w8 hematology Flashcards
what does hematology study
blood and blood forming tissue
two devisions of hematology
- hematology- studies formed elements in blood and other body fluids. rbc wbc and platelets
- coagulation studies the process of clotting
what is primary hematology test
CBC- complete blood count
automated analyzers
help diagnose anemia, leukaemia and infection
-number of rbc, wbc, and platelets
-hemoglobin concentration
-leukocyte differential (percent count of each type of red blood cell)
EDTA
CBC collected in EDTA ethylenediaminetetraacetic acid
prevents clotting and preserves cell morphology
must be clot free and homogenous
other non-blood hematology specimens
bone marrow aspirates, cerebral spinal fluid, joint fluids, semen
pre-analytical hem prep by MLA
- check volume and quantity
- clotted specimens can’t be tested
- load onto rocker-mix
- load onto cassette analyzer
- PBS
- staining other body fluid
- coverslipping
what does coag do
investigates clotting and bleeding disorders: haemophilia and thromboembolisms
-sodium citrate tubes (prevent clotting)
pre analytical-centrifuge higher speeds for longer to ensure a platelet poor specimen, plasma aliquoted off
PBS, why is it used
peripheral blood smears used for the microscopic evaluation of morphology of blood cells- it depends highly on proper cell distribution
two ways to manually make smears
- specail spreader mounted in a smear making device=consistent smears but needs to be cleaned a lot
- use another hematology slide as spreader -once slide used to smear blood it can be used for the next specimen or discarded
factors that effect quality of smear (5)
- age of specimen- morphology changes within 2-4 hrs-fridge come to room temp, prepare slides with freshly collected specimen- no longer than 24 hrs
- very clean slides with edges free of nicks-inspect for damage
- correct size of drop from well mixed tube 3mm
- quick smearing action after drop -dry=abnormal cell distribution and streaks
- correct consistent technique
correct consistent PBS
30 degree angle
spreader drawn back slowly into drop of blood, entire drop is allowed to spread along the spreader edge almost to the width of the slide then spreader is pushed away in a quick and smooth and straight motion keeping 30 degree angle
monolayer
rbc are evenly spaced while being viewed microscopically.(not overlapping)- monolayer visible under fluorescent lights- it looks like a rainbow
what does a good smear look like
- 3-4 cm
- thick to thin
- bullet or rectangular shape
- adequate monolayer
- thin edge free of tails or nicks
- not to end of slide
- no ridges wavy lines holes
- have an even wbc distribution when brewed microscopically
smear too short and thick
caused from: spreader angler greater than 30
and pushed too fast
smear too long and thin
spreader angle less than 30
or pushed too slowly
tails or streaks in smear (3 reasons)
- drying of drop creates streaks
- tails caused from lifting spreader at end
- spreader has nicks on end
monolayer too narrow on smear
this results from a smear that is too short and thick
monolayer too wide on smear
this results from a smear too long and thin
smear with holes and lumps
dirty or greasy slide results in holes
lint creates lumps
waves on smear
uneven spreader motion
chatters on smear
uneven pressure when spreading
flat sides on smear
spreader to pushed with even pressure on each edge
too narrow of a smear
spreader pushed too soon before blood can spread out
too wide of a smear
waited too long after drop was put on slide it was pushed
what to do if smear not stained right away
stabilize cells with methanol fixation so morphology preserved
biohazard of smears
hep and HIV may not be inactivated by methanol in first step of staining -back edges may not come in contact with stain
Romanowsky stain
any stain containing methylene blue and or products of oxidation and halogenated flourescein dye- usually eosin b or eosin y
types of romanowsky stains
-*wright’s
-Giemsa
-Leishmans
alcoholic solutions with acidic and basic components
polychrome stains
they can impart many colour giving typical colours to certain cell components
components of polychrome stains
acid dye component -EOSIN combine with basic cell components eosinophilic.(hemoglobin or eosinophilic granules)
basic dye components are methylene blue and azures, they combine with acidic cell components basophilic. DNA RNA azurophilic granules and basophilic granules
*when both basic and acidic stains are taken up by cytoplasmic or nuclear structures a pint to lilac colour develops and the structures are called neutrophils
eosin stain
acidic combines with basic cell components- hemoglobin or eosin granules MAKES PINK COLOUR
methylene blue or azures stain
basic combines with acidic cell components RNA DNA granules and produces a BLUE COLOUR
basic and acidic stain comp
combines with neutral cell components and makes a LILAC colour
3 steps of wrights stain
fixation in methanol
staining
washing
fixation in methanol (wrights stain)
- kills cells
- changes refractive index of cells
- makes cells more permeable to dye
- cells adhere to glass
- dehydrates cells
staining in wrights stain
- undiluted stain flooded on to smear to permeate cells
- stain diluted in buffer flooded next-from buffer wrights stain dissociates into different dyes (methylene blue, azures, alb,c, methylene violet, eosin y) each of these dyes stain different parts of cell.
washing in wrights stain
after staining rinse of stain with excess of buffer then allow to air dry
why use buffer in staining process
pH of distilled water can vary sorensens phosphate buffer most commonly used pH of 6.8
stain qc
test slide - PBS performed then stained and checked for colour, stain intensity, and artifacts.
wrights stain too blue
failure to stain eosinophilic cell components
- stain buffer too high(basic)
- staining time too long
- smear too thick
wrights stain too pinl
failure to stain basophilic cell components
- stain or buffer to low (acidic
- staining time too short
too light of a stain for wrights stain
-stain time too short
-too little stain or it dried onto slide
-wash bad, not vertical
refractive artifacts from water contamination
dip stainers
common stainer. slides loaded on rack, moved and lowered into buckets of solution-in bucket for specified time, moved to next bucket until complete
aerospray stainer
resembles centrifuge. carousel rotates then atomizing spray nozzles apply fresh reagents to slide
hema-tek stainer
stains smears by capillary action
-face down on platen or platform triggers sensing switches= activate three solution pumps which meter and deliver reagents. reagents fill space by capillary action
how are automatic stainers cleaned
daily
wipe with 70-95% alcohol or glutaraldegyde
no bleach
blood smears for malaria
Plasmodium invades liver and rbc
-detection of one parasite in blood smear is diagnostic
a thick and thin blood smear is done and Giemsa staining done to identify plasmodium
cytocentrifuge
concentrate bacteria or cells from liquid specimens– css synovial fluid, pleural fluid pericardial fluid, peritoneal fluid.
-concentrates formed elements for staining
slides-cleaned and labeled before use
ESR
erythrocyte sedimentation rate
-detect presence of an inflammatory response and a s a followup test to asses response to treatment in inflammations and injections
how does ESR work
anticoagluated blood standing= erythrocytes settle to bottom of tube– distance in mm that red cells fall through blood plasma per unit of time
modified wintrobe procedure for esr
EDTA blood drawn into self-zeroing wintrobe tube, vertical for 1 hour and the distance that red cells have fallen from top of the plasma meniscus to top of red cell column is recorded as ESR in mm/h
elevated ESR
non-specific finding associated with tissue damage and inflammation -relfecting changes in plasma proteins concentrations
women have higher ESR
automated ESR
shorten length of testing time by measuring 30 minutes then calculating mm/h- collected in sodium citrate black top tubes. evacuated to collect 1ml
