Haemolysis

Question 1

Methods used to detect free haemoglobin (2)

Answer 1

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

Question 2

Technical considerations when measuring free Hb

Answer 2

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

Question 3

Method for detecting haptoglobin

Answer 3

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.

Question 4

How is urinary haemosiderin detected?

Answer 4

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)

Question 5

Schumm Test

Answer 5

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.

Question 6

Describe methods for diagnosing HS

Answer 6

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)

Question 7

Genetics of PK deficiency

Answer 7

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

Question 8

Principle of testing for PK deficiency

Answer 8

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.

Question 9

Presentation of G6PD deficiency

Answer 9

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)

Question 10

Testing for G6PD deficiency

Answer 10

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.

Question 11

Management of G6PD deficiency

Answer 11

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

Question 12

How do you test for reduced glutathione?

Answer 12

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).

Question 13

What is P5’N deficiency

Answer 13

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

Question 14

Diagnosis of Hereditary Elliptocytosis

Answer 14

> 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

Question 15

Ddx of hereditary stomatocytosis

Answer 15

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

Question 16

Blood film findings in Hereditary Stomatocytosjs

Answer 16

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

Question 17

Causes of acanthocytes

Answer 17

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

Question 18

Technical points for PK enzyme testing

Answer 18

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

Question 19

Pathogenesis of Cold Agglutinin Disease

Answer 19

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.

Question 20

Causes of CAD

Answer 20

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

Question 21

Clinical features of CAD

Answer 21

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

Question 22

Diagnosis of CAD

Answer 22

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

Question 23

Management of CAD

Answer 23

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

Question 24

Management of cold antibodies with cardiac surgery

Answer 24

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.

Question 25

Describe the Donath Landsteiner test.

Answer 25

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.

Question 26

Causes of WAIHA

Answer 26

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

Question 27

Methods to measure ADAMTS13

Answer 27

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)

Question 28

What is the PLASMIC score?

Answer 28

``` 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 ```

Question 29

Classification of PNH (clinical)

Answer 29

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%

Question 30

Causes of anaemia in PNH (4)?

Answer 30

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

Question 31

Clinical symptoms of PNH

Answer 31

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

Question 32

What is the Ham test?

Answer 32

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)

Question 33

PNH flow testing

Answer 33

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

Question 34

How do you calculate the MCH and MCHC?

Answer 34

``` 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 ```

Question 35

Diagnosis of DIC

Answer 35

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

Question 36

Pathogenesis of DIC

Answer 36

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

Question 37

Causes of DIC

Answer 37

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