blood collection transport and processing

Blood collection transport and processing : 

Blood collection transport and processing Dr.Naseel Salim

General concepts : 

General concepts 1 appropriate collection technique .collect during haute phase .before antimicrobial therapy .Provide collection manual 2 specimen transport .within two hours Container should be leak proof

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3 specimen preservation Holding Medias Anticoagulants. To prevent clotting of specimens such as blood and bone marrow Eg :sodium polyanethol sulphonate 0.025%{w/v} Heparin for viral culture 4 Specimen storage Refrigerator 4 c Room 22 c Body temp 37c Freezer -20 to -70 c Csf kept in room temperature and serum for serological studies may frozen for upto 1 week -20 c

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CSF kept in room temperature and serum for serological studies may frozen fro up to 1 week in -20 c

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5An over view of blood collection transport and storage Container . blood culture media set or vacutainer tube with SPS Pt. preparation. Disinfect veni puncture site with 70% alcohol and betadine Special instructions Draw blood at febrile episodes . draw two sets from right and left arm . draw >20ml/set [adult] or 1-2 ml /set peadiatric depending on wt .. Transport . with in 2 hours Storage before processing at 37 c Media . blood culture bottle direct examination gram stain AO giemsa

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6specimen labeling Name Age Hospital number

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7Specimen requisition Name Age Sex Hospital number Collection date and time Ordering physician Exact nature and site of specimen history and Diagnosis Immunization history Current antimicrobial therapy

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8 rejection of unacceptable specimen information on the label does not match the information on the requisition .transported in improper temperature , medium Insufficient quantity Leaking If received in a fixative Specimen is dried up

Why we take blood samples for culture : 

Why we take blood samples for culture Microbial invasion cause Shock, multiple organ failure , dic death

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.positive blood cultures help to provide a clinical diagnosis , specific etiologic diagnosis for specific treatment with antimycrobials

General consideration : 

General consideration Successful recovery of organism Types of bacteraemia Specimen collection method blood volumes Number and timing Interpretation of results Type of patient population served by the lab

General consideration : 

General consideration Organisms commonly isolated from blood culture Staphylococcus aureus E coli Coagulase negative staphylococci Entecoccus spp Candida albicans Pseudomonas aerugenosa Klebsiella pneumonia Viridans streptococci Streptococcus pneumoniae Enterobacter cloacae Proteus spp Beta hemolytic streptococci Anaerobic bacteria – bacterioids and clostridium sp Plasmodium Trypanosome Babesia Epstien barr virus \cytomegalovirus H I V

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Types of bacteremia Transient Brushing teeth Instrumentation surgeries Continuous septic shock bacterial endocarditis typhoid fever brucellosis leptospirosis Intermittent abscess meningitis pneumonia pyogenic arthritis osteomyelitis

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types of blood steream infections intravascular infections extravascular infection

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intravascular infections That originate from the cardiovascular system infective endocarditis mycotic aneurisms suppurative thrombophlebitis intravenous catheter associated

Agents causing infective endocarditis : 

Agents causing infective endocarditis viridens streptococci enterococci streptococcus bovis staphylococcus aureus staphylococci (coagulase negative ) enterobacteriacea pseudomonas sp heamophilus aphrophilus HACEK YEAST

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Iv catheter : 1 catheter skin entry site 2migration of organism along the lumen to the tip Staph epidemidis Coagulase negative staphylococcui Staphylococcus aureus enterobacteriacea pseudomonas sp Candida sp Corynbacterium spp ther gram –ve rods

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extravascular infection portal of entry genitourinary surgical wound infection respiratory tract \biliary tract organism commonly associated Organism site… heamophilus influenza b meninges staphyloccus pneumonia lung neisseria meningitidis meninges Listeria meninges salmonella typhi intestine entering the blood through the lymphatic system

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clinical manifestations septiceamia fever / hypothermia chills hyper ventilation septic shock dic

Immuno compramised pts : 

Immuno compramised pts malignancy drugs aids

Detection of bacteremia : 

Detection of bacteremia Drawing blood for culture Principle Organisms found in circulating blood can be enriched in culture for isolation and further studies . Method Note: universal precaution require that phlebomists wear gloves for this procedure

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1 choose the vein to be drawn by touching the skin before it has been disinfected 2 using 70% alcohol ,cleanse the skin over the venipunture site in a circle approximately 5 cm in diameter rubbing vigorously allow to air dry . 3 starting in the centre of the circle apply 2% povidone iodine in ever widening circles until the entire circle has been saturated with iodine .allow the iodine to dry on the skin for at least 1 minute the timing is critical a watch or timer should be used . 4 if the site must be touched by the phlebotomist after preparation. the phlebotomist must disinfect the gloved finger used for palpation in identical fashion. 5 insert needle into the vein and withdraw blood . do not change needles before injucting blood into the culture bottle . 6 After the needle has been removed the site should be cleansed with 70% alcohol again because many patients are sensitive to iodine

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Standerdised blood collection system Instructions Guaze pads Alcohol prep pads Latex free tourniquet Vacutainer Blood culture media Gloves Swabs

Specimen volume : 

Specimen volume Adults direct relation ship between the volume and the yield Children calculated from body weight and total blood volume Number of blood culture Two or three b c adequate eg in pt with endocarditis who have not received antibiotics , a single blood cultureis positive in 90 to 95% and the second will establish the diagnosis for has received antibiotics three separate collection of 16-20 ml and additional one or two samples next day will detect most etiological agents

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Blood volume suggested for culture with wt

Timing of collection : 

Timing of collection Take two to three samples in one hour interval and transport immediately within 2 hours to the incubator

Miscellaneous matters : 

Miscellaneous matters Anticoagulants inoculate directly into the culture broth or into sterile blood collection tube with anticoagulant Sps sodium polyanethol sulphonate0.025%to0.05% Antiphagocytic Anticomplementary interfere Antimycrobial( aminoglycosides) Inhibits the growth of neisseria ,gardnella vaginalis,streptobacillus moniliformisand peptostreptococcus anearobius

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Dilution more blood and less mediunm 1:5 blood:medium blood culture media basic contain nutrient broth and anticoagulant brain heart infusion broth trypticase soy broth supplemented peptone thioglycolate broth columbia or brucella broth

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additives hypertonic broth adding sucrose mannitol or sorbose for cellwall deficient bacteria resin containing media will inactivate most antibiotics eg BACTEC SYSTEM and Bact/ALERT

culture technique : 

culture technique CONVENTIONAL BLOOD CULTURES Incubation condition ; the atmosphere in commercially prepared blood culture bottles is at a low oxidation reduction potential permitting growth of Facultative anaerobe Some anaerobes or Aerobes Detecting growth after 6 -18 hours of incubation gs and subculture Daily visual examination Culture negative bottles reincubated for 5 to 7 days

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SELF CONTAINED SUBCULTURE SYSTEM BD septi-check system biphasic blood culture medium 1 convensional blood culture broth bottle with attached chamber containing a slide coated with agar . sp media for fungi and mycobacteria also available

Lysis centrifugation system : 

Lysis centrifugation system

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Lysis centrifugation isolator commercially available Stoppered tube containing saponin to lyse blood cells Polypropylene glycol to decrease forming SPS anticoagulant EDTA to chelate calcium and inhibit complement cascade and coagulation Inert flurochemical to cushion and concentrate bacteria 30 minutes centrifugation the supernatant discarded sediment containing pathogen is vigorously vertexed and plated to the solid agar Benefits rapid detection of fungi The presence of actual colonies for direct identification and susceptibility test after initial incubation Quantify the colony forming units Limitation Greater plate contamination and decreased ablity to detect certain bacteria Streptococcus pneumoniae Listeria monocytogens Heamophilus influenza

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Instrument- based system Cost effective Fast And less labor

BACTEC system : 

BACTEC system Becton Dickson microbiology system 1 SEMIAUTOMATED,vials containg c14 labeeled substrate and measure the c14 in the gas which accumulated above the liquid medium 2BACTEC NR [NON RADIOACTIVE] Measure Co2 by infrared spectro photometry 3 BACTEC 9240 AND BACTEC 9120 Florescence to measure Co2 and continuous monitoring

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BacT/ALERT Measures Co2 derived pH changes by a colorimetric sensor. The sensor is separated from broth with a membrane that is only permeable to Co2 and dissolves in the water present in the matrix of sensor And free H ions are released which changes th pH and color in the sensor Blue to light green to yellow

ESP system . ESP culture system II : 

ESP system . ESP culture system II Detects the production of gas by monitoring changes in head space pressure by sensitive detector

handling positive blood cultures : 

handling positive blood cultures visual examination for growth gram stain subculture blood agar /chocolate agar /macConkey agar biochemical test antimicrobial susceblity 1 disc diffusion 2 broth dilution Interpretations

Special consideration : 

Special consideration Mycobacteria middle brook7H9+.05% SPS Bhib+.5%polysorbate Brucella trypticase soy broth Borrelia giemsa stain Leptospira fletchers medium Hacek

Thank you : 

Thank you