an evaluation of centrifuged vs electronically …
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Virginia Commonwealth University Virginia Commonwealth University
VCU Scholars Compass VCU Scholars Compass
Theses and Dissertations Graduate School
1975
AN EVALUATION OF CENTRIFUGED vs ELECTRONICALLY AN EVALUATION OF CENTRIFUGED vs ELECTRONICALLY
DETERMINED HEMATOCRITS IN ASSESSING THE DEGREE OF DETERMINED HEMATOCRITS IN ASSESSING THE DEGREE OF
POLYCYTHEMIA IN CYANOTIC CONGENITAL HEART DISEASE POLYCYTHEMIA IN CYANOTIC CONGENITAL HEART DISEASE
Marsha Rene Jones
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AN EVALUATION OF CENTRIFUGED y!. ELECTRONICALLY DETERMINED
HEMATOCRITS IN ASSESSING THE DEGREE OF
POLYCYTHEMIA IN CYANOTIC CONGENITAL HEART DISEASE
Marsha Rene Jones
B.S., Old Dominion Univeraity, 1972
TbeaiJS
S.ba1t •• d 1A part1al fulfill.ent at t� require.ant for the
Degrea of
of •• d1e&! T.c�oleg1 M ... er of Soleace 1A the Depart.ant
.. t •• lI.die&! Cel1ege of Vlrginia
Iealt. Sclanoe D1Ti.1o� Virginia C .... nwealtb UniTerslt7
Rlu..a4. V1r�n1&-
June, 1915
ii
This thesis by Marsha Rene Jones is accepted in its
present form as satisfying the thesis requirement for the
degree of Master of Science.
Date,
"1)?�!�$.-,,, .-. . . , . ,'I /: � !. i; r. . . , · • · >. t � :; J.t. � , ·
£. -1 ...-/ .... . .. \(.?) ... 1I12tY/. r
. . . . Y.:. � /.?� • . .
Approved, of the School of Graduate Studies
.
iii
CURRICULUM VITAE
iv
ACKNOWLEDGEMENTS
I wish to express sincere a�preciation to Dr. Harold
M&urer and Dr. Charles. Johnston who were influential in the
choice of my research topic� and offered patient guidance
and enthusiastic a�B1atance throughout.
I should like to thank the other members of my committee,
Dr. Carolyn McCue, and especially my advisor, Dr. Joanne
Stepaens for their advice and constructive criticisms. I
wish &lso to acknowledge Dr. George Vennart.
I wish to thank Dr. W. I. Rosenblum for the use of the
micro-viscometer and his helpful hints. Acknowledgement is
made of the invaluable technical assistance rendered by Mr.
Guy Nelson.
Lastly I wish to thank my parents for their patient
understanding, cooperation and belief in me as proven by their
love and support.
v
TABLE OF CONTENTS
I NTROD UCTION • • • • • • . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
REVIEW OF LITER ATURE
Secondary P o lycyt hemia • • • • • • • • • • • • • • • • • • • • • t • • I • •• • •
Cardiac Malformat i ons and Second�ry Pol ycyt hemia • • • • . • . . . . • • . • • • • . • • • • • • • •• • . • • • • • • • • • • • 3
Viscos ity and Secondary P ol yc ythemia · . . . . . . . . . . . . . . 4
Vis c os ity and Antic oagulants . . . . . . . . • • • • . • . • . • • . • • • 7 Fa ctor s Affecting t he Centrifuged va Electronically Determined Hemato c1'its • • • • . . . . . . . :-:- . . . . . . . . . . . . . . . . . 8
METHODS A ND MATER I ALS
S eleetion o f D onors . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Micro-hemato crit Centrifugat i on Meth o d • • • • • • • • •• • •• • 14 Coulter Model �Hemat o er i t Metho4 • • . . · . . . . . . . . . . . . . . 15
Vis c o s i t y Studies • • • • • • • • • . . . . • • . . • • . • • • t • • • • • • • • • • • 16
Fibri nogen Determination-Fibr ometer Method • • • • • . . • • • 18
Collection and S t orage of B lo od • • • • . • • • • . . . . . . . . . . . .
Preparation of Test S amples • • • • • . . • . . . • . . . . . . . . . . . . .
RESULTS
Hematocrit Stud ies • • • . . . . . . . . . . . . . . . . , . . . . .
Vi s c o s i t y S tud ies • • • . . . . , . . . . , . . . . . .
Fibrinogen S tudy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mean Corpuscular Volume and R ed Cell Count !E. Vis cosity • • . • • • • • • • • • . • • . • . • . . . . . .
DISCUSSION • • • . • • • . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .
SUMMARY AND CONCLUSIONS • • • . • • • . • • . . . . . , . .. . . . . .
REFERENCES . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,
20 21
2 5
48
50
5 2
58
59
l�
3 .
vi
LIST OF F I GURE S
Mi cro-hemat o crit vs Coulter S® Hematocrit of All Cyanotio Congenital Heart D i s ease D onors • • • • • • • • 30
A Compar i s on of the Me al\ Ratio of Microhemat o crit t o C oulter �ematocrit of the R andom and C CHD D onors • . • • • . • . . . . . • . • • • • • • • . • • • • • • • 32
A Comp ar i s on of t h e Mean Cal culated Diff erence with t he Ob s erved Micro-h ematocrits • • • • • • • • • • • • • • • • 37
4� Mi cro-hematocrit vs V is c os i t y Normal Curve • • . • • • . • • • . � . . • . . • . • . • • • • . • . • . . • • • . . . • • • • . • • • 42
5. Coult er S�Hematocrit � Vis c oSi t y Normal
6 .
7.
Curve . • . <II • • • • • • • • • • • • • • • • • • , • • " • • • • • • • • • • • • • • • • • • • • 4;
A C omparison o f t he Micro- and Mod e l'S® H emat o crits and Vis c o s i t y in the Phle-botomy Subje cts . . . . . . . . . . . . • • . . . • . . . . . . . . • . . • . . . . . • 45 The Effect of V i s co s i t y on t�e D iffer ence Betwe en Micro- and C o ul t er �Hemat ocrit s • . . • • • • • • . • 47
8 . The Effe ct o f Fibr inog en on the �ifferen c e B etwe en t he Mioro- and Coult er S Hematocrits • . • • • • • 49
9. Erythr o c yte Count � Vis co 8i ty • • • • . • • • • . . • • • • • • • • • • 51
vU
LIST OF TABLES
I. V1scos1ty Data of Reco�tltuted Normal Ruman Blood Measured at Various Levels of Ilematocri t and Shear Rate • • • • • • • • . • . • • • • • • • • . • • 19
II. Pre.-phlebotomy Clinioal &nd Hematologica.l Data • . • . • . • . • . • . • . . . • • • . • • . . • • . • . . • • • • . . . • . • . . • • • • 2 4
III. Phlebotomy Da.ta. • •• • • • • • •• . • . . • • . . . . • • • . • • • . • " • • • • 2 6
IV. Statiatlcal A nalysis of the 2 Populations Examln.ed • • • • . . . . • . • • . • . . • . . . • • . . • . . . , . . . . . . . . • . . •• :; 3
V. Observed I.!- Calculated Coul ter �ematocr1 ts Compared to Unadjusted M1oro-hematocrits of Cyanotic Congenital Heart Defect Donors • • • • • • • • • • • 35
VI.. V!.scos! ty Da.ta • • • • . • • • • • • • . • . . . . . . • • • • • . • . • • • . • • • • 39
Secondary polycythemia i s defined as an absolute erythro-
cyto sls caused by an enhanced stimulation of red blood cell
production. One of the most common methods of monitoring
the degree of polycythemia is the measurement of the bemato-
crit.- The hematocrit is the mea surement of the volume of red
blood cellEL_ expressed a s a percentage of the volume of whole
blood in a sample. This- measurement is u sually done using
one of two methods. (1) the Coulter Model s® or I,,�) the
centrifuged micro-hematocrit method. However, we have noted
a significant cifference in the hematocrit values determined
by the se two methods when the hematocrit s exceed 54 per cent.
As many as 10 hematocrit units of difference have been reported
\ 1 hematocrit unit = 1 ml of packed erythrocytes/lOO �l of
whole blood ) .
Several questions arisel 1,,1) does thi s variation in
hematocrit thct ) values increase in proportion to the in
crease in hematocrit;; \,) i s there a relationship between
the fibrinogen level and the amount of trapped plasma in the
centrifuged micro-hct;, \ 3) is there a relationship between
the red blood cell count tREC), mean corpuscular volume tMCV),
and whole blood viscosity; (4) which measurement is a better
indicator of the degree of polycythemia and the increa se in
viscosity.
To answer these questions data from the Coulter Model
s� centrifuged micro-hct, whole blood viscosity and fibrino-
gen determination were evaluated. With these data it was
planned to determine if it would be possible (1) to con-
2
struct a nomogram establishing the relationship between the
micro-het and Coulter S� hct greater than 50 per cent for
the conversion of one value to the other. and �2) to
asasss the degree of polycythemia and the need for phlebotomy
by the use of each measurement.
LITERATURE REVIEW
3
SECONDARY POLYCYTHEMIA
A significant increase in total red cell mass may occur
in any situation which results in tissue hypoxia. Thia lack
of oxygen leads to an increase in the level of erythropoietin,
a humoral sUbstance that is believed to be primarily respon
alble for the regulation of erythropoiesis.
In secondary polycythemia the increase in the red cell
mass la often accompanied by a decrease in plasma volume.
The excessive production of erythrocytes may lead to an ele
vatio� of the red blood cell mass sufficient enough to pro
duce alterations of the viscosity of the blood, thereby in
creasing the danger of thrombosis ,4).
CARDIAC MALFORMA�ION AND SECONDARY POLYCYTHEMIA
Any cardiac malformation which results in a high grade
veno-arterial shunt may produce secondary polycythemia \77.
78. 79, 80). Due to the mixing of venous and arterial blood,
poorly oxygenated arterial blood reaches the peripheral tis
suea. As a res�ut, it is thought that an increase in erythro
poLetln occurs. Subsequently. the increased red blood cell
count that results increases the o�ygen carrying capacity of
the blood. According to Kontras t6), the increased red cell
count serves as a compensatory mechanism for decreased oxy
genation until the hematocrit levels are greater than 70 per
cent. When this occurs, the value of the increased oxygen
carrying capacity is offset by the increa�ed blood viscosity.
OTHER CAUSES OF SECONDARY POLYCYTHEMIA
S e condary po lyc ythemia may result from many hypoxi c
st imul i such as decreased atmospheric pr essure , impaired
p ulmonary ventilat ion or t he presence o f c ertain abnormal
hemog lobins (methemo globin or a hemogl obin variant wit h an
increased o x ygen affinity' .
VISCOSITY AND SECONDARY POLYCYTHEMIA
4
Dint enfass ( 2 9 ) s howed that if t he viscos ity o f blood
wer e t e sted at high s hear rat e s (high f lo w velo cit ies) , in
t h e ab s e n c e of aggregation of t he red cells, blood viscosity
was approxima tely a semi - logar i thmi c function of the h e mato
crit r eading .
A d es cription o f s e veral fa ctor s that influence Viscosity
was given by C harm , � !1 (30)� So me of these factors were
t h e s hape and elastic1ty 01 � h e red cell, c hanges in its
rigid i t y , deformab i li ty and interna l vi sco s ity . These were
l i sted as erythroc y tic factors. A separa te category o f
p lasmatic fac tors was mad e t o d i s cuss t he mo lec ular a s pect
o f viscos ity .
Erythrocytic Factors
The i nternal vis c osity o f the erythrocyte has been c a l
culated to range b etween I and 6 c entipoise ( 24, 41). The
centipo i s e is a unit of v is cosi ty derived by di viding t he
s hear s tress (d yn e/cm2 ) b y t he shear ra te. The estima te
gi ven by Dintenfass includes cell memcrane effects as well
as hemoglobin viscosity. The internal viscosity of the
erythrocyte was found by Dintenfass (24) to become sig
nificant at hematocrit values of 49 per cent and to be of
major importance at values of 70 per cent and above. A
pronounced increase in blood viscosity can therefore be
5
caused by crenation of the red cells or by hypoxia, both
resulting in an increase in the internal red cell viscosity.
The mechanism for the increased viscosity associated with
crenation is the reduction in volume without a compensatory
decrease in the hemoglobin content of the cell. In the case
of hypoxia, significant changes in the viscosity are noted
when the arterial oxygen saturation is 66-75 per cent or less.
At this saturation the pH is decreased resulting in an in
creased red cell membrane rigidity.
According to Kurland and Charm (30, 54), cell rigidity
is related to the characteristics of the cell contents as
well as the cell membrane. The cell membrane is capable
of moving around the cell contents. The evidence for this
lies in the fact that packed cells possess a viscosity
much lower than would be expected ( 2 4). At a low pH the
hemoglobin becomes ionized. In this case, the viscosity of
the cell contents, i.e., hemoglobin, is the controlling
viscosity factor (33, 27) .
Bircher ( 28 ) states that this movement of the cell
membrane around the contents is the mechanism by which
red cells adapt to flow. In pathological states, deformaT
6
bility i s reduc e d d u e to e ith er membran e d e f e c ts or h e moglo
bin ab no rmality. Abnormal mo l e c ular int erac tio ns with r e
ciprocal b ind i ng of c e l l ular cons titu e n t s r ep r e s e n t the
basic mechanism r espons ibl e for incr eased c e ll r igid ity.
Intrac e llular r ed uced glut athio n e r ed uc es the int erac tio ns
and is th e p r i mar y protective age n t aga i n st loss of cell
d eformability.
Jandl, � a l (46) found that a low e r ed intrac e l lu lar
ATP le vel allo ws the fo rmation of c alcium bridges b etw e e n
cell constituents c ausing incr eas e d c e l l rigi dity .
Accor d i ng to Bircher ( 28 ) the d egr ee o f red c ell ag
gr egatio n as w e ll as t h e s i z e of the aggr e gate d e p end on t h e
f low velo city (sh ear rate ) and o n the i ntr i n s i c pro p erti es
of blood . The m e c han ism o f aggr e gatio n is d ep end e nt upo n
t h e in teraction of blo od p roteins with t h e r e d ce ll m embrane.
The pr esence of aggr egat ed red c e lls int erf e r e s with s tream
l ine f l o w by d ec reas i ng th e d e v e lo pm e nt of s h ear plan e s .
T his caus e s an incr e as e in th e who le blood vi s c osity and a
s ev er e handic ap to th e circ ulation (24, 2 5, 26) .
P lasmatic Factors
C hi e n, e t al ( 16) showed t hat th e r e was a correlatio n
betw e e n the mol e c ular weight of plasma prot eins and th eir
abilit y to cause aggr egatio n of r ed blood c ells . T h e heavier
proteins, s uch as macroglob u lins ( mo l ecular we ight = 1,000,000) and fibr inog en ( mo l e c ular w*ight - 32 5 , 0 00) ar e the stro ng-
e s t aggr egation i nd uc er s . Con v e r s ely , W ells ( 2 5, 28) noted
7
that a lb umin tends to decrease red cell aggregat ion, po ssib ly
becaus e o f its strong negat i ve c harge. Of t he pro teins
norma l l y found in plasma , fibrino gen , which has a neutral
c harge and is a muc h la�ger mo lec ule than albumin , has the
greatest i nf luence in promo ting red cell aggregation. Bo t h
C harm ( 28) and Well s (17) s tated that as the fibrinogen
level exceeds the upper limits of normal (500 mg/d l ) , a
s ignificant increase in red cell aggregat ion begins . However ,
it wa s demonstrated by both autho rs that macro g lobul ins were
the s tro ngest induc er s o f aggregation.
V I S C OSITY AND ANT I COAGULANTS
Ant i c oagulants were gro uped into t wo categories by
Ro senblum (22)1 ( 1) tho se which shr ink erythr o c ytes (Citrate
and o xalate) and (2) tho se having no effect o n eryt hro cytes
Bi�e or shape if in the c o rrect co ncentration and if ex
amined tmmed iately (heparin , ethylene-diamino-tetra-acet i c
a cid ( EDTA) , and a cid c i trate dextrose (ACD»)� However ,
EDT! may cause signif icant swelling o f the red cells if the
mixtur e i s l eft at room temperature for 24 ho urs� In addi
tion , if the concentration o f EDTA i s to o high, red c ell
shrinkage may res ult. Although cell s shr int i n c itrate , they
do no t in ACD ac cordin g to Gal luzzi , � a l (45) .
The first group of ant i coagulants increases vi sco s it y
event ho ugh c itrate reduc es the hemato c rit; while t ho se i n
the seco nd gro up have no effect if used in the proper c o n
c entratio n and t ime l imit (within 2 4 ho urs i f refr igerated
8
at 4°C). Britti n , et al (81) stated t hat after s torage fo r
24 ho urs at 4°C llO s ign ificant diff'erence wa s observed in
the hemoglobin, hemato crit, red eell co unt , s i ze, shape, or
content determined by the Co ulter Model S® or by eo nvention-
al method s� However, when these same measurements were made
on dup l icate sampl e s stored at ro o m temperature for 24 ho urs
s ignificant swel ling of t he red c ells was seen.
Blood in ACD can be maintained for two days at 4°0 with
no c hange in vise o s ity� However, the vi s co s i t y of blo o d in
po tas sium o xalate c hanges in two or t hree ho urs . Gal luzzi,
� !l (45) we t on to say that blood in "balanced" o xalate ,
i . e . , ammo nium oxalat e and po tassium oxalate mixture, EDTA ,
o r heparin may a lso be s tab le for o ne to two days at 4°C .
FACT OR S AFFECT I NG T HE CENTRIFUGED VB ELECTRONICALLY
DETERMINED HEMAT OCRI T S
T h e Effec t of Ant i c o agulant s
The type o f ant ico agulant used i n blood co llect io n
affect s the centri fuged micro-het and the mean c o rpusc ular
vo lume ( MC V) . Although s alts of EDTA pro vide excellent ant i -
eo agulants for hemato logi¢ determinat ions , hi gh co ncentratio ns
o f EDTA ( greater t han 2 mg/ml who le blood) produce errors
in t he hematocr i t determined b y the micro-centrifuged method
( 73). This e levated EDTA co ncentrat ion caus es s hrinkage of
the red c ells� This error may be seea in incomp letely fil led
evacuated tubes c o ntaining a fixed amount of anti coagulant .
9
Lampas so (73) reported t hat the error o f the hemat o c rit may
reach 5 p er cent belo w the true value when a Vacutainer�
t ube des igned to draw 7 ml o f blood is filled with 2 ml , and
it may reach 10 per c ent i f the tube c o ntains o n ly I mI. He
o b s erved a pro gre s s i ve d e crease in hemato crit value s as the
concentratio n of EDTA increa s ed . R e liabl e micro -hct es t ima-
tions canno t be performed when the concentration o f EDTA
exceeds 2 mg/ml of who l e blood �
Britt�n, � a l (70) showed t h e advantage o f us ing the
C o ulter Model S® o ver the c entrifuged micro -hct method�' The
Co ulter �e limina t ed the errors in hcts produc ed by exc essive
EDTA; This Was don e by making a 1150,000 d ilut i o n i n an
is o to nic m edium. This 1150,000 dilution i n an iso tonic
m edium restored the shrunken red b lood c e lls to their initial
siz:e (70, 74).
A comparison o f the effec t o f ant ic o agulant conc entra-
t io n o n eentr ifuged and elec tronica lly determined hcts was
made by Ferro (72). I n his stud y three m ethods o f measuring
hct s wer e e xamined 1 (1) micro -c entrifuged hc t , (2) macro
centrifuged hc t , and (3) C o ulter Counter Model F ®methods .
Prior to determining the hct wi th the IJo del F®a 1150,000
dilutio n of b lo o d i n an i s ot o nic so lution waS made. The
conc entrat io n o f EDT A us ed in this eva luation ranged from
1�3 t o 9�0 mg p er ml of who l e bloo d� When the latter c o n-
tration o f EDTA was used , it decreased t h e micro- and macr o -
hct s b y a n average o f 4 hc t unit s . Thes e hc t units were
d efined as the equivalen c e of I ml packed erythrocytes/
10
1 C ml whole blood . T h e s e same s p e c imens wer e co ncurr ently
ch ecked fo r hct values by elec tro nic comp utat ion us ing the
C o ulter C o unt er Mod el �. No e ffe ct from exc ess ive amo unts
o f EDTA was o b s erved . In a comparable stud y b y Bri t t i n , et
al ( 7 1 ) no effe c t o n the comput ed hct using the C o ult er � Mod e l S was ob s er ved .
T he dat a pres ented c o nfirmed the fact that the c o nc en-
tration of ant icoagulant must be pro p er l y c ontrolled to
avo id decr ea s e s in paoked c ell volume by micro - and macro -
c entrifuged hemato c r it s . I n addit i o n , t h e us efulnes s o f the
C o ult er C o unt er elec tronic t e c hnique for hemat ocrit est ima-
t io n was s uppo rt ed . The maximum d ifference in values for
hemat o c r i t s by the t hr e e methods us i ng the pro p er proportion
o f anticoagulant and whole b lo o d ( 1 . 3 mg EDTAI 7 �l whole
blo o d) wa s I hc t unit . The hemato c r it s examined rang ed fro m
17 to 54 p er cent ( 7 2)�
Other Fac to r s Affe c t i ng C ent ri fuged Hemat ocrits
Daci e and Lewis (50) stat ed t hat plasma prot eins , red
blo od cell c o unt , s i z e , and morpho logy all affect the micro -
hct� According to Williams , et !l ( 2) the d ifferenc e s i n
hemat ocrit value s ar e dep end ent upo n t h e amo unt o f trapped
pla sma whi c h ma y rang e from 2 to 8. 5 p er cent , whi c h in turn,
i s aff ect ed by the r e la t i ve c entr i fugal for c e emp lo yed and
t he duration of centrifugat io n�
11
Fac tors Affec ting t h e Electronically Det ermined H ematocrits
S ignific ant erro r s in the m ean c o rpuscular vo lume (MeV) and c o ns equent l y , the h ema tocrit will o c c ur with the C o ult er
Count er Mo del S®wh en t h e l euko c yt e c o unt is markedly incr eas e d .
In addition , i t was point ed o ut b y s ever al authors ( 2, 47 .
48 , 49. 5 0 , 5 1) t ha t as the numb er of c e l l s counted is in -
cr eas ed, t h er e is an inc r eas ed c hanc e t hat two or more c ells
wil l b e s ens ed as one by the d et ec ting d e vic e. Ther efor e ,
t e c hniques for minimizing or c o rr ecting for coincid ence
error have been a r e quirement of electro nic c el l c o unting
d e vic es �
METHODS AND MATERIALS
12
SELECTI ON OF DONOR S
I t ha s b e e n noted t hat the number , mo rpbo logy and hemo -
g lobin content o f the red blood c e lls c an affect their aggre-
gatlo n tende n c i e8� I n addit io n , plasma prot eins, espec ially
fibrinogen , tend to influence aggregati o n . When the hemato -
crit exceeds 5 0 per c ent , there i s an expo nent ial increase
in the who l e blo od vis co s it y ac companies b y an increase i n
the ano unt o f trapped pla sma in the c entrifuged m i c ro -hct
s ample� This trapped plasma res ults in a 4 to 10 per c ent
increase i n the hc t values deter mined by the m icro -hc t met hod.
Because c yano ti c c o ngen ita l heart disease (CCHn) c hi ldres
frequent l y have elevated hemato c rit s , t hey were selected as
the primary donor sro up for s t udy (with t he perm i s s i o n o f
the pediatric Cardio l o gy Department o f the Med i c a l C o llege
o f V irginia Ho s pitals ) . Th ese donors had had varying
amo unts of t herapy.
S inc e there wa s a limited n umber of OCHD c hi ldren ava i l-
ab le for s t ud y , it was tho ught that a s im ulated c o nd i t i o n o f
secondary po lyc ythemia co uld b e produced b y t he fo l lowing
methodl ( 1 ) c entrifugati o n ( 1800 rpm for 10 minutes ) o f
rando mly s elec ted mo nlipemic , no nhem o l yzed b lo od; ( 2 ) a fter
c e ntrifuga t i o n the plasma �o u ld be remo ved unt i l the desired
micro-hc t was obtained. Howe ver, prior to c entrifugatio n an
® initial m i c ro-hct and C o ulter S ( S ) evaluat ion w�s made.
Only samp l es with normal h emo glob i n (heb), hematocrit (het),
red blood cell co un t s (RBC ) , and red c el l indices (MeV ,
MCH, MCHC ) were s e lected for study.
13
It was no t e d that adult s were the primary donors in
this group . Howe ver , no literature reViewed indicated that
there wa s any signiricant d i f f er ence between the red Dlood
cells of adults as compare to those of children exc ept to
reflect the differences in reticulo cyte counts. �ormally
about 0.5 to 1.5 per cent of all erythrocytes in adult� are
reticulo cyt es . Norma l value s at birth range from 2.5 to 6.5
per cent, falling to normal adult level by the end of the
second wee� .
I n a t ext by M ia le (1) i t wa s no t ed t hat t he d e gre e of
ret iculo cyt o s i s was proport ional to erythropo iet ic actiVity.
It was s tat ed that Significant increases in the reticulocyte
count ware observed in re�ponse to increases in erythropo ietin .
Dintenfass (27) found no s ignific ant differ enc e betwe en the
Vi s c o s i t y values obtained comparing fetal and ad ult b loods .
that had b een adjusted to the same micro-hct despite the
differences in reti c ulo cyte c oun t s. He found t hat the pH
of the bloods had more effect on the V i s c o s i t y . Furthermore,
it was shown that the acid-b a s e values of infants and c hi ld
r en are mo r e variab l e than those of adults ( 3)�
Another gro up of donors was s e le c t ed for t h e study of
the effe c t s of e l e vat ed fibrino gen levels on the difference ®
between the micro- and Coulter S hematocrits. The blood
from any pat i ent who had had a fibrinog en d e t ermina t io n and
C o ult er S� evaluation performed was selected for study. The
fibrino gen l e ve l and initial Co ult er s� data were recorded.
The same c entrifugation a nd micro-hct adjustment procedures
wer e done as pre vious ly d e s cribed . The micro -hc t adjust
ment was fo llowed b y a Co ulter S® measurement of the
hemato crit �
T EST S PERF ORMED
Micro-hematocrit C entr ifugation Method
The vo lum e of the r ed c el l s o c cup i ed in who le blood
14
was det ermined by sub j ec t ing t h e blood to suff i c i ent c entri-
fuga l for c e to pack the c el l s in as smal l a volum e as po s -
s ib le� This mea sur em ent was expr e s s ed as a p erc entage
d erived b y the ra tio o f t h e vo lum e o f pa cked REC's (nl)/ 100 ml of who l e b lood: The amo unt of p lasma remaining in
the packed c e l l s var i e s som ewhat with the m etho d us ed , but
i s usually abo ut 1 to 4 p er c ent for micro-hcts l e s s t han
5 0 p er c ent� For t h e standard micro-hct method cap i l lary
tub es by C laY-Adamsqpw er e used � The y wer e 75 mm in l ength
with an internal diam eter o f abo ut Im�. No ant i c o agulant
c o ating the int erior of t he c ap i l lary t ub es was us e d . The
EDTA anti c oagulat ed b l o o d was a llowei to enter t h e t ub e by
c ap illary a c t io n , l e aving at l east 15 mm unfi lled � T h e
tub e was t hen s ea led b y t h e us e o f a p la stic s ea l� Aft er
c entrifugation ( Adams Autocrit C entrifUge � b y C la y-Adams)
at 12.000 rpm for 5 minute s the packed c e ll vo lume was
m easur ed using a r eading d e vi c e . Micro -hcts gr eat er t han
50 p er c ent wer e c entrifuged an add itiona l 5 minut e s at
1 2,000 rpm to d et ermine if further packing of t h e red c e l l s
wo uld r esult �
15
C o ult er Mo del � emat o crit Method
By us e of the C o ult er Mo de l �o ne is able to make s i x
measurements r e lating t o t he red b l o o d c ell"; Thes e measure
m ent s are as fo l lo ws , RBC co unt , hemo g lobin (hgb ) , hema
to crit ( hc t ) , m ean c o rpuscular volume (MCV) , mean co rpuscu
lar hemog lobin ( MC H�, and Mean c orpus c ular hemoglobin co n
centratio n ( MCHC ) . I n addit ion o ne may measure the leuko
c yt e c ount� B y the use o f a pneumatic and vacuum s ystem
approximately 1 m l o f who l e b lo od was aspirat ed . 44 . 7 lambda
o f this blo0d wer e initial ly diluted 1,224 with 10 m l o f
an iso t onic solution. A sec o nd dilution ( 115 0 , 000 ) was
made from a 44. 7 lambda s ample fro m the first dilutio n and
mixing wit h another 10 ml o f is o to nic solutio n . B y m eans o f
a vac uum RBe susp ens ion was forc ed to flow through t hr e e
apertur e s o f s p e cifi c d i m ension"'� The samp l e was drawn
t hrough all t hr e e ap ertur e tub e s simultaneo usly for a period
of 4 seconds . I n the c hamber with the R BC susp e n s i o n two
ap ertur e s were us ed for measuring the siz e o f ea c h cell
passing through . Th e values o btained wer e then averaged .
Eac h aperture tub e had an internal ele c trod e , and there was
a c ommon electrode in the bath acting a s a ground. As t h e
c e l l s passed through two o f t h e thre e ap erture s , they , being
nonconductors , d i splac ed the electrolyt e, t hus c hanging the
r e s i stanc e between t he two electro d e s , producing a voltage
pulse magnitude proport i o nal to the v lum e cf the cell.
The voltage puls e s were amplifi ed and displayed on an
16
osci llo s c o p e s cr e en as vertical spik e s . The number of spik e s
represent ed t h e numb er of c e lls pass ing through the ap ertur e.
All thr e e ap ertur e s wer e used in enumerating the RBCs. ihe
thr e e value s obtained were t hen averaged, T h e hemato crit
was c omput ed b y the pro duct o f the MCV and the RBC co unt (I, 47 , 51).
One may a lso count c ap i l lary bloo d using the Model S •
44. 7 lambda o f capillary b lo o d wa s dilut ed with 10 ml o f
iso tonic d i luent , the who le blo od 11224 d i l ution switc h was
turned to the 112 2 4 p o sit io n and blood was asp irat ed thro ugh
the c apillary blood aspirator . The r e sults of both pro
c edur e s ( c ap i llary and who l e blood ) wer e provid ed in the
fo rm o f print -o uts.
Sinc e the Model �wa s limi t ed by t he number of d igits
it co uld print o ut, RB� c o unts gr eater than 9 . 9 9 millionjmm 3
had to b e d i lut ed to a m easurabl e �BC range . When this
probl em wa s encount ered with a few of the samp l e s fro m the
CCHD c hildren , a 112 dilut i o n o f an aliquot o f t he patient's
b lood was made wit h the i so t o nic so lutio n , and t h e samp le
wa s t hen r un as the who le b lood woul have b e e n . The
print ed o ut r e s ult was then multip l i ed b y 2. Dup licate
de terminations wer e p erformed and averaged�
Vi s c o s ity Stud i es
. ® The Bro okfi e ld c o n e -p lat e vis c o m et er was used i n thes e
exp erim ents. The principle of the cone -plat e viscometer in-
vo lves the rotatio n of a flat c one upo n a p lane Burfac e (the
17
p late) at di�ferent selec ted speeds of rotatio n ( different
shear rates ) � A t orque measuring d evice c o nnected to the
drivi ng mechanism to a verti cal sp indle from whic h the cone
was susp ended . A 2 ml sample of fluid between the eone and
p late o ffered a resistance to the rotation of the co ne and
developed a torque to a degree that was a funct ion of the
shear stres s in the f luid·� Kno wing the geometric constants
of the cone, and ob serving the rate of rotation and the tor -
que , one c ould d etermine separately the shear s tres s (dyne
cm2) and the shear rate ( number of revo lution s per minute)
i n the liqui d � B y changing the shear rate a ser i e s o f values
o f t he shear ing stre s s co uld be obtai ned. By plottin� these
values against one another the rheological characteristics
of the fluid could then have been defi ned d irectly in terms
of a shear stress-shear rate dlabram�
The fo l l o win£ rheo lo �ical experiments usinc EDTA anti-
c o agulated b lood were carried out: ( 1 ) the v i s c o s ity was
m easured o n the p re- and po s t -phlebo tomy samp les and o n a
few mid-p hlebo tomy samp les; ( 2 ) the vi scos ity o f randomly
s elected b loods with ad justed Co ult er S�hematoc r i ts o f
45 � 5 p er c ent was m easured . Blood viscometry waS done
at various shear rates of 212 s ee-1 ( 60 rp m ) , 106 s e c-l
( 30 rpm ) and 42 s ee-l (12 rpm ) . At least 3 determinations
o f shear s tress were made at each s hear rate sett ing. All
mea s urements were made at 37 °C . The a c c uracy o f the model
used was guaranteed to have been within � 1% whatever the
s hear rate emp loyed . By definition, t he v i s c o s i ty, n, was
shear str e s s divid ed by shear rat e in dyne/cm 2 or p o i s e.
The r e s ult mult iplied by 100 £ave the more commo nly used
unit of c ent ip o i s e ( cp ) .
18
To evaluate the r elatio nship b etween the vi s c o s ity and I
the h emato logi cal data obtained from the micro-hct and the
Coul t er S®graphs we re c onstructed. The Coult er �hct and
micro-hct were c ompared to t h e normal v i s co s i t y c urve e stab-
lished b y us ing EDT A anticoagulat ed b lood re const ituted by
each pati ent s own p lasma� I nd ividual who le blood samp l es
antic o agulate d with EDT A wer e eentrifug ed at 1800 rpm
for 10 m inut e s. The p lasma and RBC s wer e s eparated and re-
mixed to pro duc e mi cro -hct s of 20 , 40 , 60 and 80 p er c ents �
( Se e Tab l e I )
Fibrinogen D eterminat ion-Fibr�meter M ethod
The fibrinogen metho d cho s en was the o ne c urrently in
use at the Medical Co l l ege of Virginia. This pro cedur e was
based o n an automate d thrombin time using the fibromet er�
The t hrombin co nvert ed fibrino g en to fibri n , the tim e tak en
fo r the react ion d ep ending on the amount of fibrino g en
p r e s ent�
A pre - and p o s t -P?l ebo tomy aliquo t of b l o od wa� mixed
with c itrate (1 part 3 � 8% c itrate t o 9 part s b lo od)� The
ant i c o agulated samp l e was c entr ifuged at 1800 rpm for 10
minut e s and the p lasma remo ved. The randomly s e l e c t ed
s p e c im ens were tr eat ed in the same manner. A 1110 dilu-
t i o n o f pati ent's p lasma Was mad e with Verona l b uffer.
V�8coslty Data for
Plas ....
Hot-20�
liot-40%
Hct-60%
at Various
RPM
60
30
12
60
,0
12
60
30
12
60
,0
12
60
30
12
1 9
TABLE I
Reconstituted Normal Human Blood Me�sured Levels ot Hematocrit and Shear Rate
Sec-l
212
106
42
212
106
42
212
106
42
212.
106
42
212
106
42
VISCOSITY 0 (c�l Ikt 21 c
1.4-
1.5
1.5
,..8 4.4
5·,
6.5
7.2
8.8
14.4
21.7
.� 2 std. lrevla-tlons
0.22
0.26
0.28
0.24
0 •. 68
0.76
1.24
0.68 0.96
0.96
.--
20
0 . 2 ml o f diluted plasma was incubated at 37°C for 2 m inutes.
T he tlm in£ was started with the simultaneous additio n of
O�l ml t hromb in reagent . The dupl i c ate results c hecked
within 0 . 5 seco nds . T he c lotting times for eac h specimen
wer e averaged. The thromb in times of purified fibrinogen of
known c o n c entrat ions were used to con struct a s tandard c urve.
T he t hrombin t�mes o f each pat ient were compared to thi s
standard c urve. The normal fibrino gen s a 88,using thi s
method was 170-410 mg/dl.
Col lec t ion and storage of Blo o d
T h e minimum amount o f b loo d needed fo r hct and v i s c o s ity
te sting was 3 ml anticoagulated with EDTA. From the donors
with CCHD the amount of venous b lo od withdrawn ranged from
250 to 460 ml dur ing the phlebotomy with plasma exc hange
(15 -20 ml/kg body weight ) . The method s of blood c o l lecti o n
wer e either by venipuncture us ing a vac utainerfD System or
by an I.V . -butterfly i nfus ion set� with a stop-c o ck s ystem �
Usually during the phlebotomy an initial 7 m l sample was
taken fo llowed by 7 ml aliquot samples from eac h s ubsequent
30 to 5 0 ml o f blood removed �
Vi sco s ity studies were perfo rmed once weekly� Some
samples required sto rage at 4° C for 24 to 7 2 hours before
viscos i t y studi es were performed. Other samples had v i s c o s ity
measurem ents performed within 2 to 4 ho urs o f the phlebotomy
(see res ults ) � T he exc hange plasma anti c oagulated wi th ACD
whi c h does not affect the RBC or viscos ity was used for
21
ad justing the micro -hct values from the p hleboto mized do nors .
10 to 15 m l o f the e�change p lasma wer e saved fo r use in
ad j usting the final b l o o d sample to a norma l micro-het range
(42.!: 6%).
Preparatio n of Test Samples
The ACD p lasma used in the phleboto my and exchange as
stated before was used to dilute the fina l p hleboto my samp le
to a normal miero -hct range. 1he EDTA blood fro m the random
patients was centrifuged at 180 0 rpm for 10 minute s . The
p la sma was removed and the he�atvcrits were adjusted to a
level greater than 5 0 p er cent� Since temperature was c ritical
in vis c o sity determinatio ns , the refrigerated spec imens
were allowed to reach room t emperature prior to testing .
F urthermore, eac h samp le was thoro ughly mixed befo re any
testing was done� This eliminated any red c el l aggregates
caused b y th e lo wered temp erature and settling o f the blood .
As stated ear lier a l l vis c o sity measurements were made after
the samp le temperature reac hed 3 7 °C . T his temperature was
maintained by a c ir c ulatine 37 ° C water j a.�ket inco rpora-
ted in t he structure of the vis cometer.
RESULTS
22
This is a comparat ive s t udy of t he Co ulter lodel S$
hematocrit � the centrifuged micro-hematocrit methods to
determine the relative va lue of each method in assessing
polycythemia in cyanotic congenital heart disease chi ldren�
T he accep ted treatment for po lycyt hemia observed in these
children i s phlebotomy wit h plasma exchange ( 1 5-20 m l/kg
b o d y weight) . This procedure is usually done when t he micro
hematocri t exceeds 65 per cent .
Tab le II s ummarizes the clinical and hemato logica l
data o n t he CC HD children . The ages o f t hese children ranged
from 7 months to 8 years . No other known disease was present
excep t for hgb S-A fo und in T . N • • D ue to transfers from
t he ho spital the charts and diagno ses of t he type of t he
t ype of ceHD o f two of t he children could not be obtained � With the except ion of S.� al l sub jects had normocytic
(MCV-�6�5� �ormochromic (MC H � 29�3; MCHC- 31�4) 'red blood
cel l s . S�P� 's red cel ls were microcytic and hypochromic .
This was probab l y due to long s tanding t herapy which had
pro duced a n iron deficient s tate caused by the rapid pro
duct ion and removal o f t he red cells (by phlebot o my ) after
his hematocrit reached 65 per cent �
The p hlebotomy data ind icate t he average micro-hct
was 67 per cent which was approximately 1.7 times the
normal value . Centrifugation for a n additiona l 5 minutes
at 12,000 rpm caused no further packing of the red cel l s.
The hematocrit units of dif ference were calcul�ted b y
subtractinc the Mode l �hematocrit value (S) from the micro-
hemato crit (mi c ro -hct) va lue. Ideally ther e w0uld have
been no differ en c e betwe e n the two va lue s.
2 3
The init i a l visc oSity ( c p ) was about 2. 4 times the
norma l l e v e l in a l l o f thes e c hildr en who had , as mentioned
previous l y , had varying amo unt s cf t h erapy for their c on
ditions�
24
TABLE II Pre-Phlebotomy Clinical and Hematologic&l Data.
Donor S P :� • • s P ' . .
�ge k �. 5yrs..· 7yrs..
M 1ero h1i. 67 68 ��
,el c� 57.0 64 .. 0
IIrnc 9.99* J.l....38*
lMev 58 57 MOB 16.2 16.8
MCHO 2& •. 7 29.8 lU:b 16 •. 7 19.0
Cn ... 12.8 1?)�5 lDefect Mitral Atresia.,
Single ventricle Single &tria. Banded pu.lmo-nary artery
A H • .. T T • • T N • •
� mos. � yrs. � yrs.
74 67 65
..
67.7 61.2 62.4
7.22 6.8.5 6.96
91) 90 90
31.1 31.3 29 • .1 �3 .. 4 35.2 32.7
22.& 21. 5 20.5 14 .. 15 12.9 1.2.1
»-trans- �rlcus- . .. _-
�sltioJl p1.d of greai: Atres.ia vessels, with su'b-pul. .. Bl&l.o>ck m.onic Shunt stenos1e COm.mOD atriua. common ventricle pateat Ductus Arterio-sus
*---See Methods and Materials.
•
4 yra.
67
57.7 6.45
90
29.9
33.6
1.9.3
10.1
.--....
S B • •
4.5yrs ..
62
58.8 7.26
8.2 27.8 .. ,
�4.�
20 ... 5 .. ---
S1ngl.e ventrlcl.E pulm.onarJ &tenosis with J?�" monary artery banded in 1'0&1-t10n sub-a-ortic stenosis
HEMATOCRIT STUDIES
The hemat o logical data obtained from each sp ec imen
co llected dur i ng the phlebotomy and plasma exc hange ar?
p r e s e nt e d in Tab l e I I I . Fo l lo wi ng the phlebo tomy an 8 � 4
2 5
to 21 . 1 per c e nt r educ t i o n o f t h e REe c o unt , mic ro- and
Co ult er S hemat o c rits was o b s e rved � The phlebotomies wer e
ac companied by a r educ t ion in the d iffer ence b etwe e n the
t wo hemato crit value s . The pre-phlebo tomy diff erenc e s
ranged fro m 2� 6 to 10 � 0 hemato crit units; whi le the po st
phlebotomy d i ffer e n c e s ranged fro m 0.9 t o 8 . 0 hemat o c r i t
uni t s � Additio nal centri fugat io n did no t r e s ult in any
c hange in the m i c ro -hematoc ri t.
Donor � .P.
S.P.
!-.H.
Total VQ1Ul1le Reaoved
]..0 ml 70
120
170
220
270
320
40 0
20
100
150
20 0
250
30 0
350
400
420
430
10
55
85
100
105
TABLE III
Phlebotolll3' Data
Total Plasu Re'D1aced
o ml
0
80
120 170
220
270
;80
0
20
10 0
150
200
250
;00
350
400
420
0
10 ,.
55
85
100
Miero- �de1 Ret Hot
67 57. 0
63 53.2
62 51.a 62 52.1
59 50.2
57 48.5
55 46.3
53 45. 0
68 64.0
66 60.2
64 60 .4
62 55.2
60 55.8
59 55.2
57 53.2
56 52.0
54 51.2
54 51.0
14 61 .7
62 57.6
64 66.5
63 58. 4
56 53.1
26
Micro-S RBC Rct UU:t&
9.99 �o.o
9.61 9.8 9. 35 10.2
9.}5 9.9
8.94 8.8
8.72 8.5
8.28 8.7
S.0 2 8. 0
11.38 4.0
10.96 5. 8
11.0 6 ;.6
10.12 6.8.
10.24 4.2
10.18 3.8
9.70 3.8
9. 48 4.0
9.32 2.8
9.30 3.0
1.22 6.;
6. 29 4.4 7.17 -2.5
6.;2 4.6
5.70 2.9
TABLE. III c ont, Total Volue
D R - d onor e move
� .T . � O JIll
6 6
8 6
1 0 6
1 5 6
2 06
2 4 6
2 1 6
281
T .N . 1 0
6 0
8 0
100
1 20
1 4 0
1 6 0
1 9 0
2 0 5
2 2 5
245
265
js .J3 . �o
5 0
1 0 0
1 5 0
Total Plasma R lac d e.J.>, e
O ml
0
7 6
86
� 06
1 5 6
2 0 6
246
27 6
0
0
1 0
a o
1 0 0
1 20
1 4 0
160
190
2 0 5
2 2 5
2 4 5
0
0
60
1 0 0
Microt Be
61
6 6
6 ,
6 ;
61
59
5 7
5 5
5 2
6 5
66
6;
6;
61
61
59
59
59
58
58
58
62
6l.
59
5 1
MRdel � H t. RBC c
61 . 2 6 .8 5
61 ,'9 6 . � 5
58 . 9 6 . 61
5 9 . 0 6 . 64
5 7 -1 6 . 4 4
5 4 , 9 6 .21
5 h7 6 . 0 6
5 0 .. 4 5. 79
5 0 ..1 5 . 6 5
6 2 . 4 6 . 9 6
6; . 0 7 . 0 4
59 . 5 6 . 68
60 . 1 6 . 1 0
58 , 9 6 . 5 6
5 8 . a 6 . 58-
5 6. 1 6 . 3 3
5 5 . 5 6 . 29
5 6 . 5 6 . 2 1
5 5 . 8 6 . 19
5 5 . 5 6 .. 11
5 1 .1 6 . 29
58.a 1 . 2 6
5 8 . 8 7 . 24
5 1 .-5 1 . 09
5 4 ..8 6 . 1 2
27
M1ero-S llc t Udt
5 ..8
4 . 1 -
4 .1-
4 . 0
; .9 4 . 1
; - :;
4 . 6
1 .9
2 . 6
3 . 0
:; . 5
2 , 9
2 . 1
2 . 2
2 , ;
3 . 5
2 . 5
2 . 2
2 . 5
0 . 9
; . 2
2 . 2
1 .-5
2 . 2
a.
TABLE III c ont .
Donar S.B .
M . S .
Total V01wae Rem&ved 200 JIll 25 0
260
20
40
60
80
100
120
�40
Total PlaSlla. Ran1ao ed
150 ml
200
250
0
20
40
6.0 80
100
120
Mloro-Rct 55
55
53
67
65
04 63
62
60
5 9
28
�del lU .. oro-S 110'\ RBC Jlct Uuts
54.6 6.13 0.4
54.5 6.68 0.5
5 2 .. 0 6. 36 1. 0
57. 7 6.45 9.3
58. 1 6.55 6.3
5 8 . 0 6 . 41 6.0
51 e 4 6. 38 5 . 6
55.4 6 .12 6.6
53. 9 5.98 6.1
5 2.9 5 . 91 6.1
F igur e 1 sho ws a c o mpar i s o n of the micro -hct and the
Co ulter S hemato cr i t s of each phlebotomy sub j e c t. The rat io
® o f micro -het to the Coulter S hct yie ld ed an average value
o f 1 � 0 8 � Id eallY t h e ratio wo uld have y i e lded a value o f
1. 0 0 , thereby ind i cat ing n o d ifferenc e be twe en t h e value s
obtained using both methods � However , theoretically it
was p o s s ib l e to o btain a micro -het sma ller than the Coulter
S h c t � I n this case the ratio wo uld have b e e n less than
1. O O � 9 5 . 5 p er c ent o f a l l the me as ur em ents fell within
� 2 standard d eviations o f the mean ratio value �
30
\..... . .... Q:::
'" � CJ �
• � • •
• • •
• •
"'" •
•
� �
q:
� �
" �';i • \I) '-J • "" \I)
w • � � • '" :::s
� 8 '"
"'- . '" IJ) ...
'" "
(%) -L1c/:J01IIW:lfl08JIt1!
31
Sinc e o n ly a l imited numb er o f C CHD donors was avail
ab l e for stud y , i t wa s tho ught that a c o ndition simi lar t o
t hat o b served i n s ec o ndary po lycyt h emia c o uld b e creat ed b y
the r emo val o f p lasma fro m randomly s e l ec t ed normal b l o ods
p r i o r t o t e sting ( Se e Methods and Mat eria ls ) . The samp l e s
were � e lected regard l e s s to t he ag e o r d i agno s i s o f t h e
d o n o r � The o n l y Jrer equi s i t e s wer e the pres enc e o f normal
RBC c o unts , hgb , hct and r ed c e ll indic e s .
Fi gur e 2 Shows a c ompar i s o n o f t h e m ean rat i o o f
m i c ro -hc t t o C o ulter S®
hct i n the random donors and i n t he
c e HD d o no r s � I n t h e random donors t h i s ratio was fo und t o
have a va lue o f l � I O .
The stat i s t i c al analysis o f th e s e data i s pre s e nted i n
Tab l e IV. A standard deviat i o n o f � 0 . 0 5 was c al culated for
t h e mean rat i o s o f b o th the C C RD c h i ld r en and the random
donors � Analys i s by the t-test gav e a va lue o f 2 . 0 6 3 in
d ic at i nb a s ignifi cant d i fferen c e betwe en the two p o p ulat io ns .
Eac h blo o d sam p l e from the phlebotomized do no r was
tr eated as a new and unique sp eci men s i n c e treatment ( p lasma
e x chang e ) had o c c urred pr ior to t he wi thdrawa l of each
b l o od samp l e . Therefo r e , i t wa s a s i f a n ew pati e nt were
b e i ng e xamin e d . The differ enc e b etwe e n the t wo popu lat ions
sugge s t s t hat there i s a unique featur e c o mmo n to the er ythro -
c yt e s o f the C C HD donors e xamined that c anno t b e d�p li cat ed
b y the simple c o n c e ntrat ion of normal eryt hr o c yt e s �
3 2
F/G /l RE G
� A COMPI/Ii/ SoN OF rHE IIfEAN RllrlO DF Cii(JL T£R8s NcT OF TN. 1?H�OIl( IINZ> CeilD .JRIHP/, E,S
j I ...
t' t
i � � � � f.
l-'""
� � '--0:: � 12 �
� ':l:: -
� � "-/
� � o � "' 5:) G
3 C'/co) .J lb' iJ O..JI/W3H O!l.)/W
TABLE IV
stat i st i c al Analysia o f the 2 populationA E xamined
aOu.J:�e CCRD Donors
Randoll Donors
t-- 2 . 063
Number of
S�l e s
6 5
4 5
NWIlbe r of
Donors
6
4 5
M ean Micro -hci; Mo de! Y!'
1 . 06
l �O
t S1UIl of Square s
0 .149 2
0 . 1090
Mean Square
0 . 0023
0 .0 0 2 5
3 3
std. Dev.
!0 . 05
!.0 . O 5
To a s s e s s the accurac y o f the averaged value o f the
rat i o of mi cro -hct to the C o ulter S@hematocrit (1 � 08 ) as
3 4
a factor b y whi c h the obs erved C o ul t er �hct c o uld b y c o n-
ver t ed t o the micro-hc t , the data i n Table V Were c ompiled
and evaluated . This factor wa s fo und to be 0� 925 for the
conver s i o n of the micro -hc t to the corr e sponding Coult er � , ® S hemat o c r it . Ho wever , the C o ult er S hemato crit had to b e
mult i p l i ed b y 1 � 08 t o o btain t h e c orr esp ond ing micro-hc t . ® The se fact ors were fo und to b e r e l iab le for C o ult er S hc t s
equal t o o r e x c e eding 50 p er c ent .
The observed diff erenc es ( o b s erved micro-hct minus the ®
observed Coult er S hct = do ) and the c a l c ulat ed d i ff er enc e s
( o b s erved micro-hct minus the c al culated Coult er �hct _ d e )
are shown in Tab le V . I n add i t io n , it wa s f ound t hat 98 . 2
® p er c ent o f all t he obs erved Co ult er S hct s (So ) f e l l with-
i n � 2 standard deViat io n s of t he m ea n c a lc ulat ed C o ulter ® S hemat o crits (S c ) '
T ABLE V
Obs erved � Calc ulated Coulter �Hemato erits Compared t o Unad just ed M i cro-hematocr its of Cyano t i c
Congenital Heart Defe ct D on or s
M i cr o Het
/7 4
s '.fi
6 7 . 7
Mean S �
68 . 5
d . ·n
62 . 9-7 5 . 5 6 � 3
Me an d ,.
5 . 6
d c . 2 SD
1. 5 -11 . 1
3 5
� 8 6 4 . 0 6 3 . 9 5 7 . 8 -69 . 4 4 . 0 5 . 1 - 1 . 4-10 . 2
67 5 7 . 0 - 61 . 2 62 . 0 5 7 . 0-68 . 3 5 . 8- 10 . 0 5 . 0 - 1 . 3-10 . 0
6 6 60 . 2 -6 3 . 0 61 . 1 5 6 . 1 - 67 . 3 3 . 0 -5 . 8 4 . 9 -l . 3-9 . 9
65 5 8 . 7 -6 2 . 4 60 . 1 5 5 . 3-6 6 . 3 2 . 6- 6 . 3 4 . 9 -1 . 3-9 . 7
64 5 8 . 0 -6 6. 5 5 9 . 2 5 4 . 4- 65 . 3 - 2 . 5 - 6 . 0 4. 8 -1 . 3-9 . 6
6 3 5 3 . 2 - 60 . 1 58 , 3 5 3 . 6-6 4 . 3 2 . 9-9 . 8 4 . 7 -1 . 3- 9 . 4
�2 5 1 . 8 -5 8 . 8 5 7 . 4 5 2 . 7 -6 3 . 2 3 . 2 - 10 . 2 4. 6 - 1 . 2 - 9 . 3
bl . 5 7 . 1 - 5 8 . 9 5 6 . 4 5 1 . 9- 62 . 2 2 . 1- 3 . 9 4 . 6 - 1 . 2 -9 . 1
�O 5 3 . 9- 5 5 . 8 5 5 . 5 5 1 . 0 -61 . 2 4 . 2 - 6 . 1 4 . 5 -1 . 2 - 9 . 0
5 9 5 0 . 2 - 5 7 . 5 5 4 . 6 5 0 . 2 -60 . 2 1 . 5 -8 . 8 4. 4 -1 . 2 -8 . 8
5 8 5 5 . 5 - 5 7 . 1 5 3 . 7 49 . 3- 5 9 . 2 O·� 9-2 . 5 4 . 3 -1 . 2 -8 . 7
� 7 48 . 5 - 5 4 . 8 5 2 . 7 48 . 5 - 58 . 1 2 . 2 -8 . 5 4 . 3 - 1 . 1-8 . 5
� 6 5 2 . 0 - 5 3 . 1 5 1 . 8 4 7 . 6- 5 7 . 1 2 · 9- 4 � 0 4 . 2 - 1 . I -B . 4
5 5 £1 6 . 3 - 5 4 . 6 5 0 . 9 46 . 8 - 5 6 . 1 0 . 4-8 . 7 4 . 1 - 1 . 1-8 . 2
� 4 5 1 . 0 --5 1 2 5 0 . 0 4 5 · 9 - 5 5 . 1 2 . 8 -3 . 0 4 . 0 - 1 . 1-8 . 1
5 3 4 5 . 0 -5 2 . 0 49 . 0 4 5 . 1 -5 4 . 1 1 . 0 -8 . 0 4 . 0 - 1 . 0 -7 � 8
5 2 5 0 . 1 48 . 1 4.1 . 2 - 5 3 . 0 1 . 9 3 · 9 - 1 . 0- 7 . 8
36
In an e ffort t e simp lify t h e c o nver s i o n of micro-hct to
Co ulter �hc t Figure 3 Was c o nstruc t e d . The ob j e c t was to
b ® , o t ain an ac c urat e e stimate of the Mod el S h c t', T h e micro -
hc t obs erved minus the calculat ed h emat o c rit units of dif
fer enc e wo uld yie ld the e stimat ed C o ult er � hemato c rit . T o
c a l c ulat e the m ean diff erence t h e fol lowing proc edur e was
used : Co ulter � hc t
T he average value o f the ratio of micro -hc t was d e t ermined and " fo und to equal O � 92 5 or the r ec iprocal of 1 � 08 �
® . The Coult er S hc t was calc ulat ed by multip l ying t he o b s erved micro -hct by O � 92 5 .
The calculated C o ult er S®hct was then subtracted from the obs erved mic ro -hc t , thus yie lding t h e c a lc ulat e� mean differen c e between the mlcro- and C o ul t er �hemat o crit s .
For examp l e . at an observed micro-hct of 60 p er c ent one
wo uld e xp ect the average calculat ed di fferenc e b etwe en the
h c t value s o f the two method s to b e 4 . 5 hc t unit s . All
calculat ed and obs erved values were bas ed on the b lo od from
the phl ebo to miz ed C C HD donor s .
A CO/H PRR lsON '� rifF MEIW CIU./Cul.RTEl> ) JFF£IIEN�£S WtrN THE OSSERV�b MICR.O H�,"ATo CIl irs
J� '1! C � "i � ,. . -§� 1 1 1 .i! .. .. � \{ �� ... ..
,� .... cI) -o CIl
\ ; ., �
- - - - _ _ _ _ _ _ _ _ ...l(
*" - - - - - - - -.--------+-------11-- _ _ - - - - - -¥
..a ...
"- - - - - - - - ...... ------\:------� - - - - - - - �
__ - - - - - ------�r__----- - - - - --
CIl I
37
�
g
3
I... ... Q:: u � � � :t i � "...... � � >J
2 � C'I) � ()
38
VISC O S IT Y STUDIE S
The day , time and numb er o f samp l e s for visc o s ity
d e t erm inat ions were r egulat ed � Therefor e , .&�y o f the
samp l e s wer e refrigerat ed at 4° C unt i l the visco sit i e s co uld
b e d et ermine d � It is c o mmon prac tic e to assay sample s within
8 ho urs o f c o l l e c tion. Yet , from the data shown in Tab le VI
there s e emed t o be no c orr elation betwe e n the age of the
r e frig erat ed samp l e s (up to 72 hour s ) and t he vis c o s i t y�
The vi s c o s i t i e s wer e mea sur e d at 3 different shear rates
(rpm ) . T h e r e sults wer e a s e xp e ct ed . Sinc e t h e b l o ods
e xamined were very viscous , the mor e c o ns is t ent readings
w er e obtained at the lower she ar rate o f 12 rpm� The maxi
mum scale r eadi�Gs ob�ained at shear rat es o f 30 and 60
rpms were 10 . 20 and 5 . 1 4 c entipois e ( c p ) respe c tive l y . Tbe
highest r ead ing at 12 rpm using the We l l s -Brookfie ld micro
vi s c o m e t er was 25 . 7 0 c p o
39
TABLE VI
V!scosi ty Data
M i cr o - Model V i s co sity ( cp ) at V i s c o s ity D onor Hct � Hct do 12rpm 30rpm 60rpm Samp le Age
S . p . 6 7 5 7 . 0 10 . 0 1 2 . 8 9� 5 - - -- - - 72 hours
6 2 5 1 . 8 10 . 2 1 0 . 7 8 . 5 - - - - - -
5 7 4 8 . 5 8 . 5 9 . 3 7 . 0 4 . 9
5 3 4 5 . 0 8 . 0 8 . 2 6 . 3 4 . 9 s . p . 68 64 . 0 4 . 0 1 3 . 5 9 . 9 - - - - - - 2 hour s
62 5 5 � 2 6 . 8 10 . 2 7 . 7 4 · 9
5 7 5 3 . 2 3 . 8 9 . 3 6 . 9 4 . 9
5 4 5 1 . 0 3 . 0 8 . 3 6 . 2 4 . 9
T . N. 66 6 3 . 0 3 . 0 12 . 1 9 . 6 - - - - - - 50 ho urs
63 60 . 1 2 . 9 10� 4 8 . 6 - - - - - -
59 5 5 . 5 3 . 5 9 . 2 7 . 5 - - - - - -
58 5 7 � 1 0 . 9 9 . 1 7 . 3 - - - - - -
�f • s . 6 7 5 7 . 7 9 . 3 10 . 1 8 . 5 - - - - - - 24 hour s
63 5 7 . 4 5 . 6 9 . 7 7 . 2 - - - - - -
5 9 52 . 9 6. 1 8 . 4 6 . 9 - - - - - -
A. H • . 7 4 6 7 . 7 6' 3 1 4 . 5 - - - - - - - 72 hours
5 6 5 3 . 1 2 . 9 8 . 2 7 . 1 - - - - - -
(D i l ) 4 9 48 . l 0 · 9 5 . 7 4 . 8
T . T . 67 6 1 . 2 5 . 8 1 2 . 9 48 hours
5 2 5 0 . 1 1 . 9 7 . 5 6 . 0
(�il ) 4 5 42 . 0 3 . 0 6 . 1 .1 . 2
M � M . 5 9 5 4 . 7 4 . 3 10 . 4 8 0 6 50 hour s P. t. (c one . ) 67 61 . 7 5 . 3 1 4 . 3 48 hours G . W. " II ( c on e . ) 72 6 3 . 9 8 . 1 1 4 . 4
40
TABLE VI c on t . Micr o-
�d e 1 Vis c osity ( cp ) at Vis c o sity D onor H c t H ot do 12rpm 30rpm 60rpm Samp le Age
J . H. ( cone. ) 7 t:. 60 . 6 l l . 4 1 4 . 8 - - - - - 48 hours
J . R . ( c one . ) 6 0 5 6 . 5 3 . 5 10 . 7 8 . 7 48 hours
W. T . 50 44 . 3 5 . 7 7 . 1 5 . 5 4 . 8 2 4 hour s
M . C . ( co ne . ) 6 1 5 6 . 4 4 . 6 12 . 4 9 . 8 - - - - - 24 hours
R . W. ( cone . ) 64 5 7 . 5 6 . 5 1 2 . 2 2 4 hours
C . M. 4 4 4 2 . 5 1 . 5 6 . 7 5 . 2 4 . 7 2 4 hour s
4 1
Figure s 4 a n d 5 show a c o mparison o f the normal hct
vs visco sity (at 12 rpm ) c urve and t he o bserved micro- and ® Co ulter S hemato crits � vi sco sity fr om the C C HD and random
donors � 94 . 4 p er c ent o f all o f t h e micro-hets � vi sco sity
po ints fell wi thin � 2 s tandard d eviations o f the normal
curve c o nstructed from the va lue s given in Table I . However ,
o n ly 38 � 9 p er c ent o f t he Coult er �hct vs vis€osity po ints
fell wit hin this s ame reg io n . I t must b e rememb er ed that
t he norma l c nrve fo r the vis come t er used was c onstruc t ed
u s in g hematocrits determined by the micro -metho d .
� Q:: � .... , '-J "-....J �
� � �
' -� b?
. -�
;, h.. . -Q:
R � � � ,� �
F I GURE" 4
'" ....
Cd:» Wd�G I 11:1 luI SOJS IA
....
. � e ·' . t& !l ! iC�
� .. � i � � I '" I : !l: �.� Ot l � IQ ct I " � �.� l � � �� A: ij � "l ... , I , I / 9 )C o .
\
\ \ \ \ ,
\ \ \
• •
42
�
� (J) )... -<: 7::!
Q I-.... .....
Q:: .....--. (.,J � e ,-�
� � =:t
FIGURE 5
In -
(d;)) WdY GI 1JJ AL!SOJStt.
o ....
4 3
44
A c o mparison o f the whole b l o od v i s c o s i t y and the micro-® and Mod el S hematocrit s o f the phlebotomy sub j ects was
s h own in Figure 6 . I t was fo und t hat a linear r elation
d h ip existed b etwe en the Mo del �ema t o c r it and the who le
blo od vis c o si t y . However , as menti oned in the Lit erature
R evi ew , t her e wa s a l inear relat i o nship b etwe en the micro
hemat o c�it unt i l a hemato crit value o f 5 0 per c ent was
r e a c hed� Aft er the micro - hemato c rit l evel exce ed ed 50 p er
c ent , the relationship b e c ame e xp onent ial .
When c o mparing the visc o s i t i e s at numeri cal ly e qu i v a l e n t
micro - a nd Mod e l S®hemato c%it s one not e s that for hemat octits
b e tween 25 and 0 p er c �nt the Model S®c o rresponds to higher
who l e blo od vi s c o s it i e s � Howev er , the c orr esponding micro -
h emato erit s r e sult in lower who le blood vi s c o si t i e s . Ye t
o n e must r em emb er t ha t the Mo d e l �hct value s greater t han
50 p er c ent c orrespond to micro -hc t values that on an aver-
age are 1 . 08 times as large . ®
For e xamp l e , a Mod e l S hct
of 55 per cent i s e quivalent to a micro -hct of appro ximate l y
60 p er c en t . ® C urve B d et ermined by the Model S hcts and vi sco sity
o ffers minimal ass i stan c e in indicat ing c linic ally when phle
botomy t he rapy i s need ed� Cur ve A appears to be o f more
value c linically since the crit i c a l point o f the e xponential
incr ease in who l e b l o od visc o s ity c an be s e e n . Therefore ,
c urve A may b e mor e use ful i n ind i cating the need for phle-J botomy t herapy .
FIGIJRE b A CaMPI/Ri SoN of 1?f£ M�D- ANlJ MOlJ£J. � HE1HIITOCR�1S'
RNb V/scos;ry /N rHE "PHt.EBOTOIHY .JIJ.s.:n-crs
Cd:»
45
The l imited data pre s e nt ed in Figur e 7 p ermit an
evaluati o n of the effe c t o f visco s it y o n the difference
between the mi ero - and C o ul t er �h ematocrits � No dir ect
46
correlation co uld be s e en in the d i ffe r enees in h emato crit
units between t he two methods and the increase o f who le
b lood v i s c o s ity . Additiona l samp l e s must b e e xamined befo r e •
d efinite conclus i o n s can be drawn .
FiGURE 7 71(£ EFF£CT OF V($COS"n; ON mE 'bIFFE,fEN,CE -aE7W££N MIC/?O_ fiN]) CO(JlTCR - S H£M.9roc/urs
III ...
•
•
•
•
•
•
•
•
•
.. "'
•
•
• •
•
•
•
•
(d») Wd2J Z I 11:1 A1 /�O:)S /"
4 7
48
F I B R I NO GEN STUDY
C harm ( 18 ) and Wel l s ( 17 ) reported that there w� s a
corr elat i o n b etwe en the aggre gating t enden c i e s o f r ed c e lls
and t he fibr i no g en l eve l � The differenc e s b etween the micro �
and C oult er S hc t s were e xam ined to determin e whet her the
cause o f the s e differ e nc e s wa s due to elevated fibr ino g en
levels . However , no such c orre lat ion c ould b e e s tabli shed
in t h i s s tudy . The amo unt o f trapp ed plasma in the c entri-
fuge d micro -hct s e emed more r e lat ed to the hematocrit leve l
t han to t h e f ibrinogen l e v e l (Fieure 8 ) .
FIGURE ' THE C-.t:".c£cr OP PiSflINO&E"N ON rN€ ZJiF"!=E,f�cc Bew� � IH� ;:W� CO{Jc,-€,f 8 1I£IHR7l.X:!Rf7S
.. . · .. , .i · ·
.. . - . :. t • •
• • •
..
, .I � " . , ,
•
I .1 -. . , . . . . . . -. � . - -,
• I
-..) 1
-
5 0
MEAN CORPUSCULAR VO LUME AND R ED CELL CO UNT � VI SC O S I T Y
The C oult er �hct i s calculat ed fro m t h e produc t o f
t he M6V and the r e d c e ll c o unt . T h e study wa s ext end ed to
t h e examination of the r e lation of the numb er of red c e ll s
and the MCV to the Vis c o sity (Figur e 9 ) . T h e data c o l l ected
showed that there was a d ir e ct ( exp onential ) re lat io nship . b e twe en the numb er of red c el ls and who le blood v i s c o sity.
In addi t io n , it was fo und t hat ther e wa s an inverse re la
tionship b etwe e n the s i z e of the r ed c ell and the numb er o f
r ed � e l ls r e quired to produc e a specific vi sco sity�
5 1
F"lSM£ 9 ERyTN!;t;)('yl1i a�{,JNr � VISCosiry
> � 'at; u t- t- 1/1 &, � � III 11'1 • • , f , t ' I I f I I � � I I I I I I ..a � t I I I I � : , I c
• I
a I I I I ' I � I I I I I I - lC
I • I I � , I , " •
I I , I J , I � � I � • I I Q , t • 0- tI
� � � 1, � , to.. , iJ! , , i T I I I If I I I I I I I , .
I I I I I I I , ,., I I I I I I I I I I I I I I I I T , : 1 ' I I. , , I I I -, I I I I I , , I I I f ' I I ' I I I I I , : l , , I I I I d I I I I I I t , I I I I I I I I I I I I I I I I • I/) I I ' I I I I I I , I I I I , I : , I ' " I I I ' , " , , I , I a::.� ,. t;� &�9. � � cl::: � l a: � � ..
ltl �
(d'" Wd� zr J.J:J Id .. , SOJS 1/1
DISCUSSION
52
There is a marked differ e n c e b etwe e n the Co ult er S� and micro -hemato crit values in c hi ldren with c yano tic congenital heart d i s ease � A c omparison of the hemat o c r it values obtained u�ing these m ethods show ed that a differ enc e a s
high a s 1 0 � 2 hematocrit units may exist �
Mo nitoring the hemato crit i s very important in the
c linical d et e rm inat ion o f the degre e of p o l yc ythemia in
t he s e c hi ldr e n . An increase in the packed c e l l vo lume d e t er
mined by e ither method was a c c o mp anied b y an increase in the
Who l e b l o od Visc o sity . However , t he data showed that the
m i c ro -hemato crit value gave a mor e ac curate indication of
t h e d egr e e of po lyc ythemia . In addition , t he inc r ease in
the micro -hematocrit was shown to more closely paral l e l the
incre as e i n who l e b l o od vis c o s i t y �
® The d ecr eas ed ac curac y o f the Coulter S hemato crit i n
c linic ally a s s e s s ing p o l yc yth emia and increas ed v i s c o s i t y
was probab l y d u e t o the 1 1 5 0 , 000 d i luti o n of the who l e b l o od
e l iminating the effects o f the factors that influenced red
c e ll packing and visc o sity ( 7 0 ) .
Sinc e o nl y 6 c yano t i c c o ng enit al heart disease dhildren
were obtained for phl ebotomi e s , another donor gro up was
s e l ec t ed ( s ee Methods and Mat erials ) . It was fo und that a
S igni f i c an t differe n c e exis t ed b etween the b lood c e l ls of
the normal donors and tho s e of the C C HD donors . C o nc e ntra-
t i o ns and d i lut ions did no t produc e c omparabl e co ndit ions
in e ither p opulat i o n . Therefor e , t h e aggr egating and pack
i ng t end enc i e s o f the r ed c ells fro m each population were
d i f ler ent .
5 3
S e v e r a l e xp lanati o n s for t h i s d i ffer enc e e x is t . Ac c o r d
i n g t o D i nt e nf a s s ( 2 9 ) the agg r e g a t i on a n d p ac k ing t e nd enc i e s
o f e r yt hr o c y t e s ar e i n f luen c e d b y t h e r i g id i t y o f t h e c e l l s ,
whi c h i n t ur n , i s r e lated t o th e c e l l c o nt e n t s a s we l l a s
t h e c e l l m embrane � Ja c o b s ( 3 3) s howed t hat t h e c e ll membra n e
i s c apa b l e o f m o v i n g aro und t h e c e l l c o nt en t � T h e evid enc e
f o r t hi s l i e s i n t h e fact that t h e p ac k ed c e l l s p o s s e s s a
Vi s c o s i t y much l owe r than wo u ld b e e xp ee t e d �
I t has b e e n s ug g e s t e d that m i cr o c y t i c hyp o c hr o m i c r ed
c e l l s d i f f er fro m normal r ed c e l l s i n t h e ir rhe o lo g i c a l pro -
p ert i e s . I n t h i s c a s e , t h e v i s c o s i t y o f t h e c e l l c on t e n t s ,
i . e . , h em o g l o b in , i s t h e c on tro l l ing vi s c o s i t y fac t o r� I n
two s t ud i e s b y D i n t en f a s s ( 24, 41 ) the i n t ernal vi s c o s i t y o f
t h e n o rm a l r ed c e l l was exam i n ed and fo und t o range b e t we e n
1 and 6 c ent ipo i s e� This e st i mat e i n lud ed t h e c e ll membrane
i , effe c t s as w e l l a s hemoglobin vi s co s i t y. An a s s e s sm ent o f
hypo chro m i a and m i cro c yt o s i s s ho uld b e made t o d e t ermine t h e
aff e c t e a c h might have o n who l e b l o o d Vi s co s i t y .
I t wa s tho ught t ha t s torage m i ght have had an adver s e
e f f e c t o n t h e i n t e gr i t y o f t h e r ed c e l l m embran e , t h ereb y . . aff e ct i n g t h e v i sc o s i t y � Howev e r , i n suff i c i en t data wer e
ava i l ab l e t o a s c er t a i n t h e e f f e c t o f s t orage o n t h e vi s c o s i t y
o f t h e b lo o d samp l e s � Who l e b l o od wa S suitab l e f or r ed
c e ll c ount s , h e m o g l ob in and hematocrit d e t ermina t i ons for
24 to 48 h o ur s i f s t ored at 4° c (1) . Ther e fo r e , wit h t h e s e
m e a sur em e n t s unc haneed b y st orage o ne wo uld e xp e c t no s i g-
n i f i c an t c ha n g e i n V i S C O S i t y .
5 4
A s indicated i n Tab le V I some samp les d id not have t he visco sity � et ermination p erformed unt i l 7 2 hours after the
phlebot o � y � I t was not ed that in ones patient who had had
two p h l ebotomi e s , there was no d iffer ence i n the who l e b lood
v i s c o s i t y of the b l o od samp l e s with comparabl e nicro -hema
t o cri t s eventhough the ages o f the samp les wer e 2 and 7 2 j
ho urs'� Ye t , d e spite the equival ent mi cro -hemat o cri t s the
Co ult er S hematocrits d iffered by 5 . 4 unit s � Furthermo re ,
the red b lo od c e l l oounts differ ed b y as m ch as 1� 28
m il H o n! mm3�
One major que stion was raised , what events o c curr ed to
c hange the packing c haract er i s t i c s o f the erythro cyt e s in
this pati e nt ? It was s ho wn that the d ifferences be tween
the m icro-hct and Coulter �hct c hanged from 8 . 3 units after
t h e first phlebotomy to 3 . 4 units after the s e co nd phlebotomy
s ix mont hs later� No medication had b e en administered prior
to e i t her phl ebot om y . B o t h p hl ebotomie s were p erformed in ® the same manner with the micro-hct and Co ult er S hct deter -
minat io n s d o n e withis thirt y m inut e s o f t h e samp l e withdrawa l s .
The only differ ence obs erved i n the pat ient during the p hle
botomies was the fac t t hat during the first p hl ebotomy the . , patient was r elative l y calm� Yet , during the s e cond phlebo-
tomy the p atient was very ups e t and t ended to hyp ervent ilat e .
Could the h yp erventilation have b een suffic i ent enough t o
e nhan c e t he oxygenation o f t h e C irc ulating �ed De ll a�ere cat es ,
t her eby c hangine t h e venous p H from acid to alka l ine?
5 5
Oxygenation o f " s ludge U c ause s a d e c r ease i n t h e numb er o f red c e l l aggregat e s b e ing formed � This e xp lanati on Was s upported by the data obtained b y Dint enfass ( 27 ) . The
ranGe o f p H cho s e n for hi s stud y wa s 6 . 9 to 7 . 4 . The lower
limit c orr e s p o nd s t o blood p H o b s erved in babies with acute
re spirat o r y d is tr e s s s yndrom e � A ct ua l l y the l o ealized blood
p H c an be s e en l ower in a numb er of c o ndi tions in whi ch
trapp ed red c e lls and l o c a l i z ed stas is exist�
H i s stud y ut i l ized both adu lt and fetal blood . 7he
fetal blood Was obtained fro m the c ord and p lac enta immedia
te l y after d e liver y . Adult blo od was drawn by venipunct ure .
I n t he fil st serie s , t he metabo lic pH c hang e s were induc ed
b y addit ions o f sodium hydro xid e , hydro chloric ac id and bi-
carbonate s o lut i o n s in i s o tonic c o ncentrat ion s . Pack ed c e ll s
w e r e p r epar ed b y centrifu[a t i o n , pH ad jus t ed , and then hema
t o crits ad jus t ed to the r equir ed o n e s . B l o od pH wa s measur ed
with a rad iom eter pH-m eter . It was shown t hat a d e cr eas e o f
p H l ed to a pronoun c ed increase of Visc o s it y a t all rat e s of
s h e ar . I t wa s also shown that a t hi£h h ematocrits t h e vis
c o s i t y e leVat io n at low p H c ould b e attributed both t o an in
c r e a s e in the ribidity of the r ed b l o od c ells and to their
a ggregat i o n . The se r e s ult s wer e c o mmon to b o t h fetal and
adult b l o o d s . A pronounced d e c rease in the f iterabi lity o f
b l o od with t he fa ll o f t he p H was observed �
There was a further eff e ct to c o nsid er t hat was men
t ioned , name l y , tha: , due to an incr ease in the rigidity of
the red b lood c e ll s , an appar ent increas e o f hemato crit t o ok
5 6
p la c e � A fall in p H of b l o od b y 0 � 5 units was accompanie d
b y a r i s e in h emat o crit (micro -method ) of abo ut 1 5 p er c ent�
The effe ct o f the p H was attribut ed to the increase in vol
ume o f the red c e l l s and a c hange of their shap e to spheri
cal , the swe l l ing b e ing due to the entry of anions a s the
pH d e cr eased in a c cordance to e qui libria wit h the degr e e of
i o n i zation of hemo globin� Human r ed c e l ls do not act a s
p erfect o smometers and r e s i s t swe l ling� The e ffects o f
t o nic ity o n t h e vi s c o s i t y o f the r e d c e ll were due t o the
fact t hat the internal viscosity of the r ed c e l l inc rea s es
during crenation whi l e swel ling of the r ed c e l ls in the
hyp o t o n i c s ystems l ead t o higher t ension in the red c ell
m embrane� Both the s e e ffe cts r esulted in an increased vis-
c o s it y and l o o s er p ack ing o f the red b lood c e ll s �
S i n c e t h e 1 1 5 0 , 000 d ilut io n i n an isotonic medium r e
s t o r e s t h e red b l o od c e ll s t o their orifi nal s i z e ( 7 0 ) and
the r itidity of t he c e lls have no effect on the m easurement ,
the Coulter S hem�tocrit wo uld not b e e l evat e d . Therefore ,
the d e gr e e of po l yc yth emia and incr eased vi s co s i t y would not
be r e f l e c t ed a cc urately by the Coulter �h ematocrit �
However , o ther data provid ed by the Coulter �mi ght b e
u s e d to a s s e s s the degr e e o f p o lycythemia and increased vis-
c o s i t y. As stated pr evious l y there is an inverse re lati o n
ship : , e twe en the m ean corpnsc ular vo lume and the �umbe r of
r ed cell requir ed to produc e a sp e c i fi c vis cosity� A traph
snch as F i gure 9 that incorp orate s the MCV , erythro cyte
c o unt and who l e bl ood visc o s i ty co uld pro ve useful in e st�-
5 7
mating t he visc o s i ty i n patients be int monitored. I n this
instanc e the r ed cell c o unt wo uld b e ind i c at ed as more valu-® ab l e than the Coulter S hemat ocrit in monitoring the d eGree
of p o l yc yt hemia in c yanot i c congenital h eart d i s ea s e childr en .
P erhap s venous pH and red c e ll filt erab i l i t y in con-
jun c t i on with who le b l o od vis c o sity , micr o -hemat o c r i ts and
red c e l l eounts miEht give a more ac curat e delin eat ion o f
t h e p atient ' s circulatory stat us and n e ed fo r p hleb ot omy�
In add it i on , the d et erminat ion o f glutathione and ATP levels
might give ind i c at i ons of the d e gr e e of r i g idity o f the c e ll s
whi c h i n turn influen c e the visc o s i t y o f t h e blood . All
o f t h e s e fac tors influenc e the ass e s sment of the d e�re e of
p o lyc ythemia and who l e blo od viscos ity
SUMMARY AND CONCLUSIO NS
Mo nit oring the he mato crit i s very important in t h e c l i n i c a l evaluation of t h e derr e e o f p o l y cythemia and the
58
n e ed for phl eboto my therap y. We have noted signific ant d iffer e nc e s b etwe en the Coulter S®and micro -hemato erit values . The s e hemato crit value s differed b y as manY a s 10 � 2 hemat o crit
unit s , wit h the C o ult er Mod el S®Yie lding the lower value�
The d ifferenc e b et we en the hemato crit value s o f the two
methods wa s virt ual l y unaffe ct ed b y t he fibr inog en c onc en
trat&nn �
The red b l o od c e ll c o unt with the MCV proved t o b e o f
more value than the Coulter S@hematocrit in assessin g the
d egre e of p o l yc ythe m i a and incr e a s ed visc o s ity in c yano t i c
c ongenital hear t d ef e c t chi ldre n . T h ere waS a n inver se r e la-
t ioRshi p b etwe en t he MCV and the number of the se red c e lls
r e quired t o produc e a partic ular vis c o s it y .
S i mp l e d i lut ions and concentrations of n o r m a l b lo od
d id not re sult in e quivalent rat io values of micro -hct to
® C oulter S hct whe n c o mp ar ed to t he values obtained using
the b lo od fro m CCHD donors . This i ndicat e s t hat the dif-
ferenc e s o b s e r ved were not s imp l e di lution or c onc entration
p he no mena�
Final l y , the data in t his study sugg e s t that the micro-
hematocrit is a more r e li ab l e and accurat e measur e of the
d e gre e of p o l y c ythemia ; b ut that other param eters may " measured and used concurr ent ly to d et ermine t h e need for
phl ebotomy therapy.
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