Thromboplastin Time (Prothrombin Time) Heinrich Sudhof

Thromboplastin Time (Prothrombin Time)

Heinrich Sudhof

Principle

By thromboplastin time (prothrombin time) is meant the time required for coagulation after the addition of an optimum amount of C a

2+

and excess thromboplastin to b l o o d plasma. Contrary to the original idea of Quick

1

>

2

> the coagulation time obtained under these conditions estimates not only the prothrombin (Factor II) content of the plasma, but also a series of other blood coagulation factors: Factor V (proaccelerin-accelerin), Factor VII (proconvertin-convertin) and Factor X (Stuart-Prower Factor). A large increase of antithrombin or a decrease of fibrinogen also affect the prothrombin time.

The "Quick value" corresponding to the thromboplastin time is read off from a standard curve.

This value gives no indication of the amounts of the individual coagulation factors contained in the plasma, but merely gives the coagulation activity as a percentage of normal: the Quick value indicates h o w much normal plasma must be diluted so as to have the same coagulation time as the patient's plasma.

The thromboplastin added to the citrated plasma, in conjunction with the above mentioned factors, acts as "prothrombin conversion factor". In the presence of C a

2+

ions, the prothrombin contained in blood plasma is converted to thrombin which initiates clot formation ("Phase I of blood coagula­

tion"). In its turn the thrombin acts on the complicated process of " Phase II of blood coagulation ", which starts with the transformation of fibrinogen and ends with the formation of the fibrin clot.

Standardized and stable thromboplastin preparations must be used for the determination of the thromboplastin time. The same plasma can have different thromboplastin times depending on the type of thromboplastin preparation. The reason for this is that particular thromboplastin prepara­

tions differ in their reactions to the individual coagulation factors.

Commercially Available Thromboplastin Preparations

Thrombokinase — "Behring-Werke":

Monkey brain thrombokinase, very strongly Factor VII sensitive; obtainable in ampoules, the contents of which should be dissolved in 1 ml. distilled water.

Thrombokinase — " G e i g y " :

Thrombokinase from lung extracts. Factor II or X sensitive; changes in the content of other factors are less detectable. It is marketed in tablet form. Each tablet contains sufficient calcium so that the suspension prepared from the tablets is 0.1 % with respect to C a C ^ .

Thromboplastin solution — " R o c h e " :

Extracts from human brain. Factor VII sensitive. Ampoules containing 1 and 5 ml. The solution contains gelatine. If stored in a refrigerator the contents solidify, so that the gel must be carefully liquified before use by allowing it to warm to r o o m temperature or by warming in a water bath at 37° C.

1) A. J. Quick, M. Stanley-Brown and M. Bancroft, Amer. J. med. Sci. 190, 501 [1935].

2) A. J. Quick, Amer. J. Physiol. 118, 260 [1937].

Determination of the Thromboplastin Time According to Quick

*

( L a b o r a t o r y M e t h o d )

Reagents

1. T h r o m b o p l a s t i n s o l u t i o n " R o c h e " *>

2. S o d i u m citrate, N a

3

6

5

2

3. C a l c i u m c h l o r i d e , C a C l

2

Preparation of Solutions

I. T h r o m b o p l a s t i n :

U s e a c o m m e r c i a l p r e p a r a t i o n u n d i l u t e d . II. S o d i u m citrate (0.1 M ) :

D i s s o l v e 3.6 g. N a

3

6

5

7

2

III. C a l c i u m c h l o r i d e (2.5 x 1 0 - 2 M ) :

D i s s o l v e 0 . 2 7 5 g. C a C b in 100 m l . distilled water. 10 m l . o f this s o l u t i o n s h o u l d be e q u i v a l e n t t o 5 m l . 0.1 N H g N 0

3

IV. S o d i u m c h l o r i d e ( 0 . 8 5 % w / v ) :

D i s s o l v e 0.85 g. N a C l in 100 m l . distilled water.

Procedure

Experimental material D r a w u p

0.2 m l . N a citrate s o l u t i o n (II)

w i t h a 2 m l . i n j e c t i o n syringe. W i t h light pressure o n the u p p e r a r m p u n c t u r e the cubital vein a n d w i t h d r a w

1.8 m l . b l o o d .

M i x the c o n t e n t s o f the syringe by carefully w i t h d r a w i n g t h e p l u n g e r s o m e w h a t further a n d tilting b a c k a n d forth. T r a n s f e r the b l o o d t o a centrifuge t u b e a n d centrifuge for 15 m i n . at 3 0 0 0 r . p . m . Carefully t a k e off t h e p l a s m a a n d store at r o o m t e m p e r a t u r e until ready for the d e t e r m i n a t i o n ( n o later t h a n 2 h o u r s after c o l l e c t i n g the b l o o d ) .

A s s a y

D u p l i c a t e d e t e r m i n a t i o n s are r e c o m m e n d e d . P r e - w a r m the t h r o m b o p l a s t i n a n d c a l c i u m c h l o r i d e s o l u t i o n t o 3 7 ° C ( w a t e r b a t h ) . P i p e t t e i n t o t w o test t u b e s ( l e n g t h : 8 — 1 0 c m . ; d i a m e t e r : 0.9 — 1.3 c m . )

0.1 m l . p l a s m a

a n d p l a c e in a w a t e r b a t h at 37° C . A f t e r e q u i l i b r a t i o n , m i x i n t o e a c h t u b e 0.1 m l . t h r o m b o p l a s t i n s o l u t i o n (I)

0.1 m l . c a l c i u m c h l o r i d e s o l u t i o n (III).

O n m i x i n g in the C a C b s o l u t i o n ( b l o w o u t the p i p e t t e ) start a s t o p w a t c h . D r a w a sterile, c o l d p l a t i n u m l o o p o n c e or t w i c e every s e c o n d t h r o u g h t h e r e a c t i o n m i x t u r e . A s s o o n as t h r e a d s o f fibrin h a n g o n the l o o p s t o p the s t o p w a t c h . T h e t i m e r e c o r d e d is the t h r o m b o ­ p l a s t i n t i m e . T h e Q u i c k v a l u e is o b t a i n e d f r o m this by m e a n s o f a s t a n d a r d c u r v e .

*> or any other commercially available thrombokinase preparation. In which case the procedure given by the manufacturer should be used.

3)

A.J. Quick: The Physiology and Pathology of Hemostasis. Henry Klimpton, London 1951, p. 125.

Standard curve

Dilute normal plasma (obtained as described in the Section "Experimental materiar') with 0.85% NaCl solution (IV) in the ratios of 20:100, 40:100, 60:100 and 80:100*). Deter­

mine the thromboplastin times for these dilutions and for the original plasma (see under

"Assay "). Plot the measured coagulation times (ordinate) against the % by volume of plasma in the dilute solutions ( = Quick value; abscissa) on double logarithmic graph paper. This permits easier reading and the detection of errors in the method on drawing the standard curve (see Fig. 1 a and b).

w-

n I 1 1 1 1 1 1 1 1 1 1— / # J - 1 1 1 1—i—i i i i

u

10 20 30 W 50 60 10 80 90100 10 20 30 kO 506070 90100

a) b) Fig. I. Standard curve (relation between the thromboplastin time and the Quick value).

a) Linear graph paper

b) Double logarithmic graph paper Ordinates: Thromboplastin time [ s e c ]

Abscissa: Quick value ( = plasma dilution) [% by volume plasma in the dilute solutions]

"Geigy" Micromethod

)

(Whole blood method, also suitable as a bedside method) Reagents

1. Thrombokinase, "Geigy"

2. Sodium citrate, N a 3 ^C 6 ^H 5 ⁰ 7 ^{- 5 V 2 H} 2 ⁰ 3. Sodium chloride

Preparation of Solutions I. Thrombokinase suspension:

Grind a "Geigy" thrombokinase tablet in a thick-walled test tube with a glass rod.

Stir the powder with a few drops of distilled water to form a homogeneous paste. Suspend this paste in 2.5 ml. distilled water and incubate for 15 min. in a water bath at 37° C (constant). Use this warm suspension.

II. Sodium citrate (3.8% w/v):

Dissolve 3.8 g. N a 3 ^C 6 ^H 5 ⁰ 7 ^{- 5*/} 2 ^H 2 0 in 100 ml. distilled water.

III. Sodium chloride (0.85% w/v):

Dissolve 0.85 g. NaCl in 100 ml. distilled water.

*) This procedure is satisfactory for routine studies. For research purposes prepare the plasma dilutions with fibrinogen solution or better still with barium adsorptive plasma

4

) instead of N a C l solution or veronal buffer.

4) P. A. Owren and K. Aas, Scand. J. clin. Lab. Invest. 3, 201 [1951].

s) C. Montigel, Therap. Umschau 9, 17 [1952].

Procedure

Collection of b l o o d

In a blood sugar pipette containing 0.01 ml. Na citrate solution (II) suck up

capillary blood to the 0.05 ml. mark

(from the finger, or with babies from the heel). Rapidly blow out the contents of the pipette into a small test tube.

A s s a y

Triplicate determinations are advisable. Pre-warm citrated blood for 2 min. in a water bath at 37° C, add

0.08 ml. of the throughly mixed and pre-warmed (37° C) thrombokinase suspension (I) and start a stopwatch. Incubate the test tube in the water bath at 37°C with continual gentle tipping until the appearance of coagulation is visible to the naked eye. Stop the stopwatch.

The time recorded is the thromboplastin time. To obtain the corresponding Quick value, see p. 910.

Standard curve

Obtaining plasma: Draw up 1 ml. Na citrate solution (II) with a 5 ml. injection syringe and with light pressure on the upper arm withdraw blood to the 5 ml. mark. Mix the contents of the syringe, centrifuge for 10 min. at ca. 2000 r.p.m., siphon off the plasma and dilute as described on p. 910 under "Standard curve".

Proceed as describea^on p. 909 under "Assay", but instead of the thromboplastin and CaCl 2 solution add only 0.1 ml. thrombokinase suspension to 0.1 ml. plasma. Plot the coagulation time against the dilution of the plasma (see p. 910 and Fig. 1).

For routine studies the prothrombin scale provided by Geigy can be used instead of a stand­

ard curve 6 )

. Other Information

Apart from the two procedures described here there are several well-tried, slightly modifiedmethods

7

).

These include the so-called bedside method which enables the thromboplastin time to be determined directly at the bedside of the p a t i e n t

8

.

9 )

.

Coagulation of the mixture can also be detected by adding a small glass bead (35 to 40 mg.) to the reaction mixture and carefully turning the sloping test tube on its longituidinal axis until the glass bead is caught by the fibrin clot. T h e paper strip method described by Studer and Winterstein has the advantage that the reaction mixture is not m o v e d

1 0

) . For large laboratories the automatic electric apparatus ( C o a g u l o m e t e r )

n

> manufactured by Heinrich Schnittger, Bracke/Lippe, Germany is suitable. With this apparatus several determinations can be carried out simultaneously and the per­

sonal error is eliminated.

6) F. K. Beller and Th. Deggelmann, Die Medizinische 1954, 395.

7) H. E. Schidtze and G. Schwick, Laboratoriumsblatter der Behring-Werke, Heft 2, Oct. 1953.

8) N. Fiechter, Schweiz. med. Wschr. 21, 259 [1940].

9

> F. K. BeUer: Uberwachung gerinnungshemmender MaBnahmen. In Th. Naegli et al.: Die throm- bembolischen Erkrankungen. 2 n d Edition, Verlag Schattauer, Stuttgart 1960, p. 404.

1 0

> See the instructions enclosed with the " R o c h e " thromboplastin solution.

n > H. Schnittger and R. Gross, Klin. Wschr. 32, 1011 [1954].

In medical literature many other names have been used for thromboplastin time, e.g. one stage coag­

ulation index, coagulation power, coagulation index, coagulation time, prothrombin content, pro­

thrombin index, prothrombin level, prothrombin value, prothrombin time, Percentage Quick. It is now generally accepted that the values are expressed as percentage activity (Quick value).

It is important that the experimental conditions should be adhered to exactly. In particular, the pipet­

ting of the solutions must be exact and the temperature of the water bath must be kept constant. A new standard curve is required for each change of the experimental conditions and for each thrombo­

plastin preparation from another source. The plasma for the standard curve must be obtained in the same way as for the routine determinations.

The determination of the thromboplastin time is used to follow the rate of clotting of blood from patients on anticoagulant therapy (anticoagulants, e.g. in myocardial infarction and thrombosis).

In addition, it is a routine determination in the diagnosis of haemorrhagic diathesis (haemophilic diseases). In the interpretation of the Quick value both the special sensitivity of the particular thrombokinase and the different half-life times of the reacting coagulation factors must be taken into account. For example, 2 5 % Quick for Geigy and Roche thrombokinase when read off from the respective standard curves need not necessarily give the same coagulation time. Factor VII sensi­

tive thrombokinase gives lower Quick values earlier than Factor II or X sensitive

1 2

).

•2) P. Matis, W. Mayer and W. Nagel, Med. Welt 1961, 891.

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