(aw\ On the day thet the first massive bombings of London took place August

16, 1941, the Blood Transfusion Association of New York, formerly known as

the Blood Transfusion Betterment Association, with the cooperationof the

American Red Crose, leunched a project unlike any other ever attempted in

the annals of medical history. This project consisted of collecting blood

from the civilian population of greater New York, removing the plassa, andshipping it across the Atlantic for use by the British in the treatment of

both civilian and military wor casualties.

A preliminary report of the difficulties as well as the favorable as-

pects of thie work wae published by the Blood Transfusion Association on

January 31, 1941. It isato recount here the things already re«

ported there in considerable detail, but it is perhaps worthwhile to recepitu-

late in brief the history of thie movement in order that some of the final re-

sults to be reported today may have more meaning.

During the winter of 1939-1940 an appropriation was made by the Blood

Trensfusion Association to Dr. John Scudder of the Presbyterian Hospital

for the purpose of carrying out studies of a laboratory and clinical type

related to the preservation of and the use of plasma as a blood substitute.

Associated in this work were Dre Charlee A. Drew and Dr. Kingsley Bishop.

While this work was being carried on Dr. Allen ©. Whipple, Chief of

the Surgical Service at the Presbyterian Hoepitel in Jew Terk, was called

upon to fly to Italy toact as & gurgicel consultant for an emissary of the

President insaeMe Fequectedthat plasma be prepared which he could ♥

take with him for use should the need arise. his was done.

Sonal meidenebte, cache D.C» Soruseiy Wedical Supervisor. Plasma DivisieaBlood Transfusion Association, New York City. es

2

During the early days of the attack on France Dr. Alexis Carreltold

to a group called together by Mir. John ¥. Bush, President of the Blood

Transfusion Association the seed of some means of treating these patients

and suggested the use of plasma or serum as the most logical way. Dr.

suggested to ir. Bush that the shipping of plasma to France was ao

Blood Transfusion Association to extend their seope to such « worthwhile

project. Thies the Heiical Board of the Association agreed with end plans

were made to enlist the aid of the voluntary hospitals in lew York City in

g and preparing plasma for trens-shipment. France, however, fell

and that it would be in line withthepolicies of the

before the German army so rapidly thet there was insufficient time to get

this aid to them.

The idea had been born, however, and the American Ned Cross had agreed

to aid in financing such a project should eny of the Allies need ouch assist~

aNCe. |

Vhen word was received of the great losses ofthe British, in their

withdrawal from Dunkerque, and informal news wae received to the effect that

the Be. & F. had sustained heavy losses in their mobile transfusion unite,

the Blood Transfusion Association attempted toescertain whether plasma from

this country would be of any assistance to them. The anawer came back in

the affirmative.

In July and early August, directed to a large extent by Mr. Tracy 3.

Voorhees, President of the Long Island College Hospitel , end legal adviser

to the Blood Transfusion Association, plans were inaugurated for the reception

of donors and collection of blood in six voluntary hospitals. These were as

follows: Presbyterian, New York, Memorial, Mte Sinai, Post Graduate, and

Long Island College. Later three others joined in this work. They were:

30

Lenox Hill, the Hospital for Joint Diseases, and the Jewish Hespital of Brooklyn.

It is impossible to give sdequate credit here to the great number of indi-

viduals, both medical end non-

ical, who gave their unstinted efforts, in many

cases at great personal sacrifice to this new type of humanitarian medical ex-

periment. Over two thousand doctors, nurses, technicians, social workers, Red

Grogs volunteers,and enthusiastic laymen cooperated to make thie dream an actuale-

be mentioned. Among them are: Mr. John F. Bush,

President of the Blood Transfusion Association, whose enthusiasm and wise gald~

itys A few, however, should

ance euch a scheme possible. He was more than ably assisted in all the

phases of the work by Mre Tracy 5. Voorhees. Dre De®itt Stetten, Vice+President

of the Association and Chairman of the Board of Medical Control, was equally

effective. The Plasma Committee had ae ite chairman Dr. ¢. FP. Bho

de, Medical

Director of the Memorial Hospital. Ably assisting him ae members of this

Committee were: Dr. David ©. Bull of the Presbyterian Hospital and Dr. Lester ♥

J. Unger of the Post Graduate Hospital and Dr. John Soudder.

When it became apparent that a central depot would be necessary in order :

to coordinate the in-coming calls, distribute the donors to the various hospi-

tals, keep a record of the entire procedure, and coordinate shipping with the |

Ameriean Red Cross, space and Zatilities were donated by the New York Academy

of Medicine in its building. Mr. Morrie Me Davidson volunteered to serve as a

fulletime director cf the many administrative detaile of this project and Dr.

Charles R. Drew was appointed as full-time Medicel Supervisor. It became the

latter's duty to coordinate the medical aspects of the program, to establish

uniform records, standard equipment, and suitable technical criteria to in-

sure the eafety of the final product.

ania

assist in controlling this phase of the work Dr. Frank L. Meleney, assisted

he

by Mies Balbina Johnson, offered the services of his laboratory as a central

testing depot of all material before final shipment.

Yorthy of especial notice aleo are: Dr. Sathan Rosenthal, Director of

tment of Hematology at Mt. Sinai Hospital, who organized a unit

009 phlebotomiesandestablished anunusualrecordfor the

tage of the blood collected which passe:

, @1l tests and was shipped

and in the form of pla line; Or. Lester J. Unger, in additionto

directing the Blood Banke at the Poet Graduate Hospital and the Hospital for

Joint Diseases, carried out important experiments throughout the project for

the purpose of improving the technique; Dr. Ralph G. Stillmandirected the

project at the tiew York Hospital, Dr. B- R. Marzullo at the Long Island

College Hospital, Dr. Perry J+ Manheime at the Lenox Hill Hospital, and Dr.

Alexander &. Weiner at the Jewish Yospital.

Der. William Thalhimer offered not only his services but in association

with Dr. Ralph 5S. Muckenfuse, Director of the Bureau of Laboratories of the

t of Health of the City of few York, offered the facilities of the

Manhattan Convalesceat Serum Laboratory for a large part of the experimental

program. |

Dr. EB. He be Corwin, Managing Direetor of the Blood Transfusion Associ-

ation, not only served on the committee with Dr. Seudder and Dr. Drew which

outlined the organization but also carried much of the administrative load

until Mr. Davidson took over thie task as a full-time responsibility.

There are many others, unnamed who probably are as worthy es the above

of special commendation but the limite of this paper do not allow a recitation

of their contributions to this work.

5.

FINAL RECORD

OF PLASHA*SALINE PREPARE!

All plasmashipped to Englend was diluted with an equal volumeof

physiological saline. This was done for several reasons, among them: (1)

tekes place more slow-

ly im the presence of saline. It was hoped that this wouldmakeit 7

precipitation of the proteins especially fibrinogen

sary to filter the solution beforeuse.

(2) Xt was felt that by filling the flasks to thetop denaturation of the

proteins would take place more slowly. (3) It was felt that since the use

of sodiwea chloride in solution has a beneficial effect upon the constricted

arteriolar vessels in shock that it might enhance the velue of this fluid

for such treatment. (44) the concensus of opinion expressed at the meeting

of the Awerican Human Serum Association held in New York June 10, 1940 was

that the safest blood substitute at that time was considered to be diluted

glaemm. All results therefore in the following Table are described in

terme of plesma-saline solution. ach 1,000 cc. Baxter Plasmavac contain-

ed 500 Gece of plasm and5 iological saline and sufficient

"Nerthiolate" (Lilly) to give s final concentration of 1:10,000. ☁The ship-

OO GeGe of phy

ments from the various hospitals were as follows:

Hospitals

Mt. Sinai

Poet Graduate

New York

Long Ieland College

Joint Diseases

Lenox Hill

Jewish

(in liters)

1,547

Te

individual hospitals as suitable for shipment and the amounts actually shipped

represents the quantity found unsuitable by the Central Laboratory. This

quantity equale 475 liters or 8.2% of the total quantity.

Thegreatestlosswasduetocontamination. There ms no hospitel in

which some contamination of the finsl

duct was not discovered. These

y to expectations and to those of investi~

aration and use of plasma. It

served to demonstrate that the mass production of hamen biologicals is not

@ simple procedure, free from danger. It soon became evident that a one-

week incubation period was mot sufficient tomene @ sterile product

several monthe later. The experience

samples of placme which had been passed afte

was hed on repeated occasions that

' @& twoeweeks observation

period were found at the end of 4 month to contain growing organisms. Many

authorities were called in to discuss this very discouraging observation

and out of it came the first steps on the part of the U. S. Public Health

Service to change the regulations regarding the preparation and testing

of human plasma which heve now been put into effect.

A gummary of the losses follows. Letters were used to represent the

cooperating hospitals rather than the names. There is no desire to indict

any particular institution but rather point out the difficulty had by all

and thereby make more strong the point that in the preparation of human

plasma great care must be exercised at each step of the procedure.

Be

☜Hospital Central Labs | cardedin(in liters) (in liters)

251-6 183-0 24.3

lg 37

4B 9-0

1h 27-3

0 265

18 ♥♥ Qike?

0

2

1-4

A

G

D

B

¥

G

H

J

☜The total loss reported includes not only infected plasma but also plasma dis- ♥

carded because of suspected contamination or known break in technique even when

cultures were returned with negative reports.

Only one boat was sunk during the life of the project which had aboard

plasma-saline solution. This was the WesternPrince which went dow off

the coast of Ireland in December carrying with it, among other things,

☁Ruglend as to what happened orwas |

sent. So wuch was this so that there were doubts in the minds of some in-

dividuals as to whether the blood which they gave for England actually ever

got there. I should like to insert at this point parte of a letter from

Mr. P. Le Oliver, Honorary Secretary of the Britich Red Grose Transfusion

Service, which is in the aature of a report to him from Dr. E. Brace White

Por many months it was very

of the National Institute for Medical Research at Hampstead, and shows with

"Zotification to expect the arrival of « consignment of Plasma is received

from the British Red Cross. On arrival the cases are checked, their seals ex~

amined, and any evidence of gross damage noted. The cases are stored - from

lack of more suitable ace ion ~ in a cool basement corridor where they

106

"At unpac!

"In the case of rare Wreakages the affected cartone are cleaned so far as

possible and dry~steam autocleved.s ☁The solidbottlesof /are sponged

with Dettol and allowed todryoff in the air before replacement. Solid

and peferring to bottles which have

been withdrawn for bacteriological testing. Zvery effort has been made to

ing: the case, carton and bottle identification marks are re

a hospital seale of the cartons

materials withdrawn from other cart

restore the affected as to the ☜chaste☝ condition in which they were

issued. ☁The empty slots are packed with paper, a record of the breakages

is pasted within and withor

☜Phe bottles are individually examined in a good light - and any abnormeli-

ties recorded. Any specimens deemed necessary for bacteriological testes

having been taken, signed notices of the removal of such bottles are pasted

outeide and inside the cartons and the vacant slots are packe

d with paper.

☜When bacteriological testing is complete the cartons are resealed with

gwaned paper tape in the original manner and cross stamped ☜National Institute

for Medical Research, Hampstead, i. ¥. 3☝ and "Medical Research Council, Bmer-

geney Public Health Laboratory Service."

"fhe Hospital label is, of course, left intact.

☜The cartons are then replaced in their appropriate case and dispatched

according to instructions received. No tranefer of bottles from carton to

carton or of cartons from case to case has been made.☝

☜According to the Hospital of origin the cartons heave been disposed of as

follows: - |

Hoe of ¢

Presbyterian Hospital 68

li.

Long Island

Memorial Hospital

Hospitel for Joint Diseases

Lennox Hill Hospital

"fhe following notes are made in no captious spirit but simply for in-

formation.

☜Phe earlier labelling in which the Presbyterian Hospital gave a serial

bottle number without the pool number while the Mount Sinai and other hos-

pitale geve a pool sumber without hospital serial bottle number has we note

with satisfaction been replaced by a system in which both pool and bottle

numbers are atated.

"It has been noted that in a number of instances where Gece of plasma

SOO cece of sodium chloride solution are declared on the label (total 1000

G-ce) the contents do not exceed 750 c+c-

tb of the Piaema Seline ☜The bottle contents have varied considerably in appearance. Some have

been almost liquid and deposit-free and, of the main contributors, the Mount

Sinai Hospital seems to have been most fortunate in this respect. Many bottles,

particularly thoee first received have shown a soft fluffy deposit forming -

layer 1/4 in. (or more) deep at the bottom; such appearances have been rare in

12.

the consignments more recently received. A few of the bottles have shown ex~

tensive clotting (see Tables I andII) and have in all cases been found heavily

contaminated with bacteria..

® - by ee 2 ; : : = ripe a 4 ste i. ~ 2 cae} is 2 z. Oa aie ae ☁| ; 2 £2. fae2 7

- . i 7 aie , ae Fe ee Nc he

"Ho attempt hes been made to neutralize the merthiolate present as this

would heve involved a preliminary research for which time is not available.

In place, a small tncoulation (062 eee) has been made into a large volume of

200 ¢.c-) to reduce the merthiolate concentration to

gh costly in media has been very ade~

nutrituve medium (

1210,000,000. We think the method thou,

☜fhe test media have been Hartley's broth for aerobic teste and for an-

aerobic culture the same medium, vaseline covered, with co:

02% of added glucose.

"fhe bottles are incubated for 5 days at 37° ani are then, if negative,

observed for aperiod at room temperature.

"Direct platings are made on agar (or blood agar) and stained filmsof

☜In a number of cases 1 cc. or 2 ¢-¢- amounts have been subcutaneously

injected into mice; it may be sald forthwith that no toxic symptoms have

been noted.

☜A list of the bottles examined is given below and the results of the

tests sumearized in Tables IfI and IY. The items are classified in what we

believe to be the manner giving the fairest presentation of the facts.

13.

"During the latter period of the testing in view of the satisfactory nature

of the results of the number of bottles taken for examination has been reduced

to « minimum and we have been guided in the choice of semples by those appear-

ances which suggested possible contamination. It has been felt improper either

rial for theseke of formal

to waste veluable

"Itwill be seen from the tables that the first period was one of some

anxiety in that, of the 17 samples received from Dr. Heyl, Dr. Harrison and

taken from the cases 7605/1M - 7605/4, no fewer than 7 and possibly 8 gave

growth in culture media. The Sth case was doubtful in that a second sample

gave negative results while in the case of 6 others tested a second time con-firmatory results were obtained. In the case of Dr. Heyl's bottle a rice

growth - confluent in direct culture on ager! - of Staph.aureus was obtained.

"fhe contents of cases 7605/1m - 7605/4M heave been withheld from circu-

lation. ee es

Ssand 35 which were all heavily contamineted with gram negative bacilli of

coliform type and showed varying degrees of clotting.

"fhe cases which we have marked OO] - and the great majority of the

arked with numbers between 1 and 96 have now been forwarded to

62 cases

the transfusion centrese It is hoped to inspect and clear the remaining

cases in hand during the next fortnight.

(Signed) P. Bruce White"

14.

12

14

17

Resulte of Some

TABLE 111.

Incorporated in Main Table IV.

146

140

157

Negative

riod)

ee

nation - sabenateeclotting

Yound contaminated ~ 3 bottles (2 being gravely infected)

(* + 1 leaking through broken needle insertedia stopper. 1 with airlead tube broken. 1 unlabelled.)

☜Hospital Wo. of lo.of No. of Bottles lo. TestedBottles Pound Contaminated ~~Cartons Bottles Bottles Found Bacterio= coae =

cose033 - 191 (anaerobic strepto~cocci)C34 ~ 198 (large gram - cocci)

Mount Sinai 6 36 - - 3 2 Cl=P8+%. T B. 02739. (@iphtheroids)

03 = PLO - MH. T & 02578(anaerobic gas forming

% (5) eocei and diphtheroids)

ey

☁ Mi "3 Pe Fg. 342 Seen ert + ae

7 % ☁ Pees is ee oe e es a

PresbyterianMount SinaiHew YorkNew Tork Postiradvate Med. Sch. 37 | -

Long Island Col. 22 132 cee 10 : = 6 bottles of Pools 29 and 33☁ in Cartons 19 and 20 heavily

Memorial 18 108 - - 2 ☝ contaminated gram-coliforn

-

Mempitel Ser ¢eims bacilli - much clotting

Q aDiseases :LennoxHil)

*(These cartons were those shipped before the establishment of » central centrol☁Uneven

4 ☁

fs ad

16-

The Blood Transfusion Association started this project with but two

1. To lend immediate aid for war casualties in Great Britain;

2. To gather information which would be of value to the arned ♥

forces of the United States in case of ao nationaleme:

This report from the British Bed Croes doaetna

part of the program. Sta

ent. Culture methods were different and pers

ly equipped te carry out such a technical procedur

. im some cases was very poor-

to the time dating from the establishment of a central edministrative office,

@ full time medical supervisor, and a central bacteriology laboratory to et

ae @ cheek on the individual hospitals. Likewise during this second phase of

the work full time technicians were prov

ded for in each hospital and the

period for observing cultures was lengthened from two weeks to three weeks

before any particular semple wae released for shipment. In this second phase

one batch of infected material did, by mistake, get shipped to England

authorities there were notified of this suspected shipment and, at shown in

the report, it was picked up on the other side. We feel that with the triple

check on this material no harm has come to any of the recipients and there is

increasing evidence, in the form of letters or direct contact with men return=

verience the following routines were determined to be adequate for

vacterial culture and were supplied to each hospital.

Aye

meet medium. This medium is p

water (distilledpreferred). This iat:

ator overnigit.

a beef heart into1 liter of

ion shouldbe left in refriger

and etir until dissolved.

eT

Adjust pH to 8.4.

Boil for 20 minutes, restore |

ih poper until clear.

The chopped cooked meat is washed in strainer under running wetes

L volume with distilled

water, filter through |

The cooked ment medium is to be distributed into tubes, 6" x 1", fille

ed { full with cooked meat, ondat least 35 ce. of broth added.

Tubes are llaved at 15 lbs. pressure for 30

should be 7+4 to 7+6-

B. Anaerobic Cultures:

5 ests of the plasma from the pool is incu

tube containing 40 cos. Brewer's dextrose Thioglyce

115:598, Auguet 24, 1940). This medium may be procured from the Balti-

☁more Biological Leboratoriesin Baltimore, Md. It contains:

Fork Infusion solids (from 37+5gms porit)sceccesesescnceesLeO®PepteNeTNLGs ss <cerorcesyresererverwesensoreserereroerverertelyDextrose OOOOH EERE THEE OTE HEHEHE O DRO HEROROOHH e eeer ee elel

Natl CHOKEEHREOH OHHHEHHREEOROOOHOEwe eeeeseeeeDe

Sodium Thioglycolate cesceseseseresecceceneseseseessonsseneOelh

Ager aeceesvesesecuetelseeseeeernreteeeton

taylene Blue (ls 500 000) eacedoesenssbseeseooccesosoccececeet

Dissolve 3-65 grams in 100 ec. of distilled water. Heat until the

solution boils and all w to boil about ome minute. Autoclave 20 minutes

G., 40 ec. to each tube. Do not store the powder in the refriger-

ator because this decreases the duration of anaerobiosis.

Both tubes are to be inoculated at 37° ©. for two weeks. Hxamination

of standard preparations of these tubes are to be made after 3, 7 and 14

are to be made to fresh 0.2% 4

rose cooked meat media. (Tubes 6" x 5/e*),

and O62 ec. is to be transferred to each of two blood ager plates, one for

aerobic incubation and the other for anaerobic incubation. The organism

If at the end of 72 hours a11 cultures from each pool are negative,

pensed into final containers. If the 7-dayor

teiaah Aeund ah. 3h been.

this should be reported to Dr. Melene

tamination.

*s Laboratory at the Presbyterian

herwise it will be assumed that the pool was free from con-

19.

Il.

If the 72-hour examination of the pool shows a contaminant in both

» If there is a contaminant in

only one of the two tubes, 22 ces. of thie pool should be recultured

cultures are sterile, the pool maybebottled.

ple of plasma from

the fresh pool for culturing, there should be added to each pool sufficient

fubbered 1-100 i olate to form a dilution of 1:10,000 Merthiolate in

the final plasma saline mixture. The safest way to be sure of the right

proportion is to add 40 cece. ofa freshly | i 1% solution to each pool-

ing flack containing 2,000 ces. of plasma. A simplermethod is to add 10ces. of a 4% solution of Merthiolate to each 2,000 ccs. pooling flack.

It hes been suggested that the Merthiolate powder be added to sterile

distilled water and bufferedby means ofchemically puresodium borate in

the ratio of 1 gm. Merthiolate to 1.4gm. sodium borate per 100ec. dig

tilled water. Or, in case of the stronger solution, 4 gm. Merthiolate to

506 gm. of sodium borate per 100 ec. distilled water.

Sueh pools are then #e@t aside in the refrig

☁etor until the 1 week

culture reports are returned. If theese reports are negative, then each

pool may be dispensed into the fimal containers. Zach Plaswavee containsec. physiological saline, to which is added 500 cc. of the pooled

- pheeua The last 35 ec. of plasma in the pool is run into a special con-

taining 35 e¢- of normal saline from the same batch ofnormal saline used in the Plasmavacs. This special bottle is to be labelled

with the date, the name of the hospital, the number of the pool, and the

trol bottle ¢o:

20.

Til.

Iv.

carton number in which the pool is packed. These control bottles willbe

sent with the carton to the warehouse where a messenger will pick them up

each day to carry them to the centrel laboratory, which is situatedin the

surgical bacteriological laboratory of the Presbyterian Hospital. Here re-

checks of the pools will be doneatone-week, and two-week intervals, be~

fore the cartons are finally releasedforshipment, a total periodof ob-

servation of one month.

POSITIVE CULTURES IN CENTRAL LABO!

IK HOSPITAL LABORATORIES

Should a positive culture be obtained in the central laboratory on a

plasma pool which has already been released by the hospital and sent to

the store-room, the whole pool will be brought to the central laboratory

and each flask in that pool will be separately cultured. The hospitals

therefore should have readily available records of the serial numbers of

of the plasmavac bottles which were put up from any givenpool.

fhe same procedure will be carried out if the two-week findings on

the individual hospitals should be positive following a negative one

week culture. This information is to be transferred to the central labora-

tery at once so that such pools may be retrieved from the storehouse and

recultured.

TOXICITY TEST.

In order to add a factor of sefety and to come within the spirit of

the law, one mouse will be injected intra~peritoneally with 1 ec. of plasma

from each control bottle, and ob: od for 72 how

☁ss It is not necessary

for this toxicity test to be done by the individua:

21s

COST OF PROJECT

The total cost of this project ing Cash receipts and cash disburse-

ments for the period from June 26, 1940 to Mareh 26, 1941 wae $42,087.2h. of

thie tetal $132.00 mes re

ing in Britain'sfight. Of ther

for shipping and for dispereal in Engl:

Red Cross. Mr. Norman Davis took a very personal interest in this unusus]

effort ae did Mr. DeWitt Smith and Dr. Williom DeKline of the National Office

in Yashington. To Capt. Charles 3. Scully of the Red Cross Lifessving Service,

sssisted by Colonel L. Booths, fell the tremendous job of supplying the hos-

piteles with an endless stream of donors, attimes asmanyas a day, for a

period of app:

from Inglend that their equipment andorganization wos sufficient to their needs

nd were taken care of by the American

tely five months. At the end of thie time word wis received

the individual hospitele spent large sume of their own funds in order te set

up a collection ceater which would meet the requirements of the Board of Medi-

cal Control of the Bleod Transfusion Association before that hospital was con-

sidered as # cooperating unit in the project. These costs, of course, do not

show and are not known exactly but the average outlay per hoepitel was in the

neighborhood of $3,000.00 for equipment.

22.

In order to lessen the burden to some extent each hospitel was paid the

sum of $1.50 for each liter of plasma-saline released for shipment. This

emounted to a total of $7,908-00 and is included as one of the items in the

total disbursement of $41,353-08- Usingthis tot

5,272 liters ae the total amount shippedthecostperliter ofpl

Was $7.84. Thie r

fhis compares very favorably with the current commercial price of plaema

which in different localities and institutions varies between 56(

$100.00 per liter. The differ

nee ie due to the fact that the blood wes given

and most of the lebor of a volunteer character.

From the very beginning of the project, its second purpos

above wes to secure all the information pos

ible concerning the preparation

and use of bleod substitutes which wight be of use to this country in case

of a national exergency. To this end Dr. Walter 3. Cannon, Chairman of the

Blood Transfusion Comaittee of the Hational Research Council, and Dr. Cyrus

G. Sturgis of Ann Arbor, Chairman of the Sub-Committee on Blood Substitutes,

were advised of each step in the procedure and their advice was sought es-

pecially in reference to the « h problems which presented themselves.

It is perhaps in the experimental work which has grow out ofthis project

that the greatest good will have been done.

Weither the literature nor past experience provided the answers to some

of the problems which arose during the project. Among these problems were:

23.

the best type of container for the collection of the blood; the safest appe-

ratus for transferring the plasma from the donor bottles to the pooling flasks;

the possibility of filtering plasma throu

ia bacteria filter; the best bac~

teriostatic agent for preserving th

urecompletelong time sterility;

num; the efficacy of plasma as a

☜ to serum; the toxicity and efficacy of dried

ed with liquid plasma or serum; the most suitable

apparatus for drying plasma or serw

about by the various methods ¢

blood substitute when compar

At this time no final answer can be given to all of the questions that

arose but certain conclusions are available as the result of work which has

been carried on by several investigators related to each of the above noted

problems. Taken in order they may be stated briefly as follows:

1. Donor Bottle. Itis the concensus of opinion of most of the men

who worked in this project that the ideal donor bottle should be es narrow

es ie compatible with stability, that it should hold between 550 and 600 ce.,

that it should be capable of being omnasi: uged, that it should be made of |

high quality glass, that it should be fitted with « stopper and attachments

which would allow the collection of blood and the removal of plasma without

exposure of the fluids to air, i-e., in s completely closed system. No such

bottle was available during the progress of this project. Several attempts

were made to create such a bottle and either the primary cost of the mold,

the inability to get centrifuge heads to fit or adequate capping devices

"prevented the realisation of this part of the progrem. Since the closing of

the program several of the larger commerciah intravenous fluid dispensing

2h.

wlves in the creation of such « bottle. The

virtues of such a bottle are: the slower rate at which the cells degenerate,

the increased yield of plasma, the incr

increased saving in refrigerator space.

freedom from infection, and the

mw. It was found that when the plasmafrom eight

| in just about the seme pro-

portion as the different bleed groups

r in the population as a whole that ♥

the resulting titre was never sufficiently high to cause coagulation of the

recipient's celle. ☁This criterion we feel to be a safe one. Complete sup-

pression of agglutinins may be brought about by using larger pools.

The Board of Medical Control at first earnestly requested and later

demanded that hospitals taking part in the program provide a dust-proof hood

or room, preferably air conditiond and bathed in ultraviolet light, for the

pooling of plasma. It was f

plasma from the blood and pooling it and thendistributingthe pooled plesne

that persone removing the

into the final containersuse surgicaltechnique throu,

out including the

wearing of sterile gloves, gown, cap and mask. It was also recommended thet

where suction for pooling is created by a motor and pwnp that there be a water

trap interposed in the line in order to prevent a backwash of contaminated air.

And that the plasma have interposed in the line between the donor bottle

the pooling bottle a stainless steel 120-mesh filter.

3. Filtration of Plesma. - Contaminated plasma picked up early in the

project suggested that an added safety factor would

be the filtering of all

plasma before final disp

neation. This proved a difficult job. The fibrine-

gen content of the plasma made filtration through the ordinary diatomaceous

earth or porcelain filter in large quentities an impossibility. Whenan

attempt was made to filter it through a Seitz filter it was found that for

25.

@ while the filtrate was clear but that later on, beginning with the last

portions filtered, not only was there the typical fibrin precipitate but

actual clotting in the absence of infection. Several investigators began

to work on thie problem. ☁Some interestingdata wcco

that the fine asbestos fibers in the Seite filters are enmeshedin a wood

pulp reticulum which is very rich in caleium in loose formation. This was

@ source of difficulty, first, by causing a rapid precipitetion of fibrino-

gen on the pads thereby clogging the pores and soon bringing the whole pro-cedure to a stop. Secondly, auch of the calcium was dissolved or washed

through by the plasma so that the calcium content of the filtered material

often was increased 200 to 300 percent above the level of the unfiltered

material. This, it is thought, played some part in the formation of thejelly-Like clot which was so often seen in the filtered material. Yinally,

it was felt that « part of the sodium citrate was removed from the plasma

by the filter pads. To i dicate this sourceof trouble three things were

done: (1) filter pads were created by the Seitz Company

ly calcium free, (2) when these pads were set Up in the filter racks a so~-

\c¢ acid was passed through the filters to fix

eny emall quantities of calcium which might har

lution of 1/10 W hydrochio:

☁® been present. These pads

were then weaehed withdistilled water to remo

f@ the excess acidity. (%) As

a final step a solution of weaksodium citrate was run through

the pads with

the hope that the suspected absorption of citrate from the plesma might be

prevented by this pre-treatment with citrate . This piece of research is to

be made the subject of a special presentation by Dr. Lester J. Uager when hie

studies are complete.

Me Separation

of

Plasma by Cream Separator Type of Centrif: <

n it

ent that the syphoning off of plasma, bottle by bottle, would always

26-

be eorather slow and tedious procedure, ways were sought in which the separation

might be done more rapidly. Having heard that a centrifuge of the Gream Sepe-

rator type had been successfully used in Snglend inquiries were made at the

creem separators modified so that itmights

& centrifuge was obtained from the Delaval Company and set up in the labora-

tories of the Manhattan Convalescent Serum Association and there tried out.

It has had insufficient trial on large scale produ

stion te unqualifiedly

recommend it for continuous mass production of plasmwe at this time but small

batches of blood have been run through it and the resultant plasma has been

free from both hemolysie ani celis. This modified cream separator was con-

nected to a specially constructed collecting chamber in which the plasma was

pooled and from which it wes forced with carbon dioxide under preseure through

the large Seite filters.

perly adjusted this series of piecesof apparatus

might well solve the problem of mass production of filteredplasmshouldthe

need arise.

5. In the beginning of the

project since bacterial filtration was not contemplated it was decided to use

some bacteriostatic agent. The agent of choice us "Herthiolate" (Sodium

Sthyl Mercuri Thisealicylate, Lilly). Ite use ws ais entirely satisfactory

and Mr. W. A. Jamieson, Director of the Biological Division of the Lilly Re-

search Laboratories, was good enough

to confer with us in Yew York on several

eccasions about thie matter. Dr. Meleney and Mies Johnson heave reported at

some length in an earlier part of thie program on this phase of the work. I

should like to correct here @ statement attributed to lr. Jamieson which oc~ ♥

curred on page 51 of the Heport of the Blood Transfusion Association. The

statement was: "I agree with Dr. Weleney that in order to have cultures of

any value, once the Merthiolate hae been added, the plasma saline mixture

would have to be neutralised and Ur. Meleney's suggestion that 1 cc. of 1s

ammonium sulfide be added toeach 10 ce. of plaaws and 50co. ofthe media,

I believe is a good one. The ammonium| ipitete mercury and

render it inert." Ur. Jamieson tells me thatthisisanincorrect statement.

Be has set up experiments to test this statement and finds that the emmonius

sulfide willnotprecipitate the mercury in ☜Werthiolate☝ and will not render

it inert in the dilution which is present in ☜Merthiolate.☝ I bring thie be-

fore you at this time in order thet your time may not be wasted in attempting

to neutralise ☜Merthiolate" with amzonium sulfide before culturing plasms so

preserved. For « more cowplete discuesion of the bacteriostatic action of

"☜Merthiolate" I refer you to the earlier paper of Dre Weleney and Hiss John:

One

When we received communication from Dr. Milan Novak of the University of

Tllinois of his successful maintene:x

ee of the sterility of blood plasma by

the use ofsulfanilemide we requested Dr. AiphonzoeDoches of the Presbyterian

Hospital to allow two of his residents and assistants, Dr. William Province

and Dr. Fred Heath, to repeat Dr. Hovaic!s work with the idea of making use

of this method in the project. This problem proved more difficult than it

first appeared. And at this time these investigators are not ready to give

a final report. Such, however, will be published in the very near future.

we ao, however, p Sthah

6 Element of in Study

in hie letter quoted above, stated: "It will be seen from the tables that the

na the works of Dre Novak for your consideration.

of Sacterial jures- Dre Pe Bruce White,

first period was one of some anxiety in that, of the 17 samples received from

Dre Heyl, ++e--+- no fewer than 7 and possibly 6 gave growth in culture media."

tf this was a period of anxiety for Dr. White it wes a period of greatest

286

distress for the Blood Transfusion Association and a matter of real concern

sat of Health, especially the Division of Biologics.

The roles concerning the culturing and testing of biclogice had been care~

fully followed and negative cultureshadbeenreportedb |

each of these bottles which was later foundcontaminated. Thiscabled re-

for the J. 5. Departm

aten the whole projectand weethe cause of

When the Central Laboratorywas established and pilot tubes from each

port from Engl

@ great raising of ste

final container were followed for at least three weeks before a negative

a for shipment, only one infected

horities there were advised ofthis posai-

report was turned in and the plasma releas

the present methods and criteria of establishing positive or negative cul~

tures, as routinely carried out in most hospital laboratories, are entirely

inadequate in such a large sealeproject as the one under discussion.

7- Plasme vs. Serum. When it became obvious that there was a differ-

ence of opinion in the minds of qualified observers as to the relative merits

of plaema and serum as blood substitutes a rather large scale experimental

program was set up in few York in an attempt to gather real date on this most

important problem. Arrangements were made whereby Capt. Charles B. Scully

and Col. Marle Boothe of the American Red Cross weve to supply donors to Dr.

Rhoads at the Memorial Hospital with the clear instruction to the donors

mt which might be of value te

the United States. This blood was to be collected at the Menorial Hospital in

thet they were giving their blood for an

uch @ manner that half of it might be used ae a source of serum and one~helf

used as a source of plasma and that each of these moieties could in turn serve

as @ source of dried muterial which wes to be prepared by Mr. Robert Folsom

in a drying unit which he hed constructed in order that comparative tests

might be run to determine the efficacy of these blood substitutes from the

same individuals on the sane recipients. The job of preparing this serum

and plasma for therapeutic trial was gra siou!

ly accepted by Dr. William

Thalhimer. Three sets of tests were to be carried out on each of these

different materials. One set consisted of chemical and electrophoretic

tests by Dr. Seudder of Presb

o Bospitel. The second type of testing

was to be carried out by Dr. Chambers of New York University and it consist-

ed of the perfusion of small mammals with serum or plasma and a recording

of the effect on the capillaries en seen under direct observat:

third series of trials were to be clinical in nature and were to be carr ed

out by Dr. Bdward B. Self and Dr. John Seudder at the Presbyterian Hospital.

There was an insufficient mumber of donors for thie specific purpose to

get reliable data before the close of the "Plasma for Britain" project, but

when the American Red Gross set up its first ble

@ center in New Yorkfor

the purpose of supplying dried plasma for our own army and navy an agreement

was made with Dr. Del

☁Line of the American Red Cross and Dr. Hhoads, repre-

senting the Mational Research Council, that the necessary bloods to continue

this experimentation might be obtained. ith thie increased supply more

definite results soon became available.

Dr. John Seudder has reported on the phase of the work which he under~

took in an earlier paper in thie program. Dr. Edward Self, associated with

Dr. Seudder, has likewise reported his results following the clinical trial

of both plasma and serum in a liquid form and dried form as well ss serum in

the fresh state as compared to serum several weeke old. I refer you to their

papers for more complete discussion of this most important question and ghell

only state here that it ie the feeling of the Blood1

fusion Association

thet both plasma end serum when properly prepared are equally innocuous

Serum, however, does have definite advantages. These are: the ☝

tentvolume for volume is row

ease through any type of bac filter, there is muchless tendency for

the material to become cloudy as the result of protein precipitation, and

since there is ac anticoagulant in serwn it may be dried much more efficient-

Ly. Por hospital bleed banks it appears the part of wisdom to continue the

use of plasma removed from blood which has become too old to use in the whole

forn.

The biological teste carried out by Drs. Robert Chambers, B. ¥. Zweifach,

u. J. Kopec, and ©. Hymanare, it enniteins of real significance in assessing the

properties of plasma and serun and with their permission I should like to in-

clude here their impressions to date. The full text of these experiments will

be the subject of a future publication. The following is an outline of their

methods and e summary of their findings to date:

"Perfusion of blood capillaries as a bio~aseay method for

the evaluation of transfusion media.

AUres

Micromanipulative investigations on the blood capillaries of the mesentery

have indicated that the responses of the small blood vessels to perfused fluids

and to micro-operations can serve as a blo-assay method in determining the ade-

quacy of various blood derivatives and substitutes which might be us

t of shock. The technique consists of the perfusion

fusion or in the t.

of artificial media through various tissues in situ permitting microscopic ob-

servations simultaneous with micro-operations of the vessels with mechanically

controlled needles. ☁The perfusions on the frog are made through the bulbus

aortae so that a circulation is maintained through the entire animal.

☜The present atudy deals with the perfusion of buman citrated plasma and

serum. Perfusions were aleo made with gelatin-2i

ra to whichcitrate, in

varying concentrations, was added inorderto rtain the specific effects

of citrate alone.

"Observations to date have been restricted to the capillary circulation

in the web, the tongue and especially the mesentery of the frog. The vessels

of the capillary bed in these tissues are striki

slight alterations in the perfusion media. The ability or inability of these

responsive to relatively

solutions to maintein the various asp

ete of the normal circulation thereby

served as a preliminary index of the relative efficiency of the two blood

substitutes.

nm Hospital and were diluted with 0.15

per cent NaCl (40 ce. plasma to 60 cc. Hall) to reduce the colloidal osmotic

pressure to the level of that of the frog. It should be noted that thispro-

cedure reduced the eonsentontion of the citrate to one-third that of the plaema

ag usually used for human transfusion purposes.

☜The citrated plasma wae obteined within a few hours after the phlebotomy

and the cells were then removed by centrifugation. An occasional sample show

ed some hemolysic, these being ted and treated as separate entities.

The serwa was obtained by allowing the fresh uncitrated plasmto clot for about

5 to 6 hours, efter which the serum wes decanted off without breaking the clot.

Care wee taken to avoid mixing the serum with cellular debris of the clot.

☜In order to obtain an adequate distritution of the perfusate, it was

necessary to use particulate matter in the perfusion fluid (fweifaeh, A. J.

30: 512, 1940). Three types were used: (1) human red cells, (2)

rooster red celle and (3) carbon particles. These were washed seve

times

and suspended in saline. Hematecrit readings were made end precautions taken

used only with homologousple

better than human cells on storage in saline and were used when human red

cells were unavailable. These cells could not be used with the serum samples

because of the marked hemolytic effect of the letter on rooster red celis.

Thus, the plasme perfusions were made with human and avien red cells and with

carbon, while ♥ was used only with carbon as the particulate component.

☜Determinations of the pH of the perfusion

medis were made with the

Beekman glase electrode. The citrated plasma samples were usually alkaline

(pH 7-9 to 8.2) and it was necessary to adjust the pil of these at least to

aboutpli 7-8 with phosphate buffers.

☜A mixture of oxygen and carbon dioxide wae bubbled throughthe perfusate

to maintein the necessary driving pressure and to serate and stir the sus~

pension. Such slow bubbling (about 1 bubble/second) produces « slight foaming

so that, wader ♥" perfusion conditions, a layer of froth about 2 em. high

rests on the surfece of the liquid.

thing, or the adsorption of proteins at oil-

water interfaces, may change proteins from a soluble to an insoluble state.

In fact, rapid frothing may actually bring about surface denaturation. It

was important, therefore, to

ine whether the bubbling of gas mixtures

through the perfusates could produce significant changes in solubility of the

plasma or serum proteins.

53¢

☜The results weres (1) The: .

id phase of the foam. (2) The

of the recovered, protein~containi

foaming was as p

The latter finding iadicates that the protein fraction remained surface

active and hence the native state predominated.

du Nouy tensiometer also failed to show any changes (significant) in eur

face tension (ligquid-gas) from untreated to bubbled plasma. The lack ef

catesthat the bubbled plasma did not gein

any increase in turbidityin

in insoluble or denatured proteins.

"It is safe to conclude, the

fore, that the amount of bubbling

6 during the course of the perfusion experi-

ment is far too low to produce any sig

"Orit

☜The specific capillary responses were: (1) effect on the rate of

blood flow and on the caliber of a, emall arteries and veins, b, arterioles

es; (2) development of a sticky

exudate and the formation of intra-capillary thrombi; (3) permeability of

Vessels in the capillary bed by observing hemoconcentration, loss of re-

and venules and ¢c, true and a-v capillar

tention of colloidal dyes, plastering of carbon and red cells against the

wall and (4) development of stasis.

"fhe observations included responses of the veesels to micromanipulative

prodding and trauma. Edema was noted groesly by observing visceral organs

and extremities.

"fhe object of the investigation thus far is to detect the direct effects

uted with the plasma of the host, on

the circulatory system. ☁Since the systemic reactions obtained by direct in-

fusion into the intect circulation are aot involved here, the results obtain-

ed may not be directly analogous with the results of transfusion studies, but

they may indicate tendencies as to the relative values of the two t

transfusion media (plasma or serum).

☜Results:

☜Citrated plasm

"Perfusion under a pressure of 30 to 40mm. Hg. maintained normal cireu-

lation for 30 to 40 minutes during which no abno

the capillaries which maintained their normal caliber end circulation. Im~

mal effects were observed on

pairment of the circulation after thie initial period was oecasioned by a fail-

ure at the venous outflow. The flow in the veins became increasingly sluggish

and the lumina gradually filled withmasses of redcells and sticky material.

These veins became enormously distended and, although a slow flow persisted,

the capillary ded wae no longer efficiently drained. The arterioles remained

patent and appeared normal. However, the perivascular snooth msecle elements

in the entire capillary bed gradually lost their normal responsiveness to

mechanical stimulation. After 15 to 20 mimutes of perfusion the skeletal

muscles went into tetany and then became non-responsive to nerve stimulation.

The unresponsiveness to prodding of the smooth muscle of the capillary bed

may be of the same order as that of the skeletal musculature.

"During the perfusion, observations in the web showed that the peripheral

arterioles become constricted and completely shut off circulation within two

to three minutes after the

☁ing of perfusion. Siwaltanecous observation

of the mesentery revealed that the mesenteric circulation was not affected

except in those cases where the concentration of citrate was raised from

0.03 to 0.3 per cent the mesenteric arterioles and larger vessels

underwent spasmodic contraction and became comp.

"No differences wereobserved in the effect

days) and stored plasma (2 to 3 weeks). The st

delicate fibrous precipitate which was always r

Use.

☜The toxic manifestations of the citrated plasma appear to be due

largely to the citrate ion and to the lack of calcium ☁fhe citrate apparent-

ly acts adversely on muscle both striated and smooth. The lack of calcium

prevents the cement elaborated by the endothelium from becoming deposited

in o stable form without apparently preventing ite secretion. As a result,

@ loose gelatinous material progressively accu

thus increasing the viscosity of the fluid and enhancing theformation of

rea cell thrombi.☜Previous experiments have shown that alkalinity stabilizes the calciun

ulates in the venous vessels,

proteinate which is a constituent of the intercellular cement. In the present

experiments, the alkalinity of the citrated plasma, which was never below pil

768, tended to cor

Cement. This is in sccordance with the known effect of pli on the caleium sslt

in alkalinity. Continued per~

fusion with the alkaline citrated plasma sctually toughens the wall of the

teract the effect of the lack of calcium on the formed

ionization which is decreased with ine:

Capilleries and makes them highly resistant to mechenical handling with micro-~

needles.

"Serum.

☜Perfusions at pressures of to 4 mm. Hg. maintained normal circulation

for several hours and proved superior to any of the previously used blood

substitutes. ♥

and even exhibited norwal rhythmic vasomotion during the period of observa-

ibited spontaneou

es and arterioles maintained their normal tonic state

| Contractions and @iletions,

tion. The arterioles alsoex?

frequently seen in intact circulation. fhe eapilleries usintained their

@ing of the capillariesproduced the usual☁sticking of carton locally, aphenomenon not found when citrated plasms was used. :

"During the severalhoursof observation(in some cases ¥ to 5 hours)

the capillaries maintained their noraal impermeability to the colloidal dye£1824 (so-called Iivans Blue).

"The skeletal muscles responded to nerve stimulation, the heart con-

tinued to beat, end no gross edema was evident in any of the organe. In

te, the 21624 cireulated through all tiseues in- |

normal tone and contractibility as e

ye perfusion experime

Cluding theskin. This effect was not obtained when citrated plasma was

☜In contrast to the above findings are the results obtained when

globin plus cellular debris) was added tothe serum

and to citrated plasma. The mixture with serum was extremely toxic, the

effects appearing within 5 to 10 minutes of pe

fusion (extreme epzem of the

arteries, vericose appearance of the veins). On the other handthe mixture

with plasme was no more toxic than the citrated placue. alone, except that

capillary stasis eppeared before venous back prese@ure became evident.

Me J* Kopac

CG. Hyman

Way 22, 1941Department of BiologyWashington Square Gollege of Arts and ScienceNew York University®

3T-

Thies biological assay gives evidence of the superiorityof serum over

plasma which is more striking than the clinical evidence though both seen

to indicate that the serum is a more physiological substance and therefore |

causes less reaction of an unfavorable nature on the part of the recipient.

One might bring thediscussion of this problem up to date by stating that

© suggests that storedplasms or serum maybe

used with freedom from reactions and with the expectation of good clinical

all of our date at this ti

results in shock, hemorrhage, end hypoproteinemia but that theadvantage

seens to lie on the side of serum rather than on the side of plasma as wethought less than o year ago. Serum from freshly clotted blood, however,

does have an increased reaction rate.

Dried serum and dried plasma, as prepared

by Mr. Robert Folsom of Menertad Hospitel in New York City by the apparatus

which he has already described to you as a part of this large scale experi-

mental program, in Dr. Self's hands have proved almost equally efficacious

in combatting the clinical conditions for which they are indicated. The re~

action rates were low in each instance and quite comparable. The electro-

phoretic and chemical changes in this material has been reported on by Dr.

Scudder in an earlier chapter. There is the t

ndency, it appears, for the

reaction rate to go up with the concentration of the material and it would

seen to be a wise precaution to use isotonic material, be it plasma or serum,

liquid or dried, unlees there be apecific indications for hypertonic fluids.

tHe UMaITRDSEATES 4oR BYTHE Al

In the report of the Blood Transfusion Association of January, 1941 a

plan was submitted for the establishment of o nationwide eystem of collecting

blood for the preparation of dried or liquid blood substitutes. Feb: a

at the Presbyterian Hospital in New York City the American Red Crosssetlp,under the direction of its ew York Chapter, the first unit in such a sys vn :

plasma and dispensing it in the 4 first mobile unit

for the ie eS eeoe end found to add so much to the

possibilities of reaching eivilis we that ;

many scattered parte of theMaumiEyeThis project 4PL ",

Ragland in a very dark} ee

mation in the fieldto our§

believe, have been sientigtey rt @

shy | program now goes on at an

a ad tts, the first, to aid

nd,OLEESOe

☁t+ and ite armed forces. Soth, we