Lab Investigation of Hemolysis Presentation 9-19-18 Ver.1

9/23/2018

Investigation of Jason Anderson, Hemolysis in the MPH, MT(ASCP) Field Product Clinical Specialist Laboratory

Objectives 1. Define hemolysis. 2. Distinguish between intrinsic and extrinsic hemolysis 3. Explain where in the body hemolysis can take place. 4. Describe how the body physiologically reacts when hemolysis is present. 5. Discuss hematological and biochemical tests, used in the laboratory investigation of hemolysis.

What is hemolysis?

• Premature breakdown of red blood cells • Where can hemolysis occur? • Within the macrophages of the Mononuclear Phagocyte System (MPS) • Within the blood vessels

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What is the normal aging process of red blood cells? • Average life span 120 days • What is the MPS? • RBCs have no nucleus – cannot • Mononuclear cells with pronounced phagocytic ability that are distributed synthesize new cellular extensively in lymphoid and other components organs. • Old/damaged RBCS are removed by macrophages within the MPS, most notably the Spleen • A small percentage break down within the circulation – cellular fragments are engulfed by macrophages.

Urobilinogen (urine) Process of normal RBC breakdown

Macrophage

RBC Protoporphyrin

Globin Reabsorbed Iron Amino acids Bilirubin

Amino acids Binds to transferrin Unconjugated bilirubin

Transported to the bone Bilirubin General protein synthesis marrow for incorporation glucoronides into erythroblasts (hgb formation Stercobilinogen (feces)

What causes hemolysis? • Some diseases and disease processes cause RBCs to breakdown prematurely • Normal response is for bone marrow to increase hematopoiesis • Causes of hemolysis can be broadly classified as being either intrinsic or extrinsic to the RBC • Intrinsic – occurs as a result of RBC defect • Most are hereditary • PNH is an exception – (acquired disorder) • Extrinsic – occurs as a result of ‘extracorpuscular’ or ‘environmental’ factors • patient’s own RBCs as well as any transfused RBCs will be affected as long as the causative factor remains in place

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Intrinsic causes of hemolysis

RBC defects Hereditary conditions RBC membrane defects 1. Hereditary Spherocytosis (HS) 2. Hereditary Elliptocytosis 3. Hereditary Stomatocytosis

Enzyme defects 1. Glucose-6-phosphate dehydrogenase deficiency (G6PD) 2. Pyruvate Kinase deficiency (PKD)

Hemoglobin defects 1. Sickle cell disease (HbS) 2. Hemoglobin C 3. Thalassemia

Extrinsic causes of hemolysis • Non-Immune • Immune • Prosethetic heart valves • Idiopathic • TTP • Autoimmune • HUS • Leukemia/Lymphoma • DIC • Drugs • Preeclampsia/HELLP • Infections syndrome • Alloimmune • Infections • Hemolytic transfusion rxn • Malaria • Hemolytic disease of the newborn • Clostridia

Extrinsic causes of hemolysis – cont. • Chemical & physical agents • Certain drugs • Burns • Snake bites • Secondary to other disease • Liver and Renal disease • Mechanical stress • March hemoglobinuria

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Where does hemolysis take place?

• Within the Mononuclear Phagocyte System (MPS) • This is referred to as ‘ Extravascular Hemolysis ’

• Directly in the circulation • This is referred to as ‘ Intravascular Hemolysis ’

• Generally speaking, intravascular hemolysis is more acute and more severe

Intravascular hemolysis • RBCs broken down directly in the circulatory system. • Free Hgb and RBC Enzymes (LDH) are released in circulation.

• Hgb tetramer broken into dimers and immediately bound by Haptoglobin which is saturated and immediately cleared by the liver • A low Haptoglobin level is a hallmark of Intravascular hemolysis.

• After Haptoglobin is saturated, excess free hgb is filtered in the kidneys and reabsorbed in the proximal tubules • Iron is recovered and converted into ferritin or hemosiderin • If rate of hemolysis is > renal tubule absorptive capacity, free hgb will be excreted in the urine (hemoglobinuria). Can be detected with urine strip.

Extravascular hemolysis

• Occurs when RBCs are phagocytosed by macrophages in the spleen, liver and bone marrow

• No free hgb is released into circulation • No hemoglobinemia/hemoglobinuria with extravascular hemolysis alone

• The breakdown of hgb within macrophages into its constituent components, heme and globin, occurs in the same manner as the normal aging process.

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The body’s reaction to hemolysis • Kidneys sensitive to changes in oxygenation of hgb. • Drop can occur due to a reduction in RBC mass (hemolysis or blood loss) • Release hormone Erythropoietin (EPO) • EPO is transported through plasma to BM where it accelerates erythropoiesis • EPO mechanism acts like a thermostat, increasing or decreasing erythropoiesis based on need

• When hemolysis is taking place, the bone marrow will increase the production of RBCs relative to the amount of EPO produced • If rate of RBC destruction is greater than ability for BM to compensate the individual will become anemic.

Detecting hemolysis in the lab • The approach in the dx of a hemolytic state involves: • Establishing that RBC destruction is accelerated • Establishing that erythropoiesis is increased

• The cause of hemolysis must be determined as well.

• If hemolytic anemia is suspected the following should be performed • Complete blood count • Reticulocyte count • Peripheral blood smear examination

Tests reflecting increased RBC destruction

Test Intravascular hemolysis Extravascular hemolysis

1. Haptoglobin Decreased/depleted Normal

2. Serum bilirubin Increased unconjugated bilirubin Increased unconjugated bilirubin

3. Urine test strip for hgb Positive Negative

4. Urine Hemosiderin Positive Negative

5. LDH Increased Normal

6. Hemopexin test Decreased/depleted Normal

7. Urine test strip for urobilinogen Positive Positive

8. Schumm’s test for methemalbumin Positive Negative

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Test reflecting increased RBC production • Reticulocyte Count • Reticulocytes remain in the BM for ~2 days before being released into the peripheral blood • # of Reticulocytes in the peripheral blood provides information about BM activity (erythropoiesis) • In the event of hemolysis, the BM will try to compensate by upregulating erythropoetic activity. • Reticulocyte population can be counted via manual or automated methods.

Impact of artefactual hemolysis • The following practices can result in RBC lysis inside the collection tube ( in-vitro hemolysis) • Poor venipuncture technique • Exposure to excessively hot or cold temperatures (freezing) • Prolonged storage prior to analysis • It is important to be aware of this, as artefactual hemolysis may be very difficult to distinguish from intravascular hemolysis. In both cases on visual inspection, the plasma will have a reddish-brown color.

Laboratory findings in artefactual hemolysis

• The following findings suggest that in-vitro hemolysis has taken place: • A low RBC count and low HCT value with a normal HGB value. As a result, MCHC and MCH will appear raised. • No reticulocytosis even in the presence of RBC fragments. • Biochemical tests for intravascular hemolysis would be ‘negative’.

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RBC Morphology in Hemolytic Anemias

RBC morphology assoc. with hemolytic conditions

RBC feature Description Underlying mechanism Disease states

Basophilic Stippling Punctate basophilic Precipitated ribosomes Thalassemia and other inclusions anemias