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
What does GDH do?
GSH is necessary for the detoxification of hydrogen peroxide and organic peroxides
What chromosome is G6PD gene located on
X chromosome
What are some symptoms of deficiency
○ Drug sensitivity e.g. antimalarials, nitrofurantoin, sulphonamides, dapsone, phenacitin
○ Favism
○ Haemolysis associated with infections
○ Chronic non-spherocytic haemolysis with anaemia
○ Neonatal jaundice
How would you carry out a 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 would no fluorescence in a G6PD sample mean?
G6P deficient
Why do we measure plasma Hb
We measure plasma Hb as an indicater of haemolysis for patients on extracorporeal devices such as ECMO
What is ECMO
a device used as a form of artifiical ciruclation for ICU patients
How does ECMO cause haemolysis
Increases the risk of negative pressure on the blood and increases the risk of the Hb being liberated into the plasma
How would you investigate query intravascular haemolysis for someone on ECMO
Plasma Hb
Other measurements should be taken to monitor the degree of haemolysis e.g. a continuously dropping haematocrit, rising potassium levels, blood in urine etc
What is the principle behind plasma Hb measurement
○ A modified azidemethemoglobin reaction takes place in the microcuvette
○ The rbcs are lysed to release the Hb, the Hb is then converted to methaemoglobin and then combined with azide to form azidemethemoglobin
○ Absorbance is measured at two wavelengths 570nm for Hb concentration and 880nm to compensate for a certain degree of turbidity
How does high levels of free Hb affect the patient
affect the kidney/liver function and also reduce the oxygen carrying capacity of the blood
How do we measure plasma Hb
○ We use a Haemocue and lithium heparin anticoagulated blood
○ Hemocue Plasma/Low Hb system is used for the quantification of low levels of Hb by using a specially ddesigned photometer and sepcially designed cuvettes
○ A modified azidemethemoglobin reaction takes place in the microcuvette
○ The rbcs are lysed to release the Hb, the Hb is then converted to methaemoglobin and then combined with azide to form azidemethemoglobin
○ Absorbance is measured at two wavelengths 570nm for Hb concentration and 880nm to compensate for a certain degree of turbidity
The absorbance is directly proportional to the Hb concentration
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
Talk about processing a sample for malaria
- We require at least four thin slides and four thick slides be made <2hours post phlebotomy
- Thin slides need only 10 mins to dry
- Thick slides need >1 hour to dry
- Stain two of the thicks with Fields
- Stain two thins with Giemsa at pH 7.2 to find and identify malaria parasites
Percentage parasitaemia must be performed if P, falciparum or P, knowlesi is identified
Rapid ICT also carried out
At what pH is the Giemsa stain carried out at?
pH 7.2 -> required to visualise the parasites
For what organisms must you carry out a parasitaemia
P. falciparum
P. knowlesi
What must be done with any positive malarias
- Any positive results are to be phoned and later confirmed by a UK malaria referal lab
All positives must also be communicated to Public Health via microbiology lab
What ICT is carried out for malaria
The CoreStart VS Rapydtest pLDH/HRP2 Combo (PAN/pf) malaria kit
How does the IHC kit for malaria work?
○ 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
What exactly does the IHC kit detect
Pan specific lactate dehydrogenase (pLDH) present on all plasmodium species
Histidine-rich Protein 2 (HRP2) which is specific to Plasmodium Falciparum
How would you interpret an IHC where line 1 = positive and line 2 = negative?
P. falciparum
How would you interpret an IHC where line 1 = negative and line 2 = positive?
Plasmodium species other than falciparum
Most likely vivax
How would you interpret an IHC where line 1 = negative and line 2 = negative but control line positive?
Negative for plasmodium species
How would you interpret an IHC where line 1 = positive and line 2 = positive?
Mixed infection
How do you carry out a Field’s Stain
○ Blood is concentrated on a slide - thick layer of many cells - increases concentration of parasites
○ The rbcs are then lysed to allow for visualisation of any parasites
○ Fields stain A and B is used
Can only be used for detecting the presence of parasites not the identification of them
When do we carry out an IMS screen
- We either fulfil requests or we can add on an IMS screen if we have a high suspicion of mono
It can be seen where a GP has sent bloods for a feverish patient, GP has not requested IMS (patient may not have normal symptoms/no swollen throat etc
When might we add on an IMS screen
Patient goes for a WDF -> histogram abnormal, elevated lymphocytes, reactive pattern seen
What shouhld we do with a positive GP IMS
if positive we must phone the result to the GP and explain what this means
What could a falsely negative IMS screen be caused by
Not everyone produces antibodies against EBV
What is the principle behind the IMS
○ 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
How do you carry out an IMS screen
○ Patient plasma+buffer is added to the reaction well
○ The mixture migrates chromatographically along the strip, reacting with both the test and control line
○ If plasma contains antibodies a coloured line will appear
The control line indicates the correct volume was used and that membrane wicking has occurred
