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Advanced Laboratory Operations and Automation > Automated Hematology Instrumentation > Flashcards

Automated Hematology Instrumentation Flashcards

1
Q

Before running the sample

A

-CBC specimens must be checked for clots (visually, with applicator
sticks, or by automated flags), significant hemolysis, and lipemia before
reporting results
-Processing either automated or manual, should be done within 8 hours
but never longer than 24 hours after sample collection
-Samples must be thoroughly mixed before testing

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2
Q

Automated Blood Cell-
Counting Instruments

1.) electrical Impedance

A

◦ Analyzes the resistance created by each cell passing through an electrical
field
◦ Coulter principle – increased resistance occurs when poorly conductive
blood cells pass through aperture.
◦ Ex: Abbott Diagnostics, Beckman Coulter Inc., and Sysmex Corporat

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3
Q

Automated Blood Cell-
Counting Instruments

2.) optical light scatter

A

◦ Analyzes the scatter of light (forward and side) detected by each cell
passing through a beam of light (optical or laser)
◦ Ex: Siemens Healthcare, Sysmex Corporation, and Abbott Diagnosti

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4
Q

Electrical impendane Instruments

1st aliquot

A

RBC/plt dilution chamber
* External electrode
* 3 apertures (ea w/ internal electrode)
3 RBC counts are obtained, compared, and evaluated. If agreement, the
reported RBC count is an average of the 3 counts.

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5
Q

If RBCs are larger then normal then

A

Shift to the right

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6
Q

If RBCs are smaller then normal then

A

Shift to the left

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7
Q

Histogram

A

Size distribution curve

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8
Q

PLT size

A

Less then 20um

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9
Q

RBC size

A

less then 36 Um

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10
Q

Blood aspirated in

A

2-4 aliquots

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11
Q

1st aliquot determinations

A

Data determines:
◦ RBC count
◦ MCV
◦ RDW-CV and RDW-SD
◦ Plt count
◦ MPV
Calculated parameters:
◦ Hct
◦ MCH
◦ MCHC
Gaussian Curve Abnormal Curve

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12
Q

2nd Aliquot determinations

Delivered to what chamber

Lytic agents lyses what and converts what

A

Delivered to WBC/hemoglobin dilution chamber
◦ Lytic agent lyses RBCs and converts released hgb to cyanmethomoglobin,
and shrinks the leukocyte cell membrane and cytoplasm… allowing WBC
count to represent cell volume rather than cell size

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13
Q

2nd aliquot

WBC count

Hgb count

A

◦ WBC count measured by electrical impedance from 3 apertures and
reported count represents the average.
◦ Hgb determined by absorbance reading at 525 nm (Beer’s La

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14
Q

Lymphocytes, Monocytes basophils and eosinophils, neutrophils curve

A

Lymphocytes on left

Mono, eosino, baso in middle

Neutrophils on right

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15
Q

3rd aliquot

steps 1,2, and 3

A

delivered to the orbital mixing chamber
1. Blood mixed with heated lysing agent to remove RBCs
2. Stabilizing agent added to preserve WBCs
3. Cells sent through the volume-conductivity-scatter (VCS) flow cell by
hydrodynamic focusing for 5-part differenti

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16
Q

3rd aliquot important cell measurements

A
  1. Cell volume – by impedance
  2. Cell conductivity – by electromagnetic probe. Determines physical and chemical components
  3. Cell’s light scatter characteristics - determines internal contents and cell surface and size
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17
Q

Optical light scatter

A

-Each cell flows in a single line through a
flow cell
-A laser device is focused
-On striking cells, light is scattered in
different directions
-Sensor captures and multiplies scatter
-Forward angle light scatter (FALS) – cell
size
-Side scatter (SS) – granularity

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18
Q

4th aliquot

Delivered to what

Mixed with what

what solution is added

A

Delivered to a heated dilution chamber
◦ Mixed with new methylene blue reagent
◦ An acidic, hypotonic solution is added

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19
Q

4th aliquot

An acidic, hypotonic solution is added to

A

◦ Elutes hemoglobin
◦ Preserves precipitated RNA
◦ Spheres RBCs (eliminates interference due to variance
of shape

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20
Q

4th aliquot

Sent to VCS for what

important cell measurement characteristics 1,2,3

A

◦ Sent to VCS for analysis to classify mature vs.
immature RBCs
1. Cell volume – by impedance
2. Cell conductivity – by electromagnetic probe.
Determines physical and chemical components
3. Cell’s light scatter characteristics - determines internal
contents and cell surface and size

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21
Q

Indicators that may appear
after the data

@

A

data is outside the linearity limit

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22
Q

*

A

Data is doubtful

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23
Q

+ or -

A

Data is outside the reference limits

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24
Q

—– minus minus minus minus

A

Data doesn’t appear due to analysis error or abnormal sample

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25
++++
Data exceeds display limit
26
Hematocrit
MCV/ RBC count
27
MCH
Hb/ RBC count
28
MCHC
Hb/ Mct
29
Automated pt count problems
1. Plt clumping - recollect in Heparin 2. Satellitosis – warm sample 3. RBC microcytes 4. Giant plts
30
What to do when controls are out of limit
out of control stop testing identify and correct problems Repeat testing on patient samples and controls Do not report results until problem is solved and controls indicate proper performance
31
Coagulation testing
Today, numerous semi-automated & fully automated instruments to perform coagulation testing Instruments today are likely to have sophisticated front end capabilities
32
Coagulation testing Modern congulation analyzers have reduced what Random what
Modern coagulation analyzers have greatly ↑ performance via improved accuracy & precision Reduced rgt & sample volumes required Random, discrete sampling capabilities Flagging (sample & instrument)
33
Electromechanical instruments Instruments
- Fibromter (BBL Microbiology Systems, Becton Dickinson) Semi-automated: reagents & samples usually added manually by operator Principle & Operation * Electromechanical clot detection Stationary & moving probes
34
Electromagnetic monitoring
* Electromagnetic monitoring of movement of a steel ball in plasma Ball moves because of applied magnetic force Clot = ↓ movement sensed by electronic sensor set to predefined limit
35
Photo optical systems
*Detection of sample optical density due to formation of fibrin *Records decreased light at the forward 180 ̊ angle
36
Nephelometry End point Detection
-Modification of photo-optical end point detection -90 degree or forward angle scatter contributes to measurement -Ag-Ab form precipitates that scatters incident light -Nephelometric instruments read loss of intensity of exiting beam as increasing amounts of impinging light scattered agglutinates form
37
Thromboelastography
Whole blood clotting assay Allows real-time comprehensive evaluation of hemostasis Can analyze fibrinogen, factor activity, platelet function, and fibrinolysis
38
Quality Control for Coag Testing specimen
Proper collection (anticoagulant, order of draw, meds) Phlebotomy technique Delays in processing Improper storage Centrifugation
39
Quality Control for Coag Testing Reagent Human error
Reagent: Shipping conditions Storage conditions Reconstitution Contamination Deterioration Lot changes Human error Choosing wrong test Analyzing wrong sample Using improper sample Using incorrect reagents The possibilities are staggering
40
Platelet testing Automated testing replaced Measures pt what detects what
the Bleeding Time Measures plt function in whole blood using: - collagen/epinephrine - collagen/ADP Detects platelet plug formation
41
Platelet aggregation studies
Patient’s PRP is warmed to 37°C, stirred, and an agonist/stimuli is added ◦ ADP ◦ Epinephrine ◦ Collagen ◦ Ristocetin – depends upon vWF and GPIb/IX/X and targets agglutination (RIPA) ◦ Arachidonic acid
42
Plt agregation is recorded on a what
Plt aggregation is recorded on a graph ◦ Biphasic curve – analyzing both waves of aggregation ADP and epinephrine ◦ Monophasic curve – only one wave of aggregation Collagen and Ristocetin
43
Flow cytometry cytometry= Flow cytometry= Multi parameter flow cytometry
-Cytometry = measurement of physical/ chemical characteristics of cells -Flow cytometry = measurements are made upon cells while suspended in a fluid stream ◦ Cells must be able to be separated and not cohesive -Multi-parameter flow cytometry = technology that simultaneously measures multiple parameters of single cells at a rapid rate ◦ Permits detailed analysis of markers of cellular differentiatio
44
Specimen processing Suspension of what
Suspension of individual cells ◦ Often after lysing RBCs
45
aspirated and injected into flow chamber containing 2 columns of fluid
◦ Cells forced into single file and sent into path of laser beam (hydrodynamic focusing) ◦ Gradient between sample and sheath fluid keeps fluids separate ◦ Light scatter is measured by photodetectors (both forward and side scatter) ◦ PMT (photomultiplier tube) detects fluorescent molecul
46
Flourochromes
Fluorochromes – molecules that are excited by light at one wavelength and emit light at another wavelength ◦ Can be attached to cells via antibodies or DNA
47
Cytometer components
Fluidics laser Electronics optics- that gather the light Light detectors- to sense the light Computer- to output the data into a form that can be used
48
Optics and electronics laser light
blue argon laser 3 fluorochromes additional fluorochromes
49
Thershold
minimal fluorescence neccessary
50
Photodetectors
adjustable for sensitivity
51
Amplification
logarithmic
52
Flourescence compensation
statistically predictable & mathematically correctable Data collection
53
Side scatter
Granularity or internal complexity
54
Forward scatter
Cell size
55
what graph is used for Flow cytometry
two dimensional histogram White cells and other cells fall into distinct populations according to their size and granularity
56
Classic flow cytometry uses
- Classic Flow Cytometry uses very specific antibodies to label cellular antigens (extra and intracellular). The antibodies are labeled in most cases directly with fluorescent dye(s) to generate a fluorescent signal. - Can take antibodies against various surface antigens and conjugate each antibody to a different fluorophore. Can determine what antigens are expressed on the cell surface and, thereby, their phenotype and function.
57
Immune panel
 Immune panel: Peripheral blood  Evaluate immune competency  Innumerate (% and absolute #) CD3+ T cells, including CD4+ and CD8+ subsets, CD19+ B cells, CD56+ NK cells, CD45RA/RO Naïve and Memory cells
58
Oxidative burst
 Oxidative Burst  Chronic granulomatous disease due to diminished or absent NADPH oxidase activity
59
Leukocyte adhension markers and what workup
 Leukocyte Adhesion Markers  Leukocyte adhesion deficiencies- poor wound healing, delayed umbilical cord separation  Leukemia/lymphoma workup
60
Satellitosis.
Platelets surrounding WBCs
61
Microcytes will cause
A shift to the left
62
Giant platelets
Some platelets will be counted as RBCs
63
Platelets clumping will
Will cause instrument to think they are RBC Red extension on length
64
Giant platelets
A extension of curve width and smaller in height in plT curve
65
check samples in heme before
Running test on them
66
Processing in heme should be done within
8 hours
67
In hematology we have what to keep the specimen mixed
rockers
68
Optical light scatter
Analyze scatter of light at a forward and side angle direction
69
in heme we have what number of aliquots
4 and 2 with four being the most common
70
difference between the external and internal electrode
Apenture measures the cells that pass through the external and internal electrode. ( current difference).
71
Histogram
Look at cell size compared to cell number
72
Width of the curve will give the
RDW
73
if cell is less then 20 then it is a
plt
74
if cell is greater then 36
RBC
75
Broad (large) range of forward scatter is dependent on
size of cell
76
Side scatter has a
Small range
77
PCW
Platelets RDW
78
Platelets aggregate around WBC will cause
An increased size measurement of the WBC or increased vol of WBC
79
12s rule
West guard rule
80
The fibrometer was the standard in
Heme for many years - the two probes worked together to detect the fibrin clot - there will be tension resistance between the two probes and that resistance will stop the test
81
Thromboelastography ROTEM
accesses the tensile strength rate of formation maximum firmness and stability clot lysing/ breakdown
82
ROTEM formula
measures claw amplitude/ time
83
ROTEM R time
reflects fibrin generation at the onset of clot formation prolonged then coagulation factor deficient short R time= hypercoagulatable
84
K time
time it takes for a clot to reach 20 min in amplitude
85
A angle
Tanget of curve at the K time decreased A angle then fibrinogen decreased Represents the speed of clot buildup
86
MA=
Represents the greastest firmness and stability of clot ( function of plts)
87
Iy30=
Fibrinolysis
88
PFA 100
primary hemostatis test Plaletet plug formation

Advanced Laboratory Operations and Automation (17 decks)

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