Past FBC/Film/End Bench VIVA Qs Flashcards
What is the x bar mean?
Moving average
- The analyser picks a stable population of sufficient size e.g. MCV, MCH or MCHC and establishes it's mean - Any shift or drift in the mean value should signal an IQC alert - At least 100 data points are needed to supply this data - The Xbar is re-established every 30 patients - Xbar doesn’t necesaarily work in the Mater all of the time as we have a large number of abnormal patients e.g. in the morning when we process all of our leukaemia samples
What controls are there on the Sysmex
○ 3 level control
○ Drift control
○ Inter analyser comparison
○ Xbar mean -> BULLS algorithm
Delta checks
What does a delta check tell you?
WBIT
Deteriorating patient?
What would you do with a low platelet?
Check sample for clot -> if clotted request repeat
Check sample for platelet clumps
If very low phone result
What might cause a lymphocytosis?
Reactive -> viral infection e.g. EBV
Inflammation -> look at ESR and CRP
Malignancy -> CLL or ALL
What blood films did you see?
Low platelets
leukaemias
Intravascular haemolysis
What red cell disorders did you see?
Sickle cell patient
Was a patient who had aged out of childrens hospital but James’ hadn’t accepted yet
Iron deficiency anaemia -> hypochromic, microcytic anaemia
Saw a few vitamin B12 deficiencies -> megaloblastic anaemia -> macrocytic anaemia
What would indicate intravascular haemolysis?
Normocytic, normochromic anaemia (low Hb)
Fragment flag
Schistocytes on blood film
Features of a vitamin B12 deficiency
Low Haemoglobin
Macrocytic rbcs
Hypochromic rbcs (due to low Hb)
Hypersegmented neutrophils
Anisopoikilocytosis + fragments in severe
Nucleated red blood cells in severe
Features of intravascular haemolysis
Low/decreasing Hb
Fragment flag
Schistocytes
Polychromasia
Increased reticulocytes
When might you see spherocytes
Hereditary spherocytosis
Autoimmune haemolytic anaemia
Burns
Causes of low platelets
Acute leukaemia
Chemotherapy
Antibiotics e.g. linezolid
ITP
TTP
DIC
Acute vs chronic leukaemia
Chronic:
- Mature cells
- Few blasts/no blasts
- Increased cells e.g. lymphocytosis/monocytosis etc
- Smudge cells in CLL
Acute:
- Immature cells
- Increased blasts
- Low Hb
- Low platelets
- Auer Rods in AML
- neutropenia also common
CD molecules for flow cytometry
Leucocytes = CD45
B lymphs = 19 + 20
T cells = 3
Myeloid lineage = CD13
Acute marker = CD34
Specifics:
CD5 = (B-CLL/Mantle cell)
CD10 = CLL
CD15 = AML
How would you diagnose acute leukaemia?
FBC -> low Hb, low platelets, possibly low lymphs/neuts, blast cells
Blood film -> features e.g. immature cells, blast cells etc
Flow cytometry
Bone marrow aspirate -> hypercellular -> blasts
When would you suspect infectious mononucleosis?
Reactive lymphs
Fever
Not improving with antibiotic treatment
Name of track
Principle of track
Principle of impedence flow cytometry
- Sample is diluted with sheath fluid and hydrodynamically focused through the middle of the flow cell
- A laser interrogates each cell individually as it passes by
- Detectors then collect either the forward scatter or side scatter of light
- A fluorescent dye is included in the reaction channel which is used to measure fluorescent light FL and side scatter side fluorescent light provides information on the RNA/DNA content of the cell
- Laminar flow ensures that cells are not counted twice
- As cells pass through the apperature, they cause an electrical resistance which is recorded as impedence pulse
- The size of the cell is proportional to the pulse height
The RBC and PLT histograms are generated from this
Features of iron deficiency anaemia
Hypochromic, microcytic anaemia
Anisopoikilocytosis
Target cells, pencil cells etc
Reticulocytes
Polychromasia
Features of sickle cell disease
Sickle shaped rbcs/poikilocytosis
Symptoms of intravascular haemolysis during times of crisis
- thrombocytopenia
- Polychromasia
- nucleated red blood cells
- howell-jolly bodies (splene)
Increased white blood cells sue to chronic inflammatory state
principle of ESR
When Anticoagulated blood left to stand undisturbed will undergo rbc sedimentation
Discrete rbcs sediment slowly while aggregates settle more quickly e.g. rouleaux
Increased in temporal arthritis + pregnancy + inflammation
How does the Sediplus S2000 work?
Light beam passes by all Sedivettes
Behind the sedivettes the light beam falls onto a detector
The surface of the erythrocyte layer is detected as a change in the light intensity during the movement
Measured at 30 mins and 1 hour
Talk about the use of the MiniCap Sebia for Haemoglobinopathy investigations
Digital electrophoresis graph showing peaks for each Hb variant
Much easier to use than HPLC but cannot report using it
Provides electrophoresis graph of Haemoglobin
Minicap doesn’t really tell you what variant is present it just tells you what variants are found in each region
○ Hence why its not reportable
HPLC or genetic testing needed to confirm results
Talk about HPLC for haemoglobinopathy investigation
Bio-Rad D-10 Hb Testing system
Cation exchange HPLC
- Provides a print out of a graph with a table of results
○ Gives us the area of each Hb variant
○ We can only report HPLC results -> not minicap results
Only gives conentrations for HbF, HbA1c and HbA2
Principle of HPLC
○ The separation of a mixture of molecules e.g. normal and variant haemoglobins with a net positive charge into its components by their adsorption onto a negatively charged stationary phase in a chromatography column, followed by their elution by a mobile phase
○ The mobile phase is a liquid with an increasing concentration of cations flowing through the column
§ The cations in the mobile phase compete with the absorbed proteins for the anionic binding sites
○ Thus the adsorbed positively charged Hb molecules are eluted from the column into the liquid phase at a rate related to their affinity for the stationary phase
When separated in this way they can be detected optically in the eluate, provisionaly identified by the elution time and quantified by computing the area under the corresponding peak in the elution profile
Sickle cell screen principle
SCS os based on the relative insolubility of Hb-S when combined with buffer and a reducing agent
Blood containing HbS when added to the reagent powder dissolved in test solution will form a cloudy, turbid suspension
Other forms of Hb are much more soluble and will form a transparent solution
SCS method
○ Make up sickle-dex reagent (powder+test)
○ Spin down patient rbcs -> take out any rbcs when pipetting -> want as little plasma contamination as possible
○ Incubate blood and controls with reagent for 15 mins then read
○ Place tubes up against straight lines
§ If you can see lines = negative
If you cannot see lines = positive
What is G6PD and what is it involved with?
a rbc enzyme called glucose-6-phosphate dehydrogenase which catalyses the initial step in the pentose phosphate pathway of glycolysis
- This pathway is responsible for the reduction of NADP to NADPH
- The pentose phosphate pathway also supplies five carbon sugars to rbcs which are required for the synthesis of adenine nucleotides
G6PD screen
○ Incubate a small volume of whole blood with glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP)
○ Drops of the mixture are removed at 0, 5 and 10 minutes
○ These drops are spotted on filter paper and viewed under long-wave ultra violet light
Fluorescence is clearly evident in mixtures prepared from whole blood, whereas deficient samples yield little or no fluorescence
What three tests do we carry out for malaria
○ Rapid immunochromatographic slide test (ICT)
○ Thick blood film stained with Fields stain
Thin blood film stained with Giemsa stain
ICT for malaria principle
○ An immunochromatographic assay for the qualitative detection of Plasmodium antigens
○ Can differentially diagnose between P.f and all other plasmodium species
○ The test contains a membrane strip pre-coated with two monoclonal antibodies
§ One is pan specific to lactate dehydrogenase (pLDH) of the plasmodium species
§ The other is a monoclonal antibody specific to Histidine-rich Protein2 (HRP2) of the Plasmodium. Falciparum species
○ The conjugate pad is dispensed with monoclonal antibodies which are pan specific to HRP2
Positive = visually detectable red line
IMS prinicple
○ Diagnosis of IMS is based on the presence of heterophile antibodies against EBV in the patient’s plasma
○ These antibodies are seen in approximately 80-90% of acute IM and are detectable in about 60-70% of patients during the first week of clinical illness
○ The Clearview IM II is used for the screening of IMS
○ The test contains a control line (+) and a test line
○ The test line contains bovine erythrocyte extracted antigen
Sysmex principle
○ RBC + PLT dilution is injected into the RBC PLT detector
○ The sample then passes through the middle of the aperature where it is hydrodynamically focused to ensure laminar flow
§ This ensures no cell is counted twice i.e. cells in single file
○ As the cells move through the aperture they cause an electrical resistance which is recorded as an impedence pulse
§ The size of the cell is proportional to the pulse height
The collective of these pulses produce the RBC histogram
SLS-Hgb method for Hb - spectrophotometry
Lysercell and fluorocell reagents for wbcs
