1. General Approach in Investigation of Hemostasis
The Islamic University – Gaza
Faculty of Health Science
Medical Technology Department
General Approach in Investigation of Hemostasis
Lecture 1: Introduction

2. Introduction
Hemostasis is a complex interaction between vessels, platelets and coagulation proteins that, when working properly, stops bleeding while maintaining blood flow in the vessel.
Specific tests are available to evaluate platelet function, coagulation proteins, natural occurring inhibitors and fibrinolysis.

3. Preanalytical Variables
To ensure that the laboratory analysis actually reflects the patient’s state we must consider what factors may influence the state of the sample and from that we may determine mechanisms to properly regulate these factors.
Pre-Analytical Variables include
Sample Collection.
Site Selection.
Storage Requirements.
Transportation of Specimen.

4. Sample Collection
Proper sample collection is of outmost importance for reliable test results to evaluate the bleeding patient, thrombosis or fibrinolysis (preanalytical phase).
All these tests are influenced by sample collection, sample processing and sample storage.
Any sample that is not obtained quickly with an immediately successful venipuncture should be discarded because of possible activation of coagulation.
The blood should not be passed back through the needle after collection into a syringe. The needle should be removed before passing the blood from the syringe into the container with anticoagulant.

5. when blood is withdrawn from a vessel, changes begin to take place in the components of blood coagulation. Some occur almost immediately, such as platelet activation and the initiation of the clotting mechanism dependent on surface contact.
There should be no delay between collection and mixing with anticoagulant.
Once blood and anticoagulant are mixed, the container should be sealed and mixed by gentle inversion five times. Avoid vigorous shaking.
The laboratory will not evaluate samples that are hemolyzed, clotted, contain fibrin strands or improperly stored.
Reference Laboratory Services will immediately notify the client of any problems with the sample.

6. Anticoagulant of choice
3.8% or 3.2% Sodium Citrate
3.2 % Preferred as the standard measure due to stability and closeness to the plasma osmolality
Anticoagulant/blood ratio is critical (1:9)
Exact amount of blood must be drawn. No short draws are acceptable, this will falsely increase results due to presence of too much anticoagulant
CLSI guideline is +/- 10 % of fill line
Purpose of the anticoagulant is to bind or chelate calcium to prevent clotting of specimen
(CLSI ) Clinical and Laboratory Standards Institute.
* CLSI : Clinical and Laboratory Standards Institute

7. Other anticoagulants, including oxalate, heparin, and EDTA, are unacceptable.
The labile factors (factors V and VIII) are unstable in oxalate, whereas heparin and EDTA directly inhibit the coagulation process and interfere with end-point determinations.
Additional benefits of tri-sodium citrate are that the calcium ion is neutralized more rapidly in citrate, and APTT tests are more sensitive to the presence of heparin.

8. Samples with High hematocrits
If the patient has a reduced hematocrit, or particularly if the hematocrit is raised, results can be affected
According to the latest CLSI (formerly NCCLS) guideline on coagulation testing, it is important to adjust the sodium citrate volume when a patient’s hematocrit is greater than 55%.
Examples of patients who may have elevated hematocrit values are newborns or people with polycythemia vera.
NCCLS* recommends adjusting anticoagulant ratio for patients with hematocrits exceeding 55%
(NCCLS) National Committee for Clinical Laboratory Standards.
* National Committee for Clinical Laboratory Standards

9. High hematocrits may cause falsely prolonged test results due to an over-anticoagulated sample
Formula correction achieves a 40% hematocrit.
X = (100–PCV)×vol.
(595–PCV)
Where:
X= volume of sodium citrate
Vol. =volume of whole blood drawn
PCV= patient’s hematocrit

10. Examples Patients Hct = 60%, V= 5 mL X = (100 - 60) ×5 (595 - 60)
535
Patient Hct = 25%, V=5 ml
X = ( ) × 5
( )
= 75 × 5 = 0.65 ml
570
HCT
Citrate (ml)
0.20
0.70
0.25
0.65
0.30
0.61
0.55
0.39
0.60
0.36
0.31
0.27

11. Site Selection
Collections from venous lines should include a process for flushing and/or discarding the initial collection volumes. Size and type of needle used may also influence results and too large (less than 16 gauge) or too small a needle bore (greater than 25 gauge) should be avoided, and heparinized needles (sometimes used for blood gas collection) not used
Untraumatic venipuncture is required
Traumatic venipuncture release tissue factor and initiate coagulation
Fingersticks/Heelsticks are not allowed
Indwelling IV line draws are discouraged
Contain heparin & diluted blood
Falsely increased results

12. Order of Draw Evacuated tube system Blue top is 2nd
If 2nd tube drawn, 1st top must be anticoagulant free (i.e. red top)

13. Storage Requirements Prothrombin Time: PT
Uncentrifuged or centrifuged with plasma remaining on top of cells in unopened tube kept at 2-4 oC or oC must be tested within 24 hours of collection
Activated Partial Thrombin Time: APTT
Uncentrifuged or centrifuged with plasma remaining on top of cells in unopened tube kept at 2-4 oC or oC must be tested within 4 hours of collection

14. Other Assays
Fibrinogen, Thrombin Time, Factor Assays
Centrifuged with plasma remaining on top of cells in unopened tube kept at 2-4 oC or oC must be tested within 4 hours of collection

15. Storage Requirements TEST PLASMA STABILITY AT RT CENTRIFUGE TO PREPARE
PLATELET-FREE PLASMA
REFRIGERATION
(Or transport on ice)
FREEZE
PLATELET-
FREE
PLASMA PT
24 hours
Do not refrigerate
If >24 hour delay in testing
PTRX
PTT
4 hours
If >4 hour delay in testing
PTTRX
2 hours
Within one hour of collection
If >2 hour delay in testing TT
OTHER
ASSAYS

16. Storage Requirements Other general notes
Perform coagulation tests ASAP
Specimen may deteriorate rapidly (especially factors V and VIII)
If the testing is not completed within specified times, plasma should be removed from the cells and placed in a frost free freezer
- 20 oC for two weeks
-70 oC for six months

17. Transportation of Specimen
Samples should be transported non-refrigerated in as short a time as possible, or can be transported on ice. Extremes of temperature (ie, both refrigerated or high) should be avoided.
Delays in transport may affect in particular the labile factors (FV, FVIII), leading to prolonged clotting times and in vitro loss of factor activity. In such cases, local centrifugation and separation of plasma followed by freezing and frozen transport of the plasma should be considered.

18. Centrifugation Platelet-Rich plasma (PRP)
Used in platelet function studies
x /L
Specimen must be centrifuged for x g

19. Platelet Poor Plasma
Platelet-poor plasma (PPP) is used for most tests of coagulation and is necessary for coagulation testing to prevent activation of platelets and release of PF4, a heparin inhibitor.
The plasma platelet count must be < 10,000 /mm3.
Specimen has been centrifuged for x g.
Why is PPP essential?
Contains platelet factor 4 (heparin neutralizer).
Contains phospholipids (affects lupus anticoagulant and factor assay testing).
Contains proteases (affect testing for vWF).

20. Platelets Poor Plasma preparation:
To prepare platelet-Poor plasma
The blood sample should be centrifuged at a minimum of 1700 g for at least 10 minutes. This can be at room temperature provided this does not exceed 25°C, in which case a refrigerated (4°C) centrifuge should be used.
The 2/3 of PPP from the first centrifugation is transferred to a plastic stoppered tube and centrifuged a second time. Care is taken not to use the bottom part of the plasma after the second centrifugation, since it may contain any platelets that remained after the first centrifugation.
remove the top ¾ of the plasma to a plastic aliquot tube with a fresh plastic pipette. Freeze the specimen within one hour of collection.

21. Samples For Immediate Testing
Samples should be tested within four hours of sample collection when possible.
More prolonged storage should be avoided for screening tests and clotting factor assays, although it has been shown that whole blood samples stored at room temperature may be stable for prothrombin time measurements.
Samples for screening tests and assay of factor VII should be maintained at room temperature to avoid the possibility of cold activation.

22. Deep-Freezing Plasma
Samples can be stored deep frozen for testing at a later stage. Storage at -70°C or lower is preferable.
Clotting factors are stable at this temperature for at least six months. Short-term sample storage at -35°C is adequate for most tests.
Storage at -20°C is usually inadequate.
Double centrifugation should be used if samples are deep-frozen prior to analysis for lupus anticoagulant.
Freezing and thawing is best avoided before APTT determinations, since results obtained by some techniques can be affected.

23. Any frozen plasmas must be transferred immediately to a 37°C water bath, thawed for four to five minutes, and mixed by gentle inversion prior to analysis. A slow thaw at lower temperature should be avoided to prevent the formation of cryoprecipitate, which reduces the FVIII, VWF, and fibrinogen content of the supernatant plasma.

24. Common Collection Problems
Error
Consequence
Comment
Short draw
<2.7 mL
PT/PTT falsely prolonged
Anticoagulant to blood ratio exceeds 1:9
Failure to mix specimen after collection
Blood clots form when anticoagulant & blood do not mix
Excess vigorous mixing
PT/PTT falsely shortened
Hemolysis and platelet activation cause start of cascade
Hemolysis
Reject specimen
Improper storage: wrong temperature or held too long
Must follow storage requirements
Chilling in refrigerator or placing on ice
PT falsely shortened
Chilling to 4 oC activates factor VII.

25. Error
Consequence
Comment
Inadequate centrifugation
PTT loses sensitivity for lupus anticoagulants and heparin.
Factor assays inaccurate
Prolonged tourniquet application
Falsely elevates vWF, factor VIII
Tourniquet causes venous stasis,
Drawing coagulation tube after to other anticoagulant tubes
PT/PTT falsely affected
Contamination
Probing the vein
PT/PTT falsely shortened
Tissue thromboplastin is released activating coagulation
Heparin contamination from line draw
PTT falsely prolonged
Heparin keeps the blood from clotting
Lipemia
Test may not work
Photo-optical methods affected

26. Principles of Laboratory Analysis
The more detailed investigations of coagulation proteins also require caution in their interpretation depending on the type of assay performed. These can be divided into three principal categories, as described in the following sections.
Immunological
Assays Using Chromogenic Peptide Substrates (Amidolytic Assays)
Coagulation Assays
Other Assays

27. Immunological
Include immuno-diffusion, immuno-electrophoresis, radioimmunometric assays, latex agglutination tests, and tests using enzyme-linked immunosorbent assays (ELISA).
Fundamentally, all these tests rely on the recognition of the protein in question by polyclonal or monoclonal antibodies. Polyclonal antibodies lack specificity but provide relatively high sensitivity, whereas monoclonal antibodies are highly specific but produce relatively low levels of antigen binding.

29. latex agglutination kit: Latex microparticles are coated with antibodies specific for the antigen to be determined. When the latex suspension is mixed with plasma an antigen–antibody reaction takes place, leading to the agglutination of the latex microparticles.
Agglutination leads to an increase in turbidity of the reaction medium, and this increase in turbidity is measured photometrically as an increase in absorbance.
Usually the wavelength used for latex assays is 405 nm, although for some assays a wavelength of 540 or 800 nm is used. This type of assay is referred to as immuno- turbidimetric.

30. Notes
Do not freeze latex particles because this will lead to irreversible clumping.
An occasional problem with latex agglutination assays is interference from rheumatoid factor or paraproteins. These may cause agglutination and overestimation of the protein under assay.

31. Chromogenic Assay
Chromogenic, or amidolytic, methodology is based on the use of a specific color-producing substance known as a chromophore.
the chromophore normally used in the coagulation laboratory is para-nitroaniline (pNA), which has an optical absorbance peak at 405 nm on a spectrophotometer.

32. Coagulation Assays
Coagulation assays are functional bioassays and rely on comparison with a control or standard preparation with a known level of activity.
In the one-stage system optimal amounts of all the clotting factors are present except the one to be determined, which should be as near to nil as possible.
The best one-stage system is provided by a substrate plasma obtained either from a patient with severe congenital deficiency or artificially depleted by immuno-adsorption.

33. Coagulation techniques are also used in mixing tests to identify a missing factor in an emergency or to identify and estimate quantitatively an inhibitor or anticoagulant.
The advantage of this type of assay is that it most closely approximates the activity in vivo of the factor in question. However, they can be technically more difficult to perform than the other types described earlier.
Mixing studies are tests performed on blood plasma used to distinguish factor deficiencies from factor inhibitors, such as lupus anticoagulant, or specific factor inhibitors, such as antibodies directed against factor VIII. Mixing studies take advantage of the fact that factor levels that are 50 percent of normal should give a normal Prothrombin time (PT) or Partial thromboplastin time (PTT) result.
If the problem is a simple factor deficiency, mixing the patient plasma 1:1 with plasma that contains 100% of the normal factor level results in a level ≥50% in the mixture (say the patient has an activity of 0%; the average of 100% + 0% = 50%). The PT or PTT will be normal (the mixing study shows correction). However, if there is an inhibitor that inactivates the added clotting factor, the resulting factor level will be low and the clotting test will be prolonged (fails to correct). Therefore, correction with mixing indicates factor deficiency; failure to correct indicates an inhibitor.

34. Other Assays
Using snake venoms (The Taipan venom time employs a reagent isolated from the venom of the Taipan snake (Oxyuranus scutellatus) that directly activates prothrombin in the presence of phospholipid and calcium.)
Aassay of ristocetin cofactor (used to diagnose von Willebrand disease )
The clot solubility test for factor XIII.
DNA analysis is becoming more useful and more prevalent in coagulation. However, this requires entirely different equipment and techniques