria in the black population.

Heinz bodies are denatured hemoglobin that adhere and weaken the RBC membrane.

110

Clinical symptoms of G-6-PDMost common clinical patterns are:• neonatal jaundice• congenital hemolytic anemia• drug-induced hemolysis• favism: severe hemolysis after exposure to the

fava bean (Mediterranean type)

Bite cells from Heinz body removal by macrophages in spleen 111

Hemolytic Anemia resulting from mechanical heart valve

Helmet cells and Schistocytes

112

Hemolytic Anemia

EXTRAVASCULAR INTRAVASCULAR

Peripheral smear Schistocytes Spherocytes

Haptoglobin Decr/absent Mild decrease

Urine Hemosiderin ++ Negative

Urine Hemoglobin ++ Negative

Direct Coombs Usually negative ++++

LDH Increase Increase

113

CAUSES OF Hemolytic Anemias

HEMATOLOGISTH: hemaglobinopathies (sickle cell most common)E: enzyme deficiencyM: medications (sulfonamides, quinine)A: antibodies (autoimmune)T: trauma to RBCsO: ovalocytosisL: liver disease

114

Hemolytic Anemias

HEMATOLOGIST ,cont.

O: osmotic fragility (hereditary spherocytosis)G: Glucose-6-dehydrogenase deficiencyI: infection (malaria, clostridium, β-hemolytic

strep septicemia)S: splenic destructionT: transfusion

115

General Laboratory Findings in Hemolytic anemia

• Hgb lowered• Marked poik• Platelets high• Retic count high• Bilirubin high (esp indirect)• Serum iron high/% sat high

116

APLASTIC ANEMIA• Caused by drugs (chloramphenicol), chemicals,

infection (esp viral), irradiation, idiopathic, hypoproliferative (immune).

• Pancytopenia on CBC with reticulopenia; ;WBC 500; plt < 20,000; RBC ct depressed.

• Bone marrow biopsy MUST be done to diagnose this condition. Shows hypocellularity of all cellular lines.

• Persistant anemia: 50% mortality rate within 6 months.

• Treatment: bone marrow transplant

117

Question

What tests would you consider if you saw this blood smear? What diagnosis are you considering?

118

Answer?

A vital stain for Heinz bodies; Bite cells suggest G-6-PD deficiency.

119

References• Laboratory Medicine, Laposta, Michael.• Platt et al, Diagnosing Anemias, Clinician

Reviews Journal Vol. 16, number 12, December 2006.

• http://medsci.indiana.edu/c602web/602/C602web/cbc/docs/introa.html

• www.labtestsonline.org/understanding/analytes/cbc/test/html

• Images from www.google.com/images• Special Thanks to Kyrus Patch for Anemia links.

120