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Practical notes TROPICAL HAEMATOLOGY

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1. ON CN CN DHT HAEMOGLOBINOMETER Developing Health Technology Ammonia technique 6 PRINCIPLE MATERIAL BLOOD COLLECTION REAGENTS METHOD PACKED CELL VOLUME BY CENTRIFUGATION or HEMATOCRIT PRINCIPLE EQUIPMENT AND SUPPLIES BLOOD COLLECTION METHOD REMARKS REFERENCE VALUES INTERPRETATION OF PCV RED BLOOD CELL INDICE MCHC SOURCES OF ERRORS QUALITY CONTROL CELL NUMBER CONCENTRATION GENERAL PRINCIPLE GENERAL MATERIALS COUNTING CHAMBERS WHITE CELL COUNT IN BLOOD _ REAGENT BLOOD COLLECTION METHOD REFERENCE RANGES QUALITY ASSURANCE SOURCES OF ERRORS RED CELL COUNT IN BLOOD RED CELL INDICES MCV MCH ADDENDUM 1 TECHNIQUES FOR ASSESSING ANAEMIA PRICE LIST ADDENDUM 2 SOME HAEMATOLOGICAL REFERENCE RANGES GUIDELINE FIGURES ADDENDUM 3 MORPHOLOGY OF BLOOD CELLS IN A MAY GRUNWALD GIEMSA STAINED BLOOD FILM 0901 22 pg tropical haematology and blood transfusion QN ON EA EN QNO lt A lt lt A Nn A A 3 77 BLOOD TRANSFUSION Short summary of basics genetics 6 Short summary of basic immunology 6 BLOOD GROUPING 6 A The ABO blood group system 6 B The Rhesus system 6 TRANSFUSIONAL RULES 6 COMPATIBLITY TESTS 6 Rapid test rapid cross match 6 Complete Major compatibility test in saline medium 6 followed by an indirect Coombs test in LISS Albumin medium 6 Auto test on the receptor
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3. Thoma counting chamber _ E Area 1 mm x 1 mm 1 mme Depth 0 1 mm Total volume 0 1 mm or ul Medium volume counting chambers Neubauer counting chamber HT ENE P Area 3 mm x 3 mm 9 mm TU rn oo Tin en Te aan Tr jd OS mm sss KNA T T 5 om E Neubauer double improved counting chamber Area 3 mm x 3 mm 9 mm Depth 0 1 mm Total volume 0 9 mm or ul 090122 pg tropical haematology and blood transfusion 28 77 Malassez counting chamber Area 2 mm x 2 5 5 mm Depth 0 2 mm Total volume 1 mm or ul Big volume counting chambers EEE LLI TEM TU OG Fe BEEBE SEER ESENE ESSE LUE CERN TU as B HSEEE pac ESE REE SERRE SES Total volume 3 2 mm or ul Fuchs Rosenthal counting chamber Area 4 mm x 4 mm 16 mm2 LI 0 554 i Nageotte counting chamber SSS Area 10 mm x 10 mm 100 Depth 0 25 mm 0 50 or 1mm Total volume 25 mm or ul ed 50 mm or yl EE 100 mm or ul ia The used type of counting chamber is a ma
4. ie Fausse an m ie vraie h m odilution Fig 3 True and false anaemia due to haemodilution PCV is decreased in anaemia PCV values are increased in case of loss of plasma dehydration cholera diarrhoea severe burns in dengue haemorrhagic fever and rarely in all forms of polycythaemia 090122 pg tropical haematology and blood transfusion 24 77 RED BLOOD CELL INDICE MCHC Red cell indices are frequently used in the investigation of anaemia If a laboratory is able to measure a PCV and to perform an accurate haemoglobin determination an MCHC Mean Corpuscular Haemoglobin Concentration can be calculated The MCHC gives the concentration of haemoglobin in red blood cells MCHC g 100 ml Haemoglobin concentration g 100 ml PCV x 100 Example A patient with a haemoglobin of 16 g 100 ml and PCV de 45 MCHC 16 45 x 100 36 g 96 Haemoglobin g 3 4 5 low incregsing anaemia haemoglobin normal 0 M C H C P di the dotted line i5 an example of how to E use this nomogram the patient s haemoglobin 10 15 20 25 30 3540 3 was 8 1 9 and his haematocrit 34 a ruler very low MCHC uos has been put across the scales as shown by De he dotted line it cuts the MCHC scale at increasing hypgchr a I t ii ss 24 the patient s MCHC is 24 Haematocrit 80 70 60 increasingly low haematocrit very low haematocrit Fig 4 MCHC Determination A guidelin
5. maarr acus doigt 57 yl de En el Me unt ue Cordata dd EDITA fa rome tim helt aka ee eee Arria mam i ee freq 4 Peuter et Perm ie CONTROLE DE Ue cnni diu i la aree Thed eit nett cr alin Jannar valid hi bant ig Dt 00 deel Are harage REMARQUES La bei ami di tee ceed ird crura caer ou pion bones la rcnt EH un ncn eru HDi m om die rper Ana iaci wire Bere Abba NTC Le Abc mii desir mcm de plasma maru tal uno Care Won pees jer wig eed pin etten an ee eism tort ens rm ar nn M pricove di Cun Can amel jor in ur wi dap EE run ede ele cuin cc D M E eM it RL pore wd na efectos ib hierin ce Ag tv atin Plt wen mr ra Y RICE ee SPECHWCITE Un er 190 chartnns de Vinum prm it provenant amet ai d d a th afud bar dee Alison Determine al After Duran iss ASHE Tabiamn i Faure ai HT Lars GE b IE erm
6. 090122 pg tropical haematology and blood transfusion 23177 INTERPRETATION OF PCV PCV is an easy simple but indirect technique to detect anaemia It is of diagnostic value in patients suffering from anaemia dehydration shock or burns The number of red blood cells the size of red blood cells and plasma volume influence the PCV If the number of red blood cell stays in the normal range 1 PCV represents more or less 110 000 red blood cells mm of blood As the ratio between the haemoglobin concentration in the red blood cells and the red blood cell volume is quite stable there is normally a linear relationship between PCV and haemoglobin concentration The formula PCV in x 0 3 2 gives roughly an idea of the haemoglobin concentration This is only true in case of normocytic or normochromic anaemia This formula will not substitute the haemoglobin determination Example the estimation of the haemoglobin concentration for a PCV of 33 is 10 1 g 100 ml 33 x 0 3 0 2 In case of a normocytic or normochromic anaemia the formula stays equal since hematocrit and haemoglobin are equally reduced Example For a hematocrit of 27 95 the haemoglobin is estimated on 27 x 0 3 0 2 or 8 4 g 100 ml In case of a macrocytic or hypochromic or megaloblastic anaemia but also in case of a microcytic anaemia this estimation does not work Volume plasmatique SE M dFe M M rM gt Volume globulaire Norm al
7. Caution Used values are only valid for the haemoglobinometer N 0931 that is used at the ITM Do not touch the clear working side areas of the cuvette Avoid contamination of these handling the cuvette only by the top of the etched surfaces at front and back non optical sides Always ensure the outer surface of the cuvette is completely dry to affect measurement A CHECK BLANK When filling cuvette ensure no air bubbles are present Install a CLEAN and DRY cuvette into the cuvette aperture Use the same kind of cuvette which is used for haemoglobin determination The value on the display must correspond with the blank value BR1 5 14 24 g l for our haemoglobinometer 0931 If this is not the case check for the following cause PROBABLE CAUSE REQUIRED ACTION again the reading for BR1 Check again the reading for BR1 Check again the reading for BR1 Value of scale factor M incorrect Zero level of the device incorrect Other type of cuvette used for the calibration Press and hold down the R button located at the back of the device The symbol HHH and after some seconds the scale factor M will appear Compare this value with the figure for M shown in certification 162 for our device If there is a difference between these values then adjust the displayed value by pressing the L button to lower the value and the R button to raise the value Check again the reading for BR1 Pla
8. Mazda T Lao POR Hood program final report 5000 2001 Japanese Red Cross Blood Programme Tokyo lapan Lin M The national blood program of Taiwan ROC letter Vox Sang 19948615 Lalezari P AF The manual Polrbrene test a dimple and rapid procedure for detection af red cell antibodies Transfusion 198r 20c2 06 11 Lin M Brnadberrr RE The modification of standard des ter peetrarefieton testing procedures far Tahran Vox Sang 194 202 c gt 2 fo min obtained from the Serum Cells and Rare Intemational Immunaohe matology Exchange Group SCARF which included antibodies rarely found in Taiwan two highly diluted commer cial MoAb anti D and anti B which were routinely used as dally positive controls far SP and and also several commerical antisera as shown In Table 1 The results show that SP detected most alloantibodies of clinical signifi cance especially anti E and anti These two antibodies are the most com mon alloantibodies of clinical signifi cance In Talwar and most likely also In the rest of southeast Asia Anti MI 18 used in Talwan to describe antibodies that react with RBCs af the phena type Other alloantibodies such as anti D K Jkt k Py and Py were also detected by SP with sensitivity similar to that of Although SP and MP are not as sensitive as LAT for the detection of anti E because most patients and dono
9. Description Macro Vue RPR Card Tests 274449 Kit No 104 300 qualitative tests contains two 3 mL amps antigen 20 G needle dispensing bottle 350 stirrers 30 cards with ten 18 mm Circle spots ea and 300 0 05 mL capillaries 275005 Kit No 110 500 qualitative tests contains three 3 mL amps antigen 20 G needle dispensing bottle 50 cards with ten 18 mm Circle spots ea and 500 0 05 mL Dispenstirs devices 275239 Kit No 112 150 quantitative tests contains five 3 mL amps antigen 20 G needle dispensing bottle 200 stirrers 50 cards with fifteen 18 mm Circle spots ea and 150 0 05 mL capillaries 275539 Kit No 115 150 qualitative tests contains one 3 mL amp antigen 20 G needle dispensing bottle 15 cards with ten 18 mm Circle spots ea and 150 0 05 mL Dispenstirs devices 275110 Bulk Kit No 510 5 000 qualitative tests 275692 Bulk Kit No 532 10 000 qualitative tests 276709 Macro Vue RPR Card Test Control Cards containing graded reactivity specimens R RM and N 18 mm circles Box of 10 272905 Dispenstirs single use plastic pipettes 0 05 mL Box of 500 REFERENCES 1 Larsen S A V Pope Johnson and E J Kennedy ed 1998 A manual of tests for syphilis 9th ed American Public Health Association Washington D C 2 Larsen S A V Pope and Quan 1992 Immunologic methods for the diagnosis of spirochetal diseases p 467 481 In Rose de Macario J L Fahey
10. Friedman G M Penn ed Manual of clinical laboratory immunology 4th ed American Society for Microbiology Washington D C Portnoy J J H Brewer and A Harris 1962 Public Health Rep 77 645 652 Portnoy J 1963 Military Med 128 414 417 Portnoy J 1965 Public Health lab 23 43 Reed E L 1965 Public Health Lab 23 96 103 Reed E L 1966 Public Health Lab 24 203 206 Reed E L 1968 Public Health Lab 26 123 133 Reed E L October 22 1968 Presented at FTA ABS Test Seminar co sponsored by Md State Dept of Public Health Bureau of Laboratories and the NCDC VDRL 10 Reed E L 1969 J Conf Public Health Lab Dir 27 8 14 11 Walker A N 1971 Br J Vener Dis 47 259 262 12 Croix J C 1975 Feuillets de Biologie 16 61 65 13 Larsen S A D E Pettit M W Perryman E A Hambie R Mullally and W Whittington 1983 J Clin Microbiol 17 341 345 14 Warner G S et al 1980 Data on file Becton Dickinson Microbiology Systems 15 National Committee for Clinical Laboratory Standards 2001 Approved Guideline M29 A2 Protection of laboratory workers from occupationally acquired infections 2nd ed NCCLS Wayne Pa 16 Garner J S 1996 Hospital Infection Control Practices Advisory COmmittee U S Department of Health and Human Services Centers for Disease Control and Prevention Guideline for isolation precautions in hospitals Infect Control Hospital Epidemiol 17 53 80 17 U S
11. prevent any income the ted result shoukd not be inizzpeeted after 20 mires B Limitations of the test negative result dees net prechide the possibility of infectie with HCV Other clinically available tests ane tespierd id questionable results are obtained As with all diagnostic tests a definitive clinical diagnosis should be based on the resudts of single teal hoali ondy he rade by the piisiciam after all clinical and laboratory findings have been evaluated 9 Performance Characteristics L Sensitivity and Specificiny The SD KIOLINE HCV have teel with positive and negative clinical samples tested by a accurate other commercial ELISA ka In camparison of the SD BIOLINE HCY versis leading commercial anti HC V ELISA ist roule gave sensltivsny of CE 206 7208 speeificsry of 99 496 419 7 500 and a val agreement of 33 155 PV TOS 2 Precisim L1 Within ren precizion was determined by asing 10 replicates of four different specimens containing different concentrations of antibody The negative and positive values were identified T0058 of thee tire ee eee eT E Comas different concentrations al in 3 differen rep icates with A different hets of ies devices Again negative amd positive results were observed of he line 10 Bibliography of suggested reading 11 CL Creslaw Wa Chao Lin Stephen M Feinsionc and Charles M Ric
12. rere allee ih melt Lini al E 1 pee eene acuti arel bieren pee wii 1 bsn pae bre doe rem achitksn be added anl alid li geni rocked Erp Band TABLE zm o a rl ctl netties darelakline meles ler ming diui mye the vie of fe equipee ing orig belden nem die rici Heeren bar nonkel rage max hs irem may er course om iem ae cde bann Ad a mumi Hand Els bere rus Hip In liene Thee DEG Le Peed Crom Peel Vien lms ara an im Tad wan hrs he in Visine TORT VE DES rente cg il Mares waa diackensd Sal very bee DEBE eee ras in ral eo servers ef ileer ceri Terror ibe derb pueri wj campanile ep ei prore dos del ui require der utar el a age oa rper ani wr magr n n sean cw pai cuba Fa Hn be ig tad blo peo gram Tad DOR rnc ra ied the rapi las of lain reple resi esl ka being uarslaxline beeing Pires Fieke bia treed relie Ea wisi cl Eon pry in Talem arl only En inc ing rrapi boos banks hej sabapa peri bee al al in jpu r ar pnr ame omg PAR Walem pretu P Wi pU rei a Pe e eb Ee a UR pie pror jet de deter of BBC acidi Hid ies Indice at Mackay Raed all Dom years
13. test and permits the demonstration of irregular incomplete antibodies of type IgG Living blood bank registered group of people living nearby the hospital with a known blood group which accept to donate freely their blood in case of need Monoclonal antibodies Very specific antibody products from one single cell or identical precursor of this cell directed against a specific epitope of an antigen As the opposite of polyclonal antibodies Natural antibodies Antibodies found in the serum without apparent pre immunisation from the corresponding antigen They appear apparently spontaneously If this is not the case they are called immune antibodies Packed cells blood bag containing mainly red blood cells obtained after elimination of the plasma and the white blood cells Regular antibodies antibodies present in all subjects in absence of the corresponding antigen As the opposite of irregular antibodies Irregular antibodies occur also naturally but not in all subjects Saline medium physiological medium that contains 9 g natrium chloride NaCl per litre of distilled water This is an isotonic solution that permits to preserve the cell volume Transfusion of full blood transfusion of a blood bag containing red blood cells white blood cells and plasma As an opposite of fractioned blood permitting to transfuse packed cells erythrocytes plasma etc Transfusion Warm transfusion of a blood bag immediately after t
14. 16 is Reactive proceed as follows 1 Prepare a 1 50 dilution of Nonreactive serum in 0 996 saline This is to be used for making 1 32 and higher dilutions of specimens to be quantitated 2 Prepare a 1 16 dilution of the test specimen by adding 0 1 mL of serum to 1 5 mL of 0 9 saline Mix thoroughly 3 Place 0 05 mL of 1 50 Nonreactive serum in circles 2 3 4 and 5 4 Using capillary place 0 05 mL of 1 16 dilution of test specimen in circle 1 5 Refill capillary to red line make serial two fold dilutions and complete tests as described under steps 3 to 6 See 18 mm Circle Quantitative Card Test Higher dilutions are prepared if necessary in 1 50 Nonreactive serum Plasma f a baseline is to be established from which changes in titer can be determined the test should be repeated on unheated serum see section Unheated Serum Reading and Reporting the Macro Vue RPR Card Tests Individual reactions should be evaluated in the wet state under a high intensity incandescent lamp or strong daylight Immediately following rotation read and record as Reactive or Nonreactive RPR 18 MM CIRCLE CARD TEST READING GUIDE QUALITATIVE TEST Reactive Reactive iMaderate Minimal Honreactive Beactiye Report as Reactive QUANTITATIVE TEST Undiluted 1 7 1 4 1 4 1 15 Reactive Reactive Reactie Monreactsre Honmreactive Quality Control Quality control requirements must be performed in accordance with applicable lo
15. 2007 Hemocue Disposable micro cuvettes Hemocue 301 200 cuvettes 75 July 2007 Hemocue Batteries 4 Type N alkaline cells 6 35 Feb 2004 DHT DHT HAEMOGLOBIN METER Haemoglobin meter DHT HB 523 543 Feb 2004 DHT Ammonia 30 96 1 litre 5 35 Feb 2004 VWR Corrosive and irritant Sahli pipette 1 34 Feb 2004 DHT Graduated glass pipette 2 ml 1 93 Feb 2004 VWHR or dispenser 2 ml Ceramus 217 Feb 2004 VWR Devices for pipetting 2 92 Feb 2004 VWR Optical cuvettes 10 mm light path 100 cuvettes 4 58 Feb 2004 DHT Batteries 4 Type N alkaline cells 6 35 Feb 2004 DHT Bottles Test tubes Test tubes rack SAHLI e Sahli kit 57 35 Feb 2004 VWR e Hydrochloric acid 37 96 9 35 Feb 2004 VWR Corrosive and irritant GENERAL Blood lancet Disinfectant Sodium hypochlorite or chlorine releasing disinfectants Cotton Alcohol 70 e 090122 pg tropical haematology and blood transfusion 34 77 ADDENDUM 2 SOME HAEMATOLOGICAL REFERENCE RANGES GUIDELINE FIGURES Wats grom So A grom e 3 12 months 100 140 30 56 26 41 284 400 70 105 5 5 17 5 3 12 months 100 140 30 56 26 41 284 400 70 105 5 5 17 5 1 12 years 10 5 15 0 3 8 5 5 32 42 320 370 72 94 50 140 1 12 years 10 5 15 0 3 8 5 5 32 42 320 370 72 94 50 14 0 Male 12 100 years Europe 13 2 17 3
16. 6 In case of positive compatibility tests in saline medium 6 Test of minor compatibility 6 Table 5 Comparison of different major compatibility tests useful on district laboratory level 6 Table 6 Use of compatibility tests on district laboratory level described in the notes 6 Table 7 Blood transfusion on district hospital level from minimum until extra possibilities 6 ANNEX 1 Wash of the red blood cells ANNEX 2 Screening of dangerous O donors ANNEX 3 Screening for infectious diseases ANNEX 4 Compatibility test on slide Polybrene method ANNEX 5 Indicative price list Diamed blood grouping http www diamed ch ANNEX 6 HIV rapid test example ANNEX 7 HBsAg rapid test example ANNEX 8 HCV rapid test example ANNEX 9 RPR test example ANNEX 10 Relation rotation speed of a centrifuge and the centrifugal power ANNEX 11 Useful internet sites GLOSSARY A OD OD NH NH 090122 pg tropical haematology and blood transfusion 4 77 BLOOD COLLECTION CAPILLARY BLOOD Capillary blood is the cheapest and the easiest method for blood collection Capillary blood is mainly used when the volume of required blood is small up to 100 ul Disadvantages in using capillary blood for blood test include e Great possibility of sampling errors particularly when the blood is not free flowing dilution of the sample with tissue fluid e Difficulty in obtaining sufficient blood for more than one test e Rapid clotting o
17. IE S art SIS Aba marem de Muni 55 dO peo tees Ebm uns Panay ln keene SE lasser ten hoo rena de SOG PRS i ded niemi publie a Chape A docte Tea Abbor fraibraaerrsen Pd pour f Apa im te 4 Mes Lang a OMIT DTA MEA t aida EWI l ee 67 77 ANNEX 8 HCV rapid test example 5 HCV 1 Explanation of the test Hepatitis C virus HCV ru ii mode aa major agent of cheonie hepatica traasfudion acquired pon A non B bepotitis and liver the werkd HCV is an esveloced penitive sense single stranded RNA vires Clinical diagnostic issues relied io de detection of HEV antibodbes in beman serum plasma cr whole blood by immumoaisay We have constructed HOV genes for the expression of recombieanr antigens in bacterium sprema moch m E coli focused on troche amd pon sbnactural regions of HCV encoded pedypensein which are definitely irmmatagenic The major irumanorscttve antigens of these have been reported as core N83 NSA and N35 region of HCV wich an known to be highly imimunodominant regions Por diagnosis of HCV infection TOM peoieins were usex a capture materials of immani himah rapid hest Compared amp the first generation HEV cb miig sin
18. Ottemberg in 1911 This compatibility in the ABO system is an essential condition for transfusions Based on this principle the blood compatibility can be resumed by the Pa scheme for blood TD NNS Moreover the blood grouping major compatibility test and the rapid cross match permits to detect the presence of this type of antibodies Qo Minor conflict It is also important to avoid to transfuse blood especially when it is complete blood containing antibodies directed against red blood cells of the receptor This problem is greatly avoided by transfusing in iso groups To determine the presence of other antibodies a test of minor compatibility can also be executed 2 Avoid as much as possible the production of antibodies Another important remark in blood transfusion is to avoid introduction of an antigen especially when it is very immunogenic which the receptor does not possess Principe non nocere n fact this introduction will bring along the production of antibodies which may have dramatical consequences for future transfusions or for future pregnancies Summary for transfusions on district hospital level For whole blood with ABO and Rhesus grouping gt Execute as much as possible transfusions in iso groups ABO and if impossible follow table 3 on next page gt A Rhesus negative person must be transfused with Rhesus negative blood 090122 pg tropical haematology and blood transfusion 46 77 Ta
19. achieved immunity depends on the prevalence of the disease in the population This is not true for young children e Some infectious agents are only present in cells and are not transmitted by cell free blood components such as plasma malaria for example this aspect is to be considered only if separation of blood components is feasible Other agents are present and infectious in cells and cell free components e Some infectious agents are killed or at least their virulence is weakened after blood storage for 72 hours at 4 to 7 C syphilis trypanosomes This could be kept in mind if storage is feasible and safe e Information given to blood donors in order to teach them about at risk behaviour and to encourage them to self deferral is less expensive less dangerous and probably as useful as testing to discard dangerous blood units more relevant for sexually transmitted diseases When financial support is limited local priority should be given to various screening tests according to the prevalence of the carrier state in the general population the consequences of infection for the recipient s health and the age of recipient 090122 pg tropical haematology and blood transfusion 61 77 WHICH TESTS CAN BE USED IN A REMOTE AREA TECHNICAL CONSIDERATION Number of blood transfusions Blood bank type Cold blood Test before during after blood unit s collection Test availability Test complexity Time Human resou
20. antigen is below the breakline snap the ampule neck and withdraw Yellow 20 30 1 drop all of the antigen into the dispensing bottle by collapsing the bottle and using it as a suction device Label the dispensing bottle with the antigen lot number expiration date and date antigen was placed in the bottle Shake the antigen dispensing bottle gently before each series of antigen droppings The needle and dispensing bottle should be discarded when the kit is used up It is imperative techniques as described herein be followed in detail 18 mm Qualitative Card Test Using Dispenstirs Devices 1 Hold a Dispenstirs device between thumb and forefinger near the stirring or sealed end Squeeze and do not release pressure until open end is below surface of specimen holding the specimen tube vertically to minimize stirring up of cellular elements when using original blood tube Release finger pressure to draw up the sample 2 Holding in a vertical position directly over the card test area to which the specimen is to be delivered not touching card surface squeeze Dispenstirs device allowing one drop to fall onto card approx 0 05 mL each Dispenstirs device is designed to expel slightly in excess of 0 05 mL to compensate for small amount of specimen retained by stirring end 3 Invert Dispenstirs device and with sealed stirring end spread the specimen filling entire surface of circle If desired sample remaining may be discharged into specim
21. blood at the 0 020 mark of Sahli pipette Do not allow air bubbles to enter Wipe the outside of the pipette with absorbent paper Check that the blood is still on the 0 02 ml mark Expel the blood into the test tube Rinse the pipette by drawing in and discharging the fluid from the test tube 3 times The dilution of blood is 1 on 20 Mix the diluted blood well and wait 3 minutes before filling the counting chamber red blood cells lysis Assemble the counting chamber Prior moistening of the chamber surface on each side of the grid areas b is necessary for the cover glass to adhere to the chamber Slide the cover glass into position over the grid areas and press down on each side until rainbow colour Newton s rings are seen Tiefe RH TO mm lt E ge E Bonsen en B Neubauer counting chamber Mix the diluted blood well Use a Pasteur pipette or a Sahli pipette to fill the counting chamber completely Take care not to overfill beyond the ruled area Caution if the liquid overflows into the channel between the two chambers you must start again remove and clean the coverslip clean the counting chamber and refill with another drop Leave the counting chamber on the bench for 3 minutes to allow the cells to settle 090122 pg tropical haematology and blood transfusion 30 77 10 Place the counting chamber on the microscope stage 11 Using the 10 x objective with the condenser
22. dominant allele If both alleles are expressed together they are co dominant A recessive allele is manifested only in the homozygote The genetic composition for a particular inherited characteristic is called the genotype gene composition and its manifestation or biological effect is called the phenotype gene expression Short summary of basic immunology Immune reactions used or involved in the blood group determinations in post transfusion reactions or compatibility tests are mostly humoral immune responses Principal characteristics of the humoral immune response Essential contact with an antigen Immune tolerance The capacity of the organism to make it unresponsive to foreign or self antigens Specific response of the antibody against the antigen Immunogenecity The capacity to induce the formation of antibodies is different for different antigens Antigenic power A step in the antibody response Production of different antibody classes Memory difference in primary and secondary response E Em Fig 1 Immune response evolution Antibody level Latency 8 to 15 days Latency 2 to 3 days Days Primary Secondary immunisatio immunisation boost PRIMARY RESPONSE SECONDARY REPOSNSE 090122 pg tropical haematology and blood transfusion 39 77 BLOOD GROUPING A blood group is a group of individuals who has a allotypical character in common which distinguishes them from other groups Thi
23. en 9 1 e Cd rri 2 Relation between centrifugal g and the number of rotations per minute rpm in function of the radius of the rotor of the centrifuge r Combining the two values r and g with a line an intersection is obtained with the line of the rotation speed of the centrifuge the value in rpm to use for this centrifuge and this rotor The relation formulae between g and rpm is g 0 00001118 xr x rpm N B the radius of the centrifuge is expressed in cm 090122 pg tropical haematology and blood transfusion 73 77 ANNEX 11 Useful internet sites World Health Organization in French English and Spanish documents can be downloaded from blood transfusion http www who int topics blood transfusion fr International Society of blood transfusion in French and English http www isbt web org Public Health Agency Canada in French and English http www phac aspc gc ca hcai iamss tti it risks f html tab2 Site of the Canadian Society of blood in French and English http www medecinetransfusionnelle ca Site de l h movigilance en francais http www hemovigilance org oite francais de l Institut National de transfusion sanguine INTS en francais WWW ints fr Site du service du sang de la croix rouge de Belgique en francais http www transfusion be 090122 pg tropical haematology and blood transfusion 74 TT GLOSSARY Agglutinins antibodies which
24. irregular antibodies of IgG type antibody anti Kell for instance so feasible in Asia but not often in Africa or in Europe Necessitates an electric centrifuge Necessitates a big enough quantity of receptor blood serum Small anaemic children Reagents rather difficult to prepare locally Rather complex Sensitivity and specificity not as good as in LISS albumin medium Necessitates a big enough quantity of receptor blood serum Small anaemic children Necessitates a centrifuge and a water bath electricity Moderate expensive Rather complex Slow 60 minutes Necessitates a big enough quantity of receptor blood serum gt Small anaemic children Necessitates a centrifuge and a water bath electricity Rather expensive Rather complex Table 6 Use of compatibility tests on district laboratory level described in the notes Type of test Rapid Cross Match Major compatibility test in saline medium Major compatibility test in indirect Coombs in LISS albumin medium Minor compatibility test in saline medium Minor compatibility test in indirect Coombs in LISS albumin medium Transfusion of whole blood or concentrated red blood cells In Iso group Transfusion of whole blood or concentrated red blood cells In iso group In non iso group Transfusion of whole blood or concentrated red blood cells In iso group In non iso group Transfusion of whole blood or concentrated red
25. is own to human and to red blood cells It is the most immunogenic of the blood group systems A subject possessing the D antigen on the surface of his red blood cells is called Rhesus positive D or Rh A subject who does not possess the D antigen is Rhesus negative D or Rh or d This system does not possess natural antibodies So a normal person does not possess any anti Rhesus antibodies in his plasma They appear by immunisation as a consequence of blood transfusions or by pregnancy The Hhesus antibodies are immune antibodies warm of the IgG type incomplete non agglutinating in saline solution Table 2 Geographical distribution of the D antigen Percentages of Rhesus positive persons South East Asia and the Pacific 98 to 100 Equator and Chilli 91to 97 Brazil and Argentina 82 to 94 Africa Bantus Ethiopians 94 to 97 Africa others 82 94 96 Western Europe and North America 80 to 85 95 Approximate average percentages Marked differences can occur between ethnic groups 090122 pg tropical haematology and blood transfusion 43 77 Technique for the Rhesus grouping on slide restricted to antigen D The determination of the Rhesus group rests on the detection of surface antigens of red blood cells Therefore known serum is used directed against these D antigens This antiserum agglutinates red blood cells which possess antigen D For the slide technique the D antiserum must be of the Ig
26. mas Geo ijn rad vel ae rem irit ein pretnansfaeRen being preceJares ten fe hee of Tifa dnce be lage duel eal an se Po Dre nee curs h BAPE pen an MI amp denied Ihe bapi ed dt ree pietas are eee and Tn be pepe me housse 18 aT ord bank pet ionge mapas gay HH Ia dg vo PRG TEE Tis Hh Be ue SF ales ees god merit Bor heb el arti blies thet dne cd nc signifi cans in io eed alei 1 joa Poh felts bas The res a wat die ite are e ohare mapa For abal niih can bet ceded deg a mgd ee Feud viua Des eye ee pee ie ceri io laar an j mtr alos das veg nr edt b n heen saw puri qa roids acd mecha a werke redes AREE KE AGIT AA HIEMS CORIN Aral AP es Lb or maid sun an eme ried arl eg nep eygene dar en gede phum engel Test eee ie ep ad hy hilii seline DAT on ger tell iti ad ATSC Few nb ds reris rg ri LE al annbagin Gt PRES DU EE Vei LUE Hr PR C Tone Al HESULTS Kenger peux dom prd si dens o ST EAT aed LAT dien been el kee ml rekenen ane d Tales E T Fein i cars em mn SP ana EAT aen rp pe semi ro ibus LAC
27. or at least the rapid cross match must be absolutely performed Verification of the ABO compatibility A The ABO blood group system R publique Francaise Minist re de la sant de la famille et des personnes handicap es CIRCULAIRE DGS DHOS AFSSAPS N 03 582 of 15 december 2003 relative la r alisation de l acte transfusionnel et CIRCULAIRE DGS SQ 3 N 99 14 du 12 janvier 1999 relative au respect de la r glementation en vigueur pour la d termination des groups sanguins ABO 090122 pg tropical haematology and blood transfusion 40 77 The ABO blood groups system was the first to be discovered This was attributed to Karl Landsteiner In 1901 For this observation which he described as the ABO 0 blood group system Landsteiner was awarded the Nobel Prize for medicine 1930 The ABO system presents an important characteristic that is at the same time at the origin of techniques of blood grouping and that explains also its crucial role in blood transfusion the constant presence of antibodies anti A and anti B corresponding with the absent antigens on red blood cells of the subject The ABO antigens are terminal sugars of complex membrane macromolecules The addition of these sugars is coded by a gene This gene bears 3 alleles 1 inactive enzyme no added sugar 2 A N Acetyl galactosamine transferase enzyme 3 B Galactose transferase enzyme The transmission of the ABO groups follows the laws of Mendel A an
28. patient heamoconcentration Secondary haemolysis to forcible passage through a fine bore needle Storing the specimen beyond 6 8 hours before performing the test Disintegration of leukocytes when specimen stands Blood collection from an arm in which an intravenous infusion is being given haemodilution Using an inappropriate anticoagulant Sample coagulation When using anticoagulated blood not mixing the blood sufficiently or not checking the sample for clots Counting chamber or cover glass dirty Incorrect measurement of blood or dilution fluid due to poor technique or using a wet or chipped pipette Dilution fluid contaminated with dust particles Inadequate mixing of blood with diluting fluid Insufficient lyses of RBC may create problems in identification of leucocytes Air bubbles in the counting chamber or in the pipette volume error Inappropriate covering of the counting chamber volume error Over filling a counting chamber or counting cells when sample contains air bubbles Counting chamber is not sufficiently filled Not allowing sufficient time for the cells to settle in the chamber Use of a too intense light source or not reducing the iris diaphragm to give good contrast Calculation error Administrative error RED CELL COUNT IN BLOOD The same kind of technique can be used for the red blood cell count Unfortunately the precision is poor and it is not recommended for clinical practice To calculate
29. procedure on reactive donor samples The RPR Card Tests cannot be used for testing spinal fluids The ideal specimen for neonatal testing is the infant s serum as obtained by heel stick procedure However cord blood may be used for baseline screening when no other specimen is available 1 With cardiolipin type antigens biological false positive reactions have been reported in diseases such as infectious mononucleosis leprosy malaria lupus erythematosus vaccines and virus pneumonia In leprosy Portnoy3 reported no false positives Achimastos19 reported 14 of 50 leprosy cases were Reactive and Scotti20 reported 1 out of 208 cases was reactive with RPR Card which were nonreactive with the FTA ABS and TPI tests Dorwart 21 studied the incidence of chronic BFP reactions in various connective tissue disorders Six out of 41 cases of systemic lupus erythematosus were reactive in the Card Test whereas only 5 were reactive in the VDRL slide test Only 1 out of 23 cases of rheumatoid arthritis was reactive with both RPR Card and VDRL slide tests In pregnancy several reports indicated the occurrence of false positive reactions 11 22 Narcotic addiction and autoimmune diseases also may give false positive reactions 23 Pinta yaws bejel and other treponemal diseases produce positive reactions in this test 1 Lipemia will not interfere with the card tests however if the degree of lipemia is so severe as to obscure the state of the antigen particles the specimen
30. provoke agglutination of the red blood cells It always concerns IgM Allo antibodies iso antibodies antibodies form against foreign antigens Antibody specialized proteins immunoglobulins which bind specifically and react with the antigen As a rule antibodies to an antigen are only formed if the corresponding antigen is missing in the antibody forming organism One Immunoglobulin will react only with a specific antigen specificity There are 5 classes IgM IgG IgA IgE 190 Antibodies warm IgG antibodies with a maximal activity at 37 C Antibodies cold IgM antibodies with a maximal activity at 4 C Antigen A substance which is recognized as foreign by a living organism and as a result induces a specific immune response Every molecule recognized by the immune system is described as an antigen This predominantly concerns proteins which occur either in the pure form or combined with other substances Auto antibodies Antibody form against own antigens NA Alloantibody against foreign Blood donor familial family member who is voluntary giving his blood Blood donor voluntary altruistic blood donor who is not compensated for it in any way Blood donor regular volontary regular altruistic blood donor who is not compensated for it in any way Blood donor Remunerated a person who is selling his blood Blood donor Replacement familial blood donor in the c
31. rotation Storage of Antigen Refrigerate at 2 to 8 C All other components of the kit should be stored in a dry place at room temperature in the original packaging See Warnings and Precautions for additional information Once placed in the dispensing bottle provided in each kit and refrigerated 2 to 8 C the antigen reactivity remains satisfactory for approximately three months or until the expiration date if it occurs sooner Label the dispensing bottle with the antigen lot number expiration date and date antigen was placed in the bottle SPECIMEN COLLECTION AND PREPARATION No special preparation of the patient is required prior to specimen collection To Test Unheated Serum Collect blood by venipuncture into a clean dry tube without anticoagulant and allow to clot Centrifuge the specimen at a force sufficient to sediment cellular elements Keep the serum in the original collecting tube or transfer the serum into a clean dry test tube if testing is to be delayed Serum removed from the clot may be refrigerated at 2 to 8 C for up to 5 days or frozen at 20 C or below in a Pyrex or equivalent vial or capped test tube 1 Avoid repeated freeze thawing of specimens 090122 pg tropical haematology and blood transfusion 69 77 To Test Heated Serum After collection and centrifugation as for unheated serum transfer to a clean dry tube and place in 56 C water bath or a heat block for 30 min To Test Unheated Plasma Collect bl
32. the MCV and the MCM indices an accurate red blood cells count is needed This needs an electronic cell analyser Most district laboratories will not therefore be able to calculate these indices However examining a well stained blood film can help to detect macrocytosis or microcytosis 090122 pg tropical haematology and blood transfusion 32 77 RED CELL INDICES MCV Mean Corpuscular Volume MOV mmn x 10 x 1075 Red blood cell count per ul in millions Reference range 82 to 92 fl for adults Age related variation for children a femtolitre fl is 10 of a litre FOR ADULTS microcytic anaemia lt 82 normocytic anaemia gt 92 macrocytic anaemia MCH Mean Corpuscular Haemoglobin Haemoglobin in g l Red blood cell count per ul in millions Reference range 28 to 32 pg for adults Age related variation for children FOR ADULTS gt 32 hyperchromic anaemia 090122 pg tropical haematology and blood transfusion 33 77 ADDENDUM 1 TECHNIQUES FOR ASSESSING ANAEMIA PRICE LIST HCS Starter kit containing 1 Cover box with 1 dispenser and 200 test strips 1 Booklet with Colour scale 1 instruction manual 4 refill dispenser with each 200 tests strips Price 21 80 Feb 2004 Copack Refill kit containing 10 dispenser boxes with each 200 test strips total 2000 tests Price 32 75 Feb 2004 Copack MICROHAEMATOCRIT CENTRIFUGE Centrifuge DHT 590 832 25 Feb 2004 DHT Centrif
33. 00 6 000 5 000 5 000 4 000 to to to to to to to to 30 000 20 000 18 800 17 500 17 000 14 500 13 500 11 000 QUALITY ASSURANCE For patient samples done in duplicate the difference between the two counts should not be more than 20 For statistical reasons the precision of the measurement will decrease with the number of counted cells To decrease the errors for low counts it may be good to repeat the count using a lower dilution or counting the cells in more than 4 large squares Caution in these cases the calculation should be adapted It can be good to compare the distribution of the cells in the 4 large squares from the Neubauer chamber The number of cells counted in each of the 4 squares should not differ by more than 10 Cf also addendum 2 References ranges vary in different population and in different laboratories different techniques District laboratories should check the above figures for the technique in use with their nearest hematology reference laboratory 090122 pg tropical haematology and blood transfusion 31 77 SOURCES OF ERRORS Position of the patient during the blood collection haemoconcentration Nucleated red cells may cause an erroneously high count of WBC This can be corrected by determining the proportion of nucleated red cells to white cells on a blood film Prolonged stasis caused by constriction with a tourniquet for more than 1 minute haemoconcentration Cells counting from a dehydrated
34. 2 to 3 to 4 to 5 mixing after each transfer Discard 0 05 mL after mixing contents in circle 5 4 Using a new stirrer broad end for each specimen start at highest dilution of serum circle 5 and spread serum filling the entire surface of circle Proceed to circles 4 2 and 1 and accomplish similar spreading 5 Gently shake antigen dispensing bottle before use Holding in vertical position dispense several drops in dispensing bottle cap to make sure needle passage is clear Place one free falling drop 20 G yellow hub needle onto each test area Do not restir mixing of antigen and specimen is accomplished during rotation Pick up the pre dropped antigen from bottle cap 6 Rotate for 8 min 30 s under humidifying cover on mechanical rotator at 100 2 rpm Following rotation to help differentiate Nonreactive from Reactive minimal to moderate RM results a brief rotating and tilting of the card by hand 3 or 4 to and fro motions must be made Immediately read macroscopically in the wet state under a high intensity incandescent lamp or strong daylight Report in terms of the highest dilution giving a Reactive including minimal to moderate reaction Examples Prozone reaction see Limitations of the Procedure H Reactive N Nonreactive u Reactive 1 4 dilution RM Reactive minimal to moderate 1 8 dilution EN LL 1 1 dilution Unheated or Heated Serum If the highest tested 1
35. 4 3 5 7 39 49 32 0 36 0 81 100 40 11 0 Female 12 100 years Europe 11 7 15 5 3 8 5 1 35 45 31 5 36 0 81 100 4 0 11 0 N B Man and women Asia 4 000 10 000 leukocytes ul Man and women Africa 2 600 8 300 leucocytes ul Differential WBC reference range CELL TYPE Percentage absolute number Polymorphonuclear EUROPE ASIA AFRICA Eosinophils 0 4 0 400 0 5 0 500 Neutrophils non segmented 0 5 0 700 0 596 0 700 Neutrophils segmented 50 75 1 800 7 000 30 40 900 4 000 Lymphocytes 30 40 96 1 000 4 000 40 60 96 1 200 6 000 10 00 sem Reticulocyte count Percentage Adults and children 2 15 1 000 RBC Infants at birth 20 60 1 000 RBC Absolute number Adults and children 25 000 160 000 ul Infants at birth up to 150 000 ul Platelet count 150 000 400 000 ul References ranges vary in different population and in different laboratories different techniques District laboratories should check the above figures for the technique in use with their nearest haematology reference laboratory 090122 pg tropical haematology and blood transfusion 35 77 ADDENDUM 3 MORPHOLOGY OF BLOOD CELLS IN A MAY GRUNWALD GIEMSA STAINED BLOOD FILM CELL TYPE SIZ NUCLEUS CYTOPLASM CHROMATIN GRANULOCYTES um FORM COLOR STRUCTURE QUANTITY COLOR GRANULES LEUKOCYTES POLYMORPHONUCLEAR GRANULOCYTES Hor
36. CTURE QUANTITY COLOR GRANULES LEUKOCYTES MONOMORPHONUCLEAR AGRANULOCYTES Big clumps of Round or oval Deep purple intensely stained Sky blue SMALL LYMPHOCYTES 7 10 Or slightly p purp or idend chromatin Often absent Clumps of deep Round or oval qd Absent or few Or slightly Red purple ie Sky blue granules azurophils are less intensely indented aned rose violet Very fine granules dusty like Vacuoles often oo azurophils rose MONOCYTES 15 25 m demonstrable Srey violet ERYTHROCYTES OUEST FUCO QVE rose none discus shape z LARGE LYMPHOCYTES 10 15 Round oval indented or Blue to slightly bean form violet 090122 pg tropical haematology and blood transfusion 37 77 Prins Leopold Instituut voor Tropische Geneeskunde Institut de Medecine Tropicale Prince Leopold Prince Leopold Institute of Tropical Medicine Instituto de Medicina Tropical Principe Leopoldo Nationalestraat 155 B 2000 Antwerpen otichting van Openbaar Nut 0410 057 701 POSTGRADUATE IN TROPICAL MEDICINE AND INTERNATIONAL HEALTH MODULE 1 BLOOD TRANSFUSION In remote areas Practical notes Philippe Gillet Luc Boel Jan Jacobs November 2007 Short summary of basics genetics The genetic information required for cell life is stored in the DNA Human cells contain a total of 23 pairs of chromosomes diploid number A chromosome is the concentration form of the chromatin DNA his
37. Department of Health and Human Services 1999 Biosafety in microbiological and biomedical laboratories HHS Publication CDC 4th ed U S Government Printing Office Washington D C 18 Directive 2000 54 EC of the European Parliament and of the Council of 18 September 2000 on the protection of workers from risks related to exposure to biological agents at work seventh individual directive within the meaning of Article 16 1 of Directive 89 391 EEC Official Journal L262 17 10 2000 p 0021 0045 19 Achimastos A G Tolis G Papadopoulos and K Kousoutzakoglou 1970 Public Health Rep 85 66 68 20 Scotti A T D M and J R Trautman 1970 Arch Dermatol 101 328 330 21 Dorwart B B and A R Myers 1974 Br J Vener Dis 50 435 436 22 Garner M F and J L Backhouse 1973 Med J Aust 1 737 739 23 Kaufman R E S Weiss J D Moore V Falcone and P J Weisner 1974 Br J Vener Dis 50 350 353 24 Falcone V H G W Stout and M B Moore Jr 1964 Public Health Rep 79 491 495 25 Portnoy J 1963 Am J Clin Pathol 40 473 479 26 Larsen S A B T Craig M E Shepherd and B McLaurin 1988 Data on file Treponema Research Branch CDC 0 100 090122 pg tropical haematology and blood transfusion 72 77 ANNEX 10 Relation rotation speed of a centrifuge and the centrifugal power Rotation speed of Radius of the the centrifuge centrifuge in rom r in cm Force centrifuge
38. HER DONOR 090122 pg tropical haematology and blood transfusion 51 77 PRINCIPLE OF THE MAJOR COMPATIBILITY INDIRECT COOMBS TEST Af e T e LME z 3 washes Incubation Binding ab RBC Patient serum Screening cells Mixture A Reading ower alight source Agagltination due to SHG 1 Addition af amp HOG Serum Only bound ab rest 1 Antigens present on the surface of the red blood cells of the donor in blue for instance the antigens Fya h 5 The fixation of the anti human antibodies on two different anti Fya antibodies are forming a bridge between the red blood cells resulting in an agglutination 090122 pg tropical haematology and blood transfusion 2 The serum of the receptor is added The antibodies anti Fya present in the serum will bind on the corresponding antigen All the other present antibodies stay free in the serum Sensibilisation step tetes 9 9 4 After adding Coombs serum the anti human antibodies are binding with the anti Fya antibody Revelation step Y 6 This agglutination is macroscopically visible in the tube as a clot Auto test on the receptor 52 77 Auto test on the receptor In case of positive compatibility tests in saline medium The autotest can be executed in case the of a positive compatibility test in saline medium in order to make the distinction between cold agglutini
39. HOD PRINCIPLE The Sahli method is based on converting haemoglobin to acid haematin brown colour and then visually matching its colour against a solid glass standard Diluted hydrochloric acid is mixed into a graduated cylinder with an accurate volume of blood sample and distilled water is added until the colour of the diluted blood sample matches the glass standard The dilution will be determined by the Haemoglobin level of the blood sample The Sahli method still used in a lot of places is not recommended The Sahli method is not an accurate way of estimating haemoglobin Not all the forms of haemoglobin are changed into acid haematin the colour changes when viewed visually are not very great and the brown colour of the glass standard is not a true match for an acid haematin solution MATERIAL Blood collection equipment and supplies Sahli haemoglobinometer small glass rod Sahli pipette safety device for pipetting Sahli tube and dropping pipette The Sahli haemoglobinometer is equipped with two glass colour standards The Sahli tube graduated until 16 g 100 ml and or in percentage 169 100 ml 100 is placed in between REAGENTS Hydrochloric acid 0 1 N Hydrochloric acid concentrated 9 5 ml Distilled water to 1 litre Caution hydrochloric acid is irritant and corrosive Handle with care in well ventilated area or in a fume cupboard Fill until half a 1 000 ml volumetric flask with distill
40. M type Samples Receptor blood obtained by capillary sampling or venous blood on EDTA Reagents Monoclonal Anti D Diaclon of Diamed for slide method Blood group cart or on a glass slide or on an opaline plate timer spirit lamp Technique 1 Preheat a glass slide to 37 C The quality of the actual antisera is such that heating is only necessary if the test is negative Put on the glass slide or on an opaline plate 1 drop of blood to be tested Put a drop of anti D near to the drop of blood Mix the blood and the anti serum with the bottom of a tube ob d du Tilt the slide during 1 minute 6 Read and note the result of the agglutinations immediately A positive reaction presence of agglutination means Rhesus positive Absence of agglutination indicates a Rhesus negative result In case of doubt observe the slide under the microscope magnification 100x to distinguish better the agglutinations In order to make lecture easier incline slightly the slide before lecture under the microscope to see the red blood cells while they are moving ze amp XO ns v gt iS lt m 4 LF red blood cells in serum Microphotography of a suspension of red blood cells in serum presenting a weak proportion of rouleaux red blood cells on a pile showing a weak agglutination red blood cells in small of plates Magnification 100x Rouleaux formation is clusters Magnific
41. Prins Leopold Instituut voor Tropische Geneeskunde Institut de M decine Tropicale Prince Leopold Prince Leopold Institute of Tropical Medicine Instituto de Medicina Tropical Principe Leopoldo Nationalestraat 155 B 2000 Antwerpen Eu ANT ERE es CAL Stichting Openbaar Nut 0410 057 701 POSTGRADUATE IN TROPICAL MEDICINE AND INTERNATIONAL HEALTH MODULE 2 CLINICAL amp BIOMEDICAL SCIENCES OF TROPICAL DISEASES Practical notes TROPICAL HAEMATOLOGY JANUARY 2009 Philippe Gillet Luc Boel Jan Jacobs pgillet itg be lboel itg be jjacobs itg be Table of Contents GENERAL HAEMATOLOGY BLOOD COLLECTION CAPILLARY BLOOD VENOUS BLOOD HAEMOGLOBIN DETERMINATION _ INTRODUCTION _ REFERENCE VALUES HCS METHOD HAEMOGLOBIN COLOUR SCALE PRINCIPLE EQUIPMENT AND SUPPLIES BLOOD COLLECTION METHOD MAINTENANCE LOVIBOND METHOD Harrison s method PRINCIPLE MATERIAL BLOOD COLLECTION METHOD SMALL PROBLEMS AND SOLUTIONS CONVERSION TABLE LOVIBOND TO g 100 ml SAHLI METHOD PRINCIPLE MATERIAL REAGENTS BLOOD COLLECTION METHOD DRABKIN METHOD _ PRINCIPLE BLOOD COLLECTION MATERIAL REAGENTS METHOD 090122 pg tropical haematology and blood transfusion Un a NNN ON oC QN ON ON ON ON lt A QN ON ON ON ON 2 77 B_ ee PRINCIPLE MATERIAL BLOOD COLLECTION METHOD
42. Rad a Bate beg en oe ks eg ie mrt rm mem inm JE DO mnl mom d Decem D THAR SPU zo dE 919 dd 204 MATERIALS AHD METHODS Lew nth olin em prepare in ke de dee Erp Liebe fey gi pereent siet phate el Fins diced eine brani agra Chemkal 5b Lai MES dn reerd salie was aber om crac Breed hy Lander ari AR prepared by ai hen old us Warrel uock chan er bri nad hen reg daar bor TETE rs eerie foliar inhi aggregation prepared hy Eire 3 nl 4 inalil Brader ae dimi 5 peren eens emile plee som rag obers gula nen dad C por BITREEWIS Seed m El da bele ree mere Lia BT SP mees SPARE ple AR ipee T L SUE B drat sen a hs eee deb wli eed ed e pee aem Bana rag eager Thee droped kee ok medias ae we io dye dike folkd by 3 drops o we xxr plane and 1 drop af pores Bele dp malige der dbal pueked The mab me nim eth amr aie wh ed ue bed a ee Le eee 1 minute phas Te drag od ib 8 Pod boss teer reg solution im they akin i the superb cn dro debe sed eth on niic orl rubis sf pom ergere FIVC agure gatnn begins i app abel 5
43. T dien eem nante Homme LAT lis mre enl Eve Man beh end Ier the doen Gland RE Voca T E cun har SP inie Ti o 71 fumes ee amd Bee Mom re andy SP deep eek adi ARO Erico i i Table 21 alic risu de iod d t riri allen mari ered Ld 4 ux ar er UNS cae HEN a a 7 141 E d E L eis Lg Er rr ab Lis T Er i y i 1 Ms E M 4 H i i 141 Ya HI LL L ink E T Mksh br univ biis si abaruos IPL Chara a misi 1 brik sisiy del Pd zel LES Fu di 441 090122 pg tropical haematology and blood transfusion LIM TABLE 2 Numbers of patients In whom antibodies were detected and methods of detection 23 E D D T B D 0 ra Fa da dn ori gu o dn gt SoM FO I oo in Mi FE TELE m mu E ambay phanotypa bodies of clinical significance Including antibodies against antigens of the Kidd and Diego blood group systems although reactions were weaker than those observed with MP and LAT Among 85 antibodies from patients that were tested 74 antibodies were detectable by SP Including 10 anti A and 10 anti B Anti Ep could only be detected by LAT Table 2 a
44. Test and was developed for field use where testing could be performed without laboratory equipment 3 4 By incorporating machine rotation ringed test surfaces and certain other technical changes the RPR Circle Card Test was developed for use in large scale testing in public health and clinical laboratories The RPR 18 mm Circle Card Test is recommended when venous blood collection is employed and a large volume of serum is available such as generally prevails in public health and clinical laboratories 5 12 When a specimen contains antibody flocculation occurs with a coagglutination of the carbon particles of the RPR Card antigen which appear as black clumps against the white background of the plastic coated card By contrast nonreactive specimens appear to have an even light gray color In special situations when nontreponemal test results are needed rapidly and the specimen is collected as EDTA plasma the RPR 18 mm Circle Card Test can be used if the test is performed within 24 h 13 14 PRINCIPLES OF THE PROCEDURE RPR Card antigen suspension is a carbon particle cardiolipin antigen1 which detects reagin an antibody like substance present in serum or plasma from syphilitic persons and occasionally in serum or plasma of persons with other acute or chronic conditions The reagin binds to the test antigen which consists of cardiolipin lecithin coated cholesterol particles causing macroscopic flocculation REAGENT The ingredients of the RPR Card
45. a MU ened Andr atie de gah residen due b First rip hn padt im REB E imm pers ere d pases rgb al pukem rc Bi Cer iii Rund CHER LA RE FT HEE Deea asl bead pou 14 diberean posl sien det marter D DEDE TENERE meme rm ipn la rouen Cut chan caus Oe dri Lab bl dete ii Edag cole de diiras Ca egi andina contes rupes dd imis miim usc eee ml reed d ferai nan n or azi Sora l chelon bes di Chr om mmr Mp Fir tee fe Ba cee paged oren rua l actu m EA ekz arie compe anicoroa codccc D CU CR Lana D rung eee lisa iai ai cas dans cae iis la rum chs weer atre ep Ted canary 90 le cur cut ares unteren eh ies Tenet a en ER wisn ir jur en ab is bin mf de che fates Cremeren Ages Vn Mel TDUZ 51 Pen opoe dace an par ra Prenom Gormless Tubes capies dec da OTA TETE PRECAUTIONS ET ERR CH de ve ATTENTION Les iced Sh partia otre dire manila Gandara dus r gl s es rue Cen pertinen Re 1 Porter dus pacte me pas aliut de pipeteges bouche Me pes m
46. agglutinating not only the erythrocytes of the child and the father but also these of 85 of the tested subjects Landsteiner and Wiener in 1940 found that the diluted serum of a rabbit immunised with erythrocytes of Macacus rhesus agglutinated the erythrocytes in the same subjects In fact taking into account that the non diluted serum of the rabbit recognised 100 des subjects it seemed that these hetero antibodies recognised a different antigen than the D antigen being present on the majority of human erythrocytes but from which the antigenic density is more important in subjects bearing the D antigen than in subjects which are deprived of it The involved antigen in this confusion was named LW Landsteiner and Wiener and the term Rhesus has been maintained to design the initially concerned system The Rhesus antigens are membrane proteins The addition of these proteins is coded by two genes One gene coded for the D one gene coding for the Cc and Ee It is a very polymorph system Nowadays 52 antigens are described Within these the DCcEe antigens are the most immunogenic Its nomenclature somewhat careless reposes on 3 genetic hypotheses the base of 3 theories of which each is used according the circumstances t is described as DCE conception of Fisher and Race it is named Rh concept of Wiener and it is exposed in figures concept of Rosenfield For the current minimal transfusion practice only the D antigen is important It
47. ansfuse if Does the receptor have antibodies IgG against red blood cells of donor Whole blood or concentrated red blood cells Do not transfuse if 4 Plasma Do not transfuse if B 56 77 Table 7 Blood transfusion on district hospital level from minimum until extra possibilities in function of possibilities listed for each activity in order of importance Blood donor e None remunerated familial blood donors Living blood bank Mini blood bank for emergencies 1 to 2 blood bags Blood bank based on none remunerated and regular voluntary blood donors Selection of donors e Questionnaire and clinical selection e Delay of reflexion between the first screening test and the first blood donation for the voluntary donors oon Type of serological screening e Rapid tests after during the taking of the blood unit e Rapid tests before the blood taking Serological screening on the donors HIV antibodies e HIV antigens HBsAg antigens e HCV antibodies Syphilis non treponemal test RPR e Syphilis Test treponemal TPPA type And in function of the region a screening for o Chagas Disease o African Trypanosomiasis o Leishmaniasis o Mlicrofilaria Cold transfusion of whole blood blood bank Transfusion of packed cells blood bank Transfusion of plasma blood bank Type de transfusion Warm transfusion of whole blood o 1 general
48. antigen suspension are1 0 00396 cardiolipin 0 020 0 022 lecithin 0 09 cholesterol 0 0125 M EDTA 0 01 M Na2HPOA 0 01 M KH2POA 0 1 thimerosal preservative 0 02 charcoal specially prepared BD 10 choline chloride w v and deionized distilled water Adjusted and or supplemented as required to meet performance criteria Warnings and Precautions For in vitro Diagnostic Use Pathogenic microorganisms including hepatitus viruses and Human Immunodeficiency Virus may be present in clinical specimens Standard Precautions 15 18 and institutional guidelines should be followed in handling all items contaminated with blood and other body fluids Antigen Refrigeration is recommended for the RPR Card antigen suspension only Storage in bright sunlight or temperatures above 30 C should be avoided such conditions may cause a rough appearance of the antigen when used with nonreactive sera If the ampule of antigen is frozen during shipment it can be reconstituted once by warming to room temperature avoid repeated freezing and thawing Immediate use of a refrigerated antigen may result in decreased sensitivity of the test Therefore upon removal from the refrigerator allow the antigen to warm to room temperature 23 to 29 C before use Do not use antigen beyond the expiration date Diagnostic Test Cards Specially prepared plastic coated cards designed for use with the RPR Card antigen In handling take care not to fingermark the card test a
49. aph BLOOD COLLECTION Capillary or venous blood For venous blood dry anticoagulant should be used to avoid dilution EDTA di potassium salt or heparin are recommended After thoroughly mixing with anticoagulant the blood can be frozen for as long as 2 years and used as control MATERIAL Blood collection equipment and supplies spectrophotometer or colorimeter that transmits light at 540 nm Sahli pipette or 20 ul automatic pipette 5 ml graduated pipette or 5 0 ml dispenser safety device for pipetting test tubes cuvettes different volumetric flasks and different calibrated pipettes for calibrator s preparation graph paper REAGENTS Drabkin s neutral diluting fluid pH 7 0 7 4 also ready to use available example Sigma D 5941 Potassium hexacyano ferrate KsFe CN e 0 20 g Potassium Cyanide KCN 0 05 g Potassium dihydrogen phosphate KH PO 0 14 9 Tween 20 1 ml Distilled water to 1 litre Tween20 can be replaced by 0 5 ml Brij 35 at 30 xe CAUTION Potassium cyanide is highly poisonous It is a pale yellow clear fluid which may no longer be used if its colour is lost or if it becomes turbid The prepared Drabkin solution is stable for at least 6 months at room temperature protected from light amber bottle Haemiglobin cyanide cyanmethaemoglobin standard for calibration HiCN reference standard solutions are stable for long periods and are commercially available as Haemoglobin s
50. arge bore fuer ri meniguier dus produits coametiques uu des che paaa deen bes lad a aide ddie her Ioden em wi de d un hel rie meekan Dionne ak M Mio en a wid TE CITES ee FER x i sa Deir Age an im jn oe E EU 2 ad DEC 1 carita bw greerde Le compose du kd por jobs in cde da prenten cond el anges hy aten jm ina RO OM ip ge Je Deir PARFLEVEHFHT DEB ECHANTRL ONT Vere ar preven apri TN meae ed uir oe eee Geert ecu Fn os ee BEMARCHE lg chos du gang 0 last lige des telen des awe oe Preheat bial ied ba Tet Aui pridie CTA Ber Do ePi Lan sia doe Se mos artan gegen at Poor iud dei da Dui an cesser m Soul dum de bemoeide cm Chania dt baatte Cos ases die Dn ete mre epee m firi ullis ict fulsit candies contenant de im du arg peg un
51. assage through a fine bore needle Haemolysis caused by heating of the blood during the sealing of the capillary Inadequate centrifugation too short or with a too low centrifugal force Blood not properly oxygenated the blood must be sufficiently but gently mixed before performing the test Haemolysis of the sample during centrifugation by an overheated centrifuge Erroneously high or low PCV may be due to Using an inappropriate anticoagulant Reading error parallax error Poor quality tubes which are not uniform bored Inadequate mixing of the blood prior to filling the micro hematocrit tube e QUALITY CONTROL opecimens run in duplicate must agree within 3 96 The complete packing of the red blood cells should be verified After reading the PCV re centrifuge the tube for 2 3 and 5 minutes more No decreasing of the PCV should be found In case of PCV value decreasing choose the centrifugation time which give a constant PCV value 090122 pg tropical haematology and blood transfusion 26 77 CELL NUMBER CONCENTRATION GENERAL PRINCIPLE The count of cells per volume unit is quite useful as diagnostic tool Electronic counter systems are often not available or affordable in district laboratories The only realistic alternative will be to use a counting chamber in which the cells are counted under the microscope Depending on the kind of liquid to be analysed expected cell number a dilution and or destructio
52. at least 30 seconds then with a dry cotton to remove any remaining ethanol 5 Using a sterile lancet make a rapid puncture sufficiently deep to allow the free flow of blood Discard immediately the lancet in a safety container 6 Execute a slight pressure on the finger to realise a better blood flow 7 Wipe away the 1 drop of blood with a dry piece of cotton wool since it may contain tissue fluid or disinfectant Except for microfilaria detection since the first drop contains more microfilaria 8 Press the finger not too hard to produce in one time the required amount of blood 9 When sufficient blood has been collected press a piece of cotton dipped in 70 alcohol over the puncture area until bleeding stops For glycaemia determination The use of ether or any disinfectant should be avoided as it may interfere with the reagent strip glucose oxidase peroxidase reaction To decrease risk of infection and food contamination fruit juice it is therefore important to clean the hands with soap before blood collection 090122 pg tropical haematology and blood transfusion 5 77 VENOUS BLOOD Venous blood is used when more than 100 ul of whole blood is required or when serum from a clotted blood sample is needed If whole blood is needed an anticoagulant should be used to prevent clotting and or morphological blood cell changes An anticoagulant acts by removing calcium example EDTA trisodium citrate or by interference wi
53. ation 100x related to plasmatic proteins concentration 13 This technique is only applicable for monoclonal Diacon Diamed anti D antiserum The technique may vary in function of the used reagent The manufacture s instructions must always be followed Check if the antiserum may be used for a reaction on slide and if it contains IgM 090122 pg tropical haematology and blood transfusion 44 77 Possible problems in the Rhesus grouping False negative reactions Weak weak expression of the antigen Partial D D Quality of anti D antibody titre to low or IgG and not IgM False positive reactions Coagulation of the blood to be determined Presence of cold agglutinins in the blood to be determined Bacterial contamination of the test reagents Chronical infection rouleaux phenomena by increased plasmatic proteins Antigenic modifications during malign pathology Infection of trypanosomiasis presence of auto agglutinins and rouleaux formation 090122 pg tropical haematology and blood transfusion 45 77 TRANSFUSIONAL RULES 1 Avoid Antigen Antibody conflicts Two types of antigen antibody conflicts can be distinguished The major and the minor conflicts The important difference between these two is based upon the quantity and concentration of the involved antibodies Major conflict The blood of the receptor may not possess antibodies directed to the antigens of the red blood cells of the donor Principle defined by
54. ation indicates the presence of the corresponding surface antigen on the red blood cells Possible problems False negative reactions Immature antigens A and B newborns Genetic variants Weak groups A or B Antiserum of bad quality antibody titre too weak False positive reactions Coagulation of the blood to be determined Presence of cold agglutinins in the tested blood Bacterial contamination of the test reagents Chronical infection rouleaux formation by increased plasmatic proteins Infection of trypanosomiasis presence of auto agglutinins and rouleaux formation Antigenic modifications during malign pathologies This technique is only applicable for the human Diacon antisera Diamed The technique may vary in function of the used reagents Always follow the particular instructions indicated in the note of the company Verify if the antisera may be used for the determination on slide 090122 pg tropical haematology and blood transfusion 42 77 B The Rhesus system The discovery of the Rhesus system as well as numerous other systems of red blood cell groups has been the result of the exploration of transfusion incidents and of haemolytic diseases of neonates The attribution of the name Rhesus to this system has its origin in a historical confusion with another system In fact in 1939 Levine and Stetson concluded that the serum of a woman who delivered a baby attacked by a neo natal haemolytic disease
55. be used to avoid dilution EDTA di potassium salt or heparin are recommended REAGENTS Ammonia solution 0 04 v v Preparation with ammonia 28 96 concentrated Ammonia 28 96 concentrated Distilled water up to 1 000 0 ml volumetric flask CAUTION Ammonia solution is a corrosive chemical with an irritating vapour Handle with care in well ventilated area or in a fume cupboard Keep the stock bottle well stoppered This solution is stable when kept in a tightly stoppered bottle Renew every 4 weeks Any concentration of any ammonia solution can be made by taking a volume of concentrated solution equivalent to the required and making this up with distilled or deionised water to a volume equivalent to the 96 of concentration C1 x V1 C2 x V2 C1 Concentration of the concentrated ammonia solution V1 Volume of the concentrated ammonia solution 090122 pg tropical haematology and blood transfusion 19 77 C2 Needed ammonia concentration V2 Needed volume of the weak ammonia solution Example C1 28 Via C2 0 04 ec 000 ml 28 x V1 0 04 x 1000 ml gt V1 1 429 METHOD It s vital to understand the factors that influence accuracy of measurement and dilution The accuracy of haemoglobin measurement depends on accuracy of proportion of blood to diluent The quality of the used water and ammonia as diluent is also quite important INITIAL CHECKS OF OPERATION AND FACTORY CALIBRATION
56. ble 3 Order of choice in the selection of a blood donor for whole blood based upon the ABO group and upon the determination of the D antigen of the Rhesus system Blood group of Preferred group of the Order of choice in case that no iso group nor iso the receptor donor Rhesus blood can be found CC AB Rhesus A Rhesus A Rhesus B Rhesus AB Rhesus AB Rhesus B Rhesus O Rhesus O Rhesus AB Rhesus AB Rhesus A Rhesus B Rhesus Rhesus omens omes N B In transfusion situations of complete blood not in iso groups the present IgM anti A or anti B cause mostly only small problems because they are in insufficient quantity for provoking important haemolysis during a standard transfusion In all cases of complete blood transfusion it is as well recommended not to mix the blood pocket to reduce the quantity of transfused plasma by sedimentation Nevertheless a situation of haemolytic accident risk exists when blood is transfused from a donor presenting anti A or more rarely anti B This type of rare haemolysin occurs mainly in donors of group O and appears by commutation from IgM anti A or anti B cf N B 2 of ABO group The haemolytic potential of an IgG is much more important than these of IgM which explains that these accidents happen with very low quantities of transfused haemolysin during a standard transfusion this is even true for concentred cells Dangerous universal donor
57. blood cells In iso group Transfusion of plasma In iso group In non iso group Transfusion of whole blood or of concentrated red blood cells In iso group Transfusion of plasma In iso group In non iso group 090122 pg tropical haematology and blood transfusion Demonstrated antibodies IgM in the receptor against the red blood cells of the donor ABO Error Cold Auto antibodies Cold Allo antibodies IgM in the receptor against the red blood cells of the donor ABO Error Cold Auto antibodies Cold Allo antibodies IgG in the receptor against the red blood cells of the donor Warm Allo antibodies IgM in the donor against the red blood cells of the receptor ABO Error Cold Auto antibodies Cold Allo antibodies IgG in the donor against the red blood cells of the receptor Warm Allo antibodies Necessary Equipment s Questions Actions m D 2 lt Error of ABO grouping Do not transfuse if Error of ABO grouping Does the receptor have antibodies IgM against red blood cells of donor If and ABO error excluded transfuse at 37 C Does the receptor have antibodies IgG against red blood cells of donor Do not transfuse if Does the receptor have antibodies IgM against red blood cells of donor Whole blood or concentrated red blood cells If and ABO error excluded transfuse at 37 C Plasma or concentrated red blood cells Do not tr
58. cal state and or federal regulations or accreditation requirements and your laboratory s standard Quality Control procedures It is recommended that the user refer to pertinent NCCLS guidance and CLIA regulations for appropriate Quality Control practices LIMITATIONS OF THE PROCEDURE The diagnosis of syphilis should not be made on a single reactive result without the support of a positive history or clinical evidence Therefore as with any serological testing procedure Reactive card test specimens should be subjected to further serologic study Serum specimens which are Reactive in qualitative testing should be quantitated to establish a baseline from which changes in titer can be determined particularly for evaluating treatment 1 The use of plasma specimens to establish a baseline from which changes in titer can be determined has not been evaluated False negative results can occur because of failure to recognize prozone reactions Prozone reactions occur in 1 to 2 of patients with secondary syphilis These specimens may exhibit a nonreactive pattern that is slightly granular or rough Upon dilution the reactivity will increase and then decrease as the endpoint titer is approached All tests with a rough appearance should be further evaluated False 090122 pg tropical haematology and blood transfusion 71 77 negative nontreponemal test results are also seen in incubating primary and late syphilis 1 It is not necessary to perfom the quantitative
59. calibration curve The colour is stable for several hours Determine the haemoglobin concentration of each test directly from the calibration curve O D Hb in g 100 ml Or Calculated x calibrator s concentration sample s Hb concentration O D Calibrator 090122 pg tropical haematology and blood transfusion 16 77 HEMOCUE B PRINCIPLE The HemoCue is an example of a robust portable and accurate haemoglobinometer readily available for use Although not affordable by most district laboratories it may be used for survey The HemoCue uses calibrated disposable cuvettes that are treated with chemicals sodium desoxycholate sodium nitrite and sodium azide which rupture the red cell wall and combine with the haemoglobin to form a compound azidemethaemoglobin which can be measured photometrically modified Vanzetti reaction Sodium nitrite converts the haemoglobin iron from the ferrous to the ferric state to form methaemoglobin The methaemoglobin then combines with azide to form azidemethaemoglobin and is measured photometrically at two wavelengths 570nm and 880nm The result in g 100 ml is displayed in digital form on the face of the instrument Web site http www hemocue co uk MATERIAL Blood collection equipments and supplies HemoCue B disposable cuvettes HemoCue B instrument HemoCue B standard control cuvette batteries BLOOD COLLECTION Capillary or venous blood For veno
60. ce a clean cuvette filled with distilled water into the cuvette window Press and hold down the L button At first the display will show the last reading followed after several seconds by a beep After this beep the LCD turns off and the new zero level is stored in the device Check again the reading for BR1 Place a clean cuvette filled with distilled water into the cuvette window Press and hold down the L button At first the display will show the last reading followed after several seconds by a beep After this beep the LCD turns off and the new zero level is stored in the device Check again the reading for BR1 Dirty or wet photocell windows Clean the glass of the photocell windows with alcohol on cotton swap stick Check again the reading for BR1 B CHECK ZERO Place a cuvette containing 1 2 ml of the weak ammonia solution 0 04 96 into the cuvette aperture Use the same kind of cuvette which is used for haemoglobin determination The value on the display should be zero If this is not the case remove the cuvette and within 2 seconds press and hold the L button until the beep Zero will automatically be reset Check again the reading for zero 090122 pg tropical haematology and blood transfusion 20 77 C CHECK CALIBRATION The permanent calibration control standard checker supplied with each instrument is numbered and matched uniquely to one instrument Place the checker into the
61. cuvette aperture The value of the displayed reading must correspond to the value 5 133 143 g l for our haemoglobinometer 0931 If this is not the case check for the following Causes PROBABLE CAUSE REQUIRED ACTION Insert the cuvette with the plastic surface in front of you Check again the reading for CR1 Check again the reading for CR1 Check again the reading for CR1 Value of scale factor M incorrect Press and hold down the R button located at the back of the device The symbol HHH and after some seconds the scale factor M will appear Compare this value with the figure for M shown in certification 162 for our device If there is a difference between these values then adjust the displayed value by pressing the L button to lower the value and the button to raise the value Check again the reading for BH1 Zero level of the device incorrect Place a clean cuvette filled with distilled water into the cuvette window Press and hold down the L button At first the display will show the last reading followed after several seconds by a beep After this beep the LCD turns off and the new zero level is stored in the device Check again the reading for BR1 Other type of cuvette used for the Place a clean cuvette filled with distilled water into the cuvette calibration window Press and hold down the L button At first the display will show the last reading followed after s
62. d take the glass stirrer out of the graduated tube and read the level of the base of the menisci of the liquid Hold the instrument about 50 cm away from your eyes on the same height under diffuse light 9 Note the reached mark that corresponds with the level on the tube 090122 pg tropical haematology and blood transfusion 14 77 DRABKIN METHOD PRINCIPLE The haemiglobin cyanide method is the most accurate method of measuring haemoglobin and is considered as the reference standard gold standard Whole blood is precisely diluted 1 on 201 in a Drabkin solution The red cells are haemolysed and the haemoglobin is oxidized by the ferricyanide to methaemoglobin This is converted by the cyanide to stable haemoglobin cyanide Hb K3Fe CN gt MHb MHb KCN gt HiCN Hb haemoglobin MHb methaemoglobin HiCN haemiglobincyanide The chemical reaction takes place at a pH stabilized by a monopotassium phosphate buffer in order to obtain a complete reaction in a reasonable time Addition of a detergent facilitates haemolysis and prevents turbidity caused by plasmatic proteins The optical density is in proportion with the haemoglobin quantity that is present in the blood Absorbance of the HiCN is read in a spectrophotometer at the wavelength 540 nm or in a colorimeter using a yellow green filter The absorbance obtained is compared with that of a reference HICN standard solution Haemoglobin values are obtained in the calibration gr
63. d B are dominant over O A and B are co dominant O is relatively recessive to A and B The ABO Antibodies are principally natural antibodies complete cold optimally at 4 C but they still agglutinate at 37 C of IgM type They appear spontaneously during young childhood by cross antigenic stimulation with surface antigens of saprophytic bacteria of the intestinal flora They appear usually between the 3 and the 6 month of life and their concentration reaches a maximum at the age of 10 years They are present on every individual who does not possess the corresponding antigen on his own red blood cells Summary ABO blood group system Iso agglutinins always present in the plasma Erythrocytes with antigens on their surface Blood groups Table 1 Estimation of the frequency and function of the skin colour Frequency of occurrence of ABO groups related to skin colour 9 Black 4 Ih 21 48 N B 1 Subgroups A and A Antigen A exists as strongly reacting antigen A and a weakly reacting antigen A Most people who are group or AB posses A antigen but up to 20 belong to the subgroup A or A B Very soon a first complexity level has been reported concerning the phenotype A The phenotype A which is found in 80 96 of subjects A and the phenotype A in 20 The phenotype is characterised by the presence of the A antigen while the phenotype A does not possess the antigen A The number of antigenic sit
64. d be teiwited EAT EDR CH EET TA POSTE Tan Durs Hel hue peni en cos aeris Pabili Casini h ml en 090122 pg tropical haematology and blood transfusion WOTE 2 ba h raat paara eren piera hus apes LR Rene M an occur pepratidly les beijei ahhaaa GERS quus local Rare and Tage Camis LIMITATIONS OF THE PROCEDURE ben Denteneer HY eia eal adt ex ies arb ew o Toni tuam en plana and miare biasi Cher body Buis rr pocipd noce may rui eer son rhe enis Une amener of the patas Dr dcc ecm ey CONTR GKR be the eer of arcade ve ite regala spin mats Deere iw mot coude the poruixAey of efector mile kV A lates cundem Ead ea wi heter Dela d aT et fee left af PEO EPROM BIE Dulce gene Mm ol Tha LET iioc eli rii El guns Peijl d lich deter r Thes erkens man FU ann coris anao es ue ee eR reek rm Dane ecanen handkng cond stuk krii f HIY clerg bayii Dnus mi dans p CPR d harmon acad b uted on ath bril redt Postes pee uid bm nena anchhenr recited ana the pete shed bee entel iri gia od e iser ee d ira Af Aion ricas EL made ele hie piema Yar ror messe offer than DOTA mag gere P
65. e Expression ar dentificatbon of Hepatitis C Virus Polypeotcin Clevape Product Kournal of Virology March 1993 p 1385 1 395 2 Young Cha Min Kyung Kyung Lib jang Chang and Voeg Chul Sung honing and Overexqaession of the Highly lmenunogerac Hapan of HEV Genome from Korean Patients Mol Celis Vol 3 407 416 115 bama Cecconaa 5 Griva F Gamio Calogero C Rosa and F Bonelli in E coli and purification of a chimeric p22 NS3 mmcomb enar aetigen of Hepatitis Vina HCV Federation of European Biochemical Societies Volume XM mumber 3 253 257 dj A K Takahashi Kishimoto Scrodiagnosit of hepatitis C virus infection ELISA for antibodies against the potative core proseim expressed in Eir herirhia eel of lmenunologscal Meihodz 14 OEF 143 180 Doise ped Pe 02 1 9 STANDARD DIAGNOSTICS INC 85 4 Paang dong Jangan a Eure Se SEL EEN 68 77 ANNEX 9 RPR test example BD Macro Vue RPR Card Tests ver 26 09 2005 18 mm Circle Qualitative and Quantitative Brewer Diagnostic Kit for the Serologic Detection of Syphilis INTENDED USE The Macro Vue RPR Rapid Plasma Reagin 18 mm Circle Card Test is a nontreponemal testing procedure for the serologic detection of syphilis 1 2 SUMMARY AND EXPLANATION The Macro Vue RPR Teardrop Card Test using finger puncture blood was the original Card
66. e reference range for MCHC in health is 30 36 g These figures should be checked locally The MCHC is used in the classification of anaemia MCHC lt 309 Hypochromic anaemia MCHC between 30 and 36 g Normochromic anaemia Hyperchromic anaemia does not really exist A red blood cell cannot contain more haemoglobin than the maximal continence with one exception for the megaloblastic anaemia Hypochrome anaemia lt 30 normochrome anaemia gt 36 hyperchrome anaemia 090122 pg tropical haematology and blood transfusion 25 77 SOURCES OF ERRORS Erroneously high PCV may be due to Patient s position during the blood collection 10 96 of difference between lying or standing Storing the specimen beyond 6 8 hours before performing the test Delay of reading after centrifugation plasma evaporation Prolonged stasis caused by constriction with a tourniquet for more than 1 minute haemoconcentration Erroneously low PCV may be due to Leakage from the tube during centrifugation due to insufficient sealing of the capillary tubes Heparin degradation in hot climate Tubes should be stored in a cool place Dilution by interstitial fluid especially where there has been difficulty in venous puncture or failure to obtain free flow of capillary blood Blood coagulation if the blood is not immediately mixed with anticoagulant EDTA in excess of 2 mg ml diminution of the red cells volume Secondary haemolysis to forcible p
67. e technique for estimating haemoglobin The red colour of blood corresponds with the amount of haemoglobin Blood is inserted directly into a special cell thickness of 0 004 without any preliminary manipulation The colour is compared with a series of reference glasses in a Lovibond comparator MATERIAL Blood collection equipment and supplies Lovibond 2000 comparator standard discs Lovibond 5 8 A and 5 8 B Lovibond cell 0 004 soft paper Beaker chlorine solution 1 96 70 96 alcohol Lovibond 2000 comparator Disc Lovibond 0 004 LALMI G Metal clip Cover plate Stud base plate The blood cell consists of a base plate and a cover plate constructed from plain white glass Fused on the cover plate are three small studs of glass which create when in position a cell of 0 004 thickness This cell is filled from the side by capillarity BLOOD COLLECTION Capillary blood For venous blood dry anticoagulant should be used to avoid dilution EDTA di potassium salt or heparin are recommended METHOD 1 Clean and decontaminate the base plate and the cover plate first with water then with 70 alcohol 2 Place the two plates in position and join them with the clip 0 004 inch 1 016 mm 0901 22 pg tropical haematology and blood transfusion 10 77 3 Check if the discs are clean Wipe with a cloth if they become dirty Select the appropriate disc evaluation of conjunctival pallor and insert it
68. ed water Add slowly 9 5 ml concentrated hydrochloric acid After cooling fill until the 1 000 mark with distilled water and mix well This reagent is stable for at least 1 year at room temperature Distilled water or filtered water BLOOD COLLECTION Capillary or venous blood For venous blood dry anticoagulant should be used to avoid dilution EDTA di potassium salt or heparin are recommended METHOD 1 Fill the graduated measuring tube up to the bottom graduation line with 0 1 N hydrochloric acid The mark level should be equal with the bottom of the meniscus formed by the liquid 2 Check the tip of the Sahli pipette Discard if broken volume error Check if the pipette is dry 3 Draw the blood a little bit further than the 20 ul mark of the Sahli pipette Do not allow air bubbles to enter Wipe the outside of the pipette with absorbent paper and adjust the blood on the 20 ul mark 4 Blow the blood from the pipette into the graduated tube of the acid solution 090122 pg tropical haematology and blood transfusion 13 77 5 Rinse the pipette by drawing in and blowing out the acid solution 6 Allow to stand for 1 minute The mixture will become dark brown and clear 7 Place the graduated tube in the haemoglobinometer compare the colour in diffused day light Add water drop by drop and mix with the glass stirrer until the colour of the solution matches the colour of the reference tube 8 When equal colours are reache
69. ei rendi al 5 20 remmen Deo not interpret test nent 20 mimics Figure Caution The alive time is based on reading the test results at room temperature of 5 to 30 degrees C your mom temperature is significantly lower than 10 depron C chon the intergecting time doit be properly increased 7 Interpretation of the test 1 A color band will appear in the section of the eowull window to there hat the beet in 21 The right section of the resol window indicates the tet results Wf another cok band appear in the section of the nesill window this hand is the est Hand 090122 pg tropical haematology and blood transfusion Negative results The presence of only oec bami within the nell wimdow indicairs a negntive result Figure 7 MW Positive results The presence of tw color hamis T band amd C haad within the result window no matter which bard appears fari indicates a resin Figure 3 Figzew 1 Invalid results 1f the purple color band is mot visible within the mot window after perforning the text the result ix considered imvalid Figure 4 Some causes of imvealid are mob tbe dineciaons comecily or the test may have deteriorated beyond the pir ies date Tr i recommended thas the specimen be weing a new rest kit Faure 4 positive result will change onec has heen cxtabliibx 20 okee never an ceder
70. en tube from which it was drawn Discard Dispenstirs device Repeat procedure for number of specimens to be tested 4 Gently shake antigen dispensing bottle before use Holding in a vertical position dispense several drops in dispensing bottle cap to make sure the needle passage is clear Place one free falling drop 20 G yellow hub needle onto each test area Do not restir mixing of antigen and specimen is accomplished during rotation Pick up the pre dropped antigen from bottle cap 5 Rotate for 8 min 30 s under humidifying cover on mechanical rotator at 100 2 rpm Following rotation to help differentiate Nonreactive from Minimally Reactive results a brief rotating and tilting of the card by hand 3 or 4 to and fro motions must be made Immediately read macroscopically in the wet state under a high intensity incandescent lamp or strong daylight Report as Reactive Showing characteristic clumping ranging from slight but definite minimal to moderate to marked and intense Nonreactive Showing no clumping See the Reading Guide Note There are only two possible final reports with the Card Test Reactive or Nonreactive regardless of the degree of reactivity Reactivity minimal to moderate showing slight but definite clumping is always reported as Reactive Slightly granular or rough reactions should be repeated using an alternative procedure For donor screening these tests may bereported as indeterminant pending furt
71. entrifuge for blood bank Immufuge Il Baxter Indicative price list serological screening us Determine HIV 1 2 Om o Determine HBsAg pm RPR card antigen suspension Becton CE Dickinson 19 Without taking into account the controls and the losses 20 Without taking into account the controls and the losses ANNEX 6 HIV rapid test example Determine HIV Z 2 AD TERE fhe Abbe Dehertens Fie UT i a in az guabizires weermee fet Ph ddr ad arme anzi 2 mn buena teres pli ie made Tha ten m mended ga med a heb daken de HE TW T avtazind breda EULA CET MATEN THE TEST imerancdalcenry Syndrome n chenecisticed bp E mmm e oe tee da ard ce cran tering Pur AGN angues al Tom mech Daher atc gis Nn D n berg d aie al am iR am Mains M MEINT HA wing wich then grtn Duas qa mater od HINA PRINCIPLES OF TIE PROCEDURE Cats HV 17 am zurmancchremaiogi mph iani ber fe quaiialies delden of amibode EEN and Bih prophet ergs puel mengde erg pk Sae Dok jeje le pd riten ard TERRI N ie viert daia aemper Then mrs kr Regie Vie phase no Tis Peoria al Lp pepodaa m oma gamer meedoen wie posee in Mo inne
72. es in phenotype A subjects is much higher 1 000 000 than in subjects of phenotype A about 200 000 per red blood cell This is resulting in weaker intensity reactions for phenotypes A than for phenotypes A this explains the importance of the quality of the used reagents in the blood grouping The practical distinction between these two phenotypes is of no importance in the transfusion context One can although observe sometimes anti A or irregular natural anti H but this concerns most often of cold antibodies of a low titter without any consequences in the transfusion aspect Other rare phenotypes have also been described phenotypes cis AB phenotype B A and A B acquired phenotype A Other variants of group A As Aera Abantu Ax etc and more exceptionally of group B weakly reacting have been reported N B 2 Occasionally IgG hyper immune anti A or Anti B can be found in the serum of group O persons in response to stimulation by A and or B like substances present in the environment following pregnancy immunization or following the injection of some vaccines immune irregular antibodies Serious haemolytic reactions can occur when Group O whole blood containing anti A and or anti B haemolysins is used to transfuse non group persons cf dangerous page 6 As IgG anti A and or anti B can cross the placenta there are also involved in some haemolytic disease of newborn Technique for ABO grouping on card Beth Vincent method o
73. es may reflect a current transmissible infection for example anti HIV antibodies The latency characterizing some infections has also to be taken into account for two main reasons first the latent phase is often infectious secondly tests detecting viral antigens before antibodies become detectable are not always available for example HCV In many developing countries the prevalence of infectious diseases in the general population is high For that reason high rates of infected blood donors can be expected and proportionally a high rate of co infections Laboratory testing of blood donors for infectious diseases is therefore an essential phase in assessing blood safety On the other hand this high prevalence will also increase the risk of missing some detection because of the latency windows period and or sensitivity limitation This problem can be partially solved by a good donor s selection 090122 pg tropical haematology and blood transfusion 60 77 LEVELS OF SCREENING STRATEGY Laboratory testing should be considered at least at three levels 1 Screening tests applied to blood units be useful in the improvement of blood safety screening tests have to be applied systematically on all blood units in order to identify any potentially dangerous blood In this context a positive test result is by itself a sufficient reason to discard the blood unit from therapeutic use Therefore for the purpose of blood safety the
74. ess like a fresh patient sample Evaluation of the utility of the Hemocue 301 haemoglobinometer for blood donor screening L D Morris A Ossei Bimpong D McKeown D Roper and S M Lewis Vox sanguinis 2007 93 64 69 090122 pg tropical haematology and blood transfusion 18 77 DHT HAEMOGLOBINOMETER Developing Health Technology Ammonia technique PRINCIPLE Cuvette Glass window Interference filter Photodiode dl MATERIAL Whole blood is diluted 1 in 101 in a weak ammonia solution The red cells are haemolysed and the amount of haemoglobin is measured over a narrow spectral band The measurement of the optical density is carried out at a wavelength of 523 nm the crossing point of absorption curves of various haemoglobin forms which are thus detected with equal sensitivity This optical density is automatically converted to read out directly as haemoglobin concentration in g l on a liquid crystal display No zero adjustment or calibration is required and no calibrating solutions are needed Web site www dht online co uk LED Diode emitting a green light Blood collection equipment and supplies DHT haemoglobinometer test tubes Sahli pipette or 20 ul automatic pipette 2 ml graduated pipette or 2 0 ml dispenser safety device for pipetting 1 000 ml volumetric flask 10 mm light path cuvettes BLOOD COLLECTION Capillary or venous blood For venous blood dry anticoagulant should
75. esults Screening and confirmation tests are expensive and complex Malaria The best method for the diagnosis of malaria is to examine a thick blood film for parasites However since this method requires microscopic examination of each sample it is not suitable on a large scale Even in endemic areas the absence of parasites in a thick blood film will not say that the blood is not infected sensitivity limitation Antibody detection is not applicable in endemic countries In endemic areas a medical history seeking evidence of recent fever and illness is essential The use of therapeutic or prophylactic anti malarial drug for transfusion recipients has to be considered 090122 pg tropical haematology and blood transfusion 62 77 Chagas disease As far as blood transmission of Chagas disease by blood is concerned the problem is most serious in South America However migration of people from endemic to non endemic areas has resulted in the presence of infectious individuals in previously non endemic areas America No systematic screening is recommended except in areas where the disease is frequent Laboratory testing in the early phase of infection is by examination of tick blood film in order to detect the protozoa In the acute phase the parasite can be cultured from blood samples None of these two methods is applicable to the screening of blood donors Several serological tests are available for the detection of antibodies that are prod
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77. everal seconds by a beep After this beep the LCD turns off and the new zero level is stored in the device Check again the reading for BR1 Dirty or wet photocell windows Clean the glass of the photocell windows with alcohol on cotton swap stick Check again the reading for BR1 TEST METHOD Attention The precision of the measurement depends for a great part on the skill and proficiency of the technician in preparing the right concentration and volume of ammonia and blood and of the quality of the distilled water el o Bring 2 0 ml of the ammonia reagent in a test tube for each sample to be measured Check the pipette on its cleanliness if it s dry and if the point is not broken Measure 20 ul of capillary blood or well mixed venous blood Aspirate the blood a bit higher than the 20 ul line of the pipette Clean the blood on the outside of the pipette and adjust the volume with a clean absorbent paper Dispense this volume into the 2 ml of the ammonia diluting fluid by rinsing the pipette 3 times aspirate and blow out the pipette Stop the tube and mix The solution can be read immediately The colour is stable for 6 8 hours Transfer the patient s sample to a clean 10 mm light path cuvette Place the cuvette into the cuvette holder wait for the audible signal and read the displayed haemoglobin value Return the sample to its tube and allow the cuvette to drain e g invert it on a paper towel Caution if the audib
78. f blood e Test cannot be repeated or further tests cannot be performed when results are unexpected Capillary blood is obtained by puncturing the skin with a lancet In adults or children the best place will be on the 3 or 4 finger of the left hand at the side of the finger which is less sensitive than the tip In infants the best place will be the side of the heel or the big toe The puncture should not be too deep because of the risk of osteomyelitis Never collect blood from an infected finger of foot Never collect blood from an arm in which an intravenous infusion is being given haemodilution The puncture should be deep enough to result in free bleeding A free flow of blood is essential and only the gentlest squeezing is permissible risk of dilution with tissue fluid resulting in unreliable values 1 Prepare all supplies in advance sterile lancet 2 pieces of cotton wool one dry the other soaked with alcohol 70 and blood collection material Sahli pipette slide capillary tube 2 f possible ask the patient to clean his hands with soap and hot water vasodilatation next dry his hands thoroughly 3 Slightly massage the place where blood will be taken Make sure the puncture area is warm enough to allow the blood to flow freely If necessary soak the hand or foot of an infant in warm water prior to collecting a sample 4 Cleanse the puncture area with a cotton swab dipped in 70 96 alcohol let alcohol react for
79. fter applying the blood then read immediately Any delay in reading the test will cause an error as the blood stain will change colour becoming lighter and unreliable Starting from the lightest shade or darkest shade slide the blood stain up and down behind the apertures in the scale until you find the best colour match When reading keep the test strip close to the back of the scale to prevent any stray of light from entering the blood stain matches one of the shades of red exactly record the haemoglobin value If the colour lies between two shades on the scale record the mid value e fthere is any doubt between two shades record the lower value Example 14 g 100 ml Too light 12 g 100 ml Correct 10 g 100 ml Too dark 8 g 100 ml Too dark 6 g 100 ml Too dark 4 g 100 ml Too dark 090122 pg tropical haematology and blood transfusion 9 77 MAINTENANCE To clean the scale wipe the back side with a humid tissue alcohol 70 then with a dry tissue The scale should be cleaned at the end of each session and during the session if the surface becomes soiled during use The scale can be used for thousands of tests but to avoid deterioration of the colours always keep the booklet closed after use and never leave it exposed to direct sunlight It should be replaced periodically LOVIBOND METHOD Harrison s method PRINCIPLE The Harrison s method is a simplified version of a visual comparativ
80. gle aapea irultipli anligens wing recombinant proteins have been skled in new serologic teste to avoid nom specific crues actitity and increase the sensitivity of dhe HCV antshody tou The ED BIOLINE HCV test is immunocchromalcpmphie rapid test for the qualitative detection of antibodies specific do HV in human seram plana or whale blood The SD HEY bes contains a membrane which i pre coated antigen core NS amd NES on test hand eegion The proicin gollmid Angus conjugue and serum sample moves along the membrane edge a sat na peotzin A gold particle complex foems wich high degree of sensitivity and specificity This iex device has a letter of T and C Test Line and Control Line on the surface of iho cas Beh the Test Line and Control Line im result window ape visible before applying any samples The Conn Line js used for procedural control Contra line should always appear if the permen is performed property anal the te reagents od control Dine ane working 2 Materials provided 1 5D BIOLINE HCV test device 2 Assay Dituent for 3 Precautions The SD MOLINE HEV test devices should be stored at room temperature The test device ix sensitive bo uliry and as well aa to beat Perform ihe test immeikaichy after romong ihe teal device fron the fioi peach Do gio eee beyond expiration 4 Specimen c
81. he taking without storing in a refrigerator This system is meanly applied for familial blood donors Transfusion Cold Transfusion of a blood bag that has been stored in the fridge This system is applied with voluntary blood donors and blood bank 090122 pg tropical haematology and blood transfusion 77 FF
82. her evaluation See Limitations of the Procedure All reactive syphilis tests should be repeated using an alternative procedure 18 mm Qualitative Card Test Using Capillaries 1 Using a new capillary attach rubber bulb to capillary and remove 0 05 mL of specimen from blood collecting tube by allowing specimen to rise to measuring line on capillary taking care not to transfer cellular elements If desired a serologic pipette may be used but do not pipette by mouth 2 Place measured specimen onto circle of diagnostic test card by compressing rubber bulb while holding one finger over the hole in the bulb 3 Using a new stirrer broad end for each specimen spread to fill entire circle Discard stirrer Repeat procedure for number of specimens to be tested 4 Gently shake antigen dispensing bottle before use Holding in vertical position dispense several drops in dispensing bottle cap to make sure the needle passage is clear Place one free falling drop 20 G yellow hub needle onto each test area Do not restir mixing of antigen and specimen is accomplished during rotation Pick up the pre dropped antigen from bottle cap 5 Rotate for 8 min 30 s under humidifying cover on mechanical rotator at 100 2 rpm Following rotation to help differentiate Nonreactive from Minimally Reactive results a brief rotation and tilting of the card by hand 3 or 4 to and fro motions must be made Immediately read macroscopically in the wet s
83. icrofilaria can be transmitted in blood and may cause allergic reactions but the larvae are unable to develop further in the recipient and therefore filariasis cannot occur Wet blood examination may be used to detect infected blood Syphilis Testing for syphilis RPR or VDRL is recommended However a positive result does not always mean that the blood unit is infectious Besides retention of blood for 3 days at 4 C inactivates the infecting agent Although the risk of post transfusion is quite low the screening of infected donors may be used as a marker of individual risk of STD infections HIV on account of their sexual behaviour If the donor is to be informed all precautions should be taken the positive result should lead to performance of a confirmation test Borreliosis In areas with endemic recurrent fever a good donor selection is the best manner to exclude the risk The best method for diagnosis of recurrent fever is to examine a thick blood film for bacteria However since this method requires microscopic examination of each sample it is not suitable for a large scale Even in endemic areas the absence of bacteria in a thick blood film doesn t mean that the blood is not infectious sensitivity limitation Distribution of some infectious markers example CONGO 2005 nz 2500 Co infections Rwanda 18 As much as 12 of blood units collected must be discarded and not transfused Congo 134 As much as 16 96 of blo
84. ies False positives ACTIONS Verify if false positive This always concerns dangerous antibodies DO NOT TRANSFUSE FIND ANOTHER DONOR 090122 pg tropical haematology and blood transfusion 50 77 PROBLEMS AND INTERPRETATION OF THE MAJOR COMPATIBILITY TESTS False negative reactions in saline medium and or in LISS albumin medium Incorrect cell concentration involving a difficult lecture Incorrect wash of the red blood cells resulting in an inhibition of the Coombs serum Unclean material bringing along an inhibition of the Coombs serum Quality of the Coombs serum expired specificity activity titre Incorrect concentration of the reagents Coombs or LISS albumin Temperature and or incubation time incorrect POSITIVE REACTION IN SALINE MEDIUM False positives ABO error Presence of fibrin or bacterial contamination of the serum Haemolysis Concentration of NaCl incorrect of physiological water Centrifugation too fast Rouleaux gt lt agglutination check agglutination under a microscope see microphotograpy page 8 for interpretation Chronical infections rouleaux formation caused by plasmatic proteins increase rypanosomiasis infection presence of auto agglutinins and rouleaux formation Do not transfuse verify the blood group of the donor and receptor and find a compatible donor Execute an auto test see page 17 Not so useful in a district laboratory Exclus
85. ill seroconvert and probably more than 50 of the persons who seroconvert will develop chronic liver disease with possible serious complications 10 to 20 years after infection liver cirrhosis hepatic cellular carcinoma HBA Hepatitis A has rarely been associated with transfusion and the infection is clinically mild screening whole blood donors is not anticipated CMV The prevalence of the CMV antibody ranges from 50 to 80 of the population Blood contaminated with CMV can cause problems in neonates or immune compromised patients Potential problems in selected patient populations can be prevented by transfusing CMV negative blood Donor blood is not routine tested for CMV Tests are expensive and complex HTLV 1 2 No systematic screening recommended except in areas where the disease is frequent epidemiological data are incomplete but there are three known high prevalence areas Central and South America and the Caribbean southern Japan and sub Saharan Africa Risk of transmission in the United States at this time is said to be 1 in 641 000 The risk of developing HTLV 1 disease adult T cell leukaemia lymphoma or tropical spastic paraparesis is estimated to 1 or 2 per 1 000 HTLV 1 positive cases per year after an incubation period averaging 20 years The actual estimates are that about 60 96 of the persons receiving blood containing HTLV 1 will seroconvert The test EIA and particle agglutination assay gives many false positive r
86. into the comparator with the values towards the front of the instrument Rotate the disc until the lowest or the highest value CHECK THE CELL BEFORE USING IT Decontamination with hydrochloric solution next with 70 96 alcohol Is it dry and dust free and clean no lines nor finger prints Does the cover plate cover properly the base plate Are the figures 004 well readable 4 Fill the cell by capillarity with capillary blood If there are air bubbles in the cell restart from point 1 5 Clean the blood excess around the cell with a soft paper 6 Remove the clip 7 Place the cell containing the prepared sample in the right compartment Using a diffuse light facing southern daylight in the southern hemisphere rotate the disc until the closest colour matches with the sample Be quick to avoid desiccation or coagulation The Lovibond value will be shown in the window in the bottom right corner of the comparator Find a suitable place for the colour comparison During the day Facing a white surface southern daylight in the southern hemisphere During the night Facing a white surface illuminated by a white lamp not fluorescent light Direct sunlight or direct artificial light gives incorrect results 8 Convert the Lovibond value in g 100 ml cf conversion table 9 Decontaminate the Lovibond cell contact with 1 chlorine solution during 30 minutes 10 To avoid deterioration of the colours store immedia
87. ion of ABO error and exclusion of false positives with positive auto test presence of cold agglutinins cold agglutinins are cold auto antibodies which are active between 4 and 22 C Practically blood may be transfused at 37 C except if the indirect Coombs is positive Exclusion of ABO error and exclusion of false positives with negative auto test Presence of cold allo antibodies these cold allo antibodies are not dangerous from the point of view of transfusion on condition that they are not active at 37 C Practically blood may be transfused at 37 C except if the indirect Coombs in LISS albumin medium is positive POSITIVE REACTION IN INDIRECT COOMBS IN LISS ALBUMIN MEDIUM False positives 2 Presence of fibrin or bacterial contamination of the serum Quality of the Coombs serum adsorption of antibodies against human red blood cells Insufficient washed red blood cells or contaminated solution by quartz Haemolysis Concentration of NaCl incorrect of physiological water Centrifugation too fast Rouleaux gt lt agglutination check agglutination under a microscope see microphotograpy page 8 for interpretation Chronical infections rouleaux formation caused by plasmatic proteins increase Trypanosomiasis infection presence of auto agglutinins and rouleaux formation If a false positive can be excluded it always concerns a dangerous antibody from the point of view of transfusion DO NOT TRANSFUSE FIND ANOT
88. iris sufficiently closed to give good contrast focus the rulings of the chamber Then using the 40 x vb Tey objective count the leukocytes in the four large corner squares of the x chamber which have a surface of 1 mm Leukocytes appear as small t transparent cells with light blue nucleus Do not take dust or unlysed red cells for leukocytes Include the cells lying on the lines of two sides of each square in the count use all the time the same lines and exclude the cells on the two other sides 12 Calculate the number of leukocytes in 1 mm of blood by multiplying the number of leukocytes counted in the four large squares by 50 Explanation of calculation e Dilution factor 20 ul of blood 380 ul Turck solution gives a 20 x blood dilution 20 380 20 e Volume factor 4 large squares counted or 4 x 0 1 mm Thus division by 4 and multiplication by 10 will give the number of leukocytes in 1 mm of diluted blood 10 4 2 5 Global factor 50 20 x 2 5 Example Large square n 25 leukocytes Large square n 2 26 leukocytes Large square n 3 24 leukocytes Large square n 4 26 leukocytes Total for 4 larges squares 101 leukocytes Number of leukocytes par mm or ul of blood 101 x 50 5 050 REFERENCE RANGES Normal leukocyte number concentration by age group per mm of blood AGE 1day 15days 2months 6 months 2 years 6 years 12 years Adults _ 9 000 6 000 5 500 6 0
89. issue Check the cleanliness of the translucent plate of the comparator and if necessary clean it with a hydrochloric solution of 1 96 next with filtered water If non of these solutions are helping and if the values are systematically too low make a comparison between the new and the old disks the colour of the disks is degrading with light 4 Too high haemoglobin values gt The upper side of the cover plate and or the under side of the base plate are soiled with blood Take of the blood on both sides with a soft paper disinfectant used for the blood taking is stained e g Betadine Restart the measurement using Ethanol 70 96 Check the cleanliness of the translucent plate of the comparator and if necessary clean it with a hydrochloric solution of 1 96 next with filtered water Thee patient is icteric jaundice His upper conjunctives are yellow Find the closest colour and note the presence of jaundice in the report The patient is dehydrated provoking a haemoconcentration No solution note the dehydration in the answer CONVERSION TABLE LOVIBOND TO g 100 ml IT IS HELPFUL TO TAKE THE COLOR MATCH UNTIL SLIGHTLY BELOW THEN SLIGHTLY ABOVE IN ORDER TO FIND THE BEST COLOR MATCH Disc N 5 8 A light colour For low Haemoglobin values Disc N 5 8 B dark colour For high Haemoglobin values 090122 pg tropical haematology and blood transfusion 12 77 SAHLI MET
90. ized distilled water 4 Serum Nonreactive to syphilis in 0 9 saline required for diluting test specimens giving a Reactive result at the 1 16 dilution Also required is the necessary equipment and labware used in preparation storage and handling of serologic specimens Preliminary Preparations Review Warnings and Precautions and Specimen Collection and Preparation prior to performance of card tests When tests are to be performed the antigen suspension should be checked with controls of graded reactivity using the particular test procedure Only those antigens which give the prescribed reactions should be used Controls RPR Card antigen suspension and test specimens should be at room temperature when used Before use vigorously shake the ampule for 10 to 15 s to resuspend the antigen and disperse any carbon particles lodged in the neck of the ampule If any carbon should remain in the neck of the ampule after this shaking no additional effort should be made to dislodge it as this will only tend to produce a coarse antigen Check delivery of the needle by placing the needle firmly on a 1 mL pipet or syringe fill the pipet or syringe with antigen suspension and holding the pipet or syringe in a vertical position count the number of drops delivered in 0 5 mL The correct number of drops is given in the table opposite Attach the aisi el ai np needle to the tapered fitting on the dispensing DAME Needle Hub in 0 5 m bottle Be sure the
91. lb pipette for physiological water haematological centrifuge vacuum pump fridge microscope mirror slides cover slips 22mm x 22mm microscope Technique Take the serum of the donor O to be tested The serum must be used within 6 hours 2 Wash the known red blood cells A and B 3 times with physiological water 3 Dilute these red blood cells at 5 in physiological water 4 Take 2 haemolysis tubes 5 Bring in a tube 1 drop of red blood cells diluted to 5 and in the other 1 drop of red blood cells B diluted to 5 95 6 Add to each tube 9 drops of serum of the donor O to be tested 7 Incubate 2 hours at 37 C 8 Bring the red blood cells back in suspension by slightly tapping the haemolysis tubes 9 Centrifuge 1 minute at 1 000 RPM 10 Control the colour of the supernatant If the serum is yellow with sediment of red blood cells this donor O can be considered as universal If the serum is pink with limited sediment of red blood cells this donor O must be considered as dangerous donor His blood may only be transfused to a receptor of group O 090122 pg tropical haematology and blood transfusion 59 77 ANNEX 3 Screening for infectious diseases Blood transfusion is known to be an efficient way for transmitting infectious diseases It is therefore important to screen blood before its potential use in order to discard any blood unit capable of infecting a recipient Viral and related disease
92. le signal accompanying the photometry process ends before the cuvette is fully seated into the device the result may be wrong Wait a few seconds for the next measurement cycle to complete 090122 pg tropical haematology and blood transfusion 21 77 PACKED CELL VOLUME BY CENTRIFUGATION or HEMATOCRIT PRINCIPLE The hematocrit level or packed cell volume is a measure of the ratio of red cells to the total volume of whole blood plasma white blood cells and red blood cells and is expressed as a percentage Volume of red blood cells PCV x 1002 x 96 Volume of whole blood In the new units the PCV is expressed as a ratio litre litre the same formula but without a multiplication by 100 The blood is placed in a standard size capillary tube and centrifuged at high speed After centrifugation the volume occupied by the red cells is measured Because of a uniform bore of the capillary the length is directly proportional to the volume Length of red cell column mm POV x 1002 x 96 Length of total column mm EQUIPMENT AND SUPPLIES Blood collection equipments and supplies micro hematocrit centrifuge radius greater than 8 cm able to achieve maximum speed within 30 seconds and to maintain a centrifugal force of at least 10 000g for 5 minutes without exceeding a temperature of 45 C Disposable heparinised capillary tubes length 75 mm diamete
93. leles of the same genetic mono factorial unit A system is composed of the total of antigenic variants of membranous components Antigens are thus immunologically defined while the systems have a genetic definition 29 blood systems are described at this moment International Society of Blood transfusion Examples ABO Rhesus Duffy Kell Lewis P Diego Lutheran Chido Rodgers In the current minimal transfusion practice only the two most important systems are taken into account e The ABO system that represents the major obstacle in all transfusions by the presence of natural antibodies it is also a system of tissular histocompatibility antigens HLA e And the Rhesus system since the D antigen is the most immunogenic of all the blood group antigens The determination of blood groups is following the 4 x 2 rule gt Two technicians gt Two series of different reagents from different producers using different techniques tubes on slides in gel Two different techniques test and counter test or forward and reverse blood grouping Two samples taken at different moments This rule of 4 x 2 permits to give a definite card of blood group In the practice of a small laboratory these 4 x 2 rule is not applicable Without giving a blood group card it is possible to realise transfusions relatively sure based upon one determination by one person on one sample But then the compatibility test
94. less reliable are available for haemoglobin determination They are based on different principles which can be classified in 3 families 090122 pg tropical haematology and blood transfusion TITT 1 Techniques based on the red colour of blood without dilution nor haemolysis Talquist HCS Lovibond 2 Techniques based on the red colour of blood after haemolysis of the red blood cells DHT 3 Techniques based on the transformation of the haemoglobin Sahli Hemocue Drabkin The PCV Packed cell volume or hematocrit may also be used to screen for anaemia The choice between these techniques will be based on reliability repeatability precision accuracy price equipment needed level of technical difficulty staff training level REFERENCE VALUES The reference ranges for haemoglobin vary by age and sex as shown in the table below Haemoglobin 12 years 100 years Men Europe 13 2 17 3 12 years 100 years Women Europe 11 7 15 5 Reference ranges vary in different population and in different laboratories different techniques District laboratories should check the figures above for the technique in use with their nearest hematology reference laboratory 090122 pg tropical haematology and blood transfusion 8 77 HCS METHOD HAEMOGLOBIN COLOUR SCALE PRINCIPLE The intensity of the red colour of blood corresponds with the amount of haemoglobin The degree of anaemia can be visually as
95. logy and blood transfusion 49 77 TUBE 1 Saline medium 2 drops of red blood cells of the donor 3 times washed and diluted at 5 in physiological water 2 drops of serum of the receptor Incubate 5 minutes at room T 22 C Centrifuge 1 minute at 1 000 RPM 1009 Head and evaluate the result a test is positive if there is presence of agglutination or haemolysis total or partial of red blood cells Demonstration of antibodies complete of IgM type ABO Error Cold allo antibodies Cold auto antibodies ACTIONS CF MORE DETAILED EXPLANATIONS ON PAGE 6 EXCLUDE THE ABO ERROR BEFORE TRANSFUSION Verify ABO group of donor Verify ABO group of receptor Verify if false positive Perform an auto test with the receptor TUBE 2 Coombs indirect LISS albumin Medium 2 drops of red blood cells of the donor 3 times washed and diluted at 5 in physiological water 2 drops of serum of the receptor 4 drops of LISS DiaLISS Albumin Incubate 5 minutes at 37 C sensibilisation step Wash three times in physiological water pour off well the supernatant after the last wash Add 2 drops of Coombs polyvalent serum demonstration step Centrifuge 1 minute at 1 000 RPM 100g Read and evaluate the result a test is positive if there is presence of agglutination or haemolysis total or partial of red blood cells Demonstration of antibodies incomplete of IgG type Warm allo antibodies Warm auto antibod
96. lood group of the donor and receptor If absence of agglutination The blood bag can be transfused In case of doubt observe the slide under the microscope magnification 100x to distinguish better the agglutinations In order to make lecture easier incline slightly the slide before lecture under the microscope to see the red blood cells while they are moving cf microphotography page 8 for interpretation Possible problems in the rapid cross match False negative reactions weak red blood cell concentration resulting in a difficult lecture p e a very anaemic patient with not much red blood cells short reaction time False positive reactions Coagulation of the blood to be determined Presence of cold agglutinins in the blood to be determined Chronical infection in the donor or the receptor rouleaux phenomena by increased plasmatic proteins Infection of trypanosomiasis presence of auto agglutinins and rouleaux formation 090122 pg tropical haematology and blood transfusion 48 77 More complete tests Complete Major compatibility test in saline medium followed by an indirect Coombs test in LISS Albumin medium Antibody examination of the receptor versus erythrocytes of the donor IgM in Saline medium and IgG in LISS Albumin Sample Serum of the receptor Red blood cells of the donor blood taken on anticoagulant Reagents Polyvalent Coombs Serum directed against hu
97. man IgG and the fractions of the complement Diaclon Coombs serum Diamed LISS Diamed medium DiaLISS albumin LISS Low lonic Strength Solution Physiological water 0 9 p v in NaCl saline or saline solution Sodium chlorite NaCI 9g Distilled Water ua dre EU oe oa 1000 ml CONSERVATION a few months CONDITIONS Brown or white flask of 1000 ml Label physiological water or saline solution and note the date of preparation Material Haemolysis tubes in plastic 10mm x 75mm Plastic Pasteur pipettes Bulb pipettes for physiological water Haematological centrifuge Vacuum pump Water bath 37 C Microscope mirror slides microscope Technique 1 Take a sample of red blood cells of the donor in a haemolysis tube 2 Wash the red blood cells 3 times with physiological water 3 Dilute the red blood cells to 5 96 in physiological water 50 ul of the pellet of the washed red blood cells 950 ul physiological water Homogenise the tube well 4 Take 2 haemolysis tubes 14 The principle of the reaction is explained on page 6 5 This technique is only applicable for Diaclon Coombs antiserum Diamed associated with DiaLISS aloumin medium Diamed The technique may vary in function of the used reagents Always follow the particular instructions which are indicated in the user manual of the producer 090122 pg tropical haemato
98. most sensitive test should be recommended for the screening of blood units Laboratory testing may acquire a role of diagnosis when blood donors ask for the results obtained by analyzing their blood for infectious agents In this context the results of screening tests have to be confirmed by confirmatory methods with high specificity The results performed in a blood bank can also be used as indicators of effectiveness of the selection criteria applied to blood donors Indeed the rate of sample found to be reactive with the screening test will give information about the prevalence in the selected population and may help to revise and or reorient the criteria used in order to recruit and select safer blood donors PARAMETERS INFLUENCING THE SCREENING STRATEGY Before dealing with technical considerations one should keep in mind that environmental parameters as well as some intrinsic characteristics of the infectious agents themselves are likely to have an effect on the prevention of transmissible diseases in developing countries e ideally any blood for transfusion purposes should be tested for the presence of all those agents which are prevalent in a given population and if transmitted can cause serious disease for the recipient e Epidemiological data if available in the local population have to be taken into account e n endemic areas the probability for an adult recipient being infected prior to transfusion and to have
99. n PAR Ml bu Nia aedis dE Deb ee a bne am pubes ei Us ie HAA andes iV ate the digen bmm pd He panni md red pi Len ad Pix pihai mrs Li fa misis dig a pemcmdhaa carr Bua rw corps gg p Hem pitas dee CONTE NTE Abbott Hiv ii SerumPlasma At Git POI 20 Fits Damira HV Mane Wi Card Faadh 10 and HIV TI recenter aigan med page Gamed Aber Deimina LV 107 ror Pcia As Leu JT 17 Mi Penis Card 2 cents 30 di HI AS recomtmnart argen and cosed 2 5 mi ios 1022 11 I proxphate Prenseestreen Agena BOCES CES equa ma pa rekt iin ick inae agua Pagan Me 1702 01 Papers 10002 81 COTA Tales Me Hor socle en D urppeges Ween ordi an y SS AND POD CAL Tar for iro Cage Use ijd peacrcas be when hath per lanen a et odo ne 4 Wear giran a rex op aat inn uke Corvin handle eran im ansa where Purse a Vier m md ol of or aged Li
100. n of undesirable cells must be done A simple calculation taking in account the volume in which the cells are counted and the dilution will give the number of cells per mm or per ul in the initial biological liquid GENERAL MATERIALS Collection material Sahli pipette or automatic pipette 1 ml graduated pipette safety device for pipetting test tubes absorbent paper diluting liquid pencil counting chamber counting chamber cover glasses plastic Pasteur pipette hand tally counter lens paper microscope objective 10 X and 40 x chlorine solution COUNTING CHAMBERS Tiafa 0 100 mm L 0 0025 mm C B A B C Neubauer counting chamber C B A B C B A B Neubauer counting chamber sectional view Neubauer counting chamber sectional view The upperside of the counting chamber is divided in 5 parts by moats or wells C B A B and C The central part A is also divided in 2 by a transversal channel Each central part of the chamber contains a specially grid area with dimensions as shown in the figures depending on the type of counting chamber Counting chambers are so constructed that the distance between the underside of the cover glass and the surface of the chamber is constant 2 depth of the chamber The area and the depth of the counting will define a precise volume 090122 pg tropical haematology and blood transfusion 27 77 Characteristics of the most common counting chambers Small volume counting chamber
101. nd higher anti K titers were obtained by LAT than by both SP and MP Table 11 Therefore the main disadvantage af the two Polybrene methods is that small number of antibodies of the Kell blood group system will not be detected However because the frequency of in oriental populations 1 very low this is not clinically significant DISCUSSION Slide methods have generally been considered inferior to tube methods with regards the detection of clinically sig nificant alloantibodies This Is mainly due to the fact that in tube methods RBCs are forced close together by cen trifugal force which thus enhances hemagglutination However In the SP method RBCs are brought close tagetherby the action of the positively charged 0 1 percent Falybrene reagent resulting In nonspecific aggrega Heparin interferes with the test and 2 to 3 times af Palybrene should be added heparinized samples are used 2 or 3 drops of Polybrene The Polybrene induced aggregation can be quickly reversed by adding 1 drop af 4 mol L citrate resuspending solution leaving any specific antibody Induced agglutination Intact In this study the sensitivity and efficacy of SP In detecting alloantibodies were compared simultaneously with MP and LAT by testing antibodies that were encoun tered during antibody screening and cross matching in the Blood Bank Mackay Memorial Hospital antibodies 412 TRANSFUSION Volume 44 March 2004 REFERENCES
102. ns and cold allo antibodies As these two types of antibodies are not very dangerous in the context of transfusion and in practice one may transfuse the blood in both situations but at 37 C this test is not very useful on district laboratory level Sample Serum of the receptor Red blood cells of receptor blood taken on EDTA anticoagulant Reagents Physiological water or saline solution cf page 6 Material Plastic haemolysis tubes of 10mm x 75mm plastic Pasteur pipettes bulb pipette for physiological water haematological centrifuge vacuum pump fridge microscope mirror slides cover slips 22mm x 22mm microscope Technique Take a blood tube of the receptor on EDTA Wash the red blood cells 3 times with physiological water Dilute the red blood cells at 5 in physiological water Take 1 haemolysis tube E o ie Bring 2 drops of red blood cells of the receptor washed 3 times and diluted to 5 96 in physiological water point 3 Add 2 drops of serum of the receptor Incubate 5 minutes at 22 C centrifuge 1 minute at 1 000 RPM read and evaluate the results Incubate 20 minutes at 4 C centrifuge 1 minute at 1 000 RPM read and evaluate the results A test is positive if there are agglutinations Table4 Interpretations of an auto test 2e Interpretations Presence of cold agglutinins if major compatibility is also positive in saline medium Presence of cold agglutinins if maj
103. od units collected must be discarded and not transfused 18 N 250 090122 pg tropical haematology and blood transfusion 63 77 ANNEX 4 Compatibility test on slide Polybrene method IMME NONE WAT OLOGY Compatibility testing without a centrifuge ihe slide Polybrene method Lin BRC aris rapid sebo Seer Fe Ku a comp pre lo E erie DJ s psp s bee gn PS eria qii ETT AP ia nin Bn i RF rn Lon i i no Pal bae Din Paal ET UIEN ARNE LL THERE Dus Endpin maen eee rn in irem cri penes rom mp ey si Hue recen Eau bei den mri Tie ei ey od SD ku thar maja io RL nipan aom eee Mad cd ME arr er LAT CM La Ud de EE sen Eos ae NP Ta LII lj ARD DE Nl Ard CAPE RAM a ET PAL De me Jus Haie hen ien Dg Eer Eia mda Me dn sn ga Wi Dee Emi rn alt eom arn BAT eee ar ie bo jei rn ees od rennes ie ee pre Toa LURE DEF be eee ieu comm Py eis coup es ue in Cei papm E NON Mer aR sR A EN sud p TAA LL t KUUSET MIT rna Poh begren Bebo pese den eb im ad dn Bebe Kalden Wel Mee ied sql bagel Taten manere icc Mise Lies Venere Robien Laban ber Maes VST ME 2
104. odies with a maximal activity at 4 C IgM IgG IgM IN Coombs serum anti human globulin antibodies Obtained by immunisation of animals rabbit or goat The Coombs serum can be polyvalent directed against all human immunoglobulins IgM IgG IgA or mono specific directed against just one specific human antibody Direct Coombs Test or direct anti globulin test Test using the Coombs serum permitting to demonstrate non agglutinating antibodies IgG in vivo fixed on the red blood cells This test permits for instance determination of maternal allo antibodies fixed on the red blood cells of the newborn or of the foetus p i in case rhesus incompatibility Agglutinations Haemolytic disease of the newborn In vivo sensitization of the Blood sample AHG Serum erythrocytes with maternal 3x washed alloantibodies Globulins plasmatic proteins containing the antibodies or the immunoglobulins Haemolysin substance that is destroying the red blood cells Generally it concerns antibodies but this expression is also used for other substances p i the continence of the venin of certain serpents HLA Human leukocyte antigens The HLA system is a human major histocompatibility system A part located on the human genome from which the genes code especially for the major histocompatibility antigens which intervene in the control of the immune response and in the phenomena of transplantation rejection Humo
105. ollection and storage Ly whole bead Collect the whole blood using the suitable anti coagulant 2 serum plans Centrifupe whole blood 10 pet plasma or senum spesimen JIE specimens are pot immediately tested they shold be vefrigeried ai 2 degrees C Fer cinta E fo room temperature prior bo wer 4 Specimens conzaining precipitate muy yacht inconsistent tesi results Sach specimens mist be chartfied price marvin S The whole blood may be for testing inmnediaiely or mary be stored at 2 5 depre C up te three days 5 W arnings 1 Far Les vitro diagnose use only 2 De noi cat or smoke while handling speciem 5 Wear prutective gines while handling species Wash hands thoroughly afterwards 4 Avoid splashing or aercand formacion 5 Clean spelle thomughly an alisisfectant 6 Decontamina e and dispose of all specimens reaction and potestially contaminated as If they were infectious waste im a bioharan container TE Deo nost use test kir id the paschi 14 or tle seal is hokea 6 Procedure of the test 1 Rare teal device from the fex pouch and place on a flat dry surface 2 Add D al of seram plasma or whole bleed to the eunple well and them ml F drog of may diners Figure 1 As the keit bagina ter weer will see purple nerve acervos the winke ant the center of the iesi device 4 t
106. on cli au ei 1 Deecearemuia and dryers of ad ipee rm other oaan easier ais te mindere mah al T STORAGE The Aiton Deinen HEV Cats amd Cras mui be semed PMT unii rper abon dee Ks p repewwn bor ke rud s pera rias wa Paree need erected Ds Pw bris RUN I dtd iE ep seras hate St COE COLLECT EN ie Sarum Mauna acd hee Bee aber rete veren piana boc bp iat codariad n such a map l Ra BITE For bind ore sperren DTA eneen helers Has Fre arn Wein fice Lo De apr P eie ge Raters cnBecireg a berger teens pica an EDTA an x cea dry warfare 1 Chen ef Pen ce des Demn tac in the rani enn Row aul re ohie han cures guae Waren bhr hard ga r el wA emit Pla Wed win 5D ETEN Biel 5 lower thas the and appli geile leem ert me haan of fur wever liner rins tH Cade Tee ne oe Mid talit a i Capilarp Tubes F en Ruben das Pred beret n spera desi bn veered st SAP bti n v mn CSL Steg an deye mon Fun 1 dapi Her spear be boven or coder eee Ded och bey werspursders dash D
107. ontext of a blood bank If a blood bank doesn t have enough voluntary blood donors they can ask the family of a transfused patient to replace one or more transfused blood units In this case it is not the familial blood that is given to the patient but a bag of stored blood This type op donor permits to reconstitute the blood stock in the context of a non self sufficient blood bank with voluntary non remunerated donations Centrifuge for blood bank Rotor tubes and speed adapted for wash of red blood cells and for centrifugation for reading of agglutinations Examples model Immufuge ll de Baxter model Diacent 12 de Diamed There also exist automatic red blood cell washers of the type Diacent CW de DiamedO These washers permit to make the work easier and to reduce duration compatibility test at the price of a moderate fragile instrument 090122 pg tropical haematology and blood transfusion 75 77 Diacent cellwash Diacent 12 Immufuge 11 Cold agglutinins antibodies found in some persons These antibodies agglutinate the own red blood cells of the subject but only at low temperature maximal agglutination at 4 C weaker agglutination at 22 C These antibodies are not active at 37 C It always concerns about IgM They are meanly directed against and antigens Complete antibodies also called agglutinins Some antibodies of the IgM class can agglutinate erythrocytes directly in a saline medium They are cold antib
108. ood by venipuncture into a tube containing anticoagulant such as EDTA heparin potassium oxalate potassium sequestrene or sodium fluoride EDTA and heparin have the advantage of not being critical with respect to concentration as little as 1 mL of blood in a tube normally used to collect 7 mL of blood produces satisfactory results With the other anticoagulants it is advisable to collect no less than one half a tube of blood Centrifuge as above Keep plasma in the original collecting tube and if stored store the specimen at 2 to 8 C Test specimen within 24 h of blood collection PROCEDURES AND RESULTS Materials Provided Various RPR Card Test kits are available see Availability which contain sufficient card antigen suspension to perform the specified number of daily control card and card tests and the required dispensing bottle dispensing needle cards and either capillaries stirrers or Dispenstirs devices Materials Required But Not Provided 1 Controls with established patterns of graded reactivity should be included in each day s testing to confirm optimal reactivity of the antigen See Availability for Macro Vue RPR 18 mm Circle Card Test Control Cards 2 A rotator 100 2 rpm circumscribing a circle 2 cm in diameter with automatic timer friction drive and a cover containing a moistened sponge or blotter 3 Saline 0 9 for use in quantitative testing Prepare by adding 900 mg dry sodium chloride ACS to 100 mL deion
109. or compatibility is also positive in saline medium Presence of cold allo antibodies if major compatibility is also positive in saline medium agglutination absence of agglutination 0901 22 pg tropical haematology and blood transfusion 53 77 Test of minor compatibility Examination of antibodies of the donor versus red blood cells of the receptor This test makes only sense for transfusions of blood in iso group presence of antibodies anti A and anti B in the serum of a person of group O This test is only rarely executed in routine in a small laboratory Taken into account that the minor test detects the antibodies of the donor and that these are much diluted in the circulation of the receptor this test is of restricted interest Of course in case of plasma transfusion the minor compatibility test is the most important The executing of the test is similar to the major compatibility but by inversing donor and receptor Washed red blood cells of the receptor are brought in contact with the serum of the donor 090122 pg tropical haematology and blood transfusion 54 77 Table 5 Comparison of different major compatibility tests useful on district laboratory level Type of compatibility test Rapid Cross match Improved Cross match Major Compatibility in saline medium Polybrene Method on slide cf article in annex 4 page 6 Major compatibility in saline medium Coombs indi
110. ors and maybe the WHO in the future use 8 g 100 ml as cut off value for severe anaemia hematocrit below 24 Fausse an m ie h m od ilu tion Fig 1 True and false anaemia due to haemodilution In some circumstances the reduction of hemogram values is related to a haemodilution caused by plasma excess resulting in a false anemia pregnancy splenomegaly heart insufficiency monoclonal immunoglobulines especially IgM Haemoglobin is the most important part of the red blood cell It is the red pigment that gives the colour to the red blood cells It carries oxygen and carbon dioxide Each molecule of haemoglobin contains four linked polypeptide globin chains and four haem groups Fig 2 Haem is an iron containing porphyrin pigment which is the oxygen carrying part of the haemoglobin molecule Oxygen binds reversibly with ferrous ions Fe contained in each haem group More than 97 of normal adult haemoglobin is Hb A having 2 alpha chains and 2 beta chains Up to 3 5 is consisting of 2 alpha chains and 2 delta chains 450 Less than 1 96 is HbF foetal composed of 2 alpha chains and 2 gamma chains HbF is the predominant haemoglobin in foetus untill 3 6 months of life Fig 2 Haemoglobin structure Haemoglobin A 2 3 DPG 2 3 diphosphoglycerate The measurement of haemoglobin is important for the diagnosis of the severity of anaemia Different techniques more or
111. r 1 5 mm spirit lamp or clay sealant or plasticine reference chart BLOOD COLLECTION Capillary or venous blood For venous blood dry anticoagulant should be used to avoid dilution EDTA di potassium salt or heparin is recommended In this case plain capillary tubes should be used Since the hematocrit increases in function of the conservation time the examination must be executed within 6 hours METHOD 1 Fill about three quarters of either A plain capillary with well mixed EDTA anticoagulated blood or A heparinised capillary with capillary blood 2 Seal by heath the unfilled end using a small flame from a spirit lamp or seal the unfilled end using a sealant material 3 Carefully locate the filled capillary in one of the numbered slots of the micro hematocrit rotor with the sealed end against the rim gasket Write the number of the slot on the patient s form 4 Balance the diametrically opposite slot with a capillary and centrifuge at high speed for 5 minutes 090122 pg tropical haematology and blood transfusion 22 77 5 After centrifugation the capillary tube will show 3 layers gt Atthe top a column of plasma P gt n the middle a very thin white layer Buffy coat of white cells and platelets GB gt Atthe bottom a column of red cells GR Using a reference chart Line up the bottom of the red cells at the zero mark Slide the capillary tube along the scale until the top level of plasma
112. r forward ABO grouping Approximate average percentages Marked differences can occur between ethnic groups 090122 pg tropical haematology and blood transfusion 41 77 The determination of the ABO group reposes on demonstration of red blood cell surface antigens Therefore Known sera directed against these antigens are used These serums agglutinate the red blood cells possessing antigens against which they are directed There also exists the inverse determination reverse blood grouping which demonstrates antibodies that are present in the serum known erythrocytes are then used The serum assay is used as a confirmation of the red blood cell assay x e Example Serum Red blood cells Agglutination Anti A A Samples Receptor blood obtained by capillary sampling or venous blood on EDTA Reagents Anti A anti B anti AB humans Diamed Diaclon for slide method Blood group cart or on a glass slide or on an opaline plate timer Technique The manufacture s instructions must be followed Put on a card 2 or 3 drops of receptor blood Near to each blood drop depose a drop of each anti serum anti A anti B anti AB 1 2 3 Mixthe blood with the anti serum with the bottom of a tube 4 Tilt the slide during 1 minute 5 Read and note the result of the agglutinations immediately Anti AB Group ABO A agglutination mostly very on absence of agglutination Agglutin
113. r high temperatures and humidity If the 2 first model Band and 201 use the principle explained above the model 301 use a different analytical method The measurement takes place in the analyzer which measures the absorbance of whole blood at a Hb HbO2 isobestic point The analyzer measures at two wavelengths 506 and 880 nm in order to compensate for turbidity In fact it s an automatised version of the Lovibond technic B Hemoglahin EMQUE Hb 207 vina Hemocue B Hemocue 2014 Hemocue 301 Microcuvettes B and 201 are to be stored at room temperature 15 30 C The reagents contained within the HemoCue microcuvettes B and 201 are moisture sensitive Recap vial immediately after removing cuvettes and do not remove desiccant from the vial The microcuvettes 301 are to be stored at 10 40 C 50 104 F Once the seal of the vial is broken the microcuvettes are stable for 3 months An unopened vial of microcuvettes can be stored for a shorter period of time 6 weeks between 18 50 C HemoCue Hb 301 Analyzer is only to be used with correspondent HemoCue Hb microcuvettes HemoCue B HB Photometer Hemoglobin Controls HYC84665 3x3 ml 1 low 1 normal 1 high HemoCue B HB Photometer Hemoglobin Controls Features a 2 year expiration from date of manufacture at 2 8 C 60 day open vial stability at 2 8 C and a 30 day open vial stability at room temperature Utilizes stabilized whole human red cells which proc
114. r technical expertise in performing the evaluation tests and all colleagues and friends ipmolred in Transfusion Medicine This work would not hare been possible without the previous work of Richard Ercadberry SLIDE POL YBREHE METHOD Lin M Proadberry EE Inomucobematology in Taiwan Trarefusion Med Rey 1212 Broadberry RE Lin M The incidence and significance ofanti Mi in Talen Transfusion 19934 34 348 52 Ferd 05 Blood group phenotypes in Vietnam personal communication 2002 Okubo Pretrarefusion testing with special reference to blood groups in lapanese Ishiyaku Publications 1991 Lin M Wang CL Chen F5 Ho LH Fatal hemalytic traresfusion reaction due ro anti Ei in a patient Immunaobematolcgs 2003 13 18 21 64 77 ANNEX 5 Indicative price list Diamed blood grouping nttp vww diamed ch Reference Price Number of Diamed 09 2005 tests Coombs serum polyvalent anti IgG rabbit anti C3d monoclonal Diaclon 10 mi 407140 green LISS modified for red cell suspension Dialiss Liss albumin 10 ml 106510 50 Anti A or anti B blood grouping monoclonal IgM Diaclon 100810 for slide and tube test 10 ml 8 5 200 Anti AB blood grouping monoclonal IgM Diaclon 10 ml 100910 for slide and tube test Anti D blood grouping monoclonal IgG and IgM antibodies D VI Diaclon 101070 10 ml for slide and tube test Number of Package Bie feasible tests Hematological c
115. ral immunity Mechanism of defence of an organism involving antibodies 090122 pg tropical haematology and blood transfusion 76 77 Indirect Coombs Test or indirect anti globulin test Test using the Coombs serum permitting to demonstrate the presence of non agglutinating antibodies in serum IgG It is used p i for the major compatibility test Incomplete antibodies IgG their fixation on the membrane of the red blood cells is not sufficient to provoke agglutination These are warm antibodies with a maximal activity at 37 C Their detection is based upon the use of artificial techniques which permit to bring red blood cells together This is provided for example by bovine albumin photolytic enzymes Coombs serum IgG Irregular antibodies appearing after immunisation in certain subjects when the corresponding antigen does not exist on the surface of their red blood cells Iso antibodies antibodies developed by an organism as an answer to an antigen from another individual of the same species synonym for Allo antibody LISS albumin medium solution with Low lonic Strength enhancing the fixation of antibodies on the erythrocytes associated with macromolecules albumins which increase the dielectric constant of the medium decreasing so the rejection of the red blood cells This medium increases thus the possibilities of the reactions between red blood cells and antibodies It is associated with the Coombs
116. rces HIV In 2005 the WHO estimated that 5 of HIV infections in Africa might be caused by transfusion Therefore HIV screening is mandatory A single positive screening test result is sufficient to decide to discard the blood unit If the donor is to be informed all precautions should be taken the positive result should lead to performance of alternative tests according the adapted strategy of the prevalence Sensitive specific and rather cheep rapid tests are available detection of antibodies and or antibodies and antigens HBV Hepatitis B is an important transfusion hazard since it is established that blood infected with hepatitis B virus is infectious in almost 100 of cases In developing countries the rate of people infected with HBV is most of the time very high and may reach 90 in adults It is essential that every blood unit should be screened for HBsAg for the following reasons a great number of transfusion indications refers to paediatric patients who have not been immunized besides the consequences of transfusing blood infected with hepatitis B to immunized individuals are not known HCV Few epidemiological data are available for developing countries Screening for HCV antibodies is 2 times more expensive than for HIV HCV is responsible for more than 90 of post transfusion hepatitis if HBV has been excluded European data Estimates are that 80 of the persons receiving a transfusion with blood infected with HCV w
117. reaches the 100 mark The line passing through the top of the red cell column will indicate the packed cell volume Do not include the buffy coat as part of the red cell level in the reading After reading discard safety the capillary tube HEMARKS Other information from the PCV Plasma from normal blood appears straw coloured In iron deficiency it appears colourless When it contains an increased amount of bilirubin it will appear abnormally yellow If the plasma is pink red this indicates a haemolysed sample a new blood sample should be tested In thalassaemia major the red cells column appears dark red When white cell numbers are significantly increased 20 000 mm this will be reflected in an increase in the volume of the buffy coat layer The microscopical examination of the dividing line between the blood cells and the plasma is used for microfilaria or trypanosome detection Woo technique REFERENCE VALUES In a similar way to haemoglobin levels PCV values vary according to age gender and altitude PCV 96 international units Children at birth 50 to 58 0 50 to 0 58 35 to 40 0 35 to 0 40 31 to 36 0 31 to 0 36 33 to 37 0 33 to 0 37 36 to 45 0 36 to 0 45 42 to 49 0 42 to 0 49 i Reference ranges vary in different population and in different laboratories different techniques District laboratories should check the above figures for the technique in use with their nearest hematology reference laboratory
118. reas as this may result in an oily deposit and improper test results When spreading specimen within confines of test areas avoid scratching the card with the Dispenstirs device or stirrer If the specimen does not spread to the outer perimeter of test area use another test area of card Dispenstirs and Capillaries In performing the Card Tests a Dispenstirs device 18 mm Circle qualitative test only or capillary may be used to transfer the specimen to the card surface A new Dispenstirs device or capillary must be used for each test specimen When transferring from the collecting tube the specimen must not be drawn up into the rubber bulb attached to the capillary as this will cause incorrect readings on subsequent tests Needles To maintain clear passage for accurate drop delivery upon completion of the tests remove the needle from the dispensing bottle and rinse the needle with deionized distilled water Do not wipe the needle since this will remove the silicone coating and may affect the accuracy of the drop of antigen being dispensed Reading of Card Test Results Read immediately following rotation in the wet state under a high intensity incandescent lamp or strong daylight Rotation The recommended speed for mechanical rotation is 100 2 rpm The rotator should circumscribe a circle approximately two centimeters in diameter in the horizontal plane A moistened humidifying cover should be used to prevent drying of test specimens during
119. rect in albumin medium Major compatibility in saline medium indirect Coombs in LISS albumin medium Mix on a slide a drop of whole blood of the donor and a drop of whole blood of the receptor Observe eventual agglutination of the red blood cells Mix on a slide or in a tube a drop of whole blood of the donor a drop of serum of receptor Observe eventual agglutination of red blood cells Mix in a tube 2 drops of washed red blood cells of donor 2 drops of serum of the receptor Incubate 5 minutes at room T 22 C next centrifuge 1 minute at 1 000 rpm 100 Observe eventual agglutination of the red blood cells On a slide 1 drop of washed and diluted at 20 blood of the donor 2 drops of serum of the receptor 3 drops of a medium with weak ionic strength Mix for 1 minute next add 1 drop of Polybrene mix and observe eventual agglutination of red blood cells Rather complex see protocol compatibility in LISS albumin medium page 6 but when using albumin instead of LISS albumin the incubation time has to be increased Rather complex see protocol page 6 Advantages Detection of ABO errors and irregular antibodies of IgM type Fast 2 minutes Easy Feasible on the bed side of the patient Neither equipment nor electricity needed Low costs Detection of ABO errors and irregular antibodies of IgM type Also applicable for transfusions of non iso groups Moderate fast 10 minu
120. ripa Hag UF HUM E Paster CEE 37121 COLIS Eru HIV Ponie a aupra qp Tanta HI Rubrypes AG 721 2213223 Tritel Mal Teibsd Group O u Hz 0008 Mdb Tritel Tribes F Bu mimi 1000 Be ores A crab os vergen wnole waeren erre dje marre Me TM tus ef ue bcd npscinent warm colepisd ig vegan arai Tabla Fairis Come tion rl Abos pees pisei Paces ee pene Eland arj Pered penam anu Plu urbe of Paren pe men waned Hee Sai 102 Dinh Dooce Flare Toi dO Withee cod i UE ar Panan wee Pien uns FR 66 77 ANNEX 7 HBsAg rapid test example E Ure Saeco KC uae Lexur bern i M keverd maven DD Dua LM La babe des vul d dotacgr he dir garanda cas MUCH re pat eend tej ports ib eror d Ahi eerie rare BEL um OOAD pi RA dt ipid heehee vimm iras da icf Cards Be Sur h diirai e polar ln C kid Ma iar ja pete tn rn ree an Laga Lars a i jm Er ET EXPLICATION DU TEST Lies agen ce T Ale pond alan pour be beperken de Aap On dara im im bedient eem reor ape er py Agi i
121. rmined the approach of the haemolysin compatibility tests is therefore most important The aim of the compatibility tests is to prevent an immunological transfusion reaction by demonstrating the incompatibility between donor and receptor It permits thus to assure to the receptor the benefit of a transfusion with reduced immunological risk In case of a positive compatibility test presence of haemolysin the search for compatible blood will not be easy Without knowing neither the antigen or the concerning antigens neither the principal donor s blood group finding compatible blood will just depend on the perseverance in the search of a donor and in a great part of luck In this notes only the rapid cross match and the major compatibility in saline medium associated with the indirect Coombs in LISS albumin medium will be practiced The majority of the considered techniques in a district laboratory are resumed in table 5 page 19 and 6 page 20 In this table the most important advantages but also the most important inconveniences are considered for each technique 090122 pg tropical haematology and blood transfusion 47 77 Rapid test rapid cross match To avoid confusion errors of the patient it is recommended to do an ultimate bed side control of the ill person in order to detect the ABO errors In basic laboratory conditions this minimal compatibility test can be performed in the laboratory as a compatibility test Neverthele
122. rs in southeast Asia are negative then ihe incidence of anti E would be expected to be very rare This is Indeed the case and dur ing the past 20 years In Tatwan only one antibody against an antigen of the Kell blood group system has been found anti Eu In a Fo person Therefore the Implementation af a sensitive procedure for the detection of antibodies to antigens of the Kell blood group system would appear to be of low priority In routine compatibility procedures for southeast Asia SP 15 extremely rapid about 5 min cost effective reagents for the test can be prepared simply and in house and 18 easy to perform Personnel require only 1 day s training to perform the test with confidence There fore the introduction af SP in countries with limited resources and especially In countries where pretransfi slan testing ls limited to ABO grouping will help signifi canth to Improve transfusion safety In such countries many antibodies of clinical significance which until now have been undetected can now be detected Transfusion services In developing countries not only lack centrifuges but also lack finances for purchasing the reagents and training new staff In such situations SP is the method of choice for routine pretranstusion testing as to Improve patient safety a T 5 1 B 2 1 2 0 1 b 0 ab S ma ACKNOWLEDGMENTS The author thanks Ms Y S Chan from Memoria Hospital for he
123. rule o iso group exception Blood grouping ABO grouping Forward blood grouping Hhesus grouping Limited to antigen D on slide ABO grouping Reverse blood grouping ABO grouping in tube Rhesus grouping in tube Major compatibility test in saline medium associated with an indirect Coombs in LISS albumin medium Minor compatibility test Compatibility test Rapid Cross Match Auto tests 0901 22 pg tropical haematology and blood transfusion 57 77 ANNEX 1 Wash of the red blood cells The purpose of the wash of the red blood cells is to eliminate all the plasmatic antibodies which are free or non specifically fixed on the surface of the red blood cells Sample Blood taken on anticoagulant EDTA Reagents Physiological water or saline solution cf preparation page 6 Material Plastic haemolysis tubes 10mm x 75mm Plastic pipettes Pasteur Bulb pipette for physiological water Haematological centrifuge Vacuum pump Technique 1 Centrifuge sample 5 minutes at 3000 RPM 2 Aspire the plasma with a vacuum pump or take off with a Pasteur pipette 3 Bring 1 volume of the cell clot in the haemolysis tube 4 Add at least 10 volumes of physiological water to the red blood cells clot with a bulb pipette use the pressure of the pipette to bring the red blood cells in suspension 5 Centrifuge the tube 5 minutes at 3000 RPM 6 Aspire the plasma with a vacuum pump or take off with a Pas
124. s am 0 in du oun runs etum ag ol code Pass Fre alus beed wp noie crdbertesd bra Engin shed be levied eere NEET The deiei eni me bt cord can ds mre beet ered benee db ie d le WOTE of he Ham crudis sva vert Bom the wider of fhe Beah be parer ee fit migen ete pau ne des el cl eur nca Peers ma poctectiws nil cree zach iai s piam Lampes dep raris Iperen pepeema Le he campis pad debe ny Per arcam s rendue menen d 1 musa up be D matos and rad Seuil 2 Fue slede reren m cl campis precium paeem 1n thee ten s pus markar Ig em areae a gerit h Was cm emus Peur apply ee Ope Conte ibe r memmun el 15 men fue ia aeu raad er F whole blood Appey 69 pd 08 ample rp capillary Per pas dep the aera vember D Was fete alrtarbed imu the sample ped then app g iw dip ol Chase Buffer tre sample pad i Was rarus d qi menus up rerba ars regi Paus anten i H das PU ee Lend Corer H coeno ben Aci fura red By COMMON ine taut stam enal and the ampla shoul
125. s Hepatitis B C A D E G VHA VHD VHG VGB C HIV 1 2 Human Immunodeficiency Virus HTLV 1 2 Human T Lymphocytotrophic Virus CMV Cytomegalovirus EBV Epstein Barr virus TTV TT virus HHV 6 HHV 8 human herpes virus type 6 and 8 SEN V SEN virus HPB19 human parvovirus Creutzfeld Jacob disease and other prions Hemorrhagic fevers e Parasitical disease Malaria Leishmaniasis Toxoplasmosis Chagas disease African trypanosomiasis Babesiosis Microfilaria Bacterial diseases e Syphilis e Borreliosis e Brucellosis e Bacterial contamination of blood products This is another often observed risk disorder directly associated with blood transfusion Most commonly associated with contamination during blood collection or during handling of blood products and on occasion associated with bacterial infection of the donor it is sometimes recognizable by obvious changes in the appearance of the blood product When grossly contaminated blood appears haemolysed and dark in colour Sometimes infectious agents can be detected directly in blood for example HBs antigen detection reflects directly an HBV infection More often blood will be analyzed in order to detect specific antibodies For some infectious diseases the presence of antibodies may reflect a past infection and does not mean that the blood is infectious hepatitis for example in other cases on contrary antibodi
126. s characteristic which is carried by the blood elements has an antigenic activity However by language restriction the expression blood groups is only used for the red blood cell groups but there exist also blood groups for platelets for polynuclear WBC lymphocytes and proteins The antigens of the blood groups are located on the membrane of the red blood cells either exclusively either also on other types of cells They can be proteins but most of the time they consists of glucides sugar complexes Glycoproteins glycolipids etc They are essentially known by their antigenic capacity their biological functions are often hardly known Transporters and membranous channels proteins assuring the molecule transport through the membrane of enzymes of structural proteins of the membrane skeleton of the cell of adhesion molecules or of membranous receptors proteins capable to link with a signal or informative molecule The immunology of blood groups is essentially circulating immunity with antibody and complement interaction Their study must thus consider the corresponding antigens and antibodies The classification is made on two levels 1 The first level consists of all the antigens of blood groups Up to now over 650 groups are described Examples Ag Ag B Ag D Ag 2 All these antigens are grouped in a second level in systems A blood group system is the total of antigens developed by the al
127. s are individuals of group O presenting a haemolysin of the ABO system Their blood must be reserved for iso groups transfusions thus for an O receptor Since a small laboratory doesn t have the possibility to detect the dangerous O donors it is very important to privilege as much as possible transfusions in iso groups A basic technique permitting to detect a part of dangerous O is although described on page 23 This difficult operational technique is rarely used on district level since dangerous O donors are rare and the preferred iso group policy COMPATIBLITY TESTS Moreover the ABO antibodies mostly natural and regular one can find other antibodies directed against non ABO erythrocyte antigens Generally they are irregular immune antibodies which take particular techniques to be demonstrated Their presence in the blood of an individual is mostly due to an immunisation against one or more antigens during a preceding blood transfusion or in women during pregnancy The risks depend on the immunogenecity of antigens by range of importance in the Rhesus system D E c e C the of the Kell system the Fy of the Duffy system the of the Kidd system do well all these antigens must be taken into account or at least the most immunogenic antigens before executing a transfusion This is obviously impossible even for a well equipped laboratory In isolated situations where only the ABO groups and the D of the Rhesus antigen are dete
128. seshoe S Clear blue Strands of fine abundant and al purple chromatin E part of width of granules the lobes Deep blue Rather thick and purple coarse L IMMATURE NON SEGMENTED NEUTROPHILS Band forms or S shaped small granules light purple or violet 12 15 Not always present SEGMENTED NEUTROPHILS Small granules 8 12 15 2 to 5 lobes Pink or pink mauve Usually a bi lobed EOSINOPHILS 12 15 nucleus Blue purple 22s coarse Very large well t Rather hick and BASOPHILS 11 13 P Blue purple coarse covered Light rose 5 polymorph Deep purple by granules Small in number 7 Left deviation of the Arneth formula an increase over 16 96 of non segmented neutrophils yet immature forms occurring in inflammations but also in stress conditions 2 to 5 segmented neutrophils are the major fraction of the neutrophils in a normal leukocyte type Right deviation of the Arneth formula in contrast with the left deviation where segmented cells are rarely seen this image shows hyper segmented neutrophils with 5 or more lobes A hyper segmentation is characteristic for megaloblastic anaemia In the early phase more than one neutrophil with 6 lobes per 100 granulocytes is found Many large uniform granules red orange 090122 pg tropical haematology and blood transfusion 36 77 CELL TYPE SI NUCLEUS CYTOPLASM CHROMATIN AGRANULOCYTES um FORM COLOR STRU
129. sessed by matching the colour of a drop of blood on special filter paper against a standardized colour chart The colour chart is developed to represent the colour range of normal to anaemic blood on filter paper 14 12 10 8 6 and 4 g 100 ml EQUIPMENT AND SUPPLIES Blood collection equipment and supplies Kit HCS booklet of 6 shades of red instructions for use dispenser of 200 special absorbent test strips in handy box Use only the special test strips that are provided by Copack since others may give inaccurate results Keep these test strips dry clean and protected from direct sunlight at any time 70 Alcohol BLOOD COLLECTION Capillary or venous blood For venous blood dry anticoagulant should be used to avoid dilution EDTA di potassium salt or heparin are recommended METHOD Find a suitable place a room which is well lit by daylight and or artificial light Avoid direct sunlight and marked shade Do not read the scale in your own shadow 1 Place a drop of blood on one end of the test strip so that it forms a spot which is large enough to spread beyond the area of an aperture in the scale about 1 cm in diameter le Correct amount of blood Too little blood The area of an Too much blood The spread will be sufficient to spread beyond the aperture in the scale will not be too thick and the blood will take too area of an aperture in the scale covered long to dry 2 Wait about 30 seconds a
130. should be considered unsatisfactory for testing Do not test specimens that are grossly hemolyzed contaminated or extremely turbid report as Specimen unsatisfactory for testing 1 EXPECTED VALUES AND PERFORMANCE CHARACTERISTICS RPR Card antigen suspension is tested for the established pattern of reactivity against reference antigen suspensions and meets the U S Centers for Disease Control and Prevention CDC product specifications for performing the RPR 18 mm Circle Card Tests These performance characteristics were established from a large number of papers which have appeared in the scientific literature from routine daily test performances in syphilis serology testing laboratories and are in conformity with CDC specifications Reported studies show the RPR Card Tests have adequate sensitivity and specificity in relation to clinical diagnosis and a reactivity level similar to that of the VDRL slide test 6 10 24 25 Heating of serum specimens at 56 C for 30 min has been shown to have no effect on reactivity 20 A qualitative comparison of 1104 simultaneously collected serum and EDTA plasma specimens was conducted using the Macro Vue RPR 18 mm Circle Card Test There was complete agreement in test results which included 134 reactive and 970 nonreactive pairs In other studies comparable results were found between plasma and serum pairs 306 specimens with RPR Card Tests both in qualitative and quantitative procedures 14 26 AVAILABILITY Cat No
131. ss it cannot replace the major compatibility test detection of IgM and IgG It is only useful for transfusions in iso groups presence of antibodies anti A and anti B in the blood of a person of group O This test has thus only a restricted capacity since it will only detect almost exclusively an ABO grouping error restricted detection of IgM in the context of an iso group transfusion Reagents Alcohol 70 Material Lancets slides needles Technique l Verify the identity of the patient to be transfused If he is able to confirm this information by the patient loudly spoken 2 Verify if the blood group of the patient matches the blood group of the pocket 3 Prepare an object slide 4 Disinfect with alcohol 70 the tubing of the blood pocket and the arm of the patient 5 Prick by means of a sterile lancet the finger of the receptor 6 Put a drop of blood of the receptor on the slide Prick by means of needle the tubing of the blood pocked In order to avoid contamination of the blood pocket there must be pricked between two knots or between two weldings 8 Put a blood drop of the tubing on the slide 9 Mix both blood drops with the corner of another slide 10 Move and tilt during at least one minute 11 Watch if any agglutination appears If agglutination is observed ABO incompatibility or presence of antibodies directed against red blood cells of IgM type DO NOT TRANSFUSE THE BLOOD BAG Check the b
132. tandard SIGMA HICN BS 3985 Merck BDH Example with Haemoglobin standard from SIGMA 090122 pg tropical haematology and blood transfusion 157 77 Reconstitute one vial of the standard by adding 50 0 ml of the Drabkin solution well Wait at least 30 minutes before use The standard must be stored at 2 8 C in the dark and is stable for at least for 6 months Prepare a calibration graph from this reference standard Plot the absorbance readings at 540 nm of different standard dilutions against their known concentrations of haemoglobin The curve is linear passing through the origin Dilution s example Tube Volume in ml Volume in ml final concentration N Drabkin solution diluted standard ing 1 4 0 0 m 2 3S5 2 05 2225 Low 3 v T 9 METHOD 1 Setthe spectrophotometer wavelength at 540 nm 2 Setup a series of labelled test tubes for blank and tests 3 Add 4 0 ml of the Drabkin solution to all tubes 4 Add 20 ul of whole blood sample to each labelled test tube 5 Rinse the pipette 3 times with the reagent 6 Mix well and allow to stand for at least 5 minutes at room temperature Attention the reaction can take until 30 minutes for a sample containing an increased proportion of carboxy haemoglobin 7 Read and record absorbance of each test or control versus the blank as the reference at 540 nm in the same instrument used for preparing the
133. tate under a high intensity 090122 pg tropical haematology and blood transfusion 70 77 incandescent lamp or strong daylight Report as Reactive Showing characteristic clumping ranging from slight but definite minimum to moderate to marked and intense Nonreactive Showing no clumping See the Reading Guide Note There are only two possible final reports with the Card Test Reactive or Nonreactive regardless of the degree of reactivity Reactive minimal to moderate showing slight but definite clumping is always reported as Reactive Slightly granular or rough reactions should be repeated using an alternative procedure For donor screening these tests may be reported as indeterminant pending further evaluation See Limitations of the Procedure All reactive syphilis tests should be repeated using an alternative procedure 18 mm Circle Quantitative Card Test 1 For each specimen to be tested place 0 05 mL of 0 996 saline onto circles numbered 2 to 5 A capillary red line or serological pipette 1 mL or less may be used DO NOT SPREAD SALINE 2 Using a capillary red line graduated at 0 05 mL to the tip with rubber bulb attached place 0 05 mL of specimen onto circle 1 3 Refill capillary to red line with test specimen and holding in a vertical position prepare serial two fold dilutions by drawing saline and test specimen mixture up and down capillary 5 to 6 times Avoid formation of bubbles Transfer 0 05 mL from circle
134. tely the disks in a box 10 Clean and decontaminate the base plate and the cover plate first with water then with 70 alcohol 11 For practical reasons it can be advised to mount the plates in advance so that no time will be lost in case of emergency SMALL PROBLEMS AND SOLUTIONS ALWAYS CHECK THE CONCORDANCE BETWEEN THE HAEMOGLOBIN VALUE AND THE COLOUR OF THE CONJONCTIVES 1 No value appears in the inner opening of the comparator The disk is placed back to front Take out the disk and turn it round 2 No colour corresponds with the colour of the blood of the patient The chosen disk does not correspond with the expected haemoglobin value Use the other disk Thee patient is icteric jaundice His upper conjunctives are yellow Find the closest colour and note the presence of jaundice in the report The blood colour is inferior to the minimum of the comparator Give as result than 20 96 or than 3 3 g 100 ml 3 The measured haemoglobin value seems to be too low Beware that the cover plate is placed in the correct way figures 004 readable If this is not the case restart the measurement with another plate 090122 pg tropical haematology and blood transfusion 11 77 Beware there no air bubbles between base and cover plate If this is not the case restart the measurement with another plate Check the cleanliness of the comparison disk and if necessary clean it with a soft t
135. tes Moderate easy Detection of ABO errors and of irregular antibodies of IgM type Also applicable for transfusions of non iso groups Detection of ABO errors and irregular antibodies of IgM type and of certain IgG Also applicable for transfusions of non iso groups Low costs Rather fast 30 minutes Detection of ABO errors and irregular antibodies of IgM and IgG type Also applicable for transfusions of non iso groups Detection of ABO errors of irregular antibodies of IgM and IgG type Also applicable for transfusions of non iso groups Rather fast 30 minutes High sensitivity High specificity 16 Une autre am lioration suppl mentaire est d utiliser des globules rouges lav s du donor ceci complique et rallonge le test pour un r sultat quivalent 77 Marie Lin Compatibility testing without a centrifuge the slide polybrene method Transfusion 2004 44 410 413 Inconveniences Only applicable for iso group transfusions Detects only IgM so almost exclusively ABO errors Detects only IgM so almost exclusively ABO errors Necessitates an electric centrifuge Necessitates a big enough quantity of receptor blood serum gt Small anaemic children Detects only IgM so almost exclusively ABO errors Necessitates a big enough quantity of receptor blood serum Small anaemic children Necessitates an electric centrifuge Slow 4 30 minutes Rather complex Bad detection of certain
136. teur pipette 7 Repeat two times steps 4 to 6 090122 pg tropical haematology and blood transfusion 58 77 ANNEX 2 Screening of dangerous O donors It can happen in case of emergency that it is necessary to transfuse O blood to a receptor A B or AB The plasma of certain O persons may contain an important quantity of antibodies anti A or more rarely anti B which may cause a reaction with the red blood cells A B or AB of the receptor These persons dangerous O donors must be detected and may not be considered as universal donors Blood of dangerous O donors may only be transfused to receptors of group O The screening of dangerous O donors is only possible in the context of a blood bank and should be systematically performed at the moment of the arrival of the pocket of the O blood in the fridge duration of the test Nevertheless for reason of rareness of dangerous O donors and the maximal use of iso group transfusion this test is little useful The fresh serum of an donor is incubated with small quantities of red blood cells and B If there are too many antibodies anti A or anti B these red blood cells will be haemolysed and the serum will be pink stained Sample Serum of the donor Known red blood cells A and B diluted at 5 in physiological water Reagents Physiological water or saline solution cf preparation page 6 Material Plastic haemolysis tubes of 10mm x 75mm plastic Pasteur pipettes bu
137. th coagulation factors example Heparin For most haematological tests Haemoglobin PVC WBC count blood group determination dipotassium EDTA is recommended Preparation of dipotassium EDTA tubes Bring 40 ul of a dipotassium EDTA solution 10 g 100 ml distilled water in 3 ml tube Leave the open tubes to dry at room temperature protect from dust Close when dry The correct amount of blood must be added to avoid blood cell changes 2 5 ml Excess EDTA causes shrinkage and degenerative changes lack of EDTA will not prevent the coagulation For measuring the ESR trisodium citrate is used to anticoagulate the blood 4 volumes of venous blood with 1 volume of trisodium citrate 32 g l DIFFERENCE BETWEEN SEDIMENTATION AND COAGULATION Without anticoagulant With anticoagulant Sedimentation or after centrifugation Coagulation Solidification Serum Plasma WBC Buffy coat WBC RBC RBC trapped ina fibrin structure Prothrombin gt Thrombin TE Gd Fibrinogen gt Stable fibrin SERUM PLASMA WITHOUT COAGULATION PROTEINS 090122 pg tropical haematology and blood transfusion 6 77 HAEMOGLOBIN DETERMINATION INTRODUCTION Anaemia is defined as having an amount of haemoglobin below reference values Anaemia is present when haemoglobin concentration falls below 11 g 100 ml hematocrit below 33 Anaemia is described as severe when the haemoglobin is below 7 g 100 ml hematocrit below 21 Some auth
138. tones which becomes visible during cell division mitosis or meiosis During the development of male and female reproductive cells a special type of cell division occurs the meiosis This reduces the number of chromosomes in the spermatozoid or the ovum to half the number haploid number found in normal body cells So when the ovum is fertilized by one spermatozoid the zygote which results contains the full diploid number of chromosomes 46 23 from the father and 23 from the mother The chromosomes from a same pair are homologous Genes are the small units strung along the length of chromosomes A gene is the factor at a particular point or locus on the chromosome which represents a hereditary characteristic Alternative or slightly contrasting forms of the gene are known as alleles Alleles are generally represented by a character in capital for the dominant allele and in small character for the recessive allele When the locus on a pair of homologous chromosomes is occupied by the same allele the person is homozygous for the particular gene characteristic homozygote ZZ or zz If however the alleles are different the person is said to be heterozygous for the particular gene characteristic heterozygote for instance 22 The alleles inherited for any particular characteristic can be dominant co dominant or recessive A dominant allele will always show itself if it is present whereas a recessive allele will only show itself if there is no
139. tter of availability Small volume counting chambers are better for liquids with a lot of cells red blood cells count in blood for example Big volume counting chambers are better for liquids with few cells white blood cells in CSF for example Medium volume counting chambers may be used for all kind of liquids The Neubauer double improved counting chamber is the most common type 0901 22 pg tropical haematology and blood transfusion 29 77 WHITE CELL COUNT IN BLOOD REAGENT Turck solution Fill a bottle with 96 ml of distilled water Add 3 ml concentrated glacial acetic acid CH3COOH and mix Add 1 ml gentian violet 1 w v 3 Caution acetic acid is a corrosive chemical with an irritating vapour Handle with care in well ventilated area or in a fume cupboard Never pour water in pure acetic acid Addition of a small quantity of water in acid produces enough heath to cause an explosion of the bottle amp This reagent is stable for at least months in a fridge BLOOD COLLECTION Capillary or venous blood For venous blood dry anticoagulant should be used to avoid dilution EDTA di potassium salt or heparin are recommended METHOD 1 Check the tip of the Sahli pipette Discard if broken volume error Check if the pipette is clean and dry Pipette 0 38 ml of Turck solution into a test tube using the 1 ml graduated pipette Label the tube with the patient s name and or number Draw venous or capillary
140. uced in 50 of acute phase patients and in 95 with chronic infection but their sensitivity and specificity remain questionable In some T cruzi endemic areas gentian violet is added to donor blood 125 mg 500 ml blood followed by storage at 2 8 C for 24 hours to kill the parasite Guidelines regarding the most appropriate test to use in a particular area should be obtained from the nearest Chagas disease reference laboratory African trypanosomiasis African trypanosomiasis can be transmitted when donor blood contains 7 6 gambiense or less probably T b rhodesiense It can occur in areas of high prevalence but very few instances have been reported Quality of the data The CATT test may be useful in endemic areas If donor is to be informed all precautions should be taken the positive result should lead to perform of confirmation tests Leishmaniasis Cases of transfusion associated leishmaniasis are growing each year world wide This is increasingly associated with patients who are positive for HIV Transfusion associated leishmaniasis requires that the parasites be present in the peripheral blood of the donor survive processing and storage in the blood bank and infect the recipient In endemic areas for visceral leishmaniasis where the population of potentially infected individuals may be much higher a serological screening process should be used These tests are expensive they take a long time and are a little bit complex M
141. uge Transfer 1500 Feb 2004 Transfer Capillary tubes heparinised 75 mm 200 tubes 4 80 Feb 2004 DHT Sealing paste for capillary tubes 6 4 Feb 2004 DHT LOVIBOND HAEMOGLOBINOMETER Comparator Lovibond 2000 456 22 Jan 2004 Transfer Special capillary chamber for Lovibond 53 33 Jan 2004 Transfer Colour standard disc 5 8 A lower haemoglobin 90 05 Jan 2004 Transfer Colour standard disc 5 8 B higher haemoglobin 161 98 Jan 2004 Transfer DRABKIN colorimetric determination e Colorimeter WPA C0700D wavelength range 400 700 nm 734 Feb 2004 DHT Total Haemoglobin kit sigma 525 A 1000 determination with haemoglobin standard 80 74 Dec 2003 TRANSPORT AND STORAGE 2 8 C Sahli pipette 1 34 Feb 2004 DHT Graduated glass pipette 5 ml 1 93 Feb 2004 VWR or dispenser 4 ml Ceramus 217 Feb 2004 VWR Devices for pipetting 2 92 Feb 2004 VWR Optical cuvettes 10 mm light path 100 cuvettes 4 58 Feb 2004 DHT Bottles Test tubes Test tubes rack HEMOCUE Hemocue B photometer with control cuvette 555 Feb 2004 Hemocue Disposable micro cuvettes Hemocue B 50 cuvettes 60 Feb 2004 Hemocue Hemocue 201 photometer with control cuvette 327 80 July 2006 MSF Supply Disposable micro cuvettes Hemocue 201 200 cuvettes 84 66 March 2007 MSF Supply Hemocue 301 photometer with control cuvette 350 00 July
142. us blood dry anticoagulant should be used to avoid dilution EDTA di potassium salt or heparin are recommended METHOD 1 Check the stability of the calibration Place the control cuvette into the cuvette older and push it into the measuring position The displayed value should not deviate more from the assigned value on the control cuvette card than 0 3 g 100 ml for our control cuvette N 0149 003 037 the value lies between 11 0 and 11 6 g 100 ml 2 Fill the disposable cuvette with the blood drop by touching the capillary tip of the cuvette with the blood drop 3 Be sure that the cuvette is entirely filled with blood If air bubbles are present discard the cuvette and fill a new disposable cuvette 4 Wipe off the excess of blood on the outside of the cuvette tip Make sure that no blood is drawn out of the cuvette in this procedure 5 Place the filled cuvette into the cuvette holder immediately and push it into the measuring position 6 After approximately 30 to 50 seconds the result is displayed in g 100 ml or g l or mmol l The filled cuvette should be analysed at least 10 minutes after it has been filled evaporation 090122 pg tropical haematology and blood transfusion 17 77 N B 3 different models are now commercialized HemoCue B et HemoCue 201 for normal working conditions temperature lt 30 C and low humidity and The Hb 301 system is optimized for use in primary care and designed fo

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