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Disorders affecting red cells > Haemolysis > Flashcards

Haemolysis Flashcards

1
Q

Methods used to detect free haemoglobin (2)

A
  1. Direct = spectrophotometer. Compare patient sample to standard curve.
  2. Peroxidase reaction = free Hb in the presence of hydrogen peroxide causes a colour change in the substrate. The colour change is proportional to the free Hb measurement
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2
Q

Technical considerations when measuring free Hb

A
  1. Plasma sample (serum = clotted = haemolysis)
  2. Heparin anticoagulation (EDTA causes haemolysis)
  3. Large needle, avoid vacutainer if possible
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3
Q

Method for detecting haptoglobin

A

Immunoturbidometric.
Anti-haptoglobin antibodies are bound to beads. Patient plasma is added. Haptoglobin-antibody complex forms and increases turbidity of sample. Absorbance read off standard curve to give haptoglobin concentration.

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4
Q

How is urinary haemosiderin detected?

A

Urine is spun down and the deposit is put on a slide
Perform a Perls’ reaction:
Mild HCL liberates the ferric iron.
Fe3+ + ferrocyanide forms ferriferrocyanide (blue)

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5
Q

Schumm Test

A

For detecting methaemalbumin (present with intravascular haemolysis)
Plasma is covered with ether then ammonium sulphide is mixed with plasma.
If methaemalbumin is present there will be a band at 558nm in the green.

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6
Q

Describe methods for diagnosing HS

A
  1. DAT negative spherocytic haemolytic anaemia + splenomegaly + family history
  2. EMA binding (flow cytometry, EMA dye binds to band 3 causing fluorescence. Positive result <0.8 MFI compared to normal control).
  3. Osmotic fragility test (11 dilutions of NaCL + patient whole blood, measure %lysis on spectrophotometer. Compare all results to normal control = shift in curve to right = more lysis)
  4. Acidified glycerol lysis test (quick and easy, measure time for optical density to change by 50% on spectrophotometer = change in absorbance proportional to lysis)
  5. Autohaemolysis test (no glucose vs glucose - less lysis for HS when glucose added but no change in lysis when glucose added for PK deficiency)
  6. Cryohaemolysis test (incubate for 10mins at 37 degrees then 10 mins at 4 degrees)
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7
Q

Genetics of PK deficiency

A

Autosomal recessive
Compound heterozygous is more common than homozygous
>200 mutations have been described (PKLR gene)
Genetic heterogeneity results in wide variation in phenotype

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8
Q

Principle of testing for PK deficiency

A

PEP + ADP ==> Pyruvate + ATP in the presence of pyruvate kinase.
Pyurave + NADH ==> NAD + Lactate in the presence of LDH.
NADH natural fluorescence. In normal people, there will be no fluorescence under UV light. In PK deficiency, NADH will fluoresce.
N.B. Must remove WBCs and platelets as these contain >200x PK enzyme as RBCs. Must make haemolysate first.

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9
Q

Presentation of G6PD deficiency

A
  1. Neonatal jaundice (most common cause in affected areas)
  2. Favism e.g. fever, chills, loin/back pain, dark urine then jaundice
  3. Acute intermittent haemolysis due to drugs/infection
  4. Chronic haemolysis (uncommon, only occurs in those with severe enzyme deficiency)
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10
Q

Testing for G6PD deficiency

A

Better not to test during episodes of acute haemolysis as RBCs with denatured haemoglobin/low enzyme activity are haemolysed first and reticulocytes have higher enzyme activity.
1. G6PD fluorescence spot test
Pt haemolysate + G6P + NADP + GSSG. Fluorescence = normal G6PD enzyme activity.
2. Methaemoglobin reduction test
Pt haemolysate + sodium nitrite produces methaemoglobin (brown). Methylene blue is added and the PPP is stimulate. If G6PD enzyme is present, the solution will turn red again. Brown = no enzyme.
Confirm deficiency with G6PD enzyme assay using spectrophotometry.
Pt haemolysate + G6P + NADP + maleimide (inhibits 6PGD making NADPH). Measure fluorescence on spectrophotometer and read against standard curve.

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11
Q

Management of G6PD deficiency

A
  1. Avoid oxidative stress
    - education re drugs, fava beans
    - knowledge of G6PD status in high prevalence areas
    - cultivate low vicine fava beans
  2. Good hydration to avoid AKI
  3. Manage neonatal jaundice to prevent kernicterus
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12
Q

How do you test for reduced glutathione?

A

Estimate by using Ellman’s reagent which is reduced by GSH and turns a yellow colour (colour is proportional to the amount of GSH in the red cells).

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13
Q

What is P5’N deficiency

A

Deficiency of the enzyme that breaks down pyrimidines. Results in basophilic stippling and chronic mild to moderate haemolysis.

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14
Q

Diagnosis of Hereditary Elliptocytosis

A

> 25% Elliptocytes by morphology
Correlation with degree of poiks and haemolysis but not number of elliptocytes
Red cell indices are normal.
Family history
Rule out other causes eg B12/folate def, CDA, MAHA etc

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15
Q

Ddx of hereditary stomatocytosis

A
  1. Hereditary
    - SEAO
    - Rh null syndrome
    - Mediterranean stomatocytosjs/macro thrombocytopenia
  2. Acquired
    - liver disease
    - alcohol excess
    - hydroxyurea
    - chlorpromazine
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16
Q

Blood film findings in Hereditary Stomatocytosjs

A 
Hb 80-100
MCV 120
MCHC high (xerocytosis) or low (overhydrated)
Variable number of stomatocytes, targets
Raised K+
17
Q

Causes of acanthocytes

A 
Inherited
1. Abetalipoproteinaemia 
2. Neuroacanthocytosis
3. McLeod phenotype
4 PK deficiency 
Acquired
1. Hypolipoproteinaemia from malnutrition
2. Liver disease /spur cell haemolysis
3. Vitamin E deficiency of neonates
4. Hyposplenism
18
Q

Technical points for PK enzyme testing

A
  1. Remove WBCs and platelets as these can contain 200xRBC enzyme levels
  2. Take sample from bottom of packed RBCs.
  3. PK deficiency is associated with chronic haemolysis and reticulocytosis therefore a normal PK level + reticulocytosis may be suspicious
  4. Can use low substrate concentrations to increase sensitivity
    PEP + ADP ==> Pyruvate + ATP
    Pyruvate + NADP ==> Lactate + NAD+
    Normal activity = no fluorescence
19
Q

Pathogenesis of Cold Agglutinin Disease

A

Cold reacting autoantibody
IgM, usually Anti-I specificity
Most important determinant of disease severity is the thermal amplitude
The IgM binds to RBCs in the cold peripheries and fix complement. As the RBCs return to the warmer core the IgM dissociates leaving only C3d. Macrophages bind complement ==> phagocytosis and removal in liver.

20
Q

Causes of CAD

A

Usually secondary but sometimes idiopathic.
1. Clonal - IgM kappa
>90% of patients have a monoclonal population of B cells with kappa light chain restriction found by flow or IHC in the bone marrow. Also associated with LPL.
2. Polyclonal
- IgM due to crossreactivity with EBV or mycloplasma
- Usually self-limiting disease

21
Q

Clinical features of CAD

A

Patients typically experience symptoms in winter/colder climates

  1. Agglutination - acrocyanosis and livedo reticularis
  2. Haemolysis - anaemia and jaundice
    - depends on the thermal amplitude predominantly
    - also high titre, ability to fix complement and anti-I rather than anti-i specificity
22
Q

Diagnosis of CAD

A
  1. FBC - RBC agglutination and spherocytes
  2. Positive haemolytic markers
  3. DAT positive for CDb/d
  4. Thermal amplitude and antibody titres
  5. Look for secondary causes - Immunoglobulins, SPE, CT scan, BMBx with cell markers
23
Q

Management of CAD

A
  1. Mainstay of management is non-pharmacological
    - avoidance of cold weather
    - warm clothes, warm blankets during surgical procedures
  2. Rituximab (steroids and splenectomy don’t work)
  3. Give blood through a warmer
  4. Folate supplementation if chronic haemolysis
24
Q

Management of cold antibodies with cardiac surgery

A
  1. Preoperative assessment - FBC and film ?agglutination
  2. DAT
  3. Alloantibody vs autoantibody
  4. Thermal amplitude and titre
    Alloantibodies - give antigen negative blood if reactive at 37 degrees. Normal hypothermic cardioplegia.
    Autoantibodies with high thermal amplitude and/or CHAD require normothermic bypass with warm cardioplegia.
25
Q

Describe the Donath Landsteiner test.

A

9 tubes - use P antigen positive reagent RBCs in all tubes.
Patient serum, normal serum and a mixture are added to three tubes each.
Incubate on ice, at 37 degrees and on ice followed by 37 degrees.
There should be haemolysis evident for patient serum and/or mixture tested on ice followed by 37 degrees. All other tubes should be normal.

26
Q

Causes of WAIHA

A
  1. Idiopathic (30%)
  2. Secondary
    - LPD (CLL, NHL, cHL) and less commonly MDS or MPNs
    - EBV, HIV, hepatitis
    - CVID
    - SLE, RA, Sjogren’s
    - Pregnancy
27
Q

Methods to measure ADAMTS13

A
  1. Enzymatic activity
    - ELISA (lab plus) - synthetic VWF peptide is immobilised onto plate, add diluted patient plasma which cleaves peptide, add antibody against cleavage site with substrate. Colour change is proportional to ADAMTS13 activity.
    - FRETs - fluorescence is proportional to ADAMTS13 activity. Uses short (73 AA) peptide
  2. Antigen level (limited value)
28
Q

What is the PLASMIC score?

A 
Score to predict ADAMTS13 deficiency. High score = highly likely to have severe deficiency.
Plts <30
Haemolysis - MAHA
No active cancer
No solid organ or stem cell transplant
MCV <90 (lots of fragments)
INR <1.5
Creatinine <200
29
Q

Classification of PNH (clinical)

A
  1. Classic PNH = florid haemolysis, erythroid hyperplasia in BM, PNH clone > 50%
  2. PNH in setting of another bone marrow failure syndrome = mild haemolysis, BM shows AA or MDS, PNH clone variable but usually <50%
  3. Subclinical = No haemolysis, BM shows another bone marrow failure syndrome, PNH clone <10%
30
Q

Causes of anaemia in PNH (4)?

A
  1. Intravascular haemolysis
  2. Extravascular haemolysis
  3. Iron deficiency
  4. Bone marrow failure
31
Q

Clinical symptoms of PNH

A
  1. Haemolysis - mostly intravascular, acute episodes but also extravascular. Degree of haemolysis correlates with size of clone.
  2. Thrombosis - leading cause of morbidity and mortality
    Risk of thrombosis correlates with haemolysis and size of clone. Activation of platelets, NO depletion.
  3. Smooth muscle dystonia e.g. oesophageal dysmotility, Abdo pains, erectile dysfunction
  4. Progressive BM failure
  5. Chronic renal impairment
32
Q

What is the Ham test?

A

Acidified serum lysis test
- PNH and HEMPAS are both positive
Test patient RBCs against AB serum and patient’s own serum which has been acidified. Inactivated serum (by heating) is used as control for each). Measure lysis with spectrophotometer.
HEMPAS does not show lysis to own serum, only to AB serum (need anti-HEMPAS antibodies)

33
Q

PNH flow testing

A

RBCs: FSc vs SSc then CD59
Granulocytes: CD45 vs SSc then CD15 vs SSc then CD66b and FLAER
Monocytes: CD45 vs SSc then CD33 vs SSc then CD14 and FLAER

34
Q

How do you calculate the MCH and MCHC?

A 
MCH = Hb/RBC (haemoglobin per red cell) - reduced in iron def, thalassaemias
MCHC = Hb/Hct - increased in HS, HbC as cells are contracted with smaller volume but same haemoglobin
35
Q

Diagnosis of DIC

A 
Clinical and laboratory factors, no single test
DIC score
1. Thrombocytopenia
2. PT prolonged
3. Low fibrinogen
4. High FDPs
36
Q

Pathogenesis of DIC

A

Massive activation of both coagulation and fibrinolysis with increased risk of bleeding and thrombosis
Bleeding - decreased clotting factors, decreased platelets, hyperfibrinolysis
Thrombosis - reduced natural anticoagulants, activation of platelets, endothelial dysfunction

37
Q

Causes of DIC

A

VOMITS
Vascular malformations - Kasabach Meritt syndrome
Obstetric complications
Malignancy
Infection
Trauma
Serologic - haemolytic transfusion reaction, post transplant

Disorders affecting red cells (3 decks)

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