(M) Week 11: Peripheral Blood Smear Flashcards
1
Q
Familiarize the roles of the peripheral blood smear
A
- evaluation of anemia
- evaluation of thrombocytopenia / thrombocytosis
- Identification of abnormal cells
- INfections like malaria, microfilaria
- Inclusions like basophilic stippling, Howell-Jolly bodies, Cabot ring
2
Q
What is the first thing to consider in making PBS
A
how to collect the specimen (what specimen is acceptable)
3
Q
What anticoagulant tube is used for the sample needed for PBS?
A
EDTA
4
Q
what does EDTA mean?
A
ethylenediaminetetraacetic acid
5
Q
how does EDTA prevent coagulation?
A
by chelating the calcium
6
Q
what form of EDTA is more commonly prepared (preferred)
A
liquid form (easily mixed)
7
Q
PBS should be prepared within _______ hours of drawing the specimen
A
2-3 hours
8
Q
What will be found if a PBS is created with a sample that is more than five hours old
A
blood cell artifacts
9
Q
What are the advantages of EDTA samples
A
- different capacities per tube = multiple slides created
- Slides need not to be prepared immediately
- Prevents platelet clumping
10
Q
What are the disadvantages of using EDTA sample for PBS
A
- occurrence of satellitism
- pseudothrombocytopenia (false decrease of platelets)
- Pseudoleukocytosis (false increase of WBC due to platelet agglutinates)
11
Q
what is the other cause of platelet satelliotosis
A
platelet specific auto antibody
12
Q
T or F
in using EDTA blood samples, monocytes can be seen with vacuoles
A
T
13
Q
T or F
monocytes visualized with vacuoles will greatly affect the examination of the smear
A
F (will not effect)
14
Q
What anticoagulant should be used to prevent platelet satelliotosis
A
sodium citrate
15
Q
what is the ratio for blood to sodium citrate
A
9:1
16
Q
what is the WBC and platelet count dilution factor?
A
1:1
17
Q
what method of blood extraction is used when you need to make the film or smear on the patient’s side
A
finger / heel puncture (anticoagulated free blood)
18
Q
platelet clumping is expected when using what anticoagulant
A
heparinized microcollection tube
19
Q
When there’s no anticoagulant used, you can only make _____ slides, this is corrected by using _________
A
few
heparinized microcollection tubes
20
Q
Familiarize!
Blood smear preparation
A
- Manual wedge technique / wedge method
- Coverslip method / technique
- Coverslip and slide method
- automated blood smear preparation
21
Q
most convenient and commonly used method of PBS
A
manual wedge technique
22
Q
This is a dispenser of blood for wedge method, it is inserted through the rubberstopper of the tube
A
Diff-Safe dispenser
23
Q
What is the advantage of the Diff-Safe dispenser
A
no need to remove the rubber stopper to obtain a drop of blood
24
Q
what is the ideal diameter of blood used for PBS
A
2-3 mm
25
when the drop of blood is too large, what is the effect on the PBS
long thick film
26
when the drop of blood is too thin, what is the expected PBS outcome
short thin film
27
what should the angle be when you are holding the pusher slide
30 to 45 deg angle
28
What happens to the result of your PBS preparation if you push forward too slowly?
accentuation of poor leukocyte distribution
29
after smearing the blood, it is fixed with ______ vol / vol of methanol
10% volume
30
how many slides should be created ?
2 or more film / smear
31
you may save ___ unstained in cases where another one is needed
1
32
familiarize the causes of unacceptable PBS
1. shows a chip / rough edge on the spreader slide
2. when there's hesitation in forward motion
3. the spreader slide is pushed to quickly
4. too small drop of blood
5. the drop of blood didn't spread along the width of the slide
6. Due to dirt or grease or high lipid level of the patient in their blood
7. uneven pressure applied on the slide
8. due to time delay
33
the film is ___________ to ________________ the length of the slide
two-thirds
three-fourths
34
the film should be _______________ shaped, very slidhtly rounded at the feather edge
finger shaped
thumb
35
this feature of PBS is very important when examining your film/ smear microscopically
feathery edge
36
the ________ drop of blood is picked up and spread along the slide
whole
37
The thickness of the smear is determined by the (enumerate all three)
1. angle of the spreader slide
2. size of the drop of blood
3. speed of spreading
38
the (greater/lesser) the angle, the thicker and shorter the smear
greater
39
If the hematocrit is increased, the angle of the spreader slide should be (increased/decreased)
decreased
40
newborns have (increased/decreased) hematocrit
increased
41
If the hematocrit is decreased, the angle of the spreader slide should be (increased / decreased)
increased
42
what causes irregular spread with ridges and long tail in PBS
edge of spreader is dirty / chipped / dusty
43
what causes holes in film
slide is contaminated with fat / grease and air bubbles
or lipidemia
44
due to delay in fixing the smear or causes methanol contaminated with water
cellular degenerative changes
45
this technique is older, rarely used now, more common for blood marrow aspirate smears
coverslip techniue
46
what materials are needed for the coverslip technique
2 coverslips (22x22 mm)
blood sample
47
This technique produces an excellent leukocyte distribution, more accurate for differential counting
coverslip technique
48
what is the disadvantages of the coverslip technique
coverslips are too small (hard to label, transpo, stain, and store)
49
other name for the coverslip method when used for bone marrow preparation
crush preparation
50
how will too much pressure affect the performance of the coverslip method?
the film will cause cell rupture
51
what other material may be used for coverslip technique?
slides
52
this technique may cause an artifactual increase in lymphocyte counts
wedge smear
53
greater trauma in cells leads to smudge or _____________ cell formation
basket
54
what is the more common technique used in the lab (wedge smear / coverslip method)
wedge smear
55
These are the sample machines that we can use for the so called automated preparation of the film
automated wedge film maker machines
56
An advanced automated machine wherein both the
staining and making of the smear are done
Coulter LH slide maker and stainer
57
staining and making of the film can be done every how many seconds?
30
58
what is the basis of automated machines for making a film?
hematocrit level
59
type of automated machine that can only do slide preparation (not advanced)
automatic wedge film maker
60
two ways of drying the blood smear
small fan
natural air drying
61
why should blood smears be dried as quickly as possible
to avoid drying artifact
62
why is blowing breath on the slide counterproductive?
it produces echinocytic RBCs and drying artifacts
63
What causes drying artifacts?
humidity
anemic paitents
water absorebd from the air
64
how to avoid artifacts?
1. dry as quickly as possible
2. keep the stopper of the stain
3. Fix the slight using pure anhydrous methanol
65
The following are under what type of staining?
Wright’s & Giemsa
Leishman
May Grunwal Giemsa
Jenner-Giemsa
Field’s stain
Romanowsky staining
66
what is the common denominator of romanowsky stains?
eosin Y and azure B-methylene blue composition
67
Romanowsky stains are considered as what type of stain due to composition?
polychrome stain
68
What is the routinely used stain?
Wright's & Giemsa stain
69
What stain, when alone, is used for blood parasite detection?
giemsa
70
Characteristic of Eosin Y
acidic stain
71
What does Eosin Y stain?
hemoglobin and eosinophilic granules.
(ACIDIC STAIN)
72
What is the characteristic of Azure B-Methylene blue
basic stain such as RNA
73
What reagent is used for fixing PBS stains
pure methyl alcohol or methyl alcohol
74
what buffer is used for PBS
0.05 M sodium phosphate
aged distilled water
75
what is the pH of sodium phosphate
6.4
76
what is the pH of aged distilled water
6.4 to 6.8
77
why is a buffer solution needed for PBS
staining reactions are pH dependent
78
what indicates proper mixing of eosin Y and methylene blue?
metallic sheen
79
how many minutes are the stains left for?
1-3 minutes
80
State the Disadvantages and Advantages of Manual Wright Staining of Slides
Advantage: Produces a DESIRABLE STAINING QUALITY
Disadvantage: INCREASED RISK OF SPILLING THE STAIN and LONGER TIME IS REQUIRED FOR THE STAINING PROCEDURE (time consuming)
81
TOF. After 3 minutes, wash the back of the smear with TAP WATER
T
82
TOF. wipe the back of the slide to remove stain
residues and air dry in a HORIZONTAL position.
vertical
83
* useful for high volume of sample
* 5 – 10 mins you are done with the staining which is done by batch
* processes of fixing/staining and buffering are similar in practice to those of the manual method
AUTOMATED SLIDE STAINER
84
Disadvantage of AUTOMATED SLIDE STAINER
o YOU CANNOT ADD ADDITIONAL SLIDES
once the staining process has began.
o AQUEOUS SOLUTION should be MADE OFTEN.
85
Another method of staining aside from MANUAL
and AUTOMATED STAINING.
It is FAST AND EASY, you ONLY NEED 1
MINUTE and then you’re done.
QUICK STAINING
86
Used stain for Quick Staining
MODIFIED WRIGHT OR WRIGHT-GIEMSA is used as the stain with AGED DISTILLED WATER as the buffer
87
Disadvantage of quick staining
THE QUALITY OF THE STAIN
o Needs to be CHANGED every now and
then
88
Features of a Well-Stained Smear: Macroscopically
| color
PINK TO PURPLE in color
89
Features of a Well-Stained Smear: Microscopically
1. RBC
2. WBC Nuclei
3. Neutrophil's cytoplasm
4. Eosinophils
| state bawat colors
1. Orange - salmon pink
2. Purple - blue
3. Pink - tan w/ violet or lilac granules
4. Bright orange refractile granules
90
What do you do if this macrosopic and microscopic features are NOT SEEN after air drying and staining and if the smear is poorly prepared
MAKE A NEW ONE
91
how WBC’s should look like in a well-stained smear:
1. Neutrophil
2. Eosinophil
3. Basophil
4. Lymphocytes
5. Monocyte
1. Pink to tan
2. Bright orange refractile granules in its cytoplasm
3. Nuclei should be purple of blue in color
4. Small and large; reactive lymphocytes
5. kidney-shaped granules
92
Size of the central pallor
| in dia
1/3 OF THE WHOLE DIAMETER
93
Water contamination could also result to a?
HEAVILY DEMARCATED CENTRAL PALLOR.
94
The laboratory technician is consistently creating a bullet shaped blood film, to correct this he should:
A. Increase the acute angle of the pusher slide
B. Decrease the angle of the pusher slide
C.Push the pusher slide in a smooth and constant pressure
D. Allow the blood to spread across the width of the slide
D
95
When the blood film is viewed through the microscope, the RBCs appear redder than normal, the neutrophil is barely visible, the eosinophils are bright red orange? What is the most likely cause?
A. The slide was overstained
B. The stain was too alkaline
C. The buffer was too acidic
D. The slide was not rinsed adequately
C
96
Ideal angle (module based)
30-45 degree
97
# Pink to purple
A bluer overall is caused by what (2)
1. increased protein level (multiple myeloma patients)
2. rouleaux formation
98
# Pink to purple
* Due to RBC AGGLUTINATION
* It is when RBCs agglutinate with each other and seen in patients suffering from COLD AGGLUTININ DISEASE.
* Patients HAVE ANTIBODIES that could agglutinate RBCs
Grainy appearance
99
# Pink to purple
Holes over the film Indicates?
INCREASED LIPID LEVEL and
maybe the patient has LIPEMIA.
100
# Pink to purple
Blue specks out of feathery edge Indicates
MARKEDLY INCREASED WBC and PLATELET COUNTS.
101
What part of the microscope should be almost all the way up and adjusted correctly for the magnification used.
Condenser
102
What part of the microscope should be opened to allow a comfortable amount of light to the eye
iris diaphragm
103
What filter should be used to create a whiter light from a tungsten light source.
neutral density filter
104
conditions of Koehler illumination
* illuminator should be centered
* condenser all the way up
* iris diaphragm should be opened to allow a comfortable amount of light to the eye
* neutral density filter
105
# MICROSCOPIC MAGNIFICATION
* Assess overall film quality, color and cell distribution
* Feathery Edge and Lateral Etches (WBC distribution)
* RBC agglutination and Roulex formation
10x Objective Examination (LPO)
106
# MICROSCOPIC MAGNIFICATION
Select an assessment area to begin diff count and evaluate cell morphology
WBC estimate (2000x multiplication factor)
40x Objective Examination
(HPO)
107
# MICROSCOPIC MAGNIFICATION
Assess
WBC Diff count
RBC, WBC and platelet morphology
Platelet estimate
RBC (howell-jolly bodies) and WBC (Dohle bodies) inclusion
Reactive or abnormal cells
Nucleated RBCs - reported per 100 WBCs
100x Oil Immersion Objective Examination (highest)
1000x
108
# MICROSCOPIC MAGNIFICATION
Optimal assessment area: best possible area for peripheral blood film examination; look for feathery edge (depending on the organism)
WBC Diff. count and morphology exam; WBC estimate (3000x)
RBC, WBC and platelets is examined
50x Oil Immersion Objective Examination
109
abnormalities in the size of our RBC.
ANISOCYTOSIS
110
feature of most anemias.
Anisocytosis
111
(micro, normo or macrocytic) red cells are commonly
associated with certain anemia such as:
Iron deficiency
Anemia
Thalassemia
Sideroblastic anemia
Lead toxicity
Anemia of chronic disease
Micro
112
(micro, normo or macrocytic) red cells are seen on patients with MEGALOBLASTIC ANEMIA.
Macro
113
(micro, normo or macrocytic) red cell is found IN BONE MARROW AND KIDNEY FAILURE.
Normo
114
Artifacts from excess EDTA, Improper smear preparation, prolonged sample storage before blood film preparation
Echinocytes
115
an increase disproportionately to its volume that results from a decrease in hemoglobin as in IRON DEFICIENCY ANEMIA or an increase in the cell membrane
Target cell/ Codocytes
116
Fragmented RBCs/ Schistocytes
RBC injury from damaged endothelium, a feature of microangiopathic hemolytic anemia
117
do not bend or move easily and can block blood flow to the rest of the body
Sickle cell RBCs
118
dense, shrunken, irregularly shaped RBCs w/ spikes on the outside, forms from changes in fats and proteins on RBCs outer layers
Acanthocytes/Spur cells
119
ENUMARATE ALL RBCs SHAPE
TEARDROP CELL
BURR CELLS
DACROCYTE
ELLIPTOCYTES STOMATOCYTES SPHEROCYTES
Echinocytes
Target cell/ Codocytes
Fragmented RBCs/ Schistocytes
Sickle cell RBCs
Acanthocytes/Spur cells:
120
# Hemoglobin Conc.
mature RBCs w/ normal hemoglobin
Normochromic RBC
121
# Hemoglobin Conc.
central pallor more than ⅓ of the total diameter, decreased hemoglobin conc.
Hypochromic RBCs
122
# Hemoglobin Conc.
central pallor less than ⅓, increased hemoglobin conc., red cells stains deeply
Hyperchromic RBC
123
TOF. Sickle cell is reported as positive or negative, RBC grading is not used according to other textbooks.
T
124
* presence of blue-gray to pink cytoplasm tint of the red cell due to either hemoglobin presence or RNA residue.
* It is commonly SEEN IN YOUNG RED CELL.
* indicates RETICULOCYTOSIS.
* RBCs appears LARGER THAN NORMAL AND LACKS CENTRAL PALLOR WITH BLUE-GRAY TO PINK CYTOPLASM.
POLYCHROMATOPHILIA
125
what is the disadvantage of leaving the stain for more than 3 minutes
increased risk of spilling the stain
126
# MATCH
Supravital staining is not limited to reticulocyte it can also determine:
* Heinz bodies
* Howell Jolly bodies
* Hgb H
* Pappenheimer bodies
A. presence of DNA content.
B. presence of hemoglobin H in your RBCs.
C. denatured hemoglobin
D. indicates the presence of iron (hemosiderin)
CABD
127
Which among these is not graded as +1, +2,
+32
A. Sickle cell
B. Pappenheimer bodies
C. Howell jolly bodies
D. Basophilic stipplings
E. A,B,D
F. A, B,C,D
F
128
* when cells aggregate into cluster of masses when exposed to an anti-body;
* happened because of the presence of antibodies that agglutinates the RBC
Agglutination
129
coin or stacking appearance of RBC; common in increased level of RBC
Rouleau Formation
130
what should the drying position be?
vertical position
131
Size -1-3μm
Non-nucleated cells derived from cytoplasmic
fragments of Megakaryocytes Has purple red granules.
Lilac color
Platelets
132
machine used for staining, done by batch, it's useful for a high volume of sample
automated slide stainer
133
Clinically significant
Indicative of certain condition same in size of the RBC
Giant Platelets
134
what is the disadvantage of Hema-Tek 2000 slide stainer
1. you cannot add additional slides one the staining process has begun
2. aqueous solution should be made often
135
WBC count formula 1 (40x magnification)
Ave. WBC per field x 2000
136
this is another method of staining aside from manual and automated staining
it is fast and easy, you only need one minute to be done
quick staining
137
WBC count formula 2 (x50 magni)
Ave. WBC per field x 3000
138
Formula 1 and 2 for platelt count
Formula 1: Average platelet per field x 20,000
Formula 2: Average # of platelet per fields x RBC ct./ 200 RBC
139
what stains may be used for quick staining
modified wright
modified giemsa
140
what is the buffer used in quick staining
aged distilled water
141
what are the disadvantages of quick staining
quality of the stain
needs to be changed every now and then
142
# EXAMINATION OF BLOOD FILMS FOR PARASITES
when parasites are scanty
Thick film
143
what should the color of stained blood be?
pink to purple (macroscopically)
144
# EXAMINATION OF BLOOD FILMS FOR PARASITES
identification of species
Thin film
145
# EXAMINATION OF BLOOD FILMS FOR PARASITES
Staining of Film by?
Giemsa or Leishman’s stain at pH 7.2
146
what should be the color of WBC nuclei in a well stained smear
purple to blee
147
148
what is the ideal color of neutrophil's cytoplasm in a well stained smear
pink-tan with violet / lilac granules
149
