MANUAL PLATELET COUNTING PROCEDURE IN HEMATOLOGY LABORATORY.pdf

MANUAL PLATELET COUNTING PROCEDURE IN HEMATOLOGY LABORATORY

1. PLATELET COUNT
BRIAN M. DENNEY, MD, RMT, MSMT

2. Platelets
–Thin disks, 2 to 4 um in diameter and 5-7 fLin
volume
–Regularly-shaped clear cell fragments, which are
derived from fragmentation of precursor
megakaryocytes.
–Functions:
•Hemostasis
•Maintaining vascular integrity
•Process of blood coagulation

3. Platelet Count
–Technical factors:
•Method of blood collection
•Sample preparation
•Type of instrument and the calibration

4. Platelet Count
–More difficult than RBC and WBC Count:
–It is very small
–May disintegrate easily
–Unevenly distributed
–Adhere to glass surfaces or foreign bodies
–Resembles a debris, bacteria and dirt

5. Specimen: allow for a precheck
–Venous blood collected with EDTA as an anticoagulant
–Skin puncture produces variation but free flow of
blood is ideal
•Drawback:
–Lower platelet count
–Does not allow for a precheck
–Difficult to obtain accurate platelet count
–EDTA-anticoagulatedblood is satisfactory:
•For 5 hours at 20 C
•For 24 hours at 4 C

6. Platelet Clumping:
–Platelets in clump may cause the count to be
falsely low, likely caused by:
–Imperfect venipuncture
–Skin puncture technique
–Presence of EDTA
–Delay in anticoagulation
–Delay in sampling
–Initiation of clotting

7. •If platelet clumps are present:
–Redilutethe count
–Obtain a fresh specimen
•In the presence of EDTA, platelet agglutinins
responsible for platelet clumping are active.
•Platelet Satelitosis:
–Appear as neutrophilsringed with adhesive platelets
–The tendency for platelets to adhere to neutrophilsin
EDTA is falsely interpreted as thrombocytopenia.

8. INDIRECT COUNT
Dameshek
Method
Fonio
Method
Olef’s
Method
Diluent Rees-Ecker
Diluent
14% Magnesium
sulfate
Best method in
the indirect
procedure but
somewhat
cumbersome
Ratio 1:5 1:3
Normal Value 500,000-900,000250,000-500,00437,000-586,000

9. DIRECT COUNT
Brecker-Cronkite:
•Reference method that uses phase-contrast microscopy
–Green filter: platelets appear dark
–Gray filter: platelets appear pink or purple
•Uses a flat-bottomed hemacytometerand thin coverslip
•Diluting fluid: 1% Ammonium oxalate
•Dilution= 1: 100
•Platelets appear round to oval purple bodies sometimes with
dendriticprocesses and a purple sheen that allow the platelets to
be distinguished from debris, which is often refractile.
•Platelets are counted in 10 small squares on each side of the
chamber.
•N.V.: 150,000-450,000/cumm

10. DIRECT COUNT
Rees and EckerMethod:
•Diluting Fluid:
–BCB - 0.25 g
–Sodium Citrate - 0.4 g
–Distilled water - 100 mL
–Formalin - 3 drops in 1:10 dilution
•Dilution = 1:200
•N.V. : 150,000 –450,000
•Platelets are seen as highly refractilebodies that are
approximately 1/10 to the size of the surrounding cells

11. DIRECT COUNT
Guy and Leake:
•Diluting fluid:
–Crystal violet- 0.05g
–Sodium Citrate- 1.6 g
–Distilled water- 100 mL
–Formalin - 940nmL
•Dilution= 1:200
•Platelets appear as highly refractilebodies
•N.V: 150,000-450,000/ cumm

12. UNOPETTE System:
•Principle:
–Blood is diluted in buffered ammonium oxalate
which hemolyzesmature red cells and preserves
platelets and leukocytes. Platelets and leukocytes
may be counted in a standard hemacytometer.
•Diluent: 1% buffered ammonium oxalate
•Dilution= 1:100

13. PHYSIOLOGIC VARIABLES
AFFECTING PLATELETS
•Normally increased in high altitudes,
administration of epinephrine, after exercise,
trauma and excitement and in winter.
•Children have slightly higher count than adults.
•Normally decrease before menstruation and
during pregnancy.
•Oral contraceptives may cause slightly increased
platelet count.
•Platelets are normally absent in lymph and other
body fluids.

14. PHYSIOLOGIC VARIABLES
AFFECTING PLATELETS
Quantitative Platelet Disorders:
A.THROMBOCYTOSIS
–Myeloproliferativesyndrome
•Polycythemiavera
•Thrombocythemia
–Post Splenectomy
–After blood loss
–After administration of epinephrine
–Accompanying BM recovery
•After cytotoxicchemotheraphy
•After treatment of Vitamin B 12

15. Quantitative Platelet Disorders:
B.THROMBOCYTOPENIA
•Due to decrease platelet production
–Aplasticanemia
–Paroxysmal nocturnal hemoglobinuria
–Leukemia ( acute,chronic)
–Metastatic lymphoma or carcinoma
–Folateand Vit. B12 deficiency
–Cytotoxic& immunosuppressive chemotherapy
–Viral infections
–Drug-induced ( Quinine, Quinidine, Penicillin & Sulfa drugs
–Bacterial infection ( Diphtheria, Thyphoidfever)

16. B.THROMBOCYTOPENIA
•Due to Platelet Sequestration
–Massive splenomegaly
–Liver diseases
–Portal hypertension
–Lymphomas
•Due Immune Destruction of platelet
–Autoimmune thrombocytopenia
•After Massive Blood Transfusion

17. AUTOMATED PLATELET COUNT
Automated counters employ:
•Electrical methods
–Counting and sizing take place in the RBC aperture.
–Classifies particles that are greater than 2 fLor less
than 20 fLas PLTS.
•Optical methods
–Forward light scatter pattern discrimination between
RBCS and PLTS in the flow cell determines the PLT
count and frequency.

18. AUTOMATED PLATELET COUNT
•Falsely high results from contamination
carryover of samples with high counts and
fragments of red/ white blood cells.
•False low counts result from platelet
agglutination, platelet satellitismand large
platelets.

19. AUTOMATED PLATELET COUNT
QUALITY CONTROL:
•Instruments are standardized against reference
methods or against a single-channel instrument.
•Controls are usually run at the beginning of each
batch of platelet counts
•Samples from each batch should be screened for
accuracy by quick estimates.
•Clotting of the sample will invalidate the platelet
count.

20. LABORATORY
EVALUATION OF
PLATELET FUNCTION
BRIAN M. DENNEY, RMT

21. BLEEDING TIME
•Screening test for detecting disorders of
functional PLT and for vWd, and is directly
affected by platelet count and the ability of
platelets to form a plug.
•The bleeding time is prolonged in
thrombocytopenia, von Willebranddisease, and
following ingestion of aspirin, aspirin-containing
compounds, anti-inflammatory drugs,
anticoagulants, some antibiotics, and certain
other drugs.

22. BLEEDING TIME
DUKE Method
•Device: feather lancet
•Site: Earlobe or fingertip
•Incision size: lancet-size dependent
•Normal value: 1-3 min.

23. BLEEDING TIME
IVY Method
•Device: feather lancet with blood pressure
cuff inflated to 40 mmHg.
•Site: volarsurface of the forearm
approximately 5 cm below the antecubital
crease
•Incision size: parallel to antecubital: 3.5 mm
(depends on the size of lancet)
•Normal value: 2.5 –7 min

24. BLEEDING TIME
TEMPLATE Method:
•Device: disposable template device
•Site: volarsurface of the forearm approximately 5 cm
below the antecubitalcrease
•Incision size:
–Adult color white –5.0 mm long, 1 mm deep
–Pediatricscolor pink –3.5 mm long, 1 mm deep
•Normal Value: 2-8 min

26. BLEEDING TIME
Discussion:
•The aspirin tolerance test may be useful in
helping to distinguish functionally abnormal
platelets from normal platelets.
•Performing a bleeding time on infants using
SurgicuttNewborn
•The incision must be made consistently in the
same direction.

27. CAPILLARY FRAGILITY TEST
•This measures the ability of capillaries to
withstand increased stress, reflecting the
integrity of the vessel walls and the
maintenance of activities of platelets.
•A positive tourniquet test (presence of
numerous petechiae) will be found in
thrombocytopenia, decreased fibrinogen and
vascular purpura.

28. CAPILLARY FRAGILITY TEST:
TOURNIQUET TEST / RUMPEL-LEEDE TEST
•Equipment:
–Stethoscope and blood pressure cuff
•Principle:
–An inflated blood pressure cuff on the upper arm
is used to apply pressure to the capillaries for 5
minutes. The arm is the examined for petechiae.

29. CAPILLARY FRAGILITY TEST/ TOURNIQUET
TEST / RUMPEL-LEEDE TEST
•Procedure:
1.Examine the forearm, hand, and fingers
2.Apply a blood pressure cuff on the upper arm above the elbow
and take a blood pressure reading.
3.Inflate the blood pressure cuff
4.Remove the blood pressure cuff and wait 5 to 10 minutes
before proceeding
5.Examine the forearm, hands and fingers for petechaie.
•Reporting:
–Negative <10
–Borderline 10-20
–Positive >20

30. CLOT RETRACTION
•In vitro phenomenon in which blood clot
retraction can be observed after few hours,
leaving clear serum adjacent to the test tube
walls.
•When blood coagulation is complete, the clot
normally undergoes retraction.
•Directly proportional to the number of PLT and
inversely proportional to the Hctand fibrinogen
levels.

31. CLOT RETRACTION
•Reflective:
–No. and quality of PLT
–Fibrinogen concentration
–Fibrinolyticactivity
–Hct
•An abnormal clot retraction is found in:
–Glanzmann’sthrombasthenia
–Thrombocytopenia
–Hypofibrinogenemiaor dysfibrinogenemia
–Paraproteinemias(Multiple Myeloma)
–DIC
–High red cell count
–Anemic states

32. PLATELET ADHESIVENESS TEST
•The adhesiveness of blood platelets may be measured
in vitro by their ability to adhere to glass surfaces.
•DECREASED: Glanzman’sthrombasthenia, von
Willebrand’sdisease, Chediak-Higashi syndrome, in
some cases of myeloproliferativedisorders, uremia,
and following ingestion of aspirin and other drugs.
•INCREASED: venous thrombosis, pulmonary embolism,
carcinoma, during pregnancy, following splenectomy,
and in patients taking oral contraceptives.

33. PLATELET ADHESIVENESS TEST
SalzmanMethod
•Regents and Equipment:
–A double ended, 20 gauge, vacutainerneedle
–Hypodermic needle, 20 gauge
–Vacutainertubes (2) containing EDTA anticoagulant
–Siliconizedadapter
–Polyvinyl tubing
–Glass beads
–Siliconizednylon mesh
–Materials for platelet count
–Glass bead filter

34. PLATELET ADHESIVENESS TEST
•Specimen:
–One tube of whole blood collected by routine
procedure
–Second specimen of blood is collected through the
glass bead collecting system
•Principle:
–A platelet count is performed on both specimens of
blood. The number of platelets in the blood, collected
through the glass bead collecting system, will be lower
than the number obtained by routine venipuncture.

36. PLATELET ADHESIVENESS TEST
•Procedure:
1.Perform two clean venipucnturesat separate
sites, with and without the use of the glass bead
collecting system. (The blood collection rate
through the glass bead column should be 6 to 10
mL/min.)
2.Perform platelet count on both blood samples.
3.Calculate the percent of platelet adhesiveness.

37. PLATELET AGGREGOMETRY
•Principle:
–Platelet rich plasma (PRP) is placed in the test well of
the platelet aggregometer. An aggregating reagent is
added to the PRP and, at the same time, the optical
density of the sample is monitored. As the platelets in
the plasma clump (aggregate) the plasma becomes
more clear and light transmittance through the
specimen increases. These changes in optical density
are recorded by the instrument in the form of a graph.
The aggregometeralso calculates the slope of the
aggregation curve and the amount (%) of platelet
aggregation.

38. PLATELET AGGREGOMETRY
•Aggregating Agents: RECEPTORS
–ADP –P2Y1, P2Y12 (platelet membrane)
–Epinephrine –a2-adrenergic receptor
–Collagen –GP Ia/IIa, GP VI
–Thrombin -PAR1 & PAR4; GP Iba& GP V (Protease activatablerecept.)
–Ristocetin–GP Ib/V/IX in assoc with vWF
–ArachidonicAcid –TPa, TPb
•Special Patient Preparations:
–Preferably, the patient and the normal control should be fasting.
Otherwise, they should have had a relatively fat-free meal; this
eliminates the optical problem of lipemicplasma.
–The patient should not be taking aspirin or nonsteroidalanti-
inflammatory drugs.

39. PLATELET AGGREGATION
•Specimen Requirements:
–Platelet-rich plasma is obtained from both patient and
control specimens by centrifuging at RT at 200x g for
10 minutes.
–As a consequence of processing of the PRP, platelets
sometimes exhibit a “ platelet shock” phenomenon
and will be poorly or not responsive to ADP, collagen
and epinephrine for as long as 30 minutes. If a poor
response is obtained with one of these reagents, the
test is repeated 30 minutes later.

40. PLATELET AGGREGATION
Procedure:
1.Perform platelet count on patient and control PRP; adjust the platelet
counts to 200 to 300 X 10
9
/L by diluting with their respective aliquots of
PPP. Keep samples stopperedat room temperature.
2.Warm 0.5 mLaliquots of PRP in appropriate cuvettesto 37 deg Celsius
for several minutes as needed for testing.
3.Adjust 100% transmittance of the platelet aggregometerusing patient
PPP.
4.Place warmed PRP samples in the aggregometerand adjust the baseline
reading after at least 1 minute.
5.After stirring PRPP for 2 minutes with small magnetic stir bars, forcefully
add the appropriate volume of aggregating agent to ensure adequate
mixing.
6.Record the aggregation curve for 3-5 minutes or until no further changed
is noted.
7.Repeat steps 2-6 for each aggregating agent with patient and control PRP

41. PLATELET AGGREGATION
•Platelet Aggregation Patterns:

42. ADP

43. EPINEPHRINE

44. COLLAGEN & ARACHIDONIC ACID

45. THROMBIN

46. RISTOCETIN

47. PLATELET AGGREGATION
Comments:
–All testing should be complete within 3 hours of
blood collection.
–Specimens for platelet aggregation studies should
not come in contact with any type of glassware.
–Specimens should not be refrigerated.
–Stirring effects, reagent mixing and platelet
aggregate collisions has a significant effect on
platelet aggregation.