End Bench Flashcards
What is ESR used for?
- Only really diagnostic for temperal arthritis otherwise just a non specific marker of inflammation
We see lots of GP samples but rarely in house -> CRP better for urgents
What is the anticoagulant used for ESR
Sodium citrate anticoagulant 4:1 used in a long sedivette tube
What is the principle behind ESRs
○ Anticoagulated blood left to stand undisturbed will undergo rbc sedimentation
○ Discrete rbcs sediment slowly while aggregates settle more quickly e.g. rouleaux
ESR is non-specific but useful in disorders associated with increased production of acute-phase proteins
How do you carry out an ESR
○ Put tubes on yellow rack all together in a line and compare to reference tube making sure all tubes are filled correctly -> not too low or too high
○ Invert all tubes at least 6 times
§ Keep an eye out for blood clots as you do so
§ Check that all plungers are down all the way
○ Make sure all stickers are on the bottom of the tubes
§ So analyser can read level
○ Place tubes in correct spaces -> following suit of previous result
Set timer to 1 hour -> read results
Talk about Rouleaux in ESRS
The alteration in red cell zeta potential caused by proportions and concentrations of hydrophilic proteins such as fibrinogen, haptoglobin and CRP
What analyser did you measure ESRs on?
Sediplus S2000
How did the Sediplus S2000 work?
○ Sample is inserted into any measuring position -> measurement begins
○ During measurement the plate moves up and down
○ A light beam passes by all Sedivettes
○ Behind the Sedivette the light beam falls onto a detector
○ The surface of the erythrocyte layer is detected as a change of the light intensity during the movement
○ Measurement taken at 30 mins and 1 hour
After 1 hour the result is sent over to LIS
In general how were haemoglobinopathy investigations carried out in MMUH?
Minicap Sebia and HPLC
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
What is the principle behind 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
What stains do we do on our bone marrows?
MGG
Perl’s prussian blue for haemosiderin/iron studies
What is done with bone marrow trephines
Sent to histo -> not dealt with in micro
Talk about PPB for bone marrow
○ We can demonstrate iron in either bone marrow or urine using Perl’s Prussian Blue
○ A reaction between acid ferrocyanide and the ferric ion (Fe3+) of haemosiderin to form ferrocyanide which has an intense blue colour known as Prussian Blue
○ We use this stain to detect iron stored within the marrow and intracellular siderotic granules
We can use the stain on urine to investigate a suspected chronic intravascular haemolysis
Talk about Sickle Cell Disease
- HbS is an inherited characteristic which occurs either in the homozygous condition in S/S sickle cell anaemia or the heterozygous A/S sickle cell trait state
- HbS can also be seen with other abnormal Hbs or with thalassaemia
- HbS forms tactois/liquid crystals within erythrocytes under conditions of low oxygen tension resulting in “sickle shape” distortion of the rbc
- In the past SCD patients didn’t live past adolescence but thye now survive well past 40 with treatment
Certain conditions can bring about low oxygen tension which can convert SCT into a serious and potentially fatal clinical condition
What is the principle behind the Sickle Cell Screen?
- 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
- Blood containing HbS when added to the reagent powder dissolved in test solution will form a cloudy, turbid suspension
How do you carry out a SCS
○ 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 might cause interference in a SCS
○ Multiple myeloma
○ Plasma interference -> Wash rbcs
Low Hb?
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
What is NADPH involved in?
- NADPH is required for the reduction of oxidised glutatione (GSSG) to GSH as well as for the reduction of mixed disulphides of glutathione and cellular proteins