evaluation of hemolysis bbanys halfday2f82162c-6430-4e94-91b8-37855110a538... · hemolysis sequelae...
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Evaluation of Hemolysis
Matthew Elkins, MD PhD
Upstate University Hospital
Hemolysis
Definition:
Disruption of the cell membrane of RBCs resulting in the release of free hemoglobin
Intravascular hemolysis – disruption within the blood stream with release of RBC contents into the circulation
Extravascular hemolysis – disruption outside of blood stream (in reticuloendothelial system)
Hemolysis Sequelae
Anemia – decrease in hemoglobin below the patient’s usual baseline
Acute anemia – loss of RBC, usually without loss of intravascular volume
Chronic anemia – long term decrease in RBC mass, compensation
Pallor, lethargy, diaphoresis, nausea/vomiting, tachycardia, headache, dizziness, depression, shortness of breath, high‐output cardiac failure, death
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Hemolysis Sequelae
Schaer et al. 2013 Blood
Hemolysis SequelaeFree hemoglobin binds to nitric oxide to form methemoglobin
Methemoglobin induces release of proinflammatory cytokines fromproinflammatory cytokines from endothelial cells (e.g. IL‐6, IL‐8)
Fever, chills, SIRS
Decrease in NO results in increased vascular tone and hypercoagulability resulting in hypertension and stroke
Hemolysis Sequelae
Schaer et al. 2013 Blood
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Hemolysis Sequelae
Acute tubular necrosis
Damage to renal tubule epithelium
Acute, severe back/flank pain
Can result in permanent kidney damage
Hemolysis Sequelae
Schaer et al. 2013 Blood
Causes of HemolysisNon‐immune hemolysis
Mechanical shearing (artificial heart valve, fibrosis)
Trauma, toxins, infections
Abnormal RBC or hemoglobin
Microangiopathic hemolytic anemia (MAHA)
Immune hemolysis Immune hemolysis
Immune destruction of transfused RBCs due to alloantibodies produced by recipient
Immune destruction of patient RBCs due to antibodies in transfused plasma/antibody concentrate (e.g. RhoGam)
Immune destruction of patient RBCs due to autoantibody
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Hemolysis workupHistory, physical exam
—Symptoms and duration, PMHx
Hematology Lab tests
―CBC, Reticulocyte counts, Peripheral smears, Hemoglobin electrophoresis, Osmotic fragility testing, Bone marrow evaluationevaluation
Chemistry Lab tests
―Haptoglobin, Bilirubin, LDH, Serum‐free hemoglobin, Urinalysis (hemoglobinuria)
Blood Bank Evaluation
―Type and screen, DAT, Eluate, RBC antibody identification
Hemolysis Work‐up
Is the patient undergoing hemolysis?
Haptoglobin
Lactate dehydrogenase
Bilirubin
Serum‐free hemoglobin
Urinalysis
Chemistry EvaluationHaptoglobinMeasured by immuno‐turbidometricmeasurement
Mix patient’s serum with anti‐human h t l bi Abhaptoglobin Abs
Resultant aggregate blocks light transmittance on a spectrophotometer
Hpt is also an acute phase reactant, so may see increase with inflammation
Hpt is produced from the liver, so low levels can be seen due to liver failure
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Hemolysis
Anemic?
Hemolysis may occur without anemia due to the capacity of bone marrow to replenish
Patient Evaluation
the capacity of bone marrow to replenish destroyed RBCs
Anemia occurs if hemolysis exceeds erythropoiesis capacity of the bone marrow
High rate of hemolysis
Decreased erythropoiesis capacity
Hematology Lab Tests
Complete Blood Count (CBC)
Automated counting of
–RBCs
Platelets–Platelets
–WBCs
Determined by size of cellular component
Measured using either impedance or light scatter
Hematology Lab Tests Complete Blood Count (CBC)
Measured by impedance method:
Set up two chambers with maintained electrical current at set voltage
+‐
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Hematology Lab Tests Complete Blood Count (CBC)
As cells pass through aperture between chambers, resistance to electrical flow increases in relation to cell size
RBC and platelets tested in one reaction
Second sample tested after hydrolysis of RBCs
+‐
Hematology Lab Tests Complete Blood Count (CBC)
Light scatter measured using a flow cytometer
Cellular constituents pass through a laser beam one at a time
The light is refracted as it passes through the cell
The amount of refraction (spread) correlates to the size of the cell
Laser Detector
Side scatter used to quantitate WBC
Hemolysis
Anemic
What is the RBC morphology?
RBC h l id i i ht i t th
Patient Evaluation
RBC morphology can provide insight into the underlying cause of anemia/hemolysis
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Peripheral Blood Smear
Sample of whole blood is spread on a glass slide
Thin layer of blood isThin layer of blood is examined microscopically to evaluate morphology of platelets, RBCs, and WBCs
Case 1: Patient Presentation
52 year old woman
Recent upper respiratory infection
Left frontal headache
Gingival bleeding
Gross hematuria
New ecchymoses bilateral extremities and petechiae on trunk
Case 1: Laboratories
13.2
14.4 7
37.2
LDH 1698, Bilirubin 3.1
Haptoglobin <10
Blood Type: B+
Antibody Screen: Negative
DAT: Negative
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Case 2: Peripheral Case 2: Peripheral Blood Blood SmearSmear
Case 2: TTPThrombotic thrombocytopenic purpura
Anti‐ADAMTS13 antibody or congenital defect
Directly inactivates ADAMTS13 and causes increased clearance
ADAMTS13 cleaves vWF extra large multimersADAMTS13 cleaves vWF extra‐large multimers
Absence of functional ADAMSTS13 results in abundance of HMW vWF multimers
Larger multimers activate platelets multiple thromboses
Platelets used up thrombocytopenia
Case 2: TTPThrombotic thrombocytopenic purpura
ADAMTS13 assay:
1:1 Mix
Normal pooled plasma
Patient Sample
Dilution(same final conc
Normal >67% activity or <33% inhibition
Patient result <5% activity or >95% inhibition
1:1 MixResult=23%
(same final conc. as 1:1 mix)Result = 53%
Corrected 23/53=43.4%Percent Inhibition=56.6%
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Peripheral Blood Smear
Schistocytes
Remnants of RBCs from intravascular shearing
Small platelet‐fibrin clots transiently lodge in vessels, narrowing vascular channel andvessels, narrowing vascular channel and causing activation of complement cascade on vessel wall
Peripheral Blood Smear
Fibrin strands attach to endothelial walls
RBCs passing through the vessels are transiently bound by these fibrin strands
RBCs torn apart
Resultant fragments cleared by the spleen
Microangiopathic Hemolytic Anemias
Pathophysiology
Activation of platelets/coagulation cascade in small vessels resulting in fibrin stranding and microthrombimicrothrombi
Vascular occlusion, hemolysis, consumptive coagulopathy (platelets and/or coagulation factors)
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Microangiopathic Hemolytic Anemias
Causes Thrombotic Thrombocytopenic Purpura
Systemic malignancy
Malignant hypertension
Autoimmune diseases (SLE, PAN)
Severe infection (sepsis, DIC)
Drugs – Quinine best example, but many causes
Schistocytes ≠ TTP
Schistocytes = mechanical shearing of RBCs
Non‐MAHA Schistocytes
Marching hemolysis / drummer hemolysis
Stenotic heart valves
Artificial heart valves– Peri‐valvular leak
– Valvular stenosis
– Mechanical valves
LVADs, ECMO, bypass
Fibrosis of liver, bone marrow
Peripheral Smear
Spherocytes
RBC with smaller diameter and no central clearing
Caused by either:Lack of functional cytoskeletal components
A tib di ifi ll i i lf RBC tiAntibodies specifically recognizing self RBC antigens
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Peripheral SmearSpherocytes
Abs recognized by macrophages in the splenic sinusoids which phagocytize RBC membrane
Both pathways result in loss of cell membrane
RBC cytoplasm and membrane equilibratesRBC cytoplasm and membrane equilibrates
Anemia results due to clearing of the spherocytes
Peripheral SmearOther RBC morphologies
Stomatocytes – hereditary, Rh null syndrome
Acanthocytes (spur cells) – liver disease, dyslipidemias McLeod phenotypedyslipidemias, McLeod phenotype
Echinocyte (burr cells) ‐ renal failure, burns phosphate deficiency
Degmacyte (bite cells/blister cells) –oxidative hemolysis (ex. G6PD deficiency) resulting in condensation of hemoglobin which gets removed by macrophages
Peripheral Smear
Sickle cells, HgbC crystals
Condensation of abnormal hemoglobin resulting in insoluble crystalsinsoluble crystals
Distorts cell membrane
Intravascular and extravascularhemolysis
Chronic inflammatory state
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Hemolysis
Anemic
RBC morphology
I h l i ?
Patient Evaluation
Immune hemolysis?
What blood is compatible with patient?
Blood Bank TestingABO and Rh Typing ‐ Forward typing:
Patient’s RBCs with anti‐A, anti‐B and anti‐D
Blood Bank TestingABO and Rh Typing ‐ Reverse typing:
Patient’s plasma with A RBCs and B RBCs
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Blood Bank TestingAntibody Screen – non‐ABO antibodies:
Patient’s plasma with screening O cells
Case 2: Patient Presentation
45 yo woman G2P2
Admitted with a subarachnoid hemorrhage due to right carotid bifurcated aneurysm.
d f i l i hFour days after surgical repair, she was transfused two units RBC for Hgb=7.0 mg/dL.
Two weeks later she was transfused two additional units (Hgb=7.1 mg/dL).
Case 2: Patient Presentation
Early in the 2nd unit, the patient became febrile (38.140.1C), tachycardic (99106), hypertensive (128/72153/76).
The nurse also noted new “cranberry” coloredThe nurse also noted new cranberry colored urine in her catheter bag.
Her past transfusion history was significant for RBC approx. 15 yrs earlier.
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Case 2: Laboratory Workup
LDH= 821
Bilirubin (T/D)= 1.9 / 0.5
Haptoglobin= <6
Serum Free Hemoglobin= 87
UA= Hemoglobinuria
Visual hemolysis check: positive
Case 2: Laboratory WorkupPatient ABO/Rh type: O+ (pre, post samples)
ABO/Rh RBC units 1+2: O+
Direct Coombs: negative (C3, IgG: pre, post)
Repeat Antibody screen: anti‐ Lea, C, SC, Sp y , ,,(using enhanced methods)
Repeat Full Crossmatch:
Unit 2, Incompatible (C+, S+)
+
Blood Bank Testing
Direct Coombs test
Test for antibodies
Direct Agglutination Test (DAT)
or complement bound to patient RBCs
Use AHG for IgG
Anti‐C3 for IgM
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Immune‐mediated Hemolysis with Negative DAT
Antibody present, but all Ag+ cells hemolyzed
IgA IgD or IgE autoantibodyIgA, IgD, or IgE autoantibody (only IgG and C3 detected)
Low affinity binding –antibody dislodged during wash steps in DAT
Blood Bank Testing
Tests patient serum for anti‐RBC antibodies
Indirect Coombs Test
Combine serum with test RBCs
Add AHG to agglutinate if antibodies bound
Eluate Evaluation
Determine specificity of antibodies bound toof antibodies bound to the patient’s RBCs
Elute off antibodies from patient’s RBCs
Test against screen RBCs with AHG
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Antibody‐mediated Hemolysis
Alloantibody to antigen present on transfused RBCs
Transfused alloantibodies into patient with RBCs expressing antigenRBCs expressing antigen
— Transfused antibody concentrate (IVIG, RhoGam)
— Transfused non‐ABO compatible plasma
— All reported cases are type O plasma into type A or type AB patients
Antibody‐mediated Hemolysis
Autoantibody recognizing RBC antigen
Abnormal antibody recognizing normal antigen
– Warm autoantibody : IgG, reacts at 37⁰, extravascular hemolysisextravascular hemolysis
– Cold autoantibody : IgM, reacts at 24 ⁰, intravascular hemolysis
Case 3: Patient presentation
5 year‐old girl
2 week history of progressive dry cough
Afebrile 37.4 (99.4 F)
Vomiting, diarrhea
Tired, lethargic, shortness of breath
Chest pain
No significant PMHx
Multiple family members with cold symptoms
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Case 3: Patient presentation
V/S= T 37.6, HR 135, BP 98/55, RR 42
130 94 25 12
71 6.8 240
3 5 12 0 6 36 23.5 12 0.6 36.2
Imaging shows bilateral pneumonia with left empyema
Pleural fluid culture showed Streptococcus pneumoniae
Over 3 days, Hgb 6.3
Case 3 : Strep pneumo aHUS
Neuraminidase produced by S. pneumo cleaves N‐acetylneuraminic acid on glycoproteins of cell membrane proteins
Exposure of Thomsen‐F i d i h ti (T
High titers of antibodies against T‐antigens are normally found in all patients
Friedenreich antigen (T‐antigen) on glycophorins A and B on RBC and renal endothelial cell membranes Oliver et al 2010
Binds to RBC ag polyagglutination, hemolysis
Binds to renal endothelium HUS
Case 3 : Strep pneumo aHUS
Lectins T Th Tn HEMPAS Cad
Lectins T Th Tn HEMPAS Cad
Arachis hypogea
+ + 0 0 0
Glycine max (soja)
+ 0 + 0 0
Salvia sclarea 0 0 + 0 0
Salvia horminum
0 0 + 0 +
Dolichos biflorus
0 0 + 0 +
DAT + in 90%Lectin assay most sensitive/specific
Treatment may require minimization of exposure to donor plasma
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Antibody‐mediated Hemolysis
Autoantibody recognizing RBC antigen
Abnormal antibody recognizing normal antigen
– Warm autoantibody : IgG, reacts at 37⁰, extravascular hemolysisextravascular hemolysis
– Cold autoantibody : IgM, reacts at 24 ⁰, intravascular hemolysis
Normal antibody recognizing abnormal antigen
– Bacterial toxins resulting in exposure of cryptic antigens or creation of foreign antigens
Case 4: Patient presentation
54 year‐old man
History of coronary artery disease, GERDdisease, GERD
Transferred to UMHS for treatment of hemolytic anemia
Case 4: Patient presentation
6/28Sent home on antibiotics for
UTI
7/6For presumed AIHA, given IVIG, steroids,
d 2 it RBC6/25
Prostate biopsy
6/28 Presents at OSH w/ fever, chills, headache, urinary
frequency
UTI
7/5Re‐presents to OSH with same sx, and now hematuria
and 2 units RBCs
7/7Transferred to UMHS for treatment of hemolytic anemia
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Case 4: Clinical Presentation
On admission:
Hemoglobin 7.4, Hematocrit 21.8
LDH 1955LDH 1955
Total bilirubin 4.7
Haptoglobin <10
UMHS Blood Bank work‐up
DATPolyspecific= 3+
Anti‐IgG= 3+
Anti‐C3= 3+
IVIG given at OSH
UMHS Blood Bank work‐up
Serum:
Negative
Eluate:
Negative
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Antibiotics
6/28Sent home on antibiotics for
UTI
7/6For presumed AIHA, given IVIG, steroids,
d 2 it RBC6/25
Prostate biopsy
6/28 Presents at OSH w/ fever, chills, headache, urinary
frequency
UTI
7/5Re‐presents to OSH with same sx, and now hematuria
and 2 units RBCs
7/7Transferred to UMHS for treatment of hemolytic anemia
Drug‐induced hemolysis
Drug‐dependent Autoantibody
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Antibiotics
6/28Sent home on antibiotics for
UTI
7/6For presumed AIHA, given IVIG, steroids,
d 2 it RBC6/25
Prostate biopsy
6/28 Presents at OSH w/ fever, chills, headache, urinary
frequency
UTI
7/5Re‐presents to OSH with same sx, and now hematuria
and 2 units RBCs
7/7Transferred to UMHS for treatment of hemolytic anemia
Cefotetan and hemolysis
Cephamycin antibiotic with coverage similar to 2nd generation cephalosporins
Most common cause of drug‐dependent drug‐induced hemolysisinduced hemolysis
Commonly given prior to surgery as prophylactic
Detectable drug persists on RBCs up to 92 days after last dose (Davenport et al. 2004)
Treatment is supportive until drug is eliminated
DAT+ with negative eluate
Drug‐dependent antibody—Most commonly beta‐lactam antibiotics
Antibody with rare antigen specificity
False‐positive DAT (hyperviscosity, contamination by Wharton’s jelly)
Dialyzed patients – exposure to formaldehyde from tubing alters RBC antigens, eluate will react with formalin‐treated test RBCs
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Questions
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