Automatic measurement method of glycohemoglobin and sample injection valve

A number of blood samples collected in sample vessels are allowed to stand by as whole blood or blood cell layer, the sample vessels are sent into sampling part in order, the blood sample suctioned from the sampling nozzle is diluted by mixing with hemolyzing liquid containing labile HbA.sub.1c removing reagent, a part of the mixture is led to a sample-loop of sample injection valve and injected into the column of high pressure liquid chromatograph at a specified time after mixing start, hemoglobin fractions are measured in the state of removal or lowering of labile HbA.sub.1c, and the rate of stable glycohemoglobin is determined from thus obtained chromatogram.

BACKGROUND OF THE INVENTION 
The present invention relates to an improved automatic measurement method 
of glycohemoglobin based on the principle of high pressure liquid 
chromatography and to an improved sample injection valve used for a high 
pressure liquid chromatograph. 
Glycohemoglobin (HbA.sub.1) attaching glucose to hemoglobin is often found 
in diabetes patients, and particularly HbA.sub.1c has been an important 
measurement item as an index for a health screening such as a medical 
checkup or for a long-term control of diabetes. Because, HbA.sub.1c exists 
most abundantly in glycohemoglobin (HbA.sub.1) and the increase by 
diabetes is much more than in the other component, and moreover the value 
of HbA.sub.1c shows a significant correlation with an average blood sugar 
level in hunger for past 1.about.3 months. 
Glycohemoglobin includes HbA.sub.1a and HbA.sub.1b besides HbA.sub.1c, and 
these fractions are measured by colorimetry, electrophoresis, minicolumn 
method, or high pressure liquid chromatography. Among them, in the area of 
clinical tests, recently high pressure liquid chromatography (HPLC) has 
been prevalent in view of required time and separation. 
A so-called stable type in HbA.sub.1c shows a significant correlation with 
a past blood sugar level and besides a labile type does not so. The rate 
of the latter is said to be 10.about.15% in all HbA.sub.1c in hunger on 
healthy human. In this labile HbA.sub.1c, the N end of .beta.-chain of 
hemoglobin and the reductive end of glucose form a reversible Shiff-base 
combination, which generates and degrades in a relatively short time 
depending on blood sugar levels. Therefore, it exists in diabetes patients 
more than in healthy, sometimes being 10.about.20% to all HbA.sub.1c. 
After meal it exists more than in hunger, being effected largely by the 
condition in collecting blood. 
Stable HbA.sub.1c is generated from labile HbA.sub.1c gradually, 
continually, and irreversibly, reflecting the past long-term blood sugar 
levels. Thus, the separate measurement of the stable type only is 
desirable. However, both of the stable and labile types closely resemble 
structurally, being considerably difficult to separate by liquid 
chromatography. 
As a method against this, the elevation of separation has been conducted by 
using a long high-separation column. Though this method has a merit that 
the denaturalization of glycohemoglobin by chemical treatment may not 
occur, the analyzing time of ten and several min or more is necessary to 
gain a good separation, insufficiently dealing with the increase of 
samples or urgent measurement. Moreover, this type of column is long 
leading to the apparatus of large type and high cost. 
As another method for separately measuring the stable HbA.sub.1c, there is 
a method wherein the labile HbA.sub.1c is removed by degrading chemically 
in pretreatment. This is based on that the temporary combination (Shiff 
base) of labile HbA.sub.1c and glucose is easy to degrade. For instance, 
washed red cells are incubated in isotonic phosphoric acid buffer solution 
(37.degree. C., 4 hrs) or in physiological saline solution (room 
temperature, 14 hrs) to release glucose from labile HbA.sub.1c. 
Or, there is also a method wherein whole blood added a hemolyzing reagent 
is incubated at 35.degree. C. for ten and several hrs. It reduces the 
level of labile HbA.sub.1c by hemolyzing and diluting a sample, 
particularly having a large effect in the acidic area below pH 6, a fast 
reaction speed, and a large effect at an elevated temperature. Further, 
the addition of the reagent for removing labile HbA.sub.1c containing 
boric acid on the market which has been used for minicolumn method 
enlarges the effect. 
However, the pretreatment requires time, and together with the degradation 
of labile HbA.sub.1c, the degradation and change of other glycohemoglobin 
and pure hemoglobin (HbA.sub.o) proceed also at the same time. 
The hemolyzing type has had a defect that the quantity of stable HbA.sub.1c 
varies with the time elapsed after hemolyzing and the temperature 
experienced till measurement. Particularly, in the case of the automatic 
measurement of a number of samples, there has been a disadvantage that the 
dilution of blood sample by the hemolyzing liquid containing a degrading 
reagent changes the sample measured later in samples on standby by 
excessive proceeding of reaction because of longer time elapsed. 
SUMMARY OF THE INVENTION 
The object of the present invention is to provide a method for measuring 
stable HbA.sub.1c with good reproducibility and high accuracy with 
preventing the degradation and change of glycohemoglobin other than labile 
HbA.sub.1c together with the analysis of glycohemoglobin using the labile 
HbA.sub.1c removing reagent (degradation reagent) by high pressure liquid 
chromatography. In addition, the object of the invention is to provide a 
method for analyzing glycohemoglobin wherein the structure of the 
apparatus is relatively simple and the treatment and preparation of sample 
are easy. 
Further, the object of the invention is to provide a optimum sample 
injection valve to control the temperature of mixing solution of sample 
(not limited to blood sample) and reagent in a high pressure liquid 
chromatograph.

DETAILED DESCRIPTION OF THE INVENTION 
The object mentioned above is attained by allowing a number of blood 
samples as whole blood or blood cell layer to stand by, sampling a sample 
at the time of its measurement order, mixing the sample by quickly 
diluting with hemolyzing liquid containing a labile HbA.sub.1c removing 
reagent, and by injecting the mixing liquid into a high pressure liquid 
chromatograph column at a specified time after mixing start. Hereafter, 
"sample" refers to whole blood sample (or blood cell layer), and 
"specimen" refers to mixing liquid diluted the sample by hemolyzing 
liquid. 
Further, the reaction is accelerated by warming till the mixing liquid 
(specimen) is injected into column, enabling the speedier measurement. 
When this warming is made at the loop part of sample injection valve, 
preparation of other warming zone or useless warming of specimen is 
unnecessary, enabling space and energy saving. 
Further, the invention saves time of mixing reagent manually, and prevents 
errors by manual mixing and the change of sample till measurement after 
mixing. 
The procedure of the invention and the structure of the measurement 
apparatus will be described in the following. 
First, blood samples collected from each patient or subject are put into 
sample vessels such as blood tubes or sampling cups, and allowed to stand 
by at the sample holding part as whole blood or blood cell layer. Since 
whole blood or blood cell layer centrifuged whole blood is used, 
pretreatment is unnecessary and simple. Blood cell layer may be uased by 
making use of remaining plasma after the other tests, but whole blood also 
deposits blood cell components at the bottom after standing for long time. 
Accordingly, sampling is preferably made from the vicinity of the bottom. 
Though there is a variation in the rate of red cells in blood sample, the 
rate of HbA.sub.1c to HbA.sub.o or the other HbA.sub.1 is constant in each 
sample, being no problem. An anticoagulant is usually added to blood 
sample. As anticoagulants, ones on the market such as heparin or EDTA-2Na 
may be used. 
Each sample vessel is held in a holding device such as rack, snake-chain, 
or turn-table in a large number, and sent into the sampling position in 
order. 
At the sampling part, a specified quantity 1 to few .mu.l) of sample is 
suctioned from a sampling nozzle by the suction action of pump, and 
diluted by hemolyzing liquid containing a labile HbA.sub.1c removing 
reagent separately supplied, and mixed. The dilution is 
tens.about.hundreds-fold, preferably 10.about.400-fold. 
As the procedure of sampling and dilution, various structures and changes 
are considered depending on the structures and combinations of various 
types of pumps. In short, preferable is the structure wherein blood 
samples sampled within few tens sec.about.few min just before measurement 
are diluted with hemolyzing-washing liquid and injected into column 
through sample injection valve at a specified time after mixing start. It 
is necessary that these actions are made automatically and continously and 
a means is devised to prevent contamination. 
As labile HbA.sub.1c removing reagents, boric acid, phosphoric compunds, or 
reagents on the market containing them are used. Ones having acidic pH are 
preferable. As hemolyzing agents, general ones on the market are used. 
As a column, one for high pressure liquid chromatography such as filled 
with spherical ion-exchange gel (anion, cation) is used, and the treatment 
time of specimen is confined by the separation. 
The state of reaction proceeding after hemolyzing depends upon the property 
of the labile HbA.sub.1c removing reagent, and is associated with the time 
and temperature after reaction start. The time for measurement is the sum 
of the operation time for sampling, diluting, mixing, feeding, and washing 
and the warming time. The short measurement time is preferable to treat a 
number of samples. If the ability of degrading labile HbA.sub.1c of the 
reagent is low, the shortening of the reaction time is made by warming the 
specimen (mixed liquid) pass in the temperature range without trouble such 
as the change of sample. In this case, the temperature of whole or part of 
the pass including the sample injection valve part is so controlled that 
the time till measurement is constant in view of the analyzing time and 
the degradation ability of the reagent. In view of the degradation ability 
and the warming time, the warming temperature is 
30.degree..about.65.degree. C., preferably 40.degree..about.55.degree. C. 
The temperature above 65.degree. C. causes the change of protein 
undesirably. That below 30.degree. C. may require cooling in summer. 
In the following, the washing liquid will be described. The washing liquid 
washes the sampling nozzle and each pass to prevent contamination after 
measuring the late specimen. The structure of the invention, wherein the 
sample is mixed after dilution by hemolyzing liquid, requires naturally 2 
feeding systems of washing and hemolyzing liquid. As a matter of course, 
this structure may be used, but combining the both as the 
hemolyzing-washing liquid lessens one of the feeding pumps, simplifying 
the piping and feeding sequence. 
In the invention, the mixing liquid (specimen) is injected into column at a 
specified time after the mixing start of blood sample and hemolyzing 
liquid. In case of the mistimed mixing start in calculating back from the 
next injection timing for some reason such as smoothless shift of rack or 
mistimed wedging measurement, blank measurement action is preferably made 
without mixing and injection in this time. This mistimed mixing has no 
problem in the case of the use of only one type of eluting solution, but 
in the case of 2 types or more of eluting solution of different 
concentration or pH, the mistimed injection loses the balance in the 
column. 
EXAMPLE 
The present invention method will be described in detail by showing the 
following example of the measuring apparatus of glycohemoglobin embodied 
the invention method. In the example, hemolyzing liquid which also serves 
as washing liquid is used and a specimen is warmed in the sample-loop at 
the sample injection valve. 
FIG. 1 is a flow-diagram showing an example of glycohemoglobin automatic 
measuring apparatus embodied the invention method. FIG. 2 is a 
flow-diagram showing the sampling part. FIG. 3 is a rough oblique showing 
the sample injection valve. 
The apparatus is composed of a sample holding part(1) for holding and 
pooling plural sample vessels containing whole blood sample or blood cell 
layer, a sampling part(2) for sampling and diluting-mixing with hemolyzing 
liquid, a bottle-unit part(3) for receiving vessels for eluting solution, 
hemolyzing-washing liquid, and waste solution, an analyzing part(4) 
including a sample injection valve, column, and photometric means, a 
memory-control part for controlling the whole apparatus function, 
memorizing and calculating measurement values, and outputting the results 
on indication apparatus such as printer together with patient number and 
measuring date, and a keyboard for inputting working command. As the 
memory-control part, a microcomputer(5) is, for instance, used. For the 
indication apparatus, a digital indicator may be used besides. 
The structure and function of each part will be described in the following. 
In the sample holding part(1), a number of racks(7) holding plural sample 
vessels(6) are set. Each rack(7) is so driven towards the arrow direction 
that the sample vessel(6) is placed at the sampling position in order. A 
wedged measuring port(8) for urgent measurement may be prepared. 
The sampling part(2) has a sampling nozzle mechanism(11) with two 
nozzles(9, 10), a hemolyzing-washing liquid pump(P.sub.1), a sample 
suction pump(P.sub.2), a specimen injection pump(P.sub.3), and a dilution 
pouring vessel(12). The capacity of the sample suction pump(P.sub.2) is 
1.about.few .mu.l, and that of the washing liquid pump(P.sub.1) and the 
specimen injection pump(P.sub.3) is hundreds .mu.l. The sampling nozzle 
mechanism(11) moves up and down with rotation. The hemolyzing-washing 
liquid(13) is prepared by dissolving the above-mentioned labile HbA.sub.1c 
degrading reagent and hemolyzing agent in the dilution liquid of sample. 
In the example, this liquid is also used for washing pumps, nozzles, and 
other flow-lines. In the invention, a sample(14) refers to a whole blood 
sample or blood cell layer, and a specimen(15) refers to a solution 
prepared by diluting the sample(14) with the washing liquid(13) to 
tens-hundreds-fold. 
At the sampling part(2), first a specified quantity of sample(14) is 
suctioned from the first nozzle(9) by driving the sample suction 
pump(P.sub.2). Next, the sampling nozzle mechanism(11) is moved to over 
the dilution pouring vessel(12), and the washing liquid(13) is discharged 
from the second nozzle(10) to wash the outside of the first nozzle(9). For 
this object, the second nozzle(10) has the tip bent towards the first 
nozzle(9). The waste liquid is led to a drain bottle(17) at the 
bottle-unit part(3) through a waste liquid valve(16). Next, the sample(14) 
and a specified quantity of hemolyzing-washing liquid(13) are discharged 
into the dilution pouring vessel(12) by driving the pumps(P.sub.1, 
P.sub.2). The both(13, 14) are stirred by this discharge. Further, the 
both may be sufficiently stirred and mixed by repeating suction and 
discharge by the first nozzle(9). Thus mixed specimen(15) is suctioned 
from the first nozzle(9) by suctioning action of the specimen injection 
pump(P.sub.3) and fed into the sample injection valve(18) at the analyzing 
part(4). (the state in FIG. 1 and FIG. 2) Next, the dilution pouring 
vessel(12), piping, and the first nozzle(9) outside are washed by the 
hemolyzing-washing liquid(13), and the waste liquid is discarded and 
preparations for the next sample suction are made. 
On the other hand, the specimen(15) fed into the sample-loop(18a) at the 
sample injection valve(18) is warmed by being held in the warmed 
sample-loop(18a). The sample-loop(18a) is, as showed in FIG. 3, surrounded 
by a loop warmer(18b), and further the loop warmer(18b) and the main part 
of the sample injection valve(18) are covered by a temperature keeping 
cover(18c). In the figure, symbol(18d) is pipe for specimen, (18e) pipe 
for eluting solution, (18f) motor,(18g) geer box, and (18h) block for 
keeping temperature. The temperature keeping cover(18c) is screwed to the 
block(18h). 
In warming by this method, there is no useless energy and no injection of 
unwarmed specimen part into column(19) because of sure warming of specimen 
only in the sample-loop(18a). 
Next, the motor(18f) of sample injection valve(18) is rotated to the state 
showed as FIG. 4 and the specimen(15) in the sample-loop(18a) is injected 
into column(19) by being pushed out by the eluting solution sent from the 
bottle-unit part(3). Each component of specimen(15), HbA.sub.1a, 
HbA.sub.1b, HbA.sub.1c, and HbA.sub.o, is separated in column(19), and 
subjected to photometry by photometer(20) in order and discarded in drain 
vessel(21). The photometric results are sent to microcomputer(5) and each 
fraction pattern, elution time of peak, and content % of each component 
are calculated to be printed out at printer(22). When filter(24) is set 
before column(19) to remove impurities and the whole is kept in a 
thermostat(25), stable measurements are performed. 
Table-1 shows the summary of operations mentioned above based on FIG. 2 
concerning the action of pumps(P.sub.1, P.sub.2, and P.sub.3) and the flow 
state of flow-exchange valve(38, 39) for the hemolyzing-washing 
liquid(13). In each pump, .dwnarw. shows suction .uparw. discharge, and 
blank stop. 
TABLE 1 
______________________________________ 
pump. 
valve 
step P1 P2 P3 38 39 operation 
______________________________________ 
1 .dwnarw. 
.dwnarw. sample is suctioned, 
hemolyzing-washing liquid 
is suctioned 
2 .uparw. hemolyzing-washing liquid is 
discharged to wash nozzle- 
outside 
3 .dwnarw. hemolyzing-washing liquid is 
suctioned 
4 .uparw. 
.uparw. blood sample and hemolyzing- 
washing liquid are discharged 
to mix 
5 .dwnarw. 
specimen is suctioned and the 
part is fed to sample-loop to be 
warmed 
6 .uparw. 
specimen is injected into 
coloumn by exchanging sample 
injection valve and remaining 
specimen is discharged 
7 .dwnarw. hemolyzing-washing liquid is 
suctioned 
8 .uparw. sample-loop and other flow 
lines are washed 
______________________________________ 
In operation mentioned above, hemolyzing-washing liquid(13) of 450 .mu.l 
for dilution of 1.5 .mu.l of sample(14) is 300-fold in dilution. The 
optimum conditions of the warming time and temperature of specimen(15) are 
2 min 40 sec. at 48.degree. C., though depending on the degradation 
ability of a labile HbA.sub.1c removing reagent, under the above-mentioned 
dilution in the case of the use of hemolyzing liquid containing a labile 
HbA.sub.1c removing reagent of phosphoric acid compound which is on sale 
with the name of "21H" by the present applicants. The degradation of 
labile HbA.sub.1c is almost completely made with 2 min at 60.degree. C., 3 
min. at 40.degree. C., and 4 min at 33.degree. C. 
In the example, 3 types of eluting solution are used. Each eluting 
solution, (A), (B), and (C), based on the flow sequence, is sent to a 
manifold(34) in order by each exchange valve(31, 32 and 33) through 
heating coil(26), cooling coil(27), and debubbler (28, 29, and 30). Each 
eluting solution which entered one flow line at manifold(34) is sent to 
sample injection valve(18) by feed pump(35), injected into column(19), and 
flows into drain vessel(21) through photometer(20) with carrying 
specimen(15). In the figure, symbol(36) is a pressure detector, and(37) a 
damper. 
In FIG. 1 and FIG. 2, symbol (38, 39) is a exchange valve for 
hemolyzing-washing liquid(13), (40) a out of liquid sensor for 
hemolyzing-washing liquid(13), (41) a suction air pump for drain 
bottle(17), (42) a indicator, and (43) a operation keyboard. 
The above-mentioned description is for the case of measuring samples 
continously arranged, in which case the working in a constant sequence 
results in a constant time from dilution to injection. However, in case 
sample vessels are placed on rack(7) uncontinuously because of variation 
in the time in collecting blood, an excessive irregular time is required 
to find the next sample. Also in case a sample vessel is set on a wedging 
port for urgent measurement, the mistimed suction of blood sample may 
occur. Thus, in finishing injection of specimen(15) into column(19) the 
next sample is found and, by calculating back from the next injection 
time, dilution is preferably started timely. 
On dilution start, when the missing of the next injection already became 
clear, the continous operation of one sequence without the mixing and 
injection of specimen into column enables the stable measurement to 
continue without harming the column balancing condition even if in the 
case of the use of 3 types of eluting solution of different concentration 
or pH. 
COMISON WITH THE CONVENTION METHOD 
Comparison with the Blood Cell Washing Method by Isotonic Phosphoric Acid 
Buffer Solution 
The blood cell washing method by isotonic phosphoric acid buffer solution, 
which has been practiced as a standard method for removing labile 
HbA.sub.1c (pretreatment), was compared with the method of the present 
invention. Table-2 shows the results. 
As the results, the method of the invention produced the similar effects 
with the conventional method in removal of labile HbA.sub.1c fraction. 
Further, compared to the combination of the blood cell washing method and 
21H, the 
TABLE 2 
______________________________________ 
Measurement value (%) 
faction A .sub.1 a L-A .sub.1 c 
condition + A .sub.1 b 
F + S-A .sub.1 c 
A .sub.1 
______________________________________ 
21L 1.5 0.2 5.9 7.4 
21H 1.4 0.2 5.2 6.6 
"S" + 1.4 0.2 5.2 6.6 
21L 
"S" + 1.4 0.2 5.2 6.6 
21H 
______________________________________ 
F: Glycohemoglobin HbF 
LA .sub.1 c: Labile HbA .sub.1 c 
SA .sub.1 c: Stable HbA .sub.1 c 
"S": Blood cell washing method by isotonic phosphoric acid buffer solutio 
more lowering of labile HbA.sub.1c fraction and the increase of 
A.sub.1a+b by change were not observed. 
In the case of no pretreatment and no use of labile HbA.sub.1c removing 
reagent(21L), all values are high. The difference between 21L and other 
treatments in L-A.sub.1c +S-A.sub.1c is assumed to be labile HbA.sub.1c. 
In the example, labile HbA.sub.1c is ca 0.7% of whole hemoglobin. 
Measurements were practiced under the following conditions. 
21: Hemolyzing-Washing Liquid Without Labile HbA.sub.1c Removing Reagent 
nonionic surface-action agent 1 g/l 
potassium di-hydrogen phosphate 0.1 g/l 
potassium mono-hydrogen phosphate 0.3 g/l 
pH 7.5 
21H: Hemolyzing-Washing Liquid Containing Labile HbA.sub.1c Removing 
Reagent 
nonionic surface-active agent 1 g/l 
phosphoric acid compound 0.1 g/l 
KOH 0.3 g/l 
pH 6 
"S"+21L: Conventional Blood Cell Washing Method is Used 
Washed blood cells are added to isotonic phosphoric acid buffer solution, 
and labile HbA.sub.1c is removed by incubation for 6 hrs at 37.degree. C. 
with continuous rotary mixing. 
Then, the blood cell layer is collected and diluted with 21L to 
300.about.fold to measure. 
"S"+21H:21H was Combined to Blood Cell Washing Method 
Similarly, with "S"+21L, the blood cell layer treated with isotonic 
phosphoric acid buffer solution is collected and diluted with 21H to 
300.about.fold to measure. 
Subject: healthy normal human, test just after collecting blood. 
Measurement conditions: according to the example method, warming 
temperature 48.degree. C., warming time 2 min 40 sec, measurement time 4 
min, and n=5. 
As mentioned above in detail, the present invention relates to the improved 
method of automatic measurement of glycohemoglobin based on the principle 
of high pressure liquid chromatography which comprises allowing a number 
of blood samples collected from each patient as whole blood or blood cell 
layer to stand by at a sample holding part, sampling just before 
measurement (before tens sec.about.few min), mixing with dilution by 
hemolyzing-washing liquid containing labile HbA.sub.1c removing reagent, 
and injecting into column of high pressure liquid chromatograph at a 
specified time after mixing start. As necessary, warming may be made from 
dilution to injection into column. 
Therefore, the invention has the following many excellent effects. 1 
Compared to conventional method wherein a long column of high quality is 
used, more rapid measurement can be practiced and the same degree of 
accuracy can be obtained. 2 Labor of mixing reagent manually is saved, 
enabling accurate measurement without error due to manual dilution. 3 
Pretreatment is unnecessary because of the use of whole blood or blood 
cell layer centrifuged it. 4 Since the sample is not diluted by hemolyzing 
reagent in advance, the change of sample is not occur till measurement and 
stable HbA.sub.1c can accurately be measured by preventing the degradation 
or change of glycohemoglobin other than labile HbA.sub.1c. 5 Since the 
measurement value is not effected by labile HbA.sub.1c, there is no 
restriction by time of blood-collecting and it is not a burden on 
patients. 6 Further, since a sample is hemolyzed just before measurement 
and injected into column always at constant timing, stable measurements 
can be made. Particularly, since the balancing condition is not harmed 
even if samples are set uncontinuously, the chromatogram's patterns are 
stable. 
The sample injection valve warms the sample liquid and reagent at specified 
temperature for specified time. Since only specimen is warmed surely, 
there is no useless energy and no risk of injection of unwarmed part into 
column, leading to accurate control. The valve of the invention can be 
prepared by improving a conventional sample injection valve simply. Since 
the other warming device is not required, there is a merit of 
space-saving.