FBE studies + stains Flashcards
Hct/PCV
Both measurements of % of blood volume occupied by RBCs.
PCV - directly measured value by centrifuging blood sample in a microhaematocrit. (gold standard)
Hct - calculated value from pulse height of red cells as they pass through. Hct = (RBC xMCV)/10. Our analyzer automatically calculates Hct based on summation of pulses.
MCH - mean corpuscular haemoglobin
Average mass of Hb per red cell
MCH = Hb g/L / RBC (10^12) x 10
MCHC - Mean corpuscular haemoglobin concentration
Avg concentration of Hb in a given volume of packed RBC.
MCHC = (Hb / Hct ) x 10
Causes of change in MCHC
Low - iron def, thal
High - HS, burns, red cell autoagglutination
Can be a useful indicator of early iron def when using manual method (PCV measurement)
Artefact in aperture impedance of red cells
Normally, red cell is deformed into cigar shape when passing through aperture.
Lower Hb = more flexible = more elongation = lower pulse height = underestimation of cell size and thus MCV and Hct
Higher Hb (as in spherocytes) = less flexible = less elongation = higher pulse height = overestimated MCV
Assessment of red cell variation
Histograms of volume/seize distribution –> detects presence of multiple populations of cells
% of cells that fall above or below certain MCV thresholds –> flag. Allows detection of macrocytes or microcytes.
RDW
Measure of red cell variation in size/volume - aka anisocytosis
RDW SD - width at the 20% height level of red cell size distribtuion
Distinguishing iron def (inc) vs thal (normal)
Megaloblastic anaemia (inc)
Haemoglobin concentration variation
Variation of Hb conc in individual cells
- % of hypochromic cells
- distribution curve of Hb concentration
Detects iron def erythropoiesis (If >2.5% hypochromic cells)
HDW (hemoglobin distribution width)
Quantifies the degree of variation in red cell hemoglobinization.
Can distinguish between hypochromic microcytes vs hypochromic macrocytes
Increased % of hyperchromic cells
- spheros
-irregularly contracted cells
- sickles
Blood collection
EDTA - ethylene diamine tetra acetic acid
- irreversibly chelate Ca2+
Blood film prep - detailed/stains
- blood collection
- EDTA tube - prepare blood smear
- drop blood 1cm from edge, smear at 30*.
- anaemic - smear @ >30
- PV - smear @ <30
DRY - fixation
- prevents cellular deterioration by rendering cells resistant to osmotic lysis.
- absolute methanol for 10-20mins - romanowsky staining principle:
- basic dye (catitonic +ve charge) for anionic (acid) molecules ie DNA/RNA, basophil granules -> stains blue-violet/blue.
eg. Methylene Blue, Azure B,
- Acidic dye (anionic -ve charge) for cationic (basic) molecules) ie. Hb, granules of eosinophils. –> stains re-pink or orange.
eg. , Eosin, Eosin Y
Most common stains in Aus for
Film: Wright stain: mixture of eosin and methylene blue dyes
Aspirate: May Grunwald Giemsa, MG (Methylene Blue) ACIDIC & Giemsa (Methylene azure) BASIC)
pH 6.8 on most labs, use more basic ph7.2 for malaria to see Schuffners dots.
Blood film artefacts associated with excess water
- premature staining (smears not dry) –> apparent nuclear extrusion
- excess % water in fixatives –> apparent red cell osmostic swelling and hypochromia
Blood aspirate process
MGG - May Grunwald Giemsa
- fix slides in methanol 10 mins
- place slides in May Grunwalds stain for 10 mins
- Diluted Giemsa stain for 20 mins
- Sorensons phosphase buffer for 30 sec
- Air dry
Blood film process
Wrights but can also use MGG.
- spread/smear
- fixation (methanol 1 min)
- Stain (Wrights stain 2 mins, then dilute Wrights stain for 5 mins). Rinse slides in water 20 sec
- Place in Sorensons phosphate buffer for 1 min
- Air dry
Perl’s stain
Principle
- Siderotic material is a water insoluble complex of iron, lipid protein and carbohydrate
- Produces a positive Perl’s (or Prussian Blue) reaction
- Bone marrow siderotic granules may be visualised lying free or within phagocytes or erythroblasts
Sample
-Bone marrow aspirate slide with ≥1 particle (3-5 preferable)
Method
-Fix slides in methanol for 10 min
- Allow to air dry
- Mix 10 ml of 2% potassium ferrocyanide (reacts with ferric iron to form the Prussian Blue stain) and 10 ml of 0.2 mol/L HCl (releases ferric iron from haemosiderin)
- Immerse slides (at room temperature) for a minimum of 30 min
-Wash in running tap water for 5 min
-Counterstain with neutral red for 2 min
-Wash in running tap water
- Allow to air dry (completely)
Quality control (QC)
- Internal as compared with a positive control (prepared with each batch and with each patients stain)
Limitations
- Invalid results can occur if there is iron contamination in equipment and water
- Interpretation is difficult if there are inadequate marrow particles or poor staining technique (stain deposit or
‘starring effect)
Bone Marrow trephine processing
Principle
- removal of water from the sample and provides cellular structural support so that thin sections can be cut for analysis
Sample
-Bone marrow trephine in fixative (formalin)
Method
1. Fixation (≥2 hr) –> using either formalin or B5 (mecuric chloride, sodium acetate and formalin)
2. Decalcification
- Sample secured in a cassette then washed under running water
- Placed in weak organic acid (formic and hydrochloric acid) for 2 hrs and washed under running water
3. Fixation - post decalcification fixation in formalin
4. Dehydration and clearing - Sequential ethanol baths to remove any water and xylene baths to remove the ethanol
5. Embedding - infilitration with molten paraffin wax
6. Sectioning - sections cut on a microtome. 2 microns for H&E and 2 microns for IHC
7. Staining and mounting
Some limitations with trephine processing
**Formalin fixation –> Morphology poorer than that with mercurial and longer turnaround time
** B5 –> Antibody binding by IHC and DNA extraction affected due to protein denaturing by coagulation
Mecury exposure (requires disposal by incineration)
** Decalcification and paraffin-embedding –> Lead to considerable shrinkage and some loss of cellular detail. Cellular detail is harder on thicker specimens. Some cytochemical activity is lost
Romanowsky Stain troubleshooting
Haematoxylin & Eosin (H&E) stain on BM trephines
- Haematoxylin complex with aluminium salts is cationic and acts as a basic dye; basophilic cell components eg.nucleic acids (stain blue)
- Eosin is anionic and acts as an acidic dye; acidophilic
components eg. cytoplasm; Hb - Excellent for demonstrating the cellularity and pattern of changes such as fibrosis or the presence of granulomata or carcinoma cells
Reticulin Stain
- Silver impregnantion stains the glycoprotein matrix, which is associated with connective tissue
- Bone marrow always contains a small amount of this material, which is referred to as ‘reticulin’ and is an early form
of collagen
Immunohistochemistry on trephines
Method of demonstrating antigens in tissue sections by use of a monoclonal antibody or polyclonal antiserum, and a chromogenic detection system
- eg cell surface receptors,
nuclear transcription factors and cytoplasmic proteins; as well as identifiable proteins such as oncogenes and tumour suppressor genes and hybrid proteins
Method
- Binding of engineered monoclonal antigen to specific protein epitope
- Antibody-antigen complex is then made visible in tissue sections by the addition of a specific chromogen, which
binds with the complex usually via an intermediary substrate -> magnifies the amount of chromogen deposited so that is visible under the light microscope
- Tissues rich in endogenous peroxidase (granulocytes), phosphatase (macrophages) or biotin (mast cells) may give
false-positive reactions so can be blocked by hydrogen peroxide, levamisole and a biotin blocker, respectively
Quality control (QC)
- Internal
▪ Positive and negative tissue control with each sample
- External
▪ RCPA: 3 samples, 1 times a year
- Limitations
-Less sensitive than flow cytometry in cases where the amount of antigen expressed by a cell is low - Analysis of expression of more than 1 antigen within a tissue section is technically difficult and in practice limited to 2 or at most 3 antigens
-some stains not validated or as effective on trephine sampling compared to other tissues
Immunohistochemical stains for non-haematopoietic malignancy
- AE1/3 = carcinoma
- CK20+/CK7- = colorectal/lower GI
- CK7+/CK20(+/-) = upper GI
- TTF1 = lung
- GATA3 = breast (+ER/PR/HER2)
- PSA/PSAP = prostate
- Melan A, Sox10, S100 = melanoma
- Synaptophysin = neuroendocrine
