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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
Q

++++

A

Data exceeds display limit

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26
Q

Hematocrit

A

MCV/ RBC count

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27
Q

MCH

A

Hb/ RBC count

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28
Q

MCHC

A

Hb/ Mct

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29
Q

Automated pt count problems

A
  1. Plt clumping - recollect in Heparin
  2. Satellitosis – warm sample
  3. RBC microcytes
  4. Giant plts
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30
Q

What to do when controls are out of limit

A

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

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31
Q

Coagulation testing

A

Today, numerous semi-automated & fully automated instruments to perform coagulation testing
Instruments today are likely to have sophisticated front end capabilities

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32
Q

Coagulation testing

Modern congulation analyzers have

reduced what

Random what

A

Modern coagulation analyzers have
greatly ↑ performance via improved
accuracy & precision
Reduced rgt & sample volumes required
Random, discrete sampling capabilities
Flagging (sample & instrument)

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33
Q

Electromechanical instruments

Instruments

A
  • Fibromter (BBL Microbiology Systems, Becton Dickinson)
    Semi-automated: reagents & samples usually added manually by operator
    Principle & Operation
  • Electromechanical clot detection
    Stationary & moving probes
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34
Q

Electromagnetic monitoring

A
  • 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
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35
Q

Photo optical systems

A

*Detection of sample optical
density due to formation of fibrin
*Records decreased light at the
forward 180 ̊ angle

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36
Q

Nephelometry End point Detection

A

-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
Q

Thromboelastography

A

Whole blood clotting assay
Allows real-time comprehensive evaluation of hemostasis
Can analyze fibrinogen, factor activity, platelet function, and fibrinolysis

38
Q

Quality Control for Coag Testing

specimen

A

Proper collection (anticoagulant, order of
draw, meds)
Phlebotomy technique
Delays in processing
Improper storage
Centrifugation

39
Q

Quality Control for Coag Testing

Reagent

Human error

A

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
Q

Platelet testing

Automated testing replaced

Measures pt what

detects what

A

the Bleeding Time

Measures plt function in whole blood using:
- collagen/epinephrine
- collagen/ADP
Detects platelet plug formation

41
Q

Platelet aggregation studies

A

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
Q

Plt agregation is recorded on a what

A

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
Q

Flow cytometry

cytometry=

Flow cytometry=

Multi parameter flow cytometry

A

-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
Q

Specimen processing

Suspension of what

A

Suspension of individual cells
◦ Often after lysing RBCs

45
Q

aspirated and injected into flow chamber containing 2 columns of fluid

A

◦ 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
Q

Flourochromes

A

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
Q

Cytometer components

A

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
Q

Optics and electronics

laser light

A

blue argon laser
3 fluorochromes
additional fluorochromes

49
Q

Thershold

A

minimal fluorescence
neccessary

50
Q

Photodetectors

A

adjustable for sensitivity

51
Q

Amplification

A

logarithmic

52
Q

Flourescence compensation

A

statistically predictable & mathematically correctable
Data collection

53
Q

Side scatter

A

Granularity or internal complexity

54
Q

Forward scatter

A

Cell size

55
Q

what graph is used for Flow cytometry

A

two dimensional histogram
White cells and other cells fall into distinct populations
according to their size and granularity

56
Q

Classic flow cytometry uses

A
  • 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
Q

Immune panel

A

 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
Q

Oxidative burst

A

 Oxidative Burst
 Chronic granulomatous disease due to diminished or absent NADPH
oxidase activity

59
Q

Leukocyte adhension markers

and what workup

A

 Leukocyte Adhesion Markers
 Leukocyte adhesion deficiencies- poor wound healing, delayed umbilical
cord separation
 Leukemia/lymphoma workup

60
Q

Satellitosis.

A

Platelets surrounding WBCs

61
Q

Microcytes will cause

A

A shift to the left

62
Q

Giant platelets

A

Some platelets will be counted as RBCs

63
Q

Platelets clumping will

A

Will cause instrument to think they are RBC
Red extension on length

64
Q

Giant platelets

A

A extension of curve width and smaller in height in plT curve

65
Q

check samples in heme before

A

Running test on them

66
Q

Processing in heme should be done within

A

8 hours

67
Q

In hematology we have what to keep the specimen mixed

A

rockers

68
Q

Optical light scatter

A

Analyze scatter of light at a forward and side angle direction

69
Q

in heme we have what number of aliquots

A

4 and 2 with four being the most common

70
Q

difference between the external and internal electrode

A

Apenture measures the cells that pass through the external and internal electrode. ( current difference).

71
Q

Histogram

A

Look at cell size compared to cell number

72
Q

Width of the curve will give the

A

RDW

73
Q

if cell is less then 20 then it is a

A

plt

74
Q

if cell is greater then 36

A

RBC

75
Q

Broad (large) range of forward scatter is dependent on

A

size of cell

76
Q

Side scatter has a

A

Small range

77
Q

PCW

A

Platelets RDW

78
Q

Platelets aggregate around WBC will cause

A

An increased size measurement of the WBC or increased vol of WBC

79
Q

12s rule

A

West guard rule

80
Q

The fibrometer was the standard in

A

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
Q

Thromboelastography

ROTEM

A

accesses the tensile strength
rate of formation
maximum firmness and stability
clot lysing/ breakdown

82
Q

ROTEM formula

A

measures claw amplitude/ time

83
Q

ROTEM

R time

A

reflects fibrin generation at the onset of clot formation

prolonged then coagulation factor deficient

short R time= hypercoagulatable

84
Q

K time

A

time it takes for a clot to reach 20 min in amplitude

85
Q

A angle

A

Tanget of curve at the K time

decreased A angle then fibrinogen decreased

Represents the speed of clot buildup

86
Q

MA=

A

Represents the greastest firmness and stability of clot ( function of plts)

87
Q

Iy30=

A

Fibrinolysis

88
Q

PFA 100

A

primary hemostatis test

Plaletet plug formation

Advanced Laboratory Operations and Automation (17 decks)

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