LABORATORY ACTIVITY 1 AND LABORATORY ACTIVITY 2 Flashcards
Direct Platelet Count Principle:
measures the ability of the capillaries to withstand increased stress
Significance if positive = presence of petechiae
- capillary weakness
- thrombocytopenia
- both
Increased vascular fragility:
- Repeated skin trauma
- Qualitative and quantitative platelet defects
- Vitamin C Deficiency
- Purpura
: A few petechial on the anterior part of the forearm
1+
: May petechial on the anterior part of the forearm
2+
: Multiple petechial over the whole arm and back of the hand
3+
: Confluent petechial on the arm and back of the hand
4+
Vascular fragility correlates [?] with the probability of bleeding. If a patient has an increased vascular fragility, he or she has a high chance of bleeding. This is because, if a patient encounters a multiple skin trauma, his or her blood vessels are damaged which is prone to the rupture and bleeding when exposed again to pressure.
directly
Direct Platelet Count
Purpose: [?]
To confirm if px is suffering from thrombocytopenia
Direct Platelet Count 2 Factors:
o Genetic/weak capillary
o Platelet – can be evaluated as a medtech
Automated may detect platelets as:
o WBC: Normal
o RBC: Ab Low ( plt confused as microsized, fragmented rbc, small sized rbc)
o Plt: Ab High
Ab Low ( plt confused as [?])
microsized, fragmented rbc, small sized rbc
Levy chamber with improved Neubauer ruling
Hemocytometer
Levy chamber with improved Neubauer ruling Features:
o 3x3 mm square counting area
9mm2
o (9) 1x1mm squares
1mm2
o (4) WBC squares is divided into 16 smaller squares
0.0625 mm2
o (1) RBC square is divided into 25 smaller squares
0.04mm2
o Coverslip vs counting surface
0.1mm
o Total volume of the entire grid
0.9mm3
- used for counting blood cell
Hemocytometer
= 1 whole/large square (overall)
9 mm2
= 1 small (wbc square)
1 mm2
1 small (wbc square) o Volume =
1 mm3 x 0.1 = 0.1 mm3
= 1 smaller wbc square
0.0625 mm2
= 1 smaller wbc square o Volume =
0.0625 mm2 x 0.1 = 0.0625 mm3
= 1 smaller rbc square
1 mm2/25 = 0.04 mm2
= 1 smaller rbc square o Volume =
0.004 mm3/16
= 1 smaller rbc square o Area =
0.0024
= 1 smallest rbc square
0.04 mm2/16 = 0.0025
= 1 smallest rbc square o Volume =
0.025
Coverslip vs counting surface =
0.1mm3
Depth
0.1mm3
Used as height
0.1mm3
Total volume of the entire grid =
0.9mm3
Platelet count =
DF =
VCF =
Tocantins Method Reagent:
Rees-Ecker
is used to prevent adherence of blood cells to the thoma pipette to discharge equally in the hemocytometer
Sodium citrate
–fixative and preservative to prevent cells from floatingin the hemocytometer
Formalin
4 WBC squares
Tocantins Method
Brecker-Cronkite Method Reagent:
1% NH4 oxalate
5 RBC squares
Brecker-Cronkite Method
Tocantins Method
- anticoagulant
a. Sodium citrate
Tocantins Method
- fixative
b. Formalin
prevent premature lysis
b. Formalin
(preserves both red cells and platelets)
b. Formalin
Tocantins Method
- stains the platelets
c. BCB
Light Microscopy Method
Tocantins Method
Phase-Contrast Microscopy Method
Brecker-Cronkite Method
Brecker-Cronkite Method Counting chamber:
Spencer - Briteline # 1475
Platelets are counted in 5 R squares
Brecker-Cronkite Method
Criteria for a good diluent:
- Should prevent plt adherence to glass
- Should prevent plt aggregation
- Should not promote premature plt lysis
- Should make plt visible in the microscope
Advantage of Brecker-Cronkite over Tocantins
- Clearing of bg
- Plt is more distinguishable
- Label of pipette
- Moist chamber (15 minutes - reduce to 5 in the lab)
Platelet is [?] of the size of the rbc
1/10th
Normal:
150 – 450/uL
abnormally low
• ＜ 100,000/ uL
- bleeding possible
• 30,000-50,000/ uL
- spontaneous bleeding
• < 30,000 u/L
- severe spontaneous bleeding
• < 5,000/uL
In automated platelet count, platelets with large sizes may be confused as [?] and may cause increased values for the RBC count. However, this can be corrected by direct platelet count or manual counting since the appearance of the platelet and RBC can be differentiated.
red blood cells
2 description of capillaries:
weak or strong
The vascular system is composed of:
arterioles, capillary, blood vessel, vein artery
Capillary is much smaller like [?], but bigger than [?]
artery and vein
arterioles
[?], increase intravascular pressure, obstruct blood vessel
Pressure constricts
Pressure:
100 mmHg for 5 minutes
Applying pressure will constrict the blood vessels. The in-vitro pressure in the arm will increase the [?]
intravascular pressure
Relationship of capillary wall and petechiae: The thinner the capillary, the [?] it is. Therefore, more petechiae is produced
weaker
the more exposed to trauma, the more fragile the blood vessels are
Repeated skin trauma
Qualitative: ex.
abnormal shape or function
Quantitative:
number of platelet
helps to promote integrity of blood vessel allowing smoother blood flow
Vitamin C Deficiency
indication: haemorrhage
Purpura
if the same vein keeps on breaking, it is prone to fragility
Purpura
goes hand in hand w/ repeated skin trauma
Purpura
Neubauer Depth
0.1 mm
Neubauer Dimension
2 ruled areas; 3mm x 3mm each
Neubauer Each side is divided into
9 equal squares
Neubauer Volume
0.9 mm3 /sq
Neubauer Total Volume
1.8 mm3
hemolyses rbc in the background leaving wbc and platelets
1% NH4 oxalate
Platelet is more distinguishable w/
wbc than an rbc
Label of pipette
[?] is attached to the syringe barrel
Mouth piece
To allows cells to settle
Moist chamber (15 minutes - reduce to 5 in the lab)
CAPILLARY FRAGILITY TEST measures the ability of small capillaries to retain blood when subjected to
increased hydrostatic pressure and anoxia
CAPILLARY FRAGILITY TEST
It is a non-specific evaluation to measure
capillary weakness and deficiencies in platelet number and function
causes the capillaries to rupture which leads to bleeding and formation of petechiae.
Decreased capillary resistance
Procedure:
Rumpel-Leede Tourniquet Test
Procedure:
1. Examine the (?) to make certain that no petechiae are present.
2. With a blood pressure cuff, apply (?) pressure to upper arm.
To those who do not have a blood pressure cuff, use a tourniquet or rubber/cloth strip instead. Apply the tourniquet not too tight, not too loose to employ just enough pressure.
3. Maintain pressure for (?).
4. Release cuff and wait for (?) before making a final reading.
5. Examine the (?) for petechiae.
Note: Disregard any petechiae within (?) of the blood pressure cuff (tourniquet) because this may be due to pinching of the skin by the cuff.
6. Count the number of petechiae and roughly interpret
forearm, hand, and fingers
100 mmHg
5 minutes
5 – 10 minutes
forearm, hands and fingers
½ inch
Platelets are thin disks, (?) in diameter and (?) in volume.
2–4 μm
5–7 fL
Platelets function primarily in (?) and in maintaining (?).
hemostasis
capillary integrity
Platelet numbers must be sufficient for them to play their supportive role in (?).
hemostasis
When evaluating a (?) that maybe traceable to platelets, the counting of platelets is an important and logical starting point
bleeding problem
Light Microscopy:
Tocantins method
Diluting fluid: Rees & Ecker fluid
Tocantins method
RBC pipette
Tocantins method
Sucking tube
Tocantins method
Light Microscope
Tocantins method
Diluting fluid: 1% ammonium oxalate (freshly prepared)
Phase-Contrast Microscopy
Dilution vial
Phase-Contrast Microscopy
Phase-Contrast Microscope (or Light Microscope used with subdued light
Phase-Contrast Microscopy
Specimen Needed: (?) or First few drops from a (?) with freely flowing blood
EDTA-anticoagulated venous blood
deep skin puncture
A. LIGHT MICROSCOPY:
Tocantin’s method
B. PHASE-CONTRAST MICROSCOPY:
Brecker-Cronkite Method
LIGHT MICROSCOPY: Tocantin’s method
1. Do a finger puncture or venipuncture with (?).
2. Draw first diluting fluid up to 0.5 mark of pipette then draw blood to exactly (?) and dilute to (?) with Rees and Ecker diluting fluid.
3. Gently mix, discard the first (?) and charge into a hemocytometer.
4. Place moist chamber (Petri dish with moistened filter paper) over the charged hemocytometer and stand for. (?).
5. Examine under high dry objective and count platelets in (?)).
6. Calculate as follows:
EDTA tube
0.5 mark; 101 mark
2 drops
15 minutes
4 large corner squares (“W” squares
Platelets/cu mm = number of platelets counted in 4 squares x DF x VCF
From this point, the platelet count should be completed within (?) to avoid platelet disintegration.
30 minutes
Both platelets and RBCs are preserved by the (?) fluid.
Rees and Ecker
Platelets are much smaller than red cells ([?]the size of an RBC) and appear as round, oval or elongated particles which are highly refractile and stain light bluish.
1/10
B. PHASE-CONTRAST MICROSCOPY: Brecker-Cronkite Method
1. Prepare a (?) dilution of the blood sample
2. Rotate on a mechanical mixer for (?) the vial containing the suspension.
3. Load (?) of the hemocytometer in the usual manner using a separate capillary tube for each side.
4. Cover the loaded hemocytometer with moist chamber for (?)to allow settling of platelets in one optical plane.
5. Count platelets in (?) of the central large square, (?) on each side of the chamber; or in (?) small squares, (?) on each side of the chamber; or in all (?) small squares, until at least (?) platelets have been recorded.
6. Calculate as follows:
1:100
10 – 15 minutes
both sides
15 minutes
10 small squares; 5; 20; 10; 50; 100
Platelets/cu mm = total number of platelets counted x DF x VCF
Light Microscopy: Count platelets in
4 large/W squares (squares A,B,C,D)
Phase-Contrast: Count platelets in
10 RBC/small squares (colored squares)
