Past FBC/Film/End Bench VIVA Qs

Question 1

What is the x bar mean?

Answer 1

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

Question 2

What controls are there on the Sysmex

Answer 2

○ 3 level control
○ Drift control
○ Inter analyser comparison
○ Xbar mean -> BULLS algorithm
Delta checks

Question 3

What does a delta check tell you?

Answer 3

WBIT
Deteriorating patient?

Question 4

What would you do with a low platelet?

Answer 4

Check sample for clot -> if clotted request repeat
Check sample for platelet clumps
If very low phone result

Question 5

What might cause a lymphocytosis?

Answer 5

Reactive -> viral infection e.g. EBV

Inflammation -> look at ESR and CRP

Malignancy -> CLL or ALL

Question 6

What blood films did you see?

Answer 6

Low platelets
leukaemias
Intravascular haemolysis

Question 7

What red cell disorders did you see?

Answer 7

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

Question 8

What would indicate intravascular haemolysis?

Answer 8

Normocytic, normochromic anaemia (low Hb)

Fragment flag

Schistocytes on blood film

Question 9

Features of a vitamin B12 deficiency

Answer 9

Low Haemoglobin
Macrocytic rbcs
Hypochromic rbcs (due to low Hb)
Hypersegmented neutrophils
Anisopoikilocytosis + fragments in severe
Nucleated red blood cells in severe

Question 10

Features of intravascular haemolysis

Answer 10

Low/decreasing Hb

Fragment flag
Schistocytes
Polychromasia
Increased reticulocytes

Question 11

When might you see spherocytes

Answer 11

Hereditary spherocytosis
Autoimmune haemolytic anaemia
Burns

Question 12

Causes of low platelets

Answer 12

Acute leukaemia
Chemotherapy
Antibiotics e.g. linezolid
ITP
TTP
DIC

Question 13

Acute vs chronic leukaemia

Answer 13

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

Question 14

CD molecules for flow cytometry

Answer 14

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

Question 15

How would you diagnose acute leukaemia?

Answer 15

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

Question 16

When would you suspect infectious mononucleosis?

Answer 16

Reactive lymphs
Fever
Not improving with antibiotic treatment

Question 17

Name of track

Question 18

Principle of track

Question 19

Principle of impedence flow cytometry

Answer 19

• 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

Question 20

Features of iron deficiency anaemia

Answer 20

Hypochromic, microcytic anaemia

Anisopoikilocytosis

Target cells, pencil cells etc

Reticulocytes

Polychromasia

Question 21

Features of sickle cell disease

Answer 21

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

Question 22

principle of ESR

Answer 22

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

Question 23

How does the Sediplus S2000 work?

Answer 23

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

Question 24

Talk about the use of the MiniCap Sebia for Haemoglobinopathy investigations

Answer 24

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

Question 25

Talk about HPLC for haemoglobinopathy investigation

Answer 25

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

Question 26

Principle of HPLC

Answer 26

○ 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

Question 27

Sickle cell screen principle

Answer 27

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

Question 28

SCS method

Answer 28

○ 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

Question 29

What is G6PD and what is it involved with?

Answer 29

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

Question 30

G6PD screen

Answer 30

○ 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

Question 31

What three tests do we carry out for malaria

Answer 31

○ Rapid immunochromatographic slide test (ICT)
○ Thick blood film stained with Fields stain
Thin blood film stained with Giemsa stain

Question 32

ICT for malaria principle

Answer 32

○ 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

Question 33

IMS prinicple

Answer 33

○ 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

Question 34

Sysmex principle

Answer 34

○ 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