Lab investigation of hemolysis presentation 9-19-18 ver...• A low Haptoglobin level is a hallmark of Intravascular hemolysis. • After Haptoglobin is saturated, excess free hgb

9/23/2018

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Jason Anderson, MPH, MT(ASCP)

Field Product Specialist

Investigation of Hemolysis in the Clinical Laboratory

Objectives

1. Define hemolysis.2. Distinguish between intrinsic and extrinsic hemolysis3. 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.

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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• RBCs have no nucleus – cannot

synthesize new cellular components

• 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.

• What is the MPS?• Mononuclear cells with pronounced

phagocytic ability that are distributed extensively in lymphoid and other organs.

Process of normal RBC breakdown

RBC

Macrophage

Globin

Amino acids

Amino acids

General protein synthesis

Iron

Binds to transferrin

Protoporphyrin

Bilirubin

Unconjugated bilirubin

Transported to the bone marrow for incorporation into erythroblasts (hgbformation

Bilirubin glucoronides

Stercobilinogen (feces)

Reabsorbed

Urobilinogen (urine)

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 hemolysisRBC defects Hereditary conditions

RBC membrane defects 1. Hereditary Spherocytosis (HS)2. Hereditary Elliptocytosis3. 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 C3. Thalassemia

Extrinsic causes of hemolysis

• Immune• Idiopathic• Autoimmune• Leukemia/Lymphoma• Drugs• Infections

• Alloimmune• Hemolytic transfusion rxn• Hemolytic disease of the newborn

• Non-Immune• Prosethetic heart valves• TTP• HUS• DIC• Preeclampsia/HELLP

syndrome

• Infections• Malaria• 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

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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 Intravascu lar 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 destructionTest 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 conditionsRBC feature Description Underlying mechanism Disease s tates

Punctate basophilic inclusions

Precipitated ribosomes Thalassemia and other anemias


Basophilic Stippling

RBC morphology assoc. with hemolytic conditions-cont.

RBC feature Description Underlying mechanism Disease s tates

Smooth semicircle removed from the margin of the cell

Heinz bodies G6PD and drug-induced oxidant hemolysis


Bite Cells

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Small, discrete basophilic dense inclusions; usually singular

Nuclear remnant Hemolytic anemias


Howell-Jolly bodies



Cells smaller than normal (<7um)

Abnormal hgb production Thalassemia


Microcytes



Grey or blue hue frequently seen in reticulocytes

Ribosome material Reticulocytosis, premature release of RBC from BM


Polychromatophilia

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Distorted, fragementedcell, 2 or 3 pointed edges

Mechanical destruction Microangiopathic HA’s, prosthetic valves, burns


Schistocytes



Mouth- or cuplike deformity

Membrane defect with abnormal cation permeability

Hereditary stomatocytosis, Immune-hemolytic anemia


Stomatocytes

RBC morphology assoc. with hemolytic conditions-cont.RBC feature Description Underlying mechanism Disease s tates

Target-like appearance, hypochromic with central hgb

Relative membrane excess due to decreased hgb inside the cell

Thalassemia, Hb C disease


Target Cells

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Sickle-shaped, pointed at both ends

Molecular aggregation of hemoglobin S

Sickle cell disorders


Sickle Cells



Spherical cell with dense hemoglobin and absent central pallor; usually decreased in diameter

Loss of surface membrane Hereditary Spherocytosis, Immune-hemolytic anemia, incompatible blood transfusion


Spherocytes


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Case study #1: history

• A 59 y.o. woman reports to the ED with complaints of fatigue, shortness of breath and swelling in her right ankle.

• She had just returned from an out of town trip and due to mechanical issues before takeoff, she had spent 11 hours on the plane.

• Upon examination she was found to be hypoxemic with a saturation of 88%. Her d-dimer was elevated and CT angiogram of the thorax revealed a left segmental pulmonary emboli. A CBC was also performed and results revealed a hemoglobin of 7.2 g/dl. Patient was referred to local hematology/oncology center for a hematology consultation.

Case study #1: hematology results


Case study #1: hematology results - cont.


RETICULOCYTE – Final Value Unit Reference Range

Retic % 17.9 High % 0.6 – 2.0

Retic Absolute 0.3409 High x10^6uL 0.025 – 0.102

Immature Retic Fraction 40.90 High % 1.1 – 15.9

Reticulated Hgb 37.1 High pg 28.2 – 36.6

Questions1. Based on what we know so far, can we conclude that this is a hemolytic anemia?2. What can we gather from the test results thus far?

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Case study #1: hematology results - cont.• Manual differential/smear

review was performed

• What do you see?Spherocytes


Case study #1: biochemistry results

• CMP , LDH, and Haptoglobin was ordered• All chemistry results were within the normal reference range except

for the following


Result Value Units Range

Glucose 117 High Mg/dL 65 - 100

Total Bilirubin 2.4 High Mg/dL 0.2 - 1.0

LDH 771 High U/L 120 - 280

Haptoglobin < 10 Low Mg/dL 30 - 200

Case study #1: diagnosis

• Additional medical history reveals patient has Rheumatoid Arthritis (RA) and is taking Methotrexate as part of their treatment plan

• Diagnosis? Autoimmune Hemolytic Anemia

• People with one type of autoimmune disease, such as RA, are prone to develop another type of autoimmune disease. Drugs such as Methotrexate can also trigger an AIHA


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Case study #1: Treatment

• Patient was treated with Prednisone and blood transfusions• Weekly lab tests were performed to monitor progress

• Patient did not tolerate the prednisone very well, so her treatment was switched to Rituximab which resulted in a remission state of the patient’s AIHA


Case study #2: history

• 39 y.o male admitted to the hospital with complaints of epigastric pain, nausea and vomiting.

• CT scan revealed mass in the pancreatic head plus liver lesions• Biopsy positive for adenocarcinoma of the pancreas• Patient started on Folfirinox as palliative chemotherapy

• After 2 cycles of chemo, bilirubin increased and CT scan showed disease progression

• Chemotherapy switched to Gemzar and Abraxane


Case study #2: history – cont.

• After the 1st cycle of the new treatment regimen, pt’s hemoglobin dropped significantly.

• Lab tests were drawn including a CBC with manual diff, retic panel, CMP, LDH, Haptoglobin and Iron studies


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Case study #2: hematology results


Case study #2: hematology results – cont.

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Ref. RangeResult Units

Case study #2: hematology results - cont.• Manual differential/smear

review was performed

• What do you see?Schistocytes


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Case study #2: hematology results - cont.


Case study #2: biochemistry results

• CMP and Iron study results were unremarkable aside from a slightly elevated BUN, AST and moderately elevated ALP

• What is the Haptoglobin and LDH ‘telling us’?


Case study #2: diagnosis

• Diagnosis?Microangiopathic Hemolytic Anemia (MAHA)

• Additional testing done to try to find the cause of the MAHA including:

• Enteric Pathogen screen, ADAMTS13 and G6PD. All were ruled out.

• Conclusion was MAHA was drug related, most likely the Gemzar as there is documentation that hemolytic anemia can be a side effect of this drug.


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Lab investigation of hemolysis - summary

• Artefactual hemolysis can mimic intravascular hemolysis. The laboratory must make every effort to identify its presence

• An elevated reticulocyte count is essential for the diagnosis of hemolysis as this signals increased RBC production.

• RBC morphology is informative in determining the cause of hemolysis.

• Biochemical tests of hemolysis confirm the presence of RBC breakdown and are useful in distinguishing between intravascular and extravascular hemolysis.


References

• Sysmex Europe (2015, October 22). SEED: Laboratory Investigation of Haemolysis. Retrieved 9/4/18 from https://www.sysmex-europe.com/fileadmin/media/f100/SEED/Sysmex_SEED_Laboratory_investigation_of_haemolysis.pdf

• U.S National Library of Medicine (2016, November 23). Mononuclear Phagocyte System MeSH Descriptor Data 2018. Retrieved 9/4/18 from https://meshb.nlm.nih.gov/record/ui?name=Mononuclear%20Phagocyte%20System

• Bain, B.J. (2005, August 4). Diagnosis from the Blood Smear. Retrieved 9/4/18 from https://www.nejm.org/doi/full/10.1056/NEJMra043442

Documents

Lab investigation of hemolysis presentation 9-19-18 ver...• A low Haptoglobin level is a hallmark of Intravascular hemolysis. • After Haptoglobin is saturated, excess free hgb