Miscellaneous Flashcards
(39 cards)
Cold Agglutins Disease
15-30% of AIHA
AIHA with a monospecific DAT which is strongly positive for C3d (and negative or weakly positive with IgG), and cold agglutinin titre of 64 or greater at 4 degrees
No clinical or radiological evidence of malignancy HOWEVER, they may have an underlying B-cell clonal LPD detectable in the blood or the bone marrow
Increasing recognition that indolent LPD of the bone marrow is present in most cases
Often LPL/MZL phenotype. Usually MYD88 negative
Cold agglutinin syndrome:
- Similar but occurs secondary to another clinical disease
Cold antibodies:
- Nearly always IgM
- Most do not cause clinical haemolsysis
- Haemolysis due to the destruction of red cells by complement that is bound to the red cell surface due to antigen-antibody interaction
Causes of Cold agglutinin syndrome
Infection
- EBV against little i
- Mycoplasma against big I
- Onset is 2-3 weeks,
Other associations
- LPD
- WM
- NHL
- CLL
- MGUS
- MM
Autoimmune disorders
- RA
- Scleroderma
Specificity of cold agglutinins
Directed against I blood group 90% of the time
Little i in children/neonates
Anti-Pr (very uncommon)
Anti-IH (very uncommon)
haemolysis mechanism in CAD
Antibody (usually IgM) binds to antigen low temp in accordance with thermal amplitude of antibody
–> activation of the classical complement pathway
C3 covertase is formed –>Generation of C3b
C3b coated RBC phagocytosed by macrophages (typically in the liver rather than the spleen)
EXTRAVASCULAR haemolysis
Treatment of cold agglutinins disease
Anemia is often mild and no treatment is required
Patients with mild anemia or compensated haemolysis and no clinical symptoms have not been shown to benefit from treatment
Avoid cold temperatures
If transfusion required – blood warmer
Consider the implication of hypothermic surgeries (bypass etc)
Treat the underlying disorder
Cold Agglutinin Testing
Basic haemolysis work up:
- FBE and film
- Anemia, polychromasia
- Falsely elevated MCV, MCHC
- Agglutination on the film that resolves when warmed
Usually non spherocytic (bc. Opsonisation by complement causes red cells to be phagocytosed in full, rather than only parts of the membrane)
DAT (once haemolysis confirmed)– C3D, up to 20% also positive for IgG
Second:
- Identifying antibody specificity (big I, little i)
- Antibody titre
- Thermal amplitude.
G6PD deficiency - define
G6PD is an enzyme in the pentose phosphate pathway
G6PD converts G6P –> 6PG and in doing this also converts NADP+ to NADPH
- it is the rate limiting enzyme in this metabolic pathway that maintains level of reduced form of the co-enzyme NADPH
- NADPH maintains the supply of glutathione in the cells that are used to mop up free radicals that cause oxidative damage
Most common enzymatic disorder of red bloods cells in human
X-linked recessive disorder
Heterozygous females are usually clinically normally; however the red cell enzyme activity may be moderately reduced or grossly deficiency depending upon the degree of lyonisation and the degree to which the abnormal G6PD variant is expressed
Can be divided into 5 classes based on degree of deficiency and clinical symptoms of hemolysis
Clinical manifestations of G6PD
Majority are asymptomatic and do not have haemolysis in the steady state – no anaemia or evidence of haemolysis.
Episodes of haemolysis may be triggered by medications, certain foods, acute illnesses, especially infections
There are rare individuals with severe disease who have chronic haemolysis
Hence
1. neonatal hyperbilirubinaemia
2. chronic haemolytic anaemia
3. acute intermitted haemolytic anaemia
FBE + film - bites and blisters, polychromasia.
Inciting events :
- Medications (dapsone, primaquine)
- Food - fava beans
- Medical illnesses - infection
Indications for G6PD testing
Neonatal jaundice
Family screening
Unexplained DAT negative haemolysis
Asymptomatic individuals at high risk of G6PD deficiency prior to administration of certain medications
Prior to high risk therapy - ie dapsone or primaquine
Important considerations for testing
Timing of G6PD assay:
False negative results
- if performed during active haemolytic episode the RBCs with the most reduced G6PD activity with have haemolysed; and thus their G6PD activity will not be measured in the assay
- in the setting of reticulocytosis, which typically have normal G6PD activity
Repeat testing three months after any haemolytic episodes if clinical suspicion remains
May get falsely normal result if recently transfused
G6PD testing - QUANTITATIVE assay
Spectrophotometric assay
Sample - whole blood EDTA/heparin or ACD
Principle
- Activity of enzyme is assayed by following rate of NADPH
- NADPH has peak UV light absorption at 340nm, unlike NADP+
- Assay carried out in spectrometer
G6P + NADP+ –> in the presence of G6PD is converted –> to 6PG and NADPH
Method:
- sample +reaction buffer + NADP+ and MgCl2
- add G6P
- record absorbance
Reaction rate increases over first few minutes, then will become linear. Will then slow
> Linear part of curve over 10min period is used for calculation
Interpretation of G6PD quantitative assay
Male patients
> Can be either deficient or not deficient (normal) hemizygotes
> False low: very rarely occurs, but can occur with reticulocytopenia (e.g. pure red cell aplasia)
> False high: acute/chronic haemolytic states with reticulocytosis (a normal result in the setting of reticulocytosis should be suspicious for G6PD deficiency; confirm by repeating when the reticulocytosis has subsided, or conducting family studies)
Female patients
> Can be deficient heterozygotes, deficient homozygotes, or not deficient (normal)
> Heterozygosity can never be rigorously ruled out by a G6PD assay
» in heterozygote range, the actual value of the assay correlates with the risk of haemolysis
G6PD screening - qualitative assay
Not done in my lab
Principle:
- NADPH generated by G6PD present in a lysate of blood cells fluoresces under long-wave UV light
- In G6PD a lack of fluorescence is detected due to inability to produce NADPH
PK deficiency
Second most common red cell enzyme defect, but extremely rare
Inheritance is AR
No clinical manifestations occur with heterozygosity.
Pathophysiology:
- PK is an enzyme in glycolysis pathway and key in production of ATP.
- leading to reduced ATP production and premature RBC haemolysis
> PLKR - encodes liver and RBC enzyme
Clinical manifestations
- Chronic (rather than intermittent) haemolysis
- Neonatal jaundice
- Mild anaemia in childhood or adulthood
Lab findings
- DAT negative haemolysis, non spherocytic
- “prickle cell” echinocytes on blood films
PK assay
EDTA sample
> leucodepletion of sample (ensure no WBC or plts)
PK activity is measured by the rate of FALL of absorbance at 340nm (i.e. the rate of NADH reduction to NAD+, which is rate-limited by pyruvate production by PK)
2 steps:
1. PEP + ADP –> in presence of PK –> pyruvate + ADP
2. pyruvate + NADH –> in presence of LDH –> lactate + NAD+
Excess LDH is added to ensure it not the rate limiting step.
The change in absorbance (A) is measured over the first 5 minutes and the activity of the enzyme in umol of NADH reduced/min/mL haemolysate is calculated .
List the different types of Red cell membranopathies
Hereditary spherocytosis
Hereditary Elliptocytosis
Hereditary Pyropoikilocytosis
Hereditary Stomatocytosis
Hereditary Spherocytosis
Caused by loss of vertical linkages between the cytoskeleton and the lipid by-layer of the red cell membrane
Most common RBC membranopathy
AD
Defective proteins:
- ,Ankyrin, spectrin band 3, protein 4.2
Genes:
- Ankyrin ANK1 gene in 50% cases
- B spectrin (SPTB) gene in about 30%
Clinical presentation:
- can present any age
- Neonatal hyperbilirubinaemia <24 hours of life
- Haemolytic crisis or aplastic crisis later in life due to EBV/parvovirus B19
- Gallstones
- Splenomegaly
Blood film:
- spherocytes
- increased MCHC and RDW
- inc retics
Investigations:
- Osmotic fragility increased (outdated0
- Abnormal e%M binding
- Genetic testing
Rx:
- folate, supportive transfusions, EPO, splenectomy
hyposplenic changes on film
Hereditary Elliptocytosis
Alpha and beta spectrin are the largest of the erythrocyte cytoskeletal proteins
AD
Defective protein: Spectrin (a or b). Protein 4.1
Genetics:
Alpha- or beta-spectrin (SPTA1, SPTB) = majority
Blood film:
Elliptocytes (oval/elongated RBCs)
normal MCV + Hb
normal retics
Clinical sx:
Heterozygotes - Often asymptomatic and may go undiagnosed. (splenomegaly + gallstones)
Homozygotes - severe disease
> HE vs HPP based on morphology
No abnormal E5M binding
Hereditary Pyropoikilocytosis (HPP)
Autosomal recessive (severe HE variant)
Severe spectrin defect + deficiency
> usually mutated alpha or beta
> most commonly SPTA1
Blood film:
- (bizzare) poikilocytosis, microspheros, elliptocytes, fragments
- fragmentation at >40C
- markedly microcytic
- inc retics
Clinical sx:
- neonatal jaundice +/- transfusion requirment
- may improve after 1-2 yer to HE phenotype
No abnormal E5M binding
May respond to splenectomy
South East Asian Ovalocytosis
AD
Characteristic deletion in band 3 (SLC4A1)
Normal Hb
Normal retics
Homozygous state lethal in utero.
Heterozygous states:
- Newborns frequently present with neonatal hyperbilirubinaemia
- Haemolysis resolves by 3 years of age .
Blood film:
- Pathognomic “double slit” elliptocytes are seen
- Abnormal E5M binding
Hereditary stomatocytosis
AD
Mutation in –> PIEZO1, KCNN4 (Gardos channel)
Causes abnormal K+ leak and Na+ gain of the cell, leading to cellular dehydration
Blood film:
- stomatocytes, some target cells and dense cells
- low or normal MCV
- inc retics
Clinical presentation:
- any age
- mild-moderate haemolytic anaemia, which may be fully compensated
- Splenomegaly
- can be
Can be syndromic (sitosterolaemia, associated with macrothrombocytopenia)
E5M usually normal
Osmotic fragility test
Not usually done anymore, screening test for HS
Principle:
Red cells that are spherocytic lyse after taking up a lower volume of water than normal red cells.
Sample - lithium hep whole blood sample
Method - whole blood + hypotonic saline at varying dilutions and mix.
Spectrometer used to determine amount of haemolysis in each sample
Done alongside a normal control.
Highest saline concentration at which initial lysis and complete lysis have occurered are recorded.
Limitations:
- Nonspecific - causes of increased/decreased fragility are multiple and test
- Not sensitive
E5M testing - FLOW cytometry test
Flow cytometric test
- measure the fluorescence intensity of intact red cells labelled with dye eosin-5-maleimide (E5M) which reacts covalently with a lysine residue (Lys430) on band 3.
- can detect deficiency/defect in Band3, spectrin or protein 4.2
Deficiency or abnormality of band 3 is seen in HS, SEA and CDA type 2.
Sample
- EDTA, within 48h and kept at 4C
Method
- Washed red cells + EMA
- incubate for 1 hour (EMA is light senstivie)
- resuspend in PBS and anaylse in flow analyser.
- compare test with the mean value of several control samples as a ratio
-> 6 x normal controls collected the same day/time as test patient
Results - expressed as the % of fluoresence reduction of patient compared to mean fluoresecne of the six normal controls.
Normal = within 10% of controls
Equivocal = 10-19% of controls
Reduced = > 20% less than controls
E5M Flow assay limitations
> does not detect ankyrin deficiency/defect alone
performance of test depends on storage and freshness of reagents
cannot do patients <1 month old in my lab
any transfusions will interfere with assay
E5M very light and heat sensitive - strict storage requirements
