Full Blood Count and Stains Flashcards
Discuss the Perl’s Stain
- Potassium ferrocyanide and HCL
- Fe3+ reacts with soluble ferrocyanide to form the insoluble Prussian blue pigment
- Dilute HCL releases Fe3+ from protein bound tissue deposits
- Viewed microscopically as purple or blue deposits
- Gold standard for measurement of iron stores.
- Diagnostic utility in MDS
- Can distinguish pappenheimer bodies from other RBC inclusions in the peripheral blood
- Urinary haemosiderin may be the only distinguishing feature of intravascular haemolysis
Discuss Supravital Stains
- Examples include new methylene blue and methyl violet
- Stains are often oxidising agents, used to examine living cells that have been removed from an organism
- Manual reticulocyte count (rarely performed due to availability on automated analysers): New methylene blue.
- Heinz bodies: essential part of the diagnosis of oxidative haemolysis. Methyl violet.
- HbH inclusions: important in the diagnosis of alpha thalassaemia trait: Methyl violet.
Discuss the Kleihauer test
- Estimation of FMH by counting the number of fetal cells present in maternal circulation
- Cells containing HbF are resistant to acid elution whereas cells containing HbA are not.
- Films air dried, placed in the acid elution solution then counter stained.
- Fetal cells counted using the Miller Ocular
Discuss the myeloperoxidase stain
- 3,3 DAB
- MPX present in granulocytic granules splits H2O2. In the presence of 3,3 DAB, a coloured insoluble reaction product forms
- Used in the diagnosis of acute myeloid leukaemia
- More reliable indicator of lineage specificity than Sudan Black
Discuss the non-specific esterase stain
- Esterase is present in monocytes and can hydrolyse synthetic 1-alpha-napthyl butyrate . A napthyl compound is liberated and rapidly couples with a diazonium salt resulting in a brightly coloured precipitate at or near the site of enzyme activity.
- Used to diagnose leukaemia with monocytic differentiation.
Discuss the periodic acid Schiff stain
- Periodic acid Schiff and haematoxylin
- Periodic acid forms aldehydes.
- Aldehydes react with the Schiff reagent to release fuchsin (stains magenta).
- Haematoxylin= counter stain. Gives a pale cytoplasm and nucleus.
- In the bone marrow, glycogen appears to be the compound primarily responsible for a reaction with PAS
- Used in the diagnosis of erythroleukaemia (normally negative in immature erythroid cells)
Discuss the causes for a sudden change in MCV
May be spurious or explained by an underlying disease and/ or treatment
- Causes of spuriously altered MCV:
• Mislabelled specimen/ WBIT
• Preanalytical/ compromised specimen
- Hyper/ hypo-osmolar plasma, underfilled tube,
- Delayed processing/ analysis
• Analytical
-Systematic vs random error
• Post analytical
-Transcription error
- Causes of disease/ treatment as a cause:
• Blood transfusion
Recent B12/ folate or iron therapy.
What haematology analyser is used in your laboratory? What parameters does it report?
- Sysmex XN-10 and XN-20
- 88uL of blood, 100 samples per hour
- Higher analysis time for cytopenic patients, if Plt-F measured
- Haemoglobin: Cyanide free SLS. Measured spectrophotometrically.
- Nucleated red cells: Fluorescent flow cytometry
- Reticulocytes: Fluorescent flow cytometry
- Ret-He: Fluorescent flow cytometry
- Hct: Impedance/ DC-Sheath flow
- Plt-I: Impedance/ DC-Sheath flow
- Plt-F: Fluorescent flow cytometry
- WCC- 6 count differential: Fluorescent flow cytometry
(DIFF, WNR, WPC)
Calculated: MCV, MCHC, MCH
What is the difference between ‘abnormal’ and suspect’ flags on the Sysmex analyser?
- Abnormal flags are based on laboratory’s own patient reference ranges and can thus be changed to be specific for that particular laboratory.
- Suspect flags such as PLT clumps?, RBC Agglutination?, HGB interference?, HGB defect? and Iron deficiency? are based on arithmetic algorithms that cannot be changed by the user.
What is ‘interference’ and why is it important?
When substances other than the measurement parameter are included or affect the final result
- Results are erroneous and may not represent the true biological status of the patient.
- Affects accuracy and reliability of the results.
An attempt must always be made to not only minimise or avoid possible interferences, but to be aware of or detect them when they exist in order to correctly interpret the results.
List the pre-analytical variables that can cause spurious results on an automated haematology analyser?
• Prolonged application of tourniquet (haemoconcentration, increased RBC)
• Haemolysed sample
(Hct falsely low, increased MCHC).
- Choice of anticoagulant: EDTA most appropriate (more soluble: less cell swelling)
- Volume in tube: volumes <2ml increases the EDTA volume to blood ratio. EDTA is hypertonic. This results in cell shrinkage and a spuriously low haematocrit, elevated MCHC, reduced MCV.
• Adequate mixing of sample
(to avoid a partially clotted sample)
• Time to analysis: As sample age, cells swell. This can falsely increase the MCV and Hct.
How does an aged sample affect full blood count results?
□ Effects of aging are generally due to a combination of the EDTA anticoagulant and time.
□ As sample age, cells swell. This can falsely increase the MCV and Hct.
□ Reticulocyte count reduces after 6 hrs. NRBCs drop after 1-2 days. Haemoglobin stable for up to 2 days.
□ Plt count and WCC rapidly decline. WCC differential affected due to abnormalities in morphology (although affected less when fluorescence flow cytometry technique used (such as in the XN analysers –> stable differential up to 48hrs post collection provided sample stored at 2-8 degrees).
□ Morphological changes become apparent within 3 hours and become increasingly severe beyond 18 hours.
- Sample storage at 2-8 degrees slows but does not eliminate these changes.
- An underfilled tube will accelerate these changes
□ White cell morphology: Loss of nuclear detail, cytoplasmic vacuolation, blurred cytoplasmic edges. Pyknotic change within the neutrophils. Nuclear budding within lymphocytes.
□ Red cell morphology: Cellular crenation and sphering.
Discuss how plts and red cells are counted with the impedance method
- PLT and RBC are both analysed in the same channel and are discriminated according to their volume.
- Hydrodynamic focussing applied to keep diluted sample in the middle of the stream
- Cells pass through an aperture of known size
- Current is applied across the aperture
- Cells impede current as they pass through the aperture
- Size of the pulse (resistance) is proportional to the size of the cell
- For PLT, Mean PLT volume ranges from 6 to 10fl, but impedance‐type counters analyse particles ranging from 2 to 20fl
- Red cells measured from 25- 250fl
List the possible causes of interference at the lower discriminator of the red cell histiogram that may result in falsely low or high red cell and or Plt counts when an impedance method is used
- Giant platelets
- Platelet clumps
- Microerythrocytes
- RBC fragments or dysplastic RBC
List some causes of abnormal RDW results
- Dimorphic population: multiple peaks within the histogram
• Iron deficiency with recent iron replacement or post transfusion
• Vitamin B12/ folate therapy under treatment or post transfusion
• Extreme leukocytosis (>600) - Widened RDW:
• Cold agglutinins (tail of RBC >200fl)
• Rouleaux formation
• Extreme anisocytosis
