Exam 3 Flashcards
What measurements in the hemacytometer in the large squares and in small squares, what is the total volume on one side, how much depth?
large squares, 1x1
small center squares 0.2 x 0.2 mm
0.1 depth
total volume 0.9mm
What is the formula to get result from hemacytometer
total count = (cells counted x dilution factor)/ area x depth
What is the dilution in WBC counts
1:20
What is the corrected WBC count
if there are WBCs in an RBC count, they are indistinguishable,
must use calculation
corrected WBC count= (uncorrected WBCx100)/ RBCs per 100 WBC +100)
What do we use to dilute a plt count, what dilution factor do we use
ammonium oxalate, 1:20
What is the cyanmethemoglobin method for hgb
drabkin solution
hgb-> oxidized by potassium ferricyanide into methemoglobin -> potassium cyanide turns it into cyanmethemoglobin
cyanomethemoglobin is absorbed at 540nm
What are the common sources of error from a microhematocrit reader
sensitive to light
high WBC, plt, lipemia or abnormal hgb give high results
highly toxic
What are the common sources of error from a microhematocrit reader
sensitive to light
high WBC, plt, lipemia or abnormal hgb give high results
highly toxic
Define hematocrit
volume of packed RBCs in whole blood
packed cell volume
What could cause error in hematocrit test results
not sealing the capillary tube correctly
too much anticoagulant shrinks RBCs
not mixed
overcentrifuged
reading results wrong
What is the rule of three in hematology, what if it is not met
The value of the hematocrit should be 3x the hgb (+-3)
should be examined for abnormal RBCs could be lipemia, spherocytes, hypochromic and microcytic
Name the RBC indicies
MCV- mean cell volume
MCH- mean cell hemoglobin
MCHC- mean cell hemoglobin concentration
RDW- red distribution width
What are the possible indications in RBC morphology for MCV and MCHC
MCV microcytic (<80), normocytic (80-100), macrocytic (>100)
MCHC hypochromic (<32) normochromic (32-36) spherocytes (>36)
What is the MCV formula
HCT% x 10/ RBC
What is the MCH formula
Hgb x 10/ RBC
What is the MCHC
HGB x 100/ HCT
Why is a reticulocyte count performed
to assess the erythropoietic activity of the BM
What is used to stain the retic count
new methylene blue
What is the reticulocyte count calculation
retic = # retics x 100/ 1000 RBCs counted
a nonnucleated RBC with 2 or more particles of blue stained granulofilamentous material is considered
reticulocyte
What is a miller disk
for counting 1000 RBCs, has a large and small square, small square for RBCs large one for retics
What is an ARC?
absolute retic count- number of retics in 1L
What is the RPI
retic production index
used to correct hematocrit if high retics
What is the ESR
erythrocyte sedimentation rate or sed rate
to detect inflammatory conditions, infections, malignancies
What is the principle of an ESR
distance in mm the RBCs fall per 1 hour
What are the disadvantages to the ESR test
low sensitivity and specificity
What factors affect ESR
RBC factors: rouleaux, RBC mass proportional to EST
plasma factors: too many proteins cause rouleaux,
mechanical factors: tilted tube, insufficient centrifuge
Indicate whether the following examples would have an increased or a decreased ESR
Pregnant patient
Anemic patient (NOT sickle cell)
Sickle cell anemia doesn’t increase the sed rate, could be normal, or decreased
Polycythemia
Newborn
Multiple myeloma (increased proteins in blood)
Pregnant patient - increased
Anemic patient (NOT sickle cell) - increased
Sickle cell anemia doesn’t increase the sed rate, could be normal, or decreased
Polycythemia - decreased
Newborn - decreased
Multiple myeloma (increased proteins in blood)- increased
Who most often does POC testing
nurses
What are 3 POC tests
hematocrit on hematostat
Hgb conentration- HemoCue
cell and plt counts on heme analyzer
What is electronic impedance
detection and measurement of changes in the electrical resistance produced by cells as they cross a small space on instryment
What separates cells in electronic impedance
volume thresholds
What does the height of voltage pulse =
volume of cell
How are results given in impedance tests
volume distribution histogram
What type of differential in impedance tests
3 part diff
lymphs
mononuclear cells and granulocytes
What is radiofrequency
low voltage DC impedance that is used with high voltage electromagnetic radiofrequencies
Cell volume is proportional to ______
change in DC
Cell interior density is proportional to
change in RF signal
What results do radiofrequency instruments give
scatterplots
What kind of differential do radiofrequency instruments give
5 part diff
neutrophils, lymphs, monocytes, eosinophils and basos
Forward scatter light at 0 degrees relates with
cell volume
sideway scatter light relates with
internal complexity of the cell
What instrument utilizes MAPSS to perform its WBC dif?
cell-DYN
New parameters and indices may be useful for
diagnosis and treatment of anemias
Immature retic fraction or IRF shows
an early indication of erythropoiesis
IFR and absolute retic counts
help distinguish types of anemias
Match: cold agglutinins, icterus and lipemia, hemolysis, plt clumps, old specimen
-low RBs and high HCT,
-high MCV and MCHC and decreased RBC
- high MCV, high MPV, low plt
-high HGB and high MCH
-low plts high WBCs,
hemolysis- redraw
cold agglutinins- warm specimen
old specimen- redraw
icterus and lipemia- plasma replacement
plt clumps- sodium citrate tube
How much time do we have to make a blood film slide
4 hrs
What angle should the pusher slide be at
30-45 degree
How should the angle change if there is a high HCT? and a low one?
high- lower angle
low- higher angle
How much of the slide should the film take up
what shape should it have
2/3
feathered edge
What stain is used in peripheral blood smears
wright Geimsa stain
What do polychrome stains contain
eosin and methylene blue
What does eosin do, is it acidic or basic
acidic, stains cell components like Hgb, eosinophillic granules
What does methylene blue do? is it acidic or basic
stains acidic cellular components like RNA
it is basic
List the steps to staining a blood smear slide
flood with wright stain for 1-3 min
add buffer to slide, must look metallic green, 3 min
rinse with distilled water and air dry in vertical position
What should the colors of RBCs, WBCs and eosinophils have on a proper stained blood film
RBCs orange to pink
WBCs purple nuclei , pink cytoplasm and granules visible
eosinophils bright orange
What causes RBCs to appear gray, WBCs to be dark and eosinophils to look gray on a blood film
stain or buffer was too alkaline
did not rinse stain well enough
stained for too long
used heparin specimen for CBC
What causes RBCs to be too pale or red
and WBCs to be barely visible
stain or buffer is too acidic, not enough buffer, rinsed too much
what is the ideal area for reading a peripheral smear
a spot where RBCs are uniformly distributed, no touching or overlapping, normal biconcave appearance
200-250 RBCs per field
What units are used to report
WBC
RBC
HGB
HCT
PLT
SI listed first
common listed second
HGB and HCT- Common units, not in L- in uL or dL or %
RBC, WBC, PLT- SI unit, in L
What is absolute vs relative
absolute- real numbers
relative is a percentage
philia means ____ and cytosis means ____
philia- increase
cytosis- also increase
penia mean
decrease
RDW, HGB, HCT, MCV, MCH, MCHC are all parameters of
RBCs
What results could be given if RDW is out of range
variations in RBC volume
anisocytosis- if high
11.5-14.5%
If a sample has an MCV of 70?
and 110?
70- microcytic
110-macrocytic
If a sample has an MCHC of 36
or of 20
20-hypochromic
36- spherocytes
MPV is a CBC parameter for
platelets
mean platelet volume
an increased plt count is called ____
a decreased plt count is called ____
thrombocytosis
thrombocytopenia
If a sample has increased plts, WBCs and HGB this is called
if all are decreased it is called
pancytosis
pancytopenia
If WBC count is low it is called __
if it is low ___
leukopenia
leukocytosis
The best parameter that indicates a pt is anemic is
the HGB
If a sample has abnormally large platelets
the platelet histogram will be abnormal with peaks
What is the reason flow cytometry testing is performed
detection of fluorescent tags directly bound to specific molecules or through monoclonal antibodies
What is the reason flow cytometry testing is performed
detection of fluorescent tags directly bound to specific molecules
What is flow cytometry most often used for
leukemias, lymphomas, monitoring HIV
What test tubes are used in flow cytometry?
heparin and EDTA
How much time do you have to process a sample into flow cytometry after the time of collection
24-48 hrs
How should specimen for flow cytometry be stored
less than 24 hrs at RT for BM and peripheral blood
less than 24hrs at 4C for tissues and fluids
What allows a sample to be stained with flurochrome?
monoclonal antibodies
What lineage do these CD markers represent
CD34
CD117
TdT
Immature cells
What lineage do these CD markers represent
CD33, CD13, CD15, CD14
granulocytes/ monocytes
Three blind Mice
What lineage do these CD markers represent
CD71, glycoprotein A
erythroid
Rrolling stones, 70s rock
What lineage do these CD markers represent
CD41, CD42, CD61
Megakaryocytic
Four-fat albert
What lineage do these CD markers represent
CD19, CD20, CD22, kappa light chain, gamma light chain
B lymphs
Barbie in her 20s
What lineage do these CD markers represent
CD2,3,4,5,7,8
T- lymphs
Think of cells at work immature T.
consists of fluidics, a light source (laser), a detection system, and a computer
Cells must pass one by one through the illumination and detection system
flow cytometer
central core of individually aligned cells is surrounded by a sheath fluid, get them in single file line
Central alignment is essential for consistent illumination of cells as they pass before a laser light source
hydrodynamic focusing
What is forward scatter vs side scatter
forward- volume/ size
side- complexity
Explain how Flow cytometry works
cells are injected into a fluid and placed in a single file stream, they emit a color when exposed to a laser, forward and side scatter are detected
electronic boundary used by an operator to delineate cell clusters
gate
a process of selecting a population of interest as defined by one or more flow cytometry parameters
gating
acquisition of data for a specific cell pop as define by flow parameters
live gating
What is the CD45 marker
pan hematopoitic antigen
how much CD45 marker density for
lymphs
granulocytes
erythroid precursors
lymph-high density
granulocytic- intermediate
erythroid precursors- none
What cells have these markers
CD34, CD117, CD38 and HLA-DR
pluripotent stem cells
How are hematologic neoplasms diagnosed and classified with flow cytometry
based on hematopoietic maturation pathways
What cell is characterized as having low density expression of CD45
blasts
How to ID mature B and T cells
and mature lymphoid neoplasms?
surface immunoglobulin receptors, polyclonal pop
neoplasm- monoclonal B or T receptors
What is the CD4:CD8 ratio in health individuals
> 1
What is the CD4:CD8 ratio in HIV positive pts
reversed due to decrease in CD4 lymphs
PNH CD55 DAF, CD59 MIRL markers are decreased
RBCs
PNH CD24 and FLAER markers are decreased
granulocyte antigens
PNH CD14 and FLAER are decreased
monocyte antigen
What are the purine and pyrimidine nucleotides
pure-AG
pyrimid- CUT
Why is the isolation of RNA more difficult than the isolation of DNA
RNA is harder because of presence of ribonucleases RNases on mammalian epithelial surfaces
What is the principal technique in the clinical molecular laboratory
PCR
Why is it so important to prevent contamination when using molecular testing methods
RNAses- can amplify anything that contaminates the test, on any mammalian cell
enzyme that disrupts hydrogen bonds between DNA strands to allow them to separate for DNA replication
helicase
enzyme method for amplifying a specific target sequence to allow its detection even in small amounts
PCR
What is the purpose of PCR
amplification of DNA
End point vs real time PCR
end-amplicon detected at the end
real time- amplicon detected during each PCR cycle
Put into order
-first cycle of PCR complete
- repeat 25-40 times
-DNA poly binds first nucleotide to primer
-DNA denatured 95C
-cool to 40-60C, primers anneal to complimentary strands
-DNA denatured 95C
-cool to 40-60C, primers anneal to complimentary strands
-DNA poly binds first nucleotide to primer
-first cycle of PCR complete
- repeat 25-40 times
What is the purpose of RT-PCR
amplification of RNA
What is the starting material of RT-PCR
mRNA
The rate at which DNA fragments migrate through gels is proportional to the mass only not their relative charge
in gel electrophoresis
fragments sieved through an agarose or polyacrylamide gel matrix by passing through the gel as it is bathed in a conducting salt solution
the process of gel electrophoresis
smaller or larger
Agarose pores are ___
polyacrylamide gel pores are _____
larger
smaller
anode vs cathode
anode-positive charge
cathode-negative
If there is a mutation present, restriction endonucleases will not cleave the same sites, and will produce restriction fragments of different lengths than normal
RFLP restriction fragment length polymorphism
measures the change in nucleic acid amplification as replication progresses using fluorescent marker dyes
real time PCR
what types of body fluids can be analyzed in heme
CSF
serous or body cavity
synovial
bronchoalveolar lavage BAL
What is the volume of each side of the hematocytometer
9mm^3
yellow color to supernatant of CSF
xanthochromia
What tube is the cell count performed on
tube 3
the cells that line CNS, can be seen in CSF diffs
appear in clumps, look like malignant .
ependymal and choroid plexus cells
Cells in CSF if vertebral body is accidentally punctured
cartilage cells
macrophages that have ingested RBCs and have resulted in the breakdown of RBCs causing hemosiderin to be released
siderophages
large rough dark blue or black granules in cytoplasm
hemosiderin
golden yellow figures
bilirubin crystals
What does the presence of siderophages indicate
a pathologic hemorrhage
lining cells of body cavities, fried egg appearance, 1-3 nucleoli
mesothelial cells
intact neutrophil that has engulfed homogenous mass of degenerated nuclear material, which displaces the normal nucleus
lupus erythematosus cell
What cells are normal in synovial fluid
lymphs, monos/ histiocytes, synovial cells
can be seen in the upper respiratory tract, abnormal deeper inside the lungs,
ciliated epithelial cells
What cells shouldnt be seen in a BAL fluid
mesothelial cells
What forward and side scatter amounts do lymphs, monos and granulotcytes have
lymphs- small- low forward scatter, simple- low side scatter
monos-large- high forward scatter, complex medium side scatter
granulocytes- medium- medium forward scatter, high complexity- medium side scater
